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2010; 12(2) : 103

INDIAN JOURNAL OFPRACTICAL PEDIATRICS

••••• IJPP is a quarterly subscription journal of the Indian Academy of Pediatricscommitted to presenting practical pediatric issues and managementupdates in a simple and clear manner

••••• Indexed in Excerpta Medica, CABI Publishing.

Vol.12 No.2 APR.-JUN.2010

Dr. K.Nedunchelian Dr. S. ThangaveluEditor-in-Chief Executive Editor

C O N T E N T S

TOPIC OF INTEREST - INBORN ERRORS OF METABOLISM

Evaluation and management of a sick infant with suspectedInborn Errors of Metabolism 109

- Ratnakumari TL

Prenatal diagnosis and newborn screening: Relevance in India 131

- Mamta Muranjan, Shruti Agarwal

Sample collection, suitability and interpretation in suspectedInborn Errors of Metabolism 148

- Ananth N. Rao, Minakshi kosh, Sabyasachi Ghosh,Shobha G, Suresh Kumar V.

Inborn Errors of Metabolism in infancy and childhood presentingwith metabolic acidosis 155

- Chitra Prasad, Rupar CA

Recurrent hypoglycemia and Inborn Errors of Metabolism 165

- Madhulika Kabra, Neerja Gupta

Inborn Errors of Metabolism presenting as hyperammonemiain neonates 173

- Shanmugasundaram R, Lakshmi V

Fatty Acid Oxidation disorders 181

- Thangavelu S

Journal Office and address for communications: Dr. K.Nedunchelian, Editor-in-Chief, Indian Journal ofPractical Pediatrics, 1A, Block II, Krsna Apartments, 50, Halls Road, Egmore, Chennai - 600 008. Tamil Nadu,India. Tel.No. : 044-28190032 E.mail : [email protected]

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Indian Journal of Practical Pediatrics 2010; 12(2) : 104

GENERAL ARTICLES

Mitochondrial DNA (mtDNA) and Diabetes Mellitus 184

- Biswajit Mohanty, Balalsubramanian J

Approach to anasarca 188

- Maiya PP, Sharanabasavesh M

DRUG PROFILE

Macrolides in Children 194

- Jeeson C. Unni

DERMATOLOGY

Drug Eruptions - An overview 202

- Anandan V

RADIOLOGIST TALKS TO YOU

Phakomatosis 207

- Vijayalakshmi G, Elavarasu E, Venkatesan MD

CASE STUDY

Incontinentia pigmenti with macrocephaly 210

- Gopal Subramoniam, Prabhu Thanga Marthandan

CLIPPINGS 172, 180, 187, 193, 201, 206

NEWS AND NOTES 164, 209

Published by Dr.K.Nedunchelian, Editor-in-Chief, IJPP, on behalf of Indian Academy of Pediatrics,from 1A, Block II, Krsna Apartments, 50, Halls Road, Egmore, Chennai - 600 008. Tamil Nadu, Indiaand printed by Mr. D. Ramanathan, at Alamu Printing Works, 9, Iyyah Street, Royapettah,Chennai - 14.

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* Part or whole of the material published in this issue may be reproduced withthe note "Acknowledgement" to "Indian Journal of Practical Pediatrics" without prior permission.

- Editorial Board

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2010; 12(2) : 109

INBORN ERRORS OF METABOLISM

* Professor of PediatricsPost Graduate Institute of Medical Sciences andResearch - ESI Corporation,K.K. Nagar, Chennai

EVALUATION AND MANAGEMENTOF A SICK INFANT WITHSUSPECTED INBORN ERROROF METABOLISM

*Ratnakumari TL

Abstract: Inborn Errors of Metabolism(IEM) arenot very uncommon. They present as great mimicsto common diseases of children with symptomssuch as tachypnea, apnea, convulsions anddehydration. In the newborn they mimic sepsiswith non specific symptoms and most often thepresentation can be acute and catastrophic,referred to as ‘metabolic distress’. Even thoughhundreds of IEM are described, making adiagnosis has been made simpler with advanceddiagnostic tools like Tandem Mass Spectrometry(TMS) and genetic mutation studies which arecurrently available in India. With a ‘stagedevaluation’ a diagnosis can be made and someof them can be treated effectively. This article isan attempt at giving a basic diagnostic approachfor the management of a sick child, suspected tohave IEM.

Keywords: IEM, Staged evaluation, Metabolicdistress, Heritable disorder

The last two to three decades have generateda seemingly explosive interest in the field of“Inborn Errors of Metabolism” that, it becomesabsolutely essential for the practicing pediatricianto know the basics of IEM. The ways of making

a diagnosis of the same with a structured andobjective approach should be the endeavour ofevery practitioner when he / she meets with adifficult neonate or child.

Though it is impossible for anyone toremember the specific symptoms or thecomplexities of every disorder under the realmof IEM, there lies a logic which forms the basicthread or network1 and by holding on to thatthread one can make a reasonable attempt at adiagnostic approach. The following basiccharacteristics help in making such an approachand one must keep the following points in mindwhile setting out to workup a suspected IEM.

1. There are some disorders in which theerrors in the biochemical pathway may affectonly one functional organ or anatomic systemand symptoms are exclusive to that system.

2. In others, the basic biochemical pathwaymay affect many systems in the body and hencethe presenting symptoms are diverse, yet therecould be an attempt at categorizing the diversesigns or symptoms.2

3. Biochemical abonormality may be adefect in intracellular trafficking.

When we come to the type of presentationit can be any one of the three types, a) intoxicationtype, b) energy deficiency type and c) storagetype. It can be explained by going through thebasic biochemistry with the following illustration(Fig.1).3

When a precursor “A” in the body is to beconverted to product “C” through product “B”

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A B CE

E

D D 1

Fig. 1 Basic biochemistry in IEM

Fig. 2 Maternal - fetal impact in IEM

with one helping enzyme in the pathway, theinadequacy or absence of that particularenzyme (E) results in one of three things -

1. Absence of product “C”

2. Excess of substance “A” and “B”

3. A new pathway taken by product “B” toproduce new products “D” and “D1”

The result is, there is a deficiency ofproduct “C”, an excess of products “A” and “B”and new products “D” and “D1” which areprobably not needed for the system and can betoxic.

With this illustration, we can understand thatthe clinical signs and symptoms depend upon thefunction of that particular protein / enzyme ofthat particular pathway. The clinical presentationalso depends upon whether the defect is confinedto one physiologic system or has a multivariaterole. If any one of the products cannot bedegraded by the absence of a degrading enzyme,a) it will be stored (eg. Lysosomal storagedisorders) or b) it can be toxic (eg. PKU) or c) ifthe product is absolutely essential for energyproduction it will also result in energy deficientstate (eg. Mitochondrial disorders).

Age of onset

The signs and symptoms can manifest at anyage from neonatal period through infancy andchildhood to adulthood. Age of onset has a

bearing since many IEM can have a typical ageof onset. This is because the age of onset dependsupon the developmental stage of a particularorgan system,2 eg.cholesterol, peroxisomalbiogenic disorders and lysosomal disorders canpresent at birth.4 The presentation may beinsidious, eg.lysosomal storage disorders oracute, eg.peroxisomal disorders.

The IEM which involves intermediarymetabolism presents after introduction of feeds,which can be acute and catastrophic referred toas “metabolic distress”.2 Even though theessential intermediary system is involved, we donot see fetal onset in such cases because of thefact that protein, carbohydrate and fat handlingby the fetus is limited and the mother’s systemhandles it. This may not be true in disorders oflarge molecules which cannot be degraded by thesystem and they present at birth, eg.lysosomalstorage disorders and peroxisomal disorders.

IEMs affecting mother and fetus

Some maternal diseases of IEM can affectthe fetus, eg.PKU in the mother can causedysmorphology in the fetus which results incongenital malformations (Fig.2). Vice versa,some disorders of the fetus can affect the mothertoo, eg.Very long chain hydroxy acyl CoAdeficiency (VLCHAD) of fetus can manifest asacute fatty necrosis of maternal liver andhemolysis, elevated liver enymes, low platelets(HELLP) syndrome in the mother when she is

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heterozygous for VLCHAD.5 Similarly Mediumchain acyl CoA deficiency (MCAD) andcarnitine palmitoyl acyl coA transferase cancause acute fatty liver of pregnancy (AFLP).

Note:

• Energy deficiency disorders manifestwithin the first 24 hours of birth

• Neurodegenarative disorders manifest ininfancy and childhood, eg. Canavans disease

• Porphyrias can manifest at infancy,childhood, adolescence and adult asneuropsychiatric illness

Having said all this, six periods of specificonset of presentation can be considered: at birth,1 day to 1 month (neonatal), 1 month to 12months (early infancy), 1 to 5 years (late infancyand early childhood), 5 to 15 years (latechildhood and adolescence).

Three types of onset

• Acute stormy onset with rapid lifethreatening deterioration over hours, eg.some ofthe mitochondrial disorders with lactic acidosis.

• Episodic with intermittent decompen-sation and asymptomatic intervals, eg. some ofthe organic acidurias.

• Insidious onset with slow degenerationover decades, eg.neurodegenerative disorders -Canavan’s disease.

Provocative circumstances

Next step to be considered is ‘what exactlydid provoke the symptoms in the child who waswell earlier’. (Table 1)

Symptoms and signs

Symptoms and signs are myriad. Hence‘when and how to suspect IEM’ always lies inthe hands of the primary physician and it

encompasses all that is said initially starting withconception. So suspect IEM when there is:6-9

• Family history of fetal wastages,hydrops, severe IUGR, neonatal deaths and SIDS– not only for that family but for uncles, auntsboth paternal and maternal and cousins. Negativefamily history does not rule out IEM.

• Coarse features, dysmorphism

• Unexplained clinical deteriorationfollowing a period of normalcy

• Unexplained odor (abnormal urine odorcan be detected on a dry filter paper or bysuddenly opening the lid of the container ofstored urine at room temperature (Table.2).

• Feeding problems

• Persistent hiccoughs, change in tone,convulsions (hiccoughs when the child is well isnot abnormal)

• Abnormal neurological signs

• Persistent tachypnoea, respiratorydistress, apnoea

• Persistent lethargy

• Poor feeding, vomiting, diarrhea, anddehydration.

• Temperature instability.

• Unexplained shock

• Abnormal visceromegaly

• Acute Reye like syndrome

• Unexplained cardiomegaly withdecompensation

• Cholestatic jaundice, (not biliary)

• IEM mimic sepsis in the newborn. (Alsocertain IEMs are associated with risk of sepsis,eg. galactosemia, CAH, organic acidemias)

• Abnormal acute laboratoryabnormalities (biochemical and hematological)

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Table 1. Most frequent provocative circumstances observed in inbornerrors of intermediary metabolism2

Symptoms triggered by:

Weaning

* Fructose intolerance, Fructosediphosphatase deficiency, Urea cycledefects, Lysinuric protein intolerance,Triple-H syndrome, Maple syrup urinedisease, Organic acidurias

Fructose

* Fructose intolerance, Fructosediphosphatase deficiency

Galactose

* Galactosemia

Glycerol

* Glycerol intolerance

Protein

* Urea cycle defects, Lysinuric proteinintolerance,Triple H syndrome, Maplesyrup urine disease, Organic acidurias,Hyperinsulinism (with hyperammonemia)

Carbohydrate

* Pyruvate dehydrogenase deficiency,Respiratory chain disorders,Hyperinsulinism

Catabolic circumstances

* Aminoacidopathies

Infection

* Organic acidurias

Fever, fasting

* Fatty acid oxidation disorders, Urea cycledefects, Gluconeogenesis defects,Glycogenosis defects

Anesthesia

* Thromboembolic accident inhomocystinuria

Surgery

* All conditions listed above

Drugs

* Porphyria,Glucose-6-phosphatedehydrogenase deficiency

Table 2. List of IEM with unusualodours

IEM Odor

Isovaleric aciduria - Sweaty feet

Maple syrup urine diease - Maple syrup

Phenyl ketonuria - Musty

Tyrosinemia - Cabbage

Glutaric aciduria type II - Sweaty feet

Multiple carboxylase - Male catdeficiency urine

like: Persistent hypoglycemia, persistent acidosis,persistent abnormal hepatic and renalparameters, hyperammonemia, persistentabnormal coagulation profile, pancytopenia,leucopenia, thrombocytopenia.

• Hyperammonemia, ketosis, abnormalamino acid profile and positive DNPH test in theabsence of glycosuria and acetonuria.

Clinical findings in children andadolescence

When it comes to children andadolesents, failure to thrive, more expressiveneurological signs and developmental delay willbe remarkable. Dysmorphic features get more

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grotesque and systemic signs start appearing,eg.1 In Morquo’s syndrome the dysmorphicfeatures express themselves as age advances, eg.2In homocystinuria, molybdenum cofactor defect,sulfite oxidase deficiency, dislocation of ocularlens appears after 2 to 3 months of age.10

• One always has to look for ocular, skin,hematologic, visceral and endocrine symptomsand findings.

• Acute symptoms may be precipitated byminor viral infections.

Before starting with the ‘work up’ toevaluate an infant with suspected IEM, we willconsider the following simple lessons.

Lesson 1. IEM involving proteinmetabolism / aminoacidopathies1

Aminoacids (AAs) are made up of aminogroup and an organic acid backbone. When thereis a block in their metabolic pathway, two majorfindings arise - a) since both amino radical andacid component can not be degraded, there isacidosis as well as hyperammonemia (when ureacycle is overwhelmed) b) the particularaminoacid can not contribute to the pool ofneoglucogenesis and hence, there ishypoglycemia. The same is true for allaminoacids when the metabolic defect is in the

intermediary pathway. So to recapitulate, inorganic acidemias there is acidosis (usually notlactic), hypoglycemia and there could behyperammonemia since some of the by-productsinhibit urea cycle, eg.propionic acidemia. Theby-products interfere with acetyl coA synthesisand hence lactic acidosis may be there. (Fig.3)

For aminoacids which do not take part inintermediary metabolism the symptoms aremostly toxic, eg.PKU

Lesson 2. IEM involving carbohydrates

There are three points for consideration

a) Hypoglycemia can result from glycogenstorage disorders, glycolytic pathway disorders,aminoacidopathies, citric acid cycle disorders,neoglucogenic disorders, beta oxidation of fatdisorders and may accompany mitochondrialdisorders. (Hyper insulinemic state, a separateentity, is not dealt here)

b) Pure glucose is not the only carbohydratesource. Diet consists of the following sugars,namely fructose, sucrose, lactose and galactosewhich have to be converted to glucose to enterinto oxidative phosphorylation (Fig.4). Failureof conversion can lead to 1. accumulation of toxicmetabolites and 2.hypoglycemia

c) Glycogen storage disorder12 (Fig.5): Thestorage product of carbohydrate metabolism isglycogen. In times of need, stored glycogen is tobe broken down to glucose with the help ofenzymes. If it can not be broken down, it will bestored and there will be visceromegaly withhypoglycemia and usually ketosis (one exceptionis type I glycogenosis).

Lesson 3. Urea cycle disorders

In urea cycle disorders ammonia cannot beconverted to urea and hence urea levels can below and ammonia levels are grossly elevatedFig. 3. Metabolism of amino acid

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Fig. 4. Carbohydrate metabolism

Failure of conversion : 1. Accumulation of toxic metabolites

2. Hypoglycemia eg. Galactosemia

Fig. 5. Glycogen metabolism

Fig. 6. Urea cycle

(Fig.6). Usually there is no hypoglycemia. Thereis tachypnoea because ammonia is a potentrespiratory centre stimulent and the sign is oftenmistaken for acidosis.

Lesson 4. Respiratory chain /mitochondrial disorders1,3

Glucose is the energy currency for humanorganism. But at the cellular level ATP is thecurrency of energy. Stored chemical energy fromglucose has to be transferred to ATP throughseveral steps. In the final step, failure ofconversion of NADH to ATP leads toaccumulation of NADH and in turn lactic acid,because increased NADH favors lactic acid(pyruvate and lactic acid are in balancingequilibrium) (Fig.7).

Lesson 5. Beta oxidation of fat12

In the metabolism of fat, degradation startswith splitting of fat into free fatty acids andglycerol. Free fatty acid enters into the cell alongwith carnitine and subsequent beta oxidationtakes place within the mitochondria withproduction of ketone bodies. In defects of betaoxidation there is acidosis (fatty acidaccumulation) and hypoglycemia because ofineffective contribution to neoglucogenic pool,and there is striking absence of ketone bodies.Since urea cycle is inhibited by the by-products,

hyper-ammonemia can be there, but not severe.There is secondary carnitine deficiency withexcessive excretion of acyl carnitine.

Lesson 6. Cholesterol synthesisdefects

In cholesterol synthesis defects, there couldbe dysmorphism with hypoglycemia since theprecursors for cholesterol are aminoacids.

Lesson 7. Storage disorders

In storage disorders there will behepatosplenomegaly and cardiomegaly withdysmorphism.

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Fig. 7. Respiratory chain

Investigations

With the above background the investigatoryguidelines are given as below:

1.Start with routine simple tests

2.Then proceed with specific tests

3.Special tests when circumstances warrant

The Table.3 gives categories of tests to bedone. Physician should be judicious in choosingthem and be guided by the stems given in thetext.

After the basic and some of the specificinvestigations are done, atleast one of thefollowing findings are observed in most of theIEMs except in certain neurodegenerative

disorders like non ketotic hyperglycinemia,sulfite oxidase defect or Smith Lemli opitzsyndrome. They are,

(1) Hypoglycemia

(2) Acidosis

(3) Hyper ammonemia

(4) Ketosis (when concentration of ketonebodies are more than 7mM/l)

(5) Positive / negative DNPH tests

(6) Lactic acidosis

Holding on to any one stem can lead on tothe diagnosis (Figs..8, 9, 10, 11, 12, 13)

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Urine

Blood

Miscellaneous

Smell (special odor)Look (special color)Acetone (Acetest)Reducing substances (Clinitest)Keto acids (DNPH)pH (pHstix)SulfitestElectrolytes (Na+, K+)Uric acid (search for hypouricuria)

Complete blood countElectrolytes (search for anion gap)Glucose, calciumBlood gases (pH, PCO

2, HCO

3, PO

2)

Uric acidProthrombin timeTransaminases (and other liver tests)AmmonemiaLactic and pyuvic acidsâ-hydroxybutyrate, acetoacetate

Lumbar punctureChest x-rayEchocardiography, EKGCerebral ultrasound, EEGFree fatty acids

Urine collection: collect each freshmicturition separately and put it in therefrigerator.Freezing: freeze at -20oC samplescollected before treatment and,afterward, an aliquot of 24-h collectionon treatment.Do not use for specific investigationswithout expert metabolic advice.

Plasma, heparinized, 5 ml at -20oCBlood on filter paper (as “Guthrie test”)Whole blood 10 to 15 ml collected onEDTA and frozen(for molecular biology studies)

Skin biopsy (fibroblast culture)Cerebrospinal fluid, 1 ml frozenPostmortem: liver, muscle biopsies(macroscopic fragment frozen at -70oC)

Specimen Basic investigations Specific investigations

Table 3. Initial investigations2

A Stage evaluation always helps

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Fig. 8 Stem 1 - Hypoglycemia15

When persistent hypoglycemia is encountered, the following stem can be utilised(Hyper insulinemic syndromes are omitted)

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Fig. 9 Stem 2 - Hypoglycemia15

The other approach to hypoglycemia is to do DNPH, ABG and estimation ofketone bodies and then proceed with the stem.

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Fig. 10 Stem 3 - Acidosis15

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Fig.11. Stem 4 – Lactic acidemia

Fig.12. Stem 5 – Hyperammonemia15

Lactic acidemia is an important feature of respiratory chain and Kerbs cycle disorders

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Fig.13 Stem 6 - Urea cycle defect

CPS - Carbamoyl Phosphate synthetase

OTC - Ornithine trans carbamylase

ASL - Arginino succinic acid lyase

ASS - Arginino succinic acid synthase

The above said stems are over simplificationof metabolic pathways and are shown just tomake one understand, how to approach a childwith an IEM, involving the intermediarymetabolism. The exact pathway, enzymes, etc arewillfully omitted. Single metabolic pathways notinvolving the energy generation like, bile acidmetabolism of porphyrin metabolism are to beworked up as per need.

The reader should understand that this topicdoes not cover all the IEM. It covers only simpleintermediary metabolic defects and one can referto the list of books given at the end of this article.

The acute management protocol for a sickchild with suspected IEM is given below forguidance.

Acute management of sick childwith suspected IEM13,14

In the management of inborn error ofmetabolism, stabilisation is the hall mark.

Whatever be the IEM, supportive careshould be started at the emergency room itself.

I. General

1. Supportive

Take care of ABC.

Correct hypothermia, hypoglycemia anddehydration

Start arterial and central venous access

Give respiratory support, if needed.

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2. Antibiotics

Start on broad spectrum antibiotics alongwith metronidazole and neomycin to control thepossibility of production of organic acids byintestinal bacteria

3. Shock

Correct shock with fluid boluses10-20 ml/per kg of normal saline, repeat as perprotocol

Avoid Ringer Lactate (RL) if acidosis isalready there

Avoid hypotonic fluid to prevent cerebraledema

4. Prevention of cerebral edema

Restrict fluids, if signs of cerebral edemastart to appear

If cerebral edema, give IV Mannitol0.25-0.5g/kg and IV frusemide 1mg/kg.

5. Stop all proteins

Stop all proteins for 48 to 72 hrs includingTPN if the child as on TPN

6. Avoid offending sugars

Stop offending agents like galactose andfructose if the disorder is known

7. Treat hypoglycemia

With IV glucose 2ml/kg (200-400 mg/kg)of D20 / D25 if there is central line

8. Fluid volume and glucose infusion rate

Maintain with D10 with electrolytes asneeded at 1-1.5 times maintenance volume

Keep glucose infusion rate(GIR) as8-10 mg/kg/min at 1-1.5 times maintenance GIR

To prevent protein catabolism, give50 to 70 kcal / kg / 24hrs (as above)

Keep blood glucose levels at120 to 170 mg/dL

9. Promote anabolism

Give IV Insulin 0.05 to 0.1 U per kg / hrupto 0.2 to 0.3 U to further promote anabolism

10. Correct acidosis (pH <7 to 7.2)

Correct metabolic acidosis slowly andcautiously with IV sodium bicarbonate, give0.35 to 0.5 mEq of sodium bicarbonate upto1mEq/kg/hr or potassium acetate 1mEq/kg/hrthrough IV infusion.

Start hemodialysis if intractable acidosis isencountered (Peritoneal dialysis less effective).

11. Low protein

When oral feeds are started afterstabilization, give low protein diet (0.7 g/kg/per24 hrs).

II. Elimination of toxic metabolites(if disorder is suspected or known)

1. For hyperammonemia

For OTC and CPS deficiency, starton priming dose of sodium phenylacetate250 mg/kg and Sodium benzoate 250mg/kg as“ammonia trap” [(ammonul 2.5 ml) (‘ammonul’contains sodium benzoate 100 mg and sodiumphenylacetate 100mg/mL)] along with, arginineHCl 200 mg/kg in 25 ml/kg of 10% dextrosesolution over 90 minutes. This is followed bymaintenance dose of all the three drugs; 250 mgof sodium benzoate and sodium phenylacetateeach and 200 mg of arginine / day (oral).

Priming dose for Ammonia trapping

Sodium phenylacetate - 250 mg/kg

Sodium Benzoate - 250 mg/kg

Arginine Hydrochloride - 200 mg/kg

In 25 ml/kg of 10% Dextrose over 90 minutes

Followed by same dose of all drugs every dayorally

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For citrullinemia and arginino succinicacaidemia, give same drugs except increaseL-argininine HCl in a dose of 600 mg/kg forloading and sustained infusion.

For argininase deficiency, use same regimen of‘ammonul’ for loading and sustained infusion,but omit L-arginine HCl.

If neonate/infant is not critically ill andhyperammonemia is mild, arginine therapy alonemay suffice.

For all urea cycle disorders, IV therapy ofammonia scavenging drugs to be continued,while dialysis is being performed. A repeatloading dose of ammonia-scavenging drugsshould be given only in neonates with very severeillness who are receiving dialysis. Toxicity isassociated with high drug doses (750 mg/kg/dayand higher). Hemodialysis to be done when NH3> 500 micro mol/l

If the disorder is organic acidemia, givecarnitine 100mg/kg/24hrs IV (or) oral glycine250 mg/kg/24hrs (mainly for isovaleric acidemia)as diversion therapy

For presumed carnitine deficiency and lifethreatening amino acidopathies, administercofactor IV L-carnitine (25 to 100 mg/kg/day IV).

Carnitine therapy is controversial if the disorderis fatty acid oxidation defect

Hydration promotes renal excretion of toxins.

III. Stimulation of residual enzymeactivity with high dose of co-factors

Pyridoxine (B6) 100 mg IV for pyridoxinedependency. Maintain with 2 to 10mg / day

For folic acid responsive seizures 10-20mg offolic acid.

If the disorder is known

Methyl malonic Vit B12 1mg IM/dayacidemia

Biotinidase deficiency Biotin10 to 60 mg/day oral

Multiple carboxylase Biotin

deficiency 10 to 60 mg /day oral

Glutaric aciduria - Riboflavintype II 100 to 300 mg/ day

oral

Thiamine10 to 200 mg/day

Homocystinuria Pyridoxine IM/IV200 to 1000mg/24hrs

Carnitine deficiency Carnitine100 to 400 mg ofL-carnitine/kg/dayoral

MSUD (Maple syrup Thiamineurine disease) 10mg to 200mg/24hrs

Riboflavin200 to 300 mg/oralTID

Mevalonic acidemia Prednisone2mg / kg / 24 hrs

Hartnup disease Nicotinamide50 to 300mg/dayHigh protein

Chronic treatment includes a special dietsmall amount of offending aminoacids andrelated metabolites

To sum up, contrary to the myth, the realityis, IEM are quite common in the scenario of overpopulous nation like India and some of them areeffectively treatable.

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Points to Remember

• IEM are not uncommon

• Do suspect IEMs in all babies withunexplained deterioration of clinicalcondition and suspected sepsis when sepsisscreen is negative.

• Start with a simple approach to hold on toa ‘thread of logic’ which will lead on tothe diagnosis.

• Stabilization is the key to management.

• If a diagnosis is not made when the childis alive, do collect blood samples and freezeto send for subsequent analysis.

• An attempt to make a diagnosis gives thechoice to the parents in subsequentpregnancies.

• We have a long way to go in effectivetreatment and in the current scenariogenetic counseling to the parents is thecrux.

Bibliography

1. Fong CT. Principles of Inborn Errors of

Metabolism: An Exercise. Pediatr Rev1995;16:390-395

2. Saudubray JM, Charpentier C. Clinicalphenotypes: Diagnosis/Algorithms. In: Themetabolic and molecular bases of inheriteddisease, 6

th edn, Eds,Scriver CR, Beaudet AL,

Sly WS, McGraw Hill, New York,1997;pp1325-1403.

3. Murray RK. Biochemical and genitic basis ofdiseases. In: Harper”s Biochemistry, Eds.Rober K Murray, Daryl K Grams, Peter A.Mayes, victor W Rod well, 25

th Edn, Appleton

and lanse, MC Graw Hill – USA, 2000; pp812-816.

4. Champion MP, Clayton PT. Mini-symposium:Metabolic disease: Peroxisomal disorders.Current Paediatr 1997;7:114-117.

5. Zinn AB. Inborn Errors of metabolism. In:Neonatal perinatal medicine. Diseases of thefetus & infant,8

th Edn,Vol.2, Eds, Avroy A

Fanaroff, Richard J Martin,, Mosby, St louisMissourie, USA, 2006; pp1597-1658.

6. Berry GT. Introduction to the metabolic andbiochemical genetics. In: Avery’s Diseases ofthe Newborns, 8

th Edn,,Eds, Taeusch, Ballard,

Gleason, Sanunders, Philadelphia, 2005;pp217-252.

7. Sule U, catalype , Levy HL. Inborn ErrorsMetabolism. In: Manual of Neonatal Care. 6

th

Edn, Eds, John P Cloharty, Ericks CEichenwald, Ann R Stark, Wolters Kluwer.Lippincot, William & Wilkins, Philadelphia2008;pp555-586.

8. Neilan E, Marsden D. Metabolic disorders. In:Manual of Pediatric Therapeutics, 7

th Edn,

Eds,John W Graef, Joseph I wolfsdorf, DavidS Greenes, Wolter Kluver , Lippincot William& Wilkins, Philadelphia – USA, 2008;pp406-416.

9. Wilcox WR. Inborn Errors of Metabolism ofAcute onset in infancy – An approach todiagnosis and management. http://www.neonatology.org/syllabus/iem.html

10. Basley GTN, Wraith JE. Mini-symposium:Metabolic disease: Lysosomal disorders.Current Paediatr 1997;7:128-134.

11. Lee P. Mini-symposium: Metabolic disease:Glycogen storage disease. Current Paediatrics1997;7:108-113.

12. Bartlett K, Pourfarzam M. Mini-symposium:Metabolic disease: Inherited disorders ofmitochondrial fatty acid oxidation. CurrentPaediatr1997;7:118-122.

13. Rahman S, Leonar JV. Mini-symposium:Metabolic disease: Mitochondrial disorders.Current Paediatr 1997;7:123-127.

14. Walter JH. Mini-symposium: Metabolicdisease: Investigation and initial managementof suspected metabolic disease. Current Paediatr1997;7:103-107.

15. Burton BK. Inborn Errors of Metabolism inInfacny: A Guide to Diagnosis. Pediatrics1998;102: e69.

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Table.1 – Classification2,5

Errors of IEM in :

• Aminoacid metabolism

• Carbohydrate metabolism

• FA oxidation

• Fat metabolism eg; storage disorders

• Respiratory chain eg; mitochondrial disorders

• Large molecule metabolism

1. Disorder of glycoconjugate biosynthesis

2. Lysosomal storage disorder

• Small molecule metabolism

1. Cholesterol biosynthetic defects

2. Organic acidurias

3. Peroxisomal disorders

(a) Biogenesis defects

(b) Single enzyme defects

• Others: Bile acid metabolism

• Hemoglobinopathies

• Uric acid metabolism

• Mineral metabolism

• Cofactor (Vitamin) metabolism

Hyperlipidemias

TABLES HELPFUL IN THE WORKUP OFINBORN ERRORS OF METABOLISM

ANNEXURES

23


Table.2 . Laboratory findings helpful in the differential diagnosis of suspectedmetabolic disease in neonates2,5

Finding Diagnostic Considerations

Acidosis Fatty acid oxidation defetsGluconeogenesis defectsGlycogen storage diseasesKetogenesis defectsKetolysis defectsKrebs cycle defectsOrganic acidemiasRespiratory chain defects

AlkalosisRespiratory Urea cycle defectsMetabolic Steroid biosynthetic defects

Hepatic dysfunction Amino acid defectsBile acid biosynthesis defectsCarbohydrate defectsFatty acid oxidation defectsPeroxisomal disordersRespiratory chain defectsOther

Hyperammonemia Amino acid disordersFatty acid oxidation defectsOrganic acidemiasUrea cycle defects

Hypoglycemia Fatty acid oxidation defectsGluconeogenesis defectsGlycogen storage diseaseKetogenesis defectsOrganic acidemias

Ketosis/ketonuria Amino acid defectsGluconeogenesis defectsGlycogen storage diseaseKetogenesis defectsOrganic acidemias

Pancytopenia Organic acidemiasRespiratory chain defects

Proximal renal tubular dysfunction Amino acid defectsCarbohydrate defectsRespiratory chain defects

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Table.4. Specialized laboratory tests that may be required for the care ofneonates with suspected metabolic disease2,5

Body fluid or tissue Laboratory tests

Blood Amino acidsCarnitine(total, free, and acylcarnitine profile)Lactate and pyruvateVery-long-chain fattyAcids and phytanic acid (peroxisomal disorders)Transferrin isoelectric focusing – CDGImmunoelectrophoresis (carbohydrate-deficientglycoprotein syndromes)

Urine Amino acidsOrganic acidsCarnitine (total, free, and acylcarnitine profile)Glycolipids,oligosaccharides, and mucopolysaccharides(Iysosomal storage disorders)

Cerebrospinal fluid Amino acidsLactate and pyruvate

Other sources

Cultured skin fibroblasts Genetic studiesMetabolite analysis

White cells Genetic studies

Table.3. Characteristic urinary findings in inborn errors of metabolism2,5

Finding Disorder

Reducing substance Hereditary fructose intoleranceGalactosemiaHereditary tyrosinemia

Acetonuria Organic acidemias

DNP (-ketoacids) Maple syrup urine diseasePhenylketonuriaHereditary tyrosinemia

Ferric chloride PhenylketonuriaHereditary tyrosinemia

Nitrosonaphthol Hereditary tyrosinemia

-Nitroaniline Methylmalonic academia

Sulfitest Sulfite oxidase deficiency

25


Table 5. Neonatal-onset inborn errors of metabolism characterized byabnormal plasma amino acid patterns2,5

Disorder Finding

Amino acid disordersMaple syrup urine disease ↑ Isoleucine, leucine, valineNonketotic ↑ Glycinehyperglycinemia*Phenylketonuria ↑ PhenylalanineSulfite oxidase deficiency ↑ S-SulfocysteineHereditary tyrosinemia ↑ Tyrosine, methionine

Lactic acidemias ↑ Alanine and …PC deficiency ↑ Citruline, lysine

Organic acidemiasMethylmalonic acidemia ↑ GlycineIsovaleric acidemia ↑ GlycinePropionic acidemia ↑ Glycine

Urea cycle defects ↑ Glutamine, ↓ arginineSpecific disorder :Argininosuccinic aciduriat ↑ ASA, ↑ CitrullineCitrullinemia ↑↑ CitrullineCPS and OTC deficiency ↑ Citrulline

PC - Pyruvate carboxylaseCPS - Carbamyle phosphate synthataseOTC - Ornithine trans carbamylaseASAS - Arginino succinic acid synthetase

26

Table. 6. Prenatal diagnostic tests2,5

Amniocentesis

Chorionic villus biopsy

Liver biopsy

Restriction Fragment Length Polymorphism (RFLP) – CPS, OTC, Argininase deficiency.

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Table 7. Inborn Errors of Metabolism that cause dysmorphic syndromes 2,5

Inborn Error Syndrome

Large-molecule metabolismDisorder of glycoconjugate biosynthesis Carbohydrate-deficient glycoprotein syndromes

Lysosomal storage GlycolipidosesDiseases Mucopolysaccharidoses

OligosaccharidosesSmall-molecule metabolismCholesterol biosynthetic Defects Conradi-Hunermann syndrome

Desmosterolosis Smith-Lemli-Opitz syndrome

Organic acidurias 3-Hydroxyisobutyry1 deacylase deficiencyMevalonic aciduria*Multiple acy1-CoA dehydrogenase deficiency

Peroxisomal disordersBiogenesis disorders Chondrodysplasia punctata, rhizomelic type

Zellweger syndrome and its variantsInfantile Refsum diseaseNeonatal adrenoleukodystrophy

Single enzyme defectsDefects of peroxisomal fatty Acy1-CoA oxidase deficiencyacid oxidation Bifunctional enzyme deficiency

Thiolase deficiency

Table 8. Disorders associated with abnormal liver function 2,5

Category of disorder Defect

Amino acid metabolism Hereditary tyrosinemiaCarbohybrate metabolism Galactosemia

Hereditary fructose intoleranceFatty acid oxidation Carnitine

Palmitoryltransferase II deficiencyLong-chain 3-hyroxyacy1-CoA dehydrogenase DeficiencyMultiple acy1-CoA Dehydrogenase deficiency

Peroxisomal disorders Neonatal adrenoleukodystrophyZellweger syndrome

Respiratory chain Complex IVmtDNA depletion syndrome

Other á1-Antitrypsin

deficiency

Carbohydrate-deficient glycoprotein syndrome (CDG)Glycogen storage disease type IVNiemann-Pick disease type C

27


Table. 9. IEMs with Hepatomegaly / Splenomegaly2,5

• GSDs• Lipidoses - Farbers, Gauchers,

Krabbe, Wolman diseases,GM1 gangliosidosis,Niemann-Picks disease

• Mucopolysaccharidoses• Oligosaccharidoses - I cell disease,

Sialidosis,Fucosidosis

Table.10. IEMs with Cardiomegaly / Cardiomyopathy 2,5

• FA oxidation - Carnitine- acylcarnitine translocase def.Carnitine palmitoyl transferase def.LCHAD def.VLCHAD def.

• GSDs - type II and IX

• Lysosomal storaged diseases - glycolipidosesmucopolysaccharidosesoligosaccharidoses

• Respiratory chain defect

• CDG syndromes

Table.11. Investigations for suspected IEM if child dies without a diagnosis2,5

5 ml Red cells at +4*c5 ml Plasma at -20*c10-20 ml Urine at -20*cBiopsy – skin (for fibroblast culture), Liver, Muscle, Brain

Table.12- Dietary tips2,5

• Fructose intolerance - avoid fruit juices• Pyruvate dehydrogenase def. - high fat, low carbohydrate• Organic acidemias - high carbohydrate, low protein and fat• B-Oxidation defects - high carbohydrate, low fat diet

continuous feedingavoid fasting

• Ketolysis defect - low fat, low proteincarnitine supplementation

• LCHAD def. - medium Chain triglycerides• Purine and pyramidine metabolism - avoid nuts, non-veg.

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PRENATAL DIAGNOSIS ANDNEWBORN SCREENING:RELEVANCE IN INDIA

* Mamta Muranjan** Shruti Agarwal

Abstract: Inborn errors of metabolism (IEM) areprogressive disorders characterized by highfatality and permanent disability in survivors.They are growing at an alarming rate in India.Evaluation and treatment of these disorders isexpensive and not easily available. In a smallproportion of patients who have access totherapy, the outcome may not be optimum due tolate diagnosis and therapy. The option for manyfamilies affected by these disorders is prenataldiagnosis and newborn screening. Prenataldiagnosis for IEM is available with non-invasivemodalities such as ultrasonography andbiochemical, histopathological or moleculartesting of fetal tissues obtained by invasiveprocedures and is fairly well established forlysosomal storage disorders with enzymeestimation. However in many cases, diagnosisin the index case is not established. In suchsituations newborn screening is often advised forhigh risk screening. Universal newborn screeningis not yet practised except in some isolatedregions. The options for universal newbornscreening for IEM in India and the hurdles to beovercome are discussed.

Keywords: Inborn errors of metabolism,Chorion villus sampling, Amniocentesis,Ultrasonography, Fetal MRI, Tandem MassSpectroscopy

Inborn errors of metabolism (IEM) arebiochemical disorders in which an abnormalityof the enzyme or a transport protein can give riseto pathological consequences at birth or later inlife. Although individually rare, as a group thesedisorders are more common than appreciatedby most physicians. The overall incidence is1-2% of newborns.1 Over 300 IEM are nowrecognised and the number is increasing.2

Most IEM are progressive and manifest aftera variable symptom-free interval. Appearance ofsymptoms coincides with variable degree oftarget organ damage, which at times is permanent.Most IEM affect the brain and as a consequencemajority are associated with neurologicalsymptoms. Metabolic disorders account for43% of the causes of mental retardation and amajority result in permanent neurologicaldisability.3 The challenge is to diagnose thesedisorders, especially the treatable group, at anearly stage in order to prevent or minimize end-organ damage. This is often not borne out inpractice with tragic consequences.

In India, IEM rank number five ascontributors to the burden of genetic diseases.4

Investigations are expensive especially in theprivate sector. Cost of treatment is prohibitiveand many modalities are not easily available.Late diagnosis and treatment takes its toll on thequality of life. Though the health economicburden has not been estimated, it could be

* Associate Professor,

** Resident Medical Officer,

Genetic Clinic, Department of Pediatrics,KEM Hospital, Mumbai.


29


considerable in terms of direct costs and indirectcosts from loss of productivity. The thrust in Indiain the past decade was prevention of recurrenceby prenatal diagnosis (PND) and termination ofaffected pregnancies in families having anaffected child. However, recently an increasingnumber of individuals with IEM especiallylysosomal storage disorders (LSDs) are beingtreated. If the quality of life were to improve inthese children, the therapy has to commence asearly as feasible in the presymptomatic phase.Thus early diagnosis has to be facilitated.One option available is newborn screening orby prenatal diagnosis at an even earlier stage.There is an urgent need in India to examine scopefor newborn screening for IEM.

The planning of prenatal diagnostic servicesand newborn screening should take intoconsideration the common disorders prevalent inthe country. No population based data is availableand most information is from genetic centres inIndia and laboratory based results of high risktesting. Amongst the single gene disorders thecommon IEM were mucopolysaccharidosis(MPS), metachromatic leukodystrophy,oculocutaneous albinism, Wilson disease andaminoacid disorders. The common amino aciddisorders are hyperglycinemia, homocystinuria,alkaptonuria and maple syrup urine disease(MSUD). MPS (37%), LSDs (24%), Wilsondisease (14%), galactosemia (5%) and glycogenstorage disease (4%) were the commonestdisorders at KEM Hospital, Mumbai and AIIMS,New Delhi.3 The scope of this article is tofamiliarize the reader with facilities for prenataldiagnosis and status of newborn screening inIndia.

PRENATAL DIAGNOSIS

PND of a genetic disease was first reportedin 1968 when Fujimoto, et al correctly identifieda carrier female fetus of X linked hypoxanthineguanine phosphoribosyl transferase deficiency by

biochemical analysis of cultured amniotic fluidcells.5 PND has now evolved as a safe andaccurate option for almost all IEM. Since the pastdecade, prenatal diagnosis for LSDs has beenfairly well established in India. Several centres(KEM Hospital in Mumbai, AIIMS and SirGangaram Hospital in New Delhi, Centre forDNA fingerprinting and diagnostics inHyderabad and FRIGE in Ahmedabad) areroutinely offering PND services for LSDs byestimation of enzyme activity. Reports of PNDfor a few other IEM like phenylketonuria andtyrosinemia type 1 have been published from SirGangaram Hospital, New Delhi.6,7

PND offers prospective parents theassurance of having an unaffected child with anoption for medical termination for an affectedpregnancy for IEM that are lethal, incurable andresulting in chronic disability. When viewed fromthis context PND for treatable disorders such asWilson disease may raise ethical issues.However, the scope of PND has been currentlyextended by the increasing availability of diseasespecific therapies such as enzyme replacementtherapy for LSDs, as it can ‘prepare’ the parentsfor the financial and practical implications of thetherapeutic options. The advantage would becommencing therapy soon after birth, whichwould prevent target organ injury. It is expectedthat the eventual long-term outcome of suchpresymptomatic therapy would be optimum.

For an IEM, PND should be offered whenthe diagnosis has been confirmed (by estimationof enzyme activity, genotyping or histopathology)in a previously affected child or the carrier statusof the parents for an autosomal recessive diseasehas been established or a woman is known to bea carrier or is at risk of being a carrier of aX-linked recessive disease.8 In India the diagnosisis often not established in the previous affectedchild and the clinical records may not bepreserved. In fact, many couples with a

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previously affected child present to a geneticservice only when the wife is pregnant.Under these circumstances, PND has beenoffered for the most likely disorder based onclinical features and available records afteradequate counselling. The family should be madeto understand the limitation/inaccuracy inherentwith this approach. In case of enzyme estimationor histopathology as techniques for prenataldiagnosis, the defect must be expressed in fetaltissues used for sampling. For example, PND fordisorders such as PKU, ornithinetranscarbamylase deficiency or Von Gierke’sdisease cannot be performed by enzymeestimation of chorion villus or amniotic fluidfibroblasts as the enzyme is only expressed inthe liver. Genotyping will be the method of choicefor such disorders. Pregnancies with non-immunefetal hydrops are at risk for IEM and arecandidates for PND (Table 1).9 It is desirablethat parents are willing for termination of anaffected pregnancy without personal, social orreligious taboos if options for therapy are notavailable or the outcome with therapy issuboptimum. However, this may not be anabsolute requirement and the family’s decisionhas to be respected. In such cases invasive testsshould be avoided if a non-invasive option isavailable. Pre-test counselling for risk ofoccurrence / recurrence of the disorder versusrisk of the procedure (in case of an invasive test)is required. The couple should give informedconsent for the procedure.10 The legal provisionsunder the Prenatal Diagnostic Techniques Act(PNDT) should be fulfilled. According to theamendments in the PNDT Act, fetal sexdetermination may be allowed under exceptionalcircumstances.11 This would include X-linkedrecessive disorders such as Hunter syndrome,adrenoleukodystrophy or Fabry disease. For thesedisorders, if PND is not possible by estimationof enzyme activity or genotyping, an optionwould be fetal sex determination with termination

of the male fetus which has a 50% risk of beingaffected. Once the results of PND are available,a post-test counselling session has to bescheduled.

Modalities for prenatal diagnosis

I) Fetal imaging

A) Ultrasonography (USG): Informationprovided includes fetal viability, number ofgestations, duration of pregnancy and fetal/placental position to plan invasive procedures.USG also provides an assessment of the fetal wellbeing and a quantitative assessment of the fetaland the placental blood flow using Doppler.Fetal or placental structural abnormalitiessecondary to IEM can be detected byUSG (Table 1).8

Limitations of USG are that it isconsiderably operator and interpreter oriented.It depends on the fetal position and the bone andsoft tissue windows and provides a smaller fieldof vision as compared to MRI. Utility of USG inobese women or for the study of intracranialanatomy is limited.12 Fetal visualization hasimproved with recent use of 3-dimensional USGwhich allows several planes to be viewed andthe images to be rotated. 3D ultrasound iscurrently used as an adjunct to 2D ultrasoundand is particularly useful for evaluatingdysmorphic features, coarse facial features, facialclefts, spinal abnormalities, hand abnormalitiesand determination of fetal gender in counsellingpregnancies at risk for X-linked recessivedisorders.13

B) Fetal MRI: Currently, fetal MRI is an adjunctto USG. It is useful in the study of subtle orinconclusive lesions detected by USG and isespecially advantageous in studying brainmaturation and myelination.12 Advantagesoffered by MRI are that fetal visualization is not

31


limited by maternal obesity, oligohydramnios orfetal position and it offers superior visualizationof the brain. MRI is usually performed after20 weeks of gestation. Structural brainmalformations such as corpus callosum agenesis/dysgenesis can be detected in fetuses withpyruvate dehydrogenase deficiency, non-ketotichyperglycinemia and maternal phenylketonuria.Peroxisomal disorders and fatty acid oxidationdefects can produce migration defects andholoprosencephaly may be seen in defects ofcholesterol metabolism.13,14

II) Invasive modalities

A) Histopathology of fetal tissues or placenta:Prenatal diagnosis of mucolipidosis type II(I-cell disease) can be performed quickly andreliably by electron microscopy of chorionicvillus tissue.15

B) Amniocentesis

• Cell free amniotic fluid: Identificationof storage material or substrates(glycosaminoglycans, oligosaccharides, sialicacid, organic acid metabolites) in supernatant

• Amniotic fluid cells

- Uncultured amniocytes

- Cultured amniocytes

C) Chorion villus sampling (CVS)

Uncultured cells

Cultured cells

CVS and cultured amniotic fluid cells areused either for enzyme estimation or to obtainfetal DNA for genotyping.

Table 1. Ultrasonography for prenatal detection of IEM

Abnormality IEM to be suspected

Increased nuchal translucency Mucopolysaccharidosis type VII

Fetal hydrops Mucopolysaccharidosis type VII and IVMucolipidosis type I and II

GM1 gangliosidosis

Gaucher disease

Niemann-Pick disease type C and A

Infantile sialic acid storage diseaseGalactosialidosis

Farber disease

Multiple sulphatase deficiency

Wolman disease

Hepatosplenomegaly Gaucher disease type 2

Niemann-Pick disease

Fractures I-cell disease

Placenta: Pale and bulky Lysosomal storage disorders

Polydactyly/syndactyly/cleft lip Smith Lemli Opitz

Reduced or absent fetal movements Nonketotic hyperglycinemia

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If a mutation has been identified in theproband, the best option would be fetalgenotyping. However, in India there are rareinstances where the proband’s genotyping isavailable. In this case fetal DNA diagnosis is stillpossible by detecting a common mutation in agiven population, e.g. L444P in neuronopathicGaucher disease. It must be realized that for agiven IEM there are ethnic variations in diseasecausing mutations. Apart from some commonconditions like Gaucher disease and glycogenstorage disease type I, mutations in Indianpatients have not been studied. Moreover manydisorders demonstrate private mutations. If alaboratory tests only common mutations ormutations of an ethnically diverse population(e.g. N370S Gaucher mutation seen in AshkenaziJews, but rare in non-Jews), its absence may notrule out an affected fetus. In this case DNAsequencing is required to study the entire codingregion of the gene of interest. Moreover somechanges may be mere polymorphisms andtherefore non-pathogenic. In this case presenceof an alteration may not indicate disease.Knowledge of local disease genotype andphysician’s knowledge of the limitations of agiven diagnostic test is crucial to interpret resultsand counsel families accordingly.

D) Fetal blood sampling: By 18 weeks ofgestation fetal blood may be obtained bycordocentesis. An example is diagnosis ofargininemia by the estimation of fetal erythrocytearginase levels.5

E) Fetal tissues: Fetal tissues may be biopsiedduring fetoscopy for the diagnosis of certain IEMlike oculocutaneous albinism and Von Gierkedisease (liver).16,17

The pediatricians’ role would be to ensureconfirmed diagnosis in the index case.Being responsible for the long-term medical careof these children, the families develop a rapport

and a relationship of trust with the primary carepediatrician. This places the pediatrician in anideal position for counselling and referring suchcouples to a prenatal screening or diagnosticservice in time.

NEW BORN SCREENING

Newborn screening (NBS) was conceivedas a public health program in which everynewborn is tested for early identification of aselective panel of disorders based on the premisethat early intervention of affected newbornsbefore symptomatic disease will lead to asignificant reduction in mortality and associateddisabilities.

It is an integrated system of education(health professionals, parents, general public andpoliticians), screening (sample collection,transport, laboratory testing and reporting), earlyfollow-up (notification of abnormal results,tracking and confirmation), diagnosis,management (monitoring, genetic counsellingand long term follow-up) and evaluation (qualityassurance and outcome).27

Since Robert Guthrie first introduced NBSto detect PKU in 1961 followed by screening forcongenital hypothyroidism (CH) in the mid 70s,the benefits to the individual, family and societyhave been proven beyond doubt.28,29 Encouragedby the success for these two disorders manycountries adapted the program including Asiancountries like China, Korea, Thailand,Philippines and Singapore.27 Many disorders likegalactosemia, MSUD, homocystinuria,biotinidase deficiency, sickle cell disease andtyrosinemia have been added to the screeningpanel depending on the policy of an individualcountry or state.29 In contrast to conventionalNBS, expanded screening detects a large numberof disorders in a single analytical run. This waspossible by adaptation of tandem massspectrometry (MS/MS) for newborn screening

33


Table 2. IEM detected by newborn screening

Condition Incidence Type of test Analytes tested

Phenylketonuria, 1:12000 to BIA, C, F, MS/MS Phenylalanine, tyrosinehyperphenylalaninemia 1:15000

Maple syrup urine 1:250000 BIA, F, MS/MS Leucine or valine & leucine/disease isoleucine

Homocystinuria 1:250000 BIA, C, MS/MS Homocysteine ormethionine

Tyrosinemia 1:150000 BIA, C, MS/MS Tyrosine, methionine(type 1)

Galactosemia 1:60000 BIA, F Galactose, galactose-1-(classical) phosphate

Biotinidase deficiency 1:250000 F Biotinidase enzyme activity

Organic acidemias (Glutaric MS/MS Respective targetacidemia type I and II, acylcarnitine concentrationisovaleric acidemia,propionic academia,methylmalonic academia,disorders of ketogenesisand ketolysis)

Urea cycle defects MS/MS Respective target aminoacid concentrations

Medium chain acyl CoA 1:14600 MS/MS C6, C8,dehydrogenase deficiency C10, C10:1, C8/C10 ratio

Lysosomal storage disorders 1:7000 MS/MS Respective(Pompe, Gaucher, Fabry, (cumulative) enzyme activityMPS I, Krabbe, Niemann-Pick type A & B)

BIA= bacterial inhibition assay, C= chromatography, F=fluorometric,

in the 90s.30 IEM detected by newborn screeningand testing modality are given in Table 2. Prenataldiagnostic modalities for disorders ofcarbohydrate metabolism, lysosomal disorders,disorders of aminoacid / organic acid and otherdisorders are given in Annexures 1, 2, 3 and 4respectively.

In India newborn screening for IEM likeorganic acidemias, urea cycle defects andamnioacidopathies is presently practised in at riskfamilies with previous undiagnosed neonataldeaths, sudden infant deaths or acute metabolicencephalopathy. Despite the evidenceworld-over, the concept of universal NBS has not

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been accorded due priority, either by thegovernment or the neonatal and pediatricprofessional bodies. In an editorial in IndianPediatrics Morris has commented that “havingthe technology to undertake neonatal screeningfor a range of metabolic disorders does not meanthat this is the right thing to do. To answer thisquestion, one needs local information aboutincidence, natural history and effectiveness oftreatment”. He concluded that neonatal screeningfor these conditions would be less cost-effectivein India than in the UK.31 In light of these criticalobservations, there is a need for pilot programsto answer these doubts.

In the earliest study in Karnataka, 112369newborns were screened for aminoacidurias.Disorders identified most frequently weretyrosinemia (1 in 6243), MSUD (1 in 10215),glycinemia (1 in 26053) and PKU (1 in 28728).4

From the year 2000 onwards, an expandedprogram of newborn screening was performedin Andhra Pradesh. A total of 18,300 newbornswere screened for aminoacidopathies, CH,congenital adrenal hyperplasia (CAH),g a l a c t o s e m i a , g l u c o s e - 6 - p h o s p h a t edehydrogenase deficiency (G6PD), biotinidasedeficiency and cystic fibrosis. The study revealedan astounding high incidence of CH (1 in 1700)and CAH (1 in 2600) as compared to world-widedata.32 A small project to screen for CH in Kochi,Kerala has revealed an even higher incidence of2.1 per 1000.33 The comparative frequency inother parts of the world for CH is 1 in 3600 inthe USA, 1 in 7000 in Scandinavia, 1 in 3000 inEurope and 1 in 5700 in Japan and the incidencefor CAH is 1 in 288 in Yupik Eskimos, 1in 7000in Philipines, 1 in 12000 in USA, 1 in 5500 to1in 10000 in Italy, and 1 in 15000 in Japan.34

Recently, the State of Goa has taken the lead ininitiating expanded mass screening for newbornsas a public-private partnership with a Bangalorebased laboratory service. The program “Heel to

Heal” launched on 14th June 2008 will screenfor a panel of 45 disorders. Three monthsfollowing commencement of the program, theincidence rate in Goa was 1 in 400 and thedisorders detected were urea cycle defects,methylmalonic /propionic acidemia, VLCADD,G6PD and CH.

Thus there is no reason to assume that IEMare less frequent in India than elsewhere in theworld. With this overwhelming data, the Indiancouncil of Medical Research has taken theinitiative of constituting a Multicentric TaskForce Group for inborn metabolic disorders.Newborn screening was identified as a priority.A multicentric pilot project for NBS wascommenced in Chennai, Hyderabad, Kolkata,Mumbai and New Delhi with the aim of screening100000 newborns for CH and CAH to studyamong others the incidence and feasibility ofimplementing newborn screening on anationwide scale in India.

Newborn screening technology for IEM

The earliest first generation technology wasbacterial inhibition assay (BIA) pioneered byDr. Robert Guthrie. He also developed the driedblood spot (DBS) technique of collecting bloodon a special filter paper from a heel prick.This process was manual and took up to 24 hours.With introduction of automation, secondgeneration fluorometric or colorimetric assayswere developed (Table 2).35,36 Alternativetechniques are chromatographic (thin layerchromatography for amino acids or organic acidsor high pressure liquid chromatography (HPLC).Introduction of MS/MS for newborn screeningwas the third generation technology forsimultaneous assays for aminoacidurias, organicacidemias and fatty acid oxidation defects.Its application for NBS analyzes multipleanalytes in less than 3 minutes and has asensitivity of 100% and specificity of 99% for

35


some disorders.30 However, not all themetabolites detected may be analyzed orreported. Each state or country selects targetmetabolites for reporting. In Switzerland and UK,only PKU and MCAD are targeted, whereasGermany and Austria report 10 and 20respectively and in the USA 29 disorders areprimary targets whereas 25 additional disordersare secondary targets. Some disorders such asSCAD, 3-methyl-CoA-carboxylase deficiencydetected by MS/MS are clinically inconsequen-tial and may not be reported.37 The technologyhas recently been adapted to screen lysosomalstorage disorders. Simultaneous detection of apanel of six disorders (Gaucher, Fabry, Pompe,MPS I, Krabbe and Niemann-Pick disease typeA & B) is now well established from a dry bloodspot (DBS) sample (multiplex testing). Assaysare now being developed for Tay Sachs disease,MPS II, MPS VI and metachromaticleukodystrophy.38 A relative disadvantage of MS/MS is that congenital hypothyroidism andgalactosemia cannot be tested.

It must be clear that an elevated analyteconcentration from the original specimen requirestracking the child to obtain a second DBS. If theanalyte is abnormal in the second assay, the babyhas to be referred for confirmatory tests andtherapy. Thus it should be understood that merelydetecting an abnormal analyte from a DBS is notdiagnostic and must be followed by confirmatorytests. Confirmatory tests would includeaminoacid measurement by a quantitativetechnique like HPLC or estimation of enzymeactivity or genotyping.

Selection of disorders to screen

In 1968, Wilson and Jungner describedguidelines for selecting disorders for screening.37

The disorders selected for NBS should be severediseases relatively frequent in a given population,treatable or controllable, those for which a test

exists and a suitable specimen can be obtainedeasily. It is necessary that manifestations appearafter a variable symptom-free interval.29

Phenylketonuria was the prototype. It issignificantly prevalent in North America andEurope with an incidence of 1 in 12000 livebirths. Early manifestations such as seizures,eczema and depigmented hair and skin occurin the first few months of life and untreateddisease leads to severe mental retardation,hyperactivity, aggressive behaviour and autism.Blood phenylalanine levels in an affected infantare abnormally high by 72 hours of life. Simplemethods can detect blood phenylalanine levelsfrom a DBS (Table 6). Effective therapy in theform of medical foods complemented by aphenylalanine restricted diet begun by 3 weeksof life prevents mental retardation andneurological abnormalities. While PKU and CHare models fulfilling these requirements, thebenefits of screening for most other disorders bystrictly adhering to these criteria is less evident.Many newborns with MSUD, salt wasting CAH,classical galactosemia and organic acidemias likepropionic acidemia may develop life-threateningsymptoms even before results of newbornscreening are available.35,39 In the latter twodisorders though early identification by screeningprevents death and complications, it does notprevent long-term sequels.35 Well-treated childrenwith galactosemia develop speech andbehavioural abnormalities, visual perceptuallearning abnormalities and ovarian failure is notprevented.29 The experience with the severevariants of propionic acidemia is similar -neurocognitive disability is not completelyprevented and acute relapses occur, though lessfrequently. Pyridoxine-responsive homocysti-nuria may not have elevation of methionine, thetarget analyte in the first days of life, and maytherefore be missed.29 Thus the benefits of NBSfor these disorders is less evident. Nevertheless,strict adherence to these criteria cannot hold

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enough ground in today’s era. High prevalenceof a condition as a screening criterion is irrelevantif the diagnosis comes ‘free’ with multiplextesting for other disorders, as is the case withtandem mass spectroscopy that simultaneouslydetects up to 45 disorders from a single bloodspot. Moreover pressure from the public orsupport groups may be the driving force inselection of disorders for screening. It has beenrevealed that parents may want to know if theirnewborn is affected irrespective of prospects fortreatment. Their future reproductive planning, fore.g. prenatal diagnosis or undergoing tuballigation, may be based on the availability ofscreening for an untreatable disorder or they maysimply want to avoid the psychological traumaof a wrong early diagnosis or may adjustemotionally and financially to the presence andtreatment of a disease in their infant at an earlystage.

Challenges for newborn screening inIndia

Prospects for countrywide screening for IEM

From the Indian perspective, there is nodebate over the urgency for countrywidescreening for CH from the high incidence evenin areas not traditionally known to have endemiciodine deficiency. It is one of the first disordersto be screened in countries beginning NBSprograms. Similarly, incidence of G6PDdeficiency is 2% to 7.8% and accounts for 32%of neonates with hyperbilirubunemia.27,40

In common with several South Asian countries,NBS for G6PD deficiency would be an easychoice. The same would be true for betathalassemia. A decision to implementcountrywide NBS for IEM, even those with agratifying success like PKU would be lessstraightforward. This would stem from the lackof easy access to therapy in the form of medicalfoods. Difficulty in procuring medical foods dueto lack of appropriate legislation, government

subsidy or health insurance reforms has promptedcriticism for screening for some IEM in theUSA.41 The same argument would discourageNBS for lysosomal storage disorders till such atime that therapy is available and affordable inthe country. Until the natural history and benefitsof NBS for organic acidemias like propionicacidemia or MCAD is clearly evident fromcountries screening these disorders, adoptingthese as targets for screening in India may haveto be deferred.

Prospects for screening for IEM inindividual States

The wide disparity in demographics andhealth performance indicators within India makesa uniform countrywide policy for NBSimpractical. Difficulties would arise from a largegeographic area, wide variations in terrain withinaccessible areas like mountainous terrain andtribal belts, inter-state socioeconomic disparity,rural to urban migrations, illiteracy and religiousand cultural taboos. Therefore it would seemwiser for NBS to be prioritized in each stateaccording to their demographics; healthexpenditure projections; infrastructure in termsof technology, clinical and laboratory expertise,manpower and organizational capability fordelivery of the program.

From a demographic perspective, there arethree regions based on the infant mortality andliteracy rates. The first is Goa and Kerala, theonly two states in the country with an IMR of<20 and literacy rate of 83% and > 90%respectively (Census of India 2001, http://cyberjournalist.org.in/census/cenlit0.html). SinceJune 2008, Goa has initiated universal NBS as apublic-private partnership for 45 metabolicdisorders in addition to CH, CAH, G6PDdeficiency and cystic fibrosis. Kerala has yet toinitiate NBS on state-wide basis. In these statesfurther reduction in IMR to < 5 like Singaporeand Japan would be feasible only with measures

37


to eliminate mortality due to genetic disorderslike IEM and malformations. Therefore NBSmust be a priority in Kerala. Maharashtra(IMR 35, literacy rate 77%), Tamil Nadu(IMR 37, literacy rate 73%) and New Delhi(IMR 37, literacy rate 81%) fall in the secondregion. In these states, NBS is an emergingpriority. Implementing NBS in the next few yearsin these states will also ensure that the IMR of< 30 per 1000 is quickly achieved. The rest ofthe country, with an IMR > 40 comprises the thirdsection where infectious disease, perinatalasphyxia and neonatal sepsis still take a heavytoll. NBS will not be a priority in such states.

Hurdles to be overcome in India

Factors identified for successfulimplementation of a screening program aregovernment prioritization, government financing,public education and acceptance, co-operationand involvement of health practitioners,government participation in institutionalizing ascreening system and integration into the existingnational health care system.27 Delivery of healthcare in India is through the public and privatesectors. Under the public health programs healthcare delivery differs in the rural and urban areas.Rural population is served by a vast network ofprimary health centres and sub-centres in India.With training, health resources and manpowerat the primary health care level can be galvanizedfor implementation of NBS.3 This approach wasshown to be practical and feasible by Kochupillaiet al in a rural setting in Uttar Pradesh.42 On thecontrary, implementation in the city using theexisting infrastructure of a Pediatric referralhospital in Mumbai was fraught withorganizational difficulties: 28% missedcollections, a dismal 30% response to recall,delayed reporting/ follow-up examination,migratory slum population and illiteracy leadingto difficulty in recording addresses.43 The numberof institutional deliveries in India is presently

40.7%. (NFHS 3) The program will have to makeprovisions for covering 60% of the newbornsdelivered at home into the NBS program.Currently, one third of all deliveries are attendedby traditional birth attendants (TBA) and48.3% of deliveries are assisted by healthprofessionals (doctor / nurse / LHV / ANM / otherhealth personnel). These birth attendants willhave to be educated and trained for motivatingfamilies and for DBS collection and ensuring thatit reaches the laboratory. For program success,the tracking and follow-up of positive screens iscrucial. Efficient tracking of screen-positivebabies would have to be implemented on the linesof AFP surveillance. Developing infrastructurein terms of trained manpower (laboratory as wellas clinical expertise), investment in technologyfor confirmation and monitoring and easyavailability of therapeutic options is the logicalaccompaniment for success of program.On ethical grounds, it would be unreasonable toinvest in screening and diagnostic facilitieswithout ensuring easy availability of therapy inthe form medical foods (Aminoacidopathies,organic acidemias, urea cycle defects), enzymereplacement therapy (lysosomal storagedisorders) or organ transplantation (bone marrow,liver, kidney, stem cell). Opponents of newbornscreening would cite these deficiencies anddiverting health expenditure in implementationof the program in India. In countries of Asia-Pacific, the cost of screening varies from no fees(Palau) to USD 32 (Singapore). It is paid by theGovernment in Australia, Bangladesh, HongKong, Japan, New Zealand, Sri Lanka, Thailandand South Korea while the family bears the costin China, Taiwan, Indonesia, Philippines andSingapore.27 In India, the cost is borne by thefamily or in the case of Goa directly or indirectlyby the Government including cost forconfirmatory tests and medical foods for thosescreening positive and confirmed to have an IEM.Confirmatory tests for a few selected screen

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positive disorders are obtained through the testinglaboratory (personal communication). An optionwould be mandating payment for the cost ofscreening and therapy through insurance reformsby legislation. The program will also have toaddress the question – whether NBS should bemandated like in China and Philippines orvoluntary? Some states in the USA mandate NBSby legislation. If parents refuse testing citingreligious or other reasons, an “informed dissent”document is required to be signed.35

The need of the day would be to set up anExpert Group on newborn screening under theauspices of professional bodies like NationalNeonatology Forum and Indian Academy ofPediatrics. This group would have the necessaryauthority to represent the cause of NBS to thegovernment. It would also play a proactive rolein propagating the cause of newborn screeningin the country for training, awareness andeducational campaigns for pediatricians andother relevant health care professionals on thelines of breast feeding training, IMNCI andHIV programs.

Points to Remember

• Prenatal diagnosis for IEM should beoffered to families for prevention ofrecurrence when the diagnosis is confirmedin an index case.

• Abnormalities such as fetal hydrops orvisceromegaly detected by ultrasonographyshould prompt investigations for an IEMunder appropriate circumstances.

• The appropriate option for prenataldiagnosis is chorion villus sampling oramniocentesis for estimation of enzymeactivity or genotyping if the mutations havebeen tested in the index case. In other casesoptions such as substrate or metaboliteprofiling in the amniotic fluid supernatantmay be appropriate.

• Newborn screening for IEM permits earlydiagnosis and treatment resulting inprevention of disabilities, especiallyneurodisability.

• Implementing universal newbornscreening would lead to substantial gainsin decreasing the infant mortality rates.

Acknowledgement

The authors thank Dr. Sanjay Oak, Director(ME & H) for granting permission to publish thepaper.

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40. Pao M, Kulkarni A, Gupta V, Balan S. Neonatalscreening for glucose-6-phosphatedehydrogenase deficiency, Indian J Pediatr2005; 72: 835 – 837.

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36. Rhead WJ, Irons M. The call from the newbornscreening laboratory: frustration in theafternoon. Pediatr Clin North Am 2004; 51: 803– 818.

37. Pollitt RJ. Introducing new screens: why are weall doing different things, J Inherit Metab Dis2007; 30: 423 – 429.

38. Gelb MH, Turecek F, Ron Scott C, ChamolesNA. Direct multiplex assay of enzymes in driedblood spots by tandem mass spectroscopy forthe newborn screening of lysosomal storage

Annexure 1. Prenatal diagnosis for disorders of carbohydrate metabolism5,19

Disease Inheritance Sample Diagnostic assay Special remarks

Galactosemia AR AFC (E) Galactokinase

Galactosemia AR CVS, AFC, (E) Galactose Raised galactitolAF 1-phosphate uridyl using GC/MS in

transferase 1st/2nd trimester18

Glycogenosis Ia AR AFC, CVS M (E) Glucose Mutation analysis nowFetal liver 6- phosphatase obviates the need for abiopsy invasive fetal liver

biopsy17

Glycogenosis III AR CVS,AFC (E) Amylo 1,6glucosidase M

Glycogenosis IV AR CVC, AFC (E) Brancher USG- hydrops fetalis 19

enzyme M

41


Annexure 2. Prenatal diagnostic modalities for lysosomal storage disorders5, 8

Disease Functional Chromosome Gene Prenataldefect/deficiency locus diagnosis

GM1 Gangliosidosis *ß-Galactosidase 3p21 GLB1 AFS, E, M

Tay-Sachs disease *ß –Hexosaminidase A 15q HEX A AFS, E, M

Sandhoff Disease *ß-Hexosamindase A&B 5q13 HEX B AFS, E, M

GM2 Gangliosidosis GM2 activator protein 5q GM2A

AB variant

Niemann Pick Types *Sphingomyelinase 11p15 SMPD1, E, M

A and B NPD

Niemann Pick, Type C Cholesterol esterification NPC1 Cholesteroldefect NPC2/ HE1 esterification

in CC, M, H

Gaucher Disease *Glucocerebrosidase 1q21-q31 GBA E, M

Gaucher disease Saposin C -

Metachromatic *Arylsulfatase A 22q13 ARSA E, M

Leukodystrophy

Metachromatic Saposin B M, Sulphatide

Leukodystrophy loading of CC

Krabbe Disease *Galactocerebrosidase 14q31 GALC E, M

Fabry disease# A-galactosidase Xq22 a-Gal A E, M,

Farber Disease *Ceramidase 8P ASAH E, M

Wolman Disease Acid Lipase 10q24 LIPA Ea

MPS I *a-L-Iduronidase 4p16 IDUA, IDA AFS, E, M

MPS II# *Iduronate Sulfatase Xq28 IDS AFS, E, M

MPS IIIA Heparan N-Sulfatase 17q25 SGSH, AFS, E, M,MPS3A, radioactiveSFMD sulphamidase

assayMPS IIIB N-acetylglucosaminidase 17q21 NAGLU AFS, E, M

MPS IIIC Acetyl-CoA- 8p11-q13 MPS3C AFS, E, MGlucosaminide

Acetyltransferase

MPSIIID N-acetylglucosamine 12q14 GNS, G6S

-6-sulfatase

MPS IVA Galactosamine 16q24 GALNS AFS, E, M

-6-Sulfatase

MPS IVB *ß -Galactosidase 3p21 GLB1 AFS, E, M

MPS VI Arylsulfatase B 5q13 ARSB, AFS, E, MMPS6

Contd...42

2010; 12(2) : 145

MPS VII *ß -Glucuronidase 7q21 GUSB, E, M

MPS7

MPS IX Hyaluronidase 3p21 HYAL1 -

Mannosidosis a-Mannosidase 19cen-Q12 MAN2B1, E, M

MANB

Fucosidosis a-L-Fucosidase 1p34 FUCA1 E, M

Aspartylglyco- N-Aspartyl-b- 4q32 AGA E

saminuria Glucosaminidase

Sialidosis Neuraminidase 6p21 NEU1, CC, M

(Mucolipidosis I) SIAL1

Mucolipidosis II N- 4q GNPTA AFS, CC, M,(I-Cell Disease) Acetylglucosamine-1- H

Phosphotransferase

Mucolipidosis IIIA N 4q GNPTAB AFS, CC, M

(Pseudo-Hurler -Acetylglucosamine-1-

Polydystrophy) Phosphotransferase

Mucolipidosis IIIC N- 16p GNPTAG

Acetylglucosamine-1-

Phosphotransferase

Mucolipidosis IV Mucolipin-1 19p13 MCOLN1 AFC

Galactosialidosis Lysosomal 20q13 PPGB, Eprotective protein GSL,NGBE,(cathepsin A) CTSA,

GLB2Schindler Disease a-N-Acetyl- 22q11 NAGA E

GalactosaminidasePompe Disease Acid a-1, 4- 17q25 GAA E, M

GlucosidaseInfantile sialic acid Sialic acid transporter 6q SLC17A5, AFS, Mstorage disease SIASD,

SLDSalla Disease Sialic acid transporter 6q SLC17A5, AFS, M

SIASD,SLD

Cystinosis Cystine Transporter 17p13 CTNS CCDanon disease LAMP-2 Xq24 LAMP2, -

LAMBNCL infantile type Palmitoyl protein 1p32 PPT1, M

thioesterase CLN 1


Contd...43



NCL late Tripeptidyl peptidase 11p15 CLN 2 Minfantile type

Juvenile NCL Membrane protein 16p12 CLN 3 MAdult NCL Palmitoyl protein

(Kufs disease) thioesterase 1p32 CLN 4 -NCL Finnish late Membrane protein 13q CLN 5 Minfantile type

NCL early juvenile - 15q CLN 6 -

NCL - CLN 7 -

NCL Membrane protien CLN 8 -

Multiple sulphatase Sulphatase modifier protein Edeficiency (Arylsuphatase A, B,

steroid sulphataseenzyme activities)

# Fetal sexing is essential to identify unaffected female heterozygotes in whom low enzyme activity mayoverlap in the affected range.

AF = Amniotic Fluid, AFC = Cultured amniotic fluid cells, AFS - Amniohc flluid supernatant,CVS = Chorion villus sampling, CC = Cultured chorionic villi, M = Mutation, AR = Autosomal recessive E =Enzyme analysis

* Prenatal diagnosis for these disorders is available at several genetic centers in India by estimation ofenzyme activity from chorionic villus tissue or cultured amniotic fluid fibroblasts.

Annexure 3. Prenatal diagnosis for disorders of amino acid and organic acidmetabolism

Disease Inheri- Sample Diagnostic assay Special remarkstance

Phenylketonuria 1 AR AFC, CVS M 3/4th of the affected casesare likely to be geneticcompounds of PKU alleles20

Phenylketonuria 2 AR AFC (E) Dihydropter-idine reductasedeficiency

Tyrosinemia type I AR CVS (E) Fumarylacet AFS: Succinylacetoneoacetase M

Albinism, AR AFS, CVS, M, Electron Fetal skin biopsy (20 weeks ofoculocutaneous Fetal Skin microscopy gestation)16 Hair bulb analysis

biopsy for melanosome developmenthas given way to a moreaccurate molecular analysis21

Carbamoyl phosphate AR AFS, CVS, Msynthetase deficiency Fetal liver

biopsy

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Ornithine carbamoyl AR AFS, CVS, Mtransferase deficiency Fetal liver

biopsyArginosuccinic AR CVC, AFC, (E) Arginosucci-aciduria AFS nate lyase, MCitrullinemia AR CVC, AFC, M Radiolabelled (C

14) citrulline

AFS incorporation22

Argininemia AR Fetal blood (E) Arginasesample

Non ketotic AR AFS Increased glycine/ USG- abnormal fetalhyperglycinaemia serine ratio movements (reduced or absent)Homocystinuria AR AFC, CVC (E) Cystathionine Uncultured cells yield poor

beta synthetase results5

Maple syrup urine AR CVS, AFC M Release of CO2 from radio-disease labelled leucine. Uncultured

cells yield poor results23

Organic acidemias AR AFS, CVS E: AFC/ CVS Electrospray ionization TMS(propionic acidemia, (majority) M: AFC/ CVS analysis of acylcarnitinesmethylmalonic acidemia, Analytes: AFS in the AFS24

glutaric acidemia type 1,ketothiolase deficiency,and isovaleric acidemia)Fatty acid oxidation AR CVC, AFC (E) Amniocyte incubation withdefects isotope labelled precursors with

acylcarnitine analysis of interme-diate metabolites by TMS24

Disease Inheri- Sample Diagnostic assay Special remarkstance

Annexure 4. Prenatal diagnosis for other metabolic disorders5,26

Disease Inheritance Sample Diagnostic assay Special remarks

Zellweger AR CVS, CVC, (E) Perioxisome USG: Increased nuchalAFC biogenesis M translucency, fetal hypokinesia,

renal hyperechogenicity,dilated ventricles25

X Linked XR CVC (E) Very long chainadrenoleukodys- fatty acyl CoAtrophy# synthetase M

Lesch-Nyhan AR CVS, AFC (E) HGPRT M

AFS – Amniotic fluid supernatant, E – Measurement of enzyme activity, M – Mutation identification bymolecular techniques, H – Histopathology, CC – Cultured cells, NCL – Neuronal ceroid lipofuscinosis, GC/MS:gas chromatography-mass spectroscopy, TMS: tandem mass spectroscopy

# Fetal sexing is essential to identify unaffected female heterozygotes in whom low enzyme activity mayaverlap in the affected range

45



SAMPLE COLLECTION,SUITABILITY ANDINTERPRETATION INSUSPECTED INBORN ERRORSOF METABOLISM

* Ananth N Rao** Minakshi Koch

** Sabyasachi Ghosh*** Shobha G

***Suresh Kumar V

Abstract: Inherited metabolic disorders are aheterogeneous group of genetic conditions mostlyoccurring in childhood. They are individuallyrare but collectively numerous, causingsubstantial morbidity and mortality. Screeningfor inherited metabolic disorders is therefore veryimportant. The importance of screening forinborn errors of metabolism (IEM) introducesseveral decision points about specimencollection, processing, and storage for theinvestigator. The method of sampling is ofgreatest importance for precise results and hencefor earlier and accurate diagnoses.

Keywords: Inborn errors of metabolism,Specimen collection, Processing, Interpretation.

Inborn errors of metabolism (IEM)individually are rare but collectively are common.

In IEM single gene defects are responsible forthe abnormalities in the synthesis or catabolismof proteins, carbohydrates or fats by way ofdefective enzymes or transport proteins, resultingin a block of metabolic pathway. The male tofemale ratio is 1:1 for X-linked dominantif transmission is from mother to child.1

For evaluating an IEM the following fiveimportant aspects should follow:

1. History/ Family History: We should notewhether the neonate was born to consanguineousparents or not and also check history of previoussiblings like fetal deaths or miscarriages orgenetically affected siblings and also note thepedigree for two generations.

2. Physical examination like dermatitis, alopecia,facial dysmorphism, cataract, etc.

3. Initial screening tests includes complete bloodcount, electrolytes level, glucose, ammonia,lactate, lactate/ pyruvate ratio, reducingsubstances, organic acids, amino acids, ketones,mucopolysaccharides (MPS), uric acid, liverfunction tests (LFT), renal function tests (RFT)and Porphyrins.

4. Advanced screening tests: The test isperformed on basis of clinical context whichincludes long chain fatty acids, MPS separationand specification, quantitation of amino acids,organic acids, carbohydrate and othermetabolites.

5. Definitive diagnostic tests: To confirm thedisorder, specific enzyme assays in leucocytes,plasma/serum or red cells, immunoassays andDNA probe analysis are required.

* Consultant and Head

** Scientific Officer

*** Senior Scientific Officer

Department of Metabolic Diseases,Metabolism Laboratory,Apollo Hospitals, Bangalore.

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Individual IEM are rare disorders,most having an incidence of less than 1 per100,000 births. However, when consideredcollectively, the incidence may approach 1 in 800to 2500 births.

Age for presentation of clinical symptomsvaries for individual IEM and variant formswithin the IEM. The timing of presentationdepends on significant accumulation of toxicmetabolites or on the deficiency of substrate.The disorders of carbohydrate or proteinmetabolism and disorders of energy productiontend to present in the neonatal period or earlyinfancy and tend to be unrelenting and rapidlyprogressive. Less severe variants of thesediseases usually present later in infancy orchildhood and tend to be episodic. Fatty acidoxidation, glycogen storage and lysosomalstorage disorders tend to present insidiously ininfancy or childhood. Disorders manifested bysubtle neurological or psychiatric features oftengo undiagnosed until adulthood.2

Screening is a basic tool for clinicallysuspected cases of inborn metabolic diseasesby a simple economical and effective techniquelike paper chromatography, thin layerchromatography (TLC) and some biochemicaltests. However, it is not reasonable to makefirm decision on the basis of screening test.high performance liquid chromatography(HPLC), tandem mass spectrometry (TMS), gaschromatography-mass spectrometry (GC-MS)are the advanced techniques for confirmingmetabolic disorders. The acyl carnitines in bloodreflect the primary accumulating mitochondrialacyl-CoA metabolites in disorders of fatty acidand amino acid catabolism. Thus an acyl carnitine“profile” will recognize almost all of the defectsin these pathways using the advanced techniquelike TMS.3 Advanced chemical diagnosis usingGC-MS has also become an important part ofthe routine diagnostic service. Newborn

screening is the process of testing newborn babiesfor treatable genetic, endocrinologic, metabolicand hematologic diseases using blood sampleson filter paper obtained by pricking a newbornbaby’s heel on the second day of life to get a fewdrops of blood.4 The development of tandemmass spectrometry expanded newborn screeningled to a large expansion of potentially detectablecongenital metabolic diseases that affect bloodlevels of organic acids.5

Sample collection for metabolic tests

To keep in context of this review, samplecollection details for important parameters arediscussed first.

(1) Electrolytes, liver function test (LFT),renal function test (RFT), complete bloodcount (CBC), glucose

For LFT and RFT 1 ml of plasma or serumsample is collected. Sample should be stored at2-8°C if the test is to be performed later.

For CBC one 3 ml of blood is collected invials having anticoagulants like EDTA orK

2 EDTA.

Glucose: 1 ml plasma or serum is collectedand stored at 2-8°C if test is to be performed later.For cases suspected with glycosuria random urinespecimens are acceptable, but have no referenceintervals.

For electrolytes 24-hour or random urine isaccepted. Specimen must be refrigerated if thetest is to be performed later.6

(2) Collection of plasma for ammoniaestimation

It is preferable to collect this sample afterat least 6 hours of fasting. Heparin is the preferredanticoagulant, because it has been shown toreduce red blood cell ammonia production. EDTAcan also be used. Donors’ arms should be as

47


relaxed as possible, because muscle exertion mayincrease venous ammonia levels. Blood is drawninto a chilled, heparinized vacuum tube that isimmediately placed on ice, and plasma isseparated within 15 minutes. It is crucial to keepblood samples cold after collection, because theammonia concentration of standing blood andplasma increases spontaneously. Plasmaammonia levels of whole blood maintained at 4oCare stable for <1 hour. When promptly separatedfrom blood, plasma ammonia levels are stable at4oC for 4 hours and for 24 hours if stored frozenat –20oC.6

(3) Collection of blood or cerebrospinal fluid(CSF) for lactate and pyruvate assays

They are measured enzymatically in bloodor CSF as an index of impaired pyruvatemetabolism due to defects of glucose oxidationor gluconeogenesis. The ratio of lactate topyruvate reflects the NAD/NADH ratio and isuseful in distinguishing primary defects ofpyruvate metabolism from defects of electrontransport (or oxidation).

Collect venous or arterial blood withoutprolonged stress to the patient with brief use ofthe tourniquet, if needed. If collecting blood forseveral purposes/tests, quickly draw all the bloodneeded and place initially in a plain tube.Deproteinize immediately to avoid artifacts oflactate formation in red cells or loss of pyruvate.

For this blood or CSF obtained is measuredand transferred immediately into a tightlystoppered tube containing 8% w/v perchloricacid. Stopper the tube and shake vigorously forat least 15 seconds. The sample is now stablefor local transport to a laboratory where it can becentrifuged. Two centrifugations may berequired to obtain a clear supernatant.The supernatant should be removed with aPasteur pipette and transferred to a tightlystoppered polypropylene tube. Freeze the

supernatant, and pack in sufficient dry ice forshipment/ transport. Perchloric acid is preparedby mixing 7 ml of 70% perchloric acid anddistilled water to make 100 ml total volume.Refrigerate until ready to use.7

(4) Collection of plasma for amino acids

Collect 1 ml blood in an EDTA tube(lavender top), mix gently, and centrifuge.Transfer plasma into a polypropylene tube, andfreeze until assayed.

Quantitation of amino acids with theAutomated Analyzer or Reverse Phase-HighPerformance Liquid Chromatography(RP-HPLC) has been the method of choice forquick, reliable and effective interpretation of theaminoacidogram in suspected cases of metabolicdefects. Several trivial factors when notconsidered lead to significant deviations from theactual amino acid picture. A few of them arediscussed briefly,

(a) Choice of anticoagulant: Heparin has beenthe most commonly used and preferredanticoagulant for preparation of plasma for aminoacid analysis. The mucoitin polysaccharideinhibits the formation of thrombin from pro-thrombin. Normal concentration of use foranalysis is 2mg/10ml blood. However, itsindiscriminate use leads to hemolysis and mayintroduce artifacts. Another anticoagulant, EDTA(ethylene diamine tetra acetate) chelates calciumions for anticoagulant effect and is available intwo forms viz. dipotassium and dilithium salt.The former is preferred owing to greatersolubility. Ninhydrin positive artifact withheparin or EDTA co-eluting with taurine havebeen detected.8

(b) Hemolysis: Hemolysis can occur duringphlebotomy, preparation of the sample andtransport of the specimen. Even when hemolysisis not noticeable on naked eye inspection,

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spectroscopic examination may reveal bands ofoxyhemoglobin. In such samples, increases inconcentrations of taurine, glutamic acid andglutathione have been noted and decreases ofupto 50 % have been noted with respect toarginine and cystine.9

(c) Deproteinising agent: Picric acid andsulfosalicylic acid have served the purpose ofdeproteinising the sample prior to applicationonto the column. Appreciable losses oftryptophan have been observed with picric acid.Sulfosalicylic acid has the advantage of notaltering the amino acid composition and alsomakes the Dowex treatment that ensues theuse of picric acid, unnecessary afterdeproteinisation.9

(d) Delay in deproteinisation: Effect of delayin deproteinisation can be readily noticed withreference to cysteine, homocysteine and cystine,wherein they readily bind to red cell and plasmaproteins in the time gap available. Conversionof pyruvate to lactate is also favored during thedelay. Formation of urea is also consequent todelay, particularly in contaminated specimens.Errors with respect to homocysteine could provecostly when dealing with a homocystinuric ondietary restriction since actual prognosis may notbe indicated.9

(e) Buffy coat contamination: This is probablythe easiest contaminant while separating plasma.The effort of acquiring maximum plasma is agreat temptation to resist. Contamination withleucocytes and platelets leads to a high estimateof glutamic acid and aspartic acid since these cellscontain much higher levels than plasma itself.Other aminoacids are not significantly affectedas their concentration in mature erythrocytes issimilar to that in plasma. It would be wise enoughto leave a 5- 7mm layer above the buffy coatregion before aspirating plasma.9

(f) Delay in analysis: Deproteinised plasmastored for longer periods, even in a freezer, maynot result in the most accurate amino aciddeterminations. Losses in levels of glutamine andaspargine have been noted whilst increases inglutamic acid and aspartic acid have beenreported. However, it is generally opined that90-day freezer storage could still render thesample suitable for amino acid analysis.9

Plasma is preferred for aminoacid analysisover serum, which usually stands as the mostimportant sample for a large number ofbiochemical investigations.

Heparin interferes with detection ofsulphur containing aminoacids, cysteine andhomocysteine. Thus EDTA sample may be themost accurate for measurement of cysteineconcentration.10

Collection of plasma for homocysteineestimation

Fasting specimen is preferred. Plasma orserum must be separated from cells within onehour of collection. If specimen cannot beseparated from the cells within an hour specimenshould be stored in ice. EDTA or K

2 EDTA is the

preferred anti-coagulant.9

(5) Collection of plasma for carnitine,βββββ-hydroxybutyrate / acetoacetate andfree fatty acid assays

These compounds are measured in plasmaas parameters of lipolysis and fatty acid oxidation(e.g. for diagnostic testing of fastinghypoglycemia or after an oral fat tolerance test)or to monitor efficacy of ketogenic diets.The ratio of α-hydroxybutyrate/ acetoacetatereflects mitochondrial NAD/NADH, and may bea useful parameter for diagnosis of defects of theKrebs cycle.

49


Collect 1 ml blood is in an EDTA tube(lavender top), mix gently, and centrifuge.Transfer plasma into a polypropylene tube, andfreeze if test is to be performed later.

(6) Collection of urine for carnitine ororganic acid assays

Carnitine screening is recommended fordetection of primary or secondary carnitinedeficiency and monitoring of patients beingtreated with carnitine. Candidates includepatients with failure to thrive, cardiomyopathy,weakness, or possible metabolic disorders.

The patient’s perineal area should bethoroughly washed and rinsed with water toremove all dirt, oils, and soap. Collect urine in athoroughly clean container, preferably culturecontainer. Transfer 5 ml of urine (minimum1 ml) to a tightly stoppered polypropylene tubeand freeze.10 Preservatives may be required whilecollecing urine for certain substances (Table 1).

Metabolic decompensation, such as lacticacidosis, ketosis, or liver failure, gives rise toan abnormal excretion of organic acids(keto branched, dicarboxylic, or aromatic acids,respectively) that are otherwise involved inparticular IEM; this sometimes rendersinterpretation even more difficult.Poor preservation of urine samples will lead tonon-enzymatic conversion of all ketoacids to therespective hydroxyacid; for example,acetoacetate is converted to 3-hydroxybutyrate,and 2-ketoglutarate is converted to2-hydroxyglutarate.11

(7) Collection of urine for mucopoly-saccharides (MPS): 5 ml of urine is collectedand frozen if test is to be performed later.Morning void is preferred.12

(8) Collection of urine for GC-MS: 10 ml ofurine is collected and kept in refrigerator.

(9) Collection of whole blood for lymphocyteassays

The lymphocytes must be isolated within48 hours of collection. Blood must be collectedin Anticoagulant Citrate Dextrose (ACD) usingsterile technique. 5 ml of blood is preferred forpatients < 6 months of age and 10 ml for patientsfor > 6 months of age.

(10) Collection of blood spots for newbornscreening (NBS) and tandem massspectrometry (TMS)

Heel prick samples must be collected onspecial filter paper meant for newborn screeninganalysis and TMS for carnitines and acylcarnitines. For newborn screening collection of

Table 1. Preservatives for urine

Test Recommendedpreservative

Urine organic acids No additive

Urine for mucopoly-saccharides No additive

Urine for Carnitine No additive

5-HIAA 25 ml 6N HCl/15 gm Boric acid

Aminolevulonic acid No additive

Catecholamines 25 ml 6N HCl/15 gm Boric acid

Cystine No additive/25 ml6N HCl/ 15 gmBoric acid

Metanephrine 25 ml 6N HCl/15 gm Boric acid

Porphobilinogen No additive(Refrigerate andprotect from light)

Porphyrins 5 gm SodiumCarbonate

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urine sample is erroneous. Dried Blood spot isthe preferred sample source for screeningnewborns by TMS and/or Enzyme Immunoassay(EIA) or Fluorescence based Immuno Assay(FIA). Urine may not be the right choice, sinceanalytes vary in concentration over a large rangein newborns. This introduces error in reportingactual levels. Artifacts could also cause a problemin interpretation. GC-MS through which urinemay be screened is not a method of choice forNBS due to its low through put coupled with thevarious limiting factors associated with urineas a sample for Newborn screening. TMS is anFDA approved mode of NBS along withEnzyme Immunoassay (EIA)/ FluorescenceImmunoassay (FIA).

The analysis includes a quantitative HighPerformance Liquid Chromatography-MassSpectrometry-Mass Spectrometry (HPLC –MSMS) determination of over 35 differentacylcarnitines in plasma, urine, and tissuesamples, including short, medium and long-chainderivatives. The acylcarnitine profile includesquantitation of free and total carnitine.

Semi-quantitative determination of over50 non-volatile organic acids as TMS derivativescan be made by capillary dual column gaschromatography with confirmation by massspectrometry. It is useful for newborn screening,follow up, and diagnosis and monitoring of alarge variety of metabolic disorders, includingdefects of pyruvate metabolism, the Krebs cycle,amino acid, fatty acid oxidation and the electrontransport chain.

Interpretations

The diagnostic specificity of analysis underacute versus asymptomatic conditions may varyconsiderably. Identification of all relevant organicacids or compounds must be listed and quantitymay be mentioned. When no significant

abnormalities are detected, an analysis should bereported and interpreted in qualitative terms only.When abnormal results are detected, a detailedinterpretation must include; an overview of theresults and their significance in perspective ofthe index case, a correlation to available clinicalinformation, elements of differential diagnosis,recommendations for additional biochemicaltesting and in vitro confirmatory studies such asan enzyme assay or a molecular analysis.Age dependency of urinary metabolites must berecognized and must be applied duringinterpretation.

Common pitfalls that may lead to erroneousinterpretations include liver disease whichmay cause aminoacid elevations, renal tubulardefects causing generalized aminoaciduriaand hypoperfusion leading to lactic acidosis.Drugs such as valproate may cause ketonuria,topiramate and acetozolamide may cause lacticacademia. In essence, a single negative test maynot exclude an IEM in all cases, thus underliningthe significance of collecting a sample during anepisode.

A confirmatory assay is always essentialsince mere presence of an abnormal metabolitemight not be indicative of the disorder always.

Conclusion

Samples for inherited metabolic disordersshould be collected as mentioned using specificpreservatives. For both blood as well as urinethere is a minimum volume which should becollected for complete assay. Samples collectedduring acute episodes could be ‘precious’ andyield more information while screening for anIEM. For newborn screening it should preferablybe heel prick blood spot. Urine as a sample fornewborn screening is not acceptable for reasonsdiscussed earlier in this review. Clinicians andlaboratory personnel should have a clear view

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on the samples to be collected for accurate results.In general it may be accepted that EDTA plasmais a preferred choice for aminoacid and carnitineestimations. Heparinzed plasma is the preferredchoice for most enzyme assays with fewexceptions. Urine is the preferred choice foranalysis of organic acids, MPS, porphyrins,sugars and metachromatin granules. CSF ispreferred for analysis of glycine andneurotransmitters. One must remember the factthat “Results of a metabolic investigation are asgood as a sample”.

Points to Remember

• An appropriate sample according to themetabolite of interest is to be collected,processed and analysed as per standardprotocol.

• While interpreting the result, factorsinfluencing the result like whether samplehas been collected during symptomaticor asymptomatic period, co morbid areconditions, etc. are to be considered.

References

1. Choudhuri T, Sengupta S. Inborn errors ofmetabolism-An Indian Perspective. Int J HumGenet 2006;6: 89-91.

2. Ward JC. Inborn errors of metabolism of acuteonset in infancy. Pediatr Rev 1990;11: 205-216.

3. Newfeld E, Muenzer J. The mucopoly-saccharidoses. In: ScriverCR, Beaudet AL, SLYWs, Valle (Eds). The metabolic and molecularbases of inherited diseases. 8

th Edn, Newyork, Mc-

Graw Hill, 2001; PP 3421-3452.

4. Clague A, Thomas A. Neonatal biochemicalscreening for disease. Clin Chem Acta 2002;315: 99–110.

5. Chace DH, Kalas TA, Naylor EW. Use oftandem mass spectrometry for multianalytescreening of dried blood specimens fromnewborns. Clin Chem 2003; 49: 1797–1817.

6. Varley H, Gowenlock AH, Bell M. The ureasemethod using Berthelot reaction. In:PracticalClinical Biochemistry, 5

th Edn, Vol.1, CBS

Publishers & Distributors, Delhi 1991;pp 368-378.

7. John F, O’Brien. Inborn Errors of Amino Acid,Organic Acid and Fatty Acid Metabolism. In:Burtis CA, Ashwood ER, Tietz VW Eds, TeitzTextbook of Clinical Chemistry, 3

rd Edn,

New Delhi, Elsevier, 1986: 2208.

8. Rao AN. Laboratory Generated Artifacts inPlasma Amino acid Quantitation. OnlineJ Health Allied Sci. 2002;3:4.

9. Chuang CK, Lin SP, Lin YT, Huang FY. Effectsof anticoagulants in aminoacid analysis:Comparisons of Heparin, EDTA and SodiumCitrate in vacutainer tubes for plasmapreparation. Tech Briefs. Am Assoc Clin Chem1998; 44:1052-1056.

10. Sweetman, L. Organic acid analysis. In:Hommes, F.I. Techniques in Diagnostic HumanBiochemical Genetics: A Laboratory Manual.New York Wiley-Liss, 1991:pp 143-176.

11. Kumps A, Duez P, Mardens Y. Metabolic,nutritional, iatrogenic and artifactual sources ofurinary organic acids: a comprehensive table.Clin Chem 2002; 48:708-717.

12. Ferrante NM. The measurement of urinarymucopolysaccharides. Analytical Biochem1967; 21: 98-106.

In the previous issue, “Indian Journal of Practical Pediatrics” Vol.12 No.1(Jan – Mar), 2010 page no.7, 2nd para, in the second line, to read as “sensitivemethods like PCR” instead of “sensitive methods like PC31”.

E R R A T U M

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INBORN ERRORS OFMETABOLISM IN INFANCY ANDCHILDHOOD PRESENTING WITHMETABOLIC ACIDOSIS

* Prasad C** Rupar CA

Abstract: Disturbances of acid-basehomeostasis are not uncommon. These areimportant indicators of underlying disease ininfants and children. Metabolic acidosis is oneof the most common perturbations noted in acutepediatric emergencies. Frequent causes ofmetabolic acidosis include diabetic ketoacidosis,shock and tissue hypoxia, salicylate and ethanolpoisoning. However, it is important to recognizethat many inborn errors of metabolism (IEM)such as organic acidemias and primary lacticacidosis also present with a persistent metabolicacidosis. Calculation of the anion gap, presenceor absence of hyperammonemia, hypoglycemiaand ketosis are essential in the diagnosis of thesepatients. Early diagnosis and appropriatemanagement is necessary to optimize theoutcome. IEM have a genetic basis andappropriate genetic counseling needs to beprovided to the families.

Key words: Organic acidemias, Lactic acidosis,Hypoglycemia, Ketosis.

Metabolic acidosis is characterizedby decrease in arterial blood pH (pH< 7.30),plasma bicarbonate and pCO

2. It can result from

an abnormal loss of bicarbonate or anaccumulation of acid and can occur with a normal(10-15 mEq/L) or an increased anion gap(>15 mEq/L) that is measured as the differencebetween plasma [Na+] and the sum of plasma[Cl-] and [HCO

3-]. An increased anion gap can

result from the excess production of acid thatoccurs in diabetic ketoacidosis, primary orsecondary lactic acidemias and several IEM.1-4

Metabolic acidosis with a normal anion gap isalmost always hyperchloremic, caused by renalor gastrointestinal loss of bicarbonate (Fig.1).This review briefly outlines the pathogenesis,clinical presentations and approaches todiagnosis and management of IEM that presentwith metabolic acidosis. Treatment of metabolicacidosis is primarily based on the identificationof the underlying cause.

Biochemical abnormalities

Routine laboratory tests including bloodglucose, urine ketones, ammonia and lactate canbe very helpful in establishing a differentialdiagnosis of IEM that present with metabolicacidosis. For example, neonates have thecapacity to rapidly metabolize fat to produceketones and the presence of urine ketones ininfants less than a month should raise suspicionof organic acidemias. Hypoglycemia withmetabolic acidosis can be present in metabolicdisorders such as glycogen storage diseasetype 1, fatty acid β-oxidation disorders anddisorders of gluconeogenesis. Interestingly

* Associate Professor Genetics,Metabolism and Pediatrics

** Director of Laboratory Medicine

Department of Pediatrics and BiochemistryThe Children’s Health Research InstituteUniversity of Western Ontario,London, Ontario, Canada.

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organic acidemias can present with hypo orhyperglycemia.

Lactic acidosis is the most commonmetabolic acidosis. (Fig.2) It can be caused byshock or poor perfusion from a variety of causesincluding congenital heart defects (for examplecoarctation of the aorta and hypoplastic left heartsyndrome). Lactate levels less than 10 mmol/Lmay not result in an elevated anion gap howeverhigh anion gap metabolic acidosis can be due tolactic acidosis (levels>10-15mmol/L). There areprimary lactic acidosis disorders such as pyruvate

dehydrogenase deficiency, pyruvate carboxylasedeficiency and respiratory chain disorders.Elevations in lactate reflect increasedconcentrations of pyruvate, the assay of whichis not easily available in most laboratories.Accumulated pyruvate, is also converted tolactate and alanine. The ratio of plasma lactateconcentration to pyruvate reflects the redoxpotential within the cytosol. A decreased lactate/pyruvate ratio of <10 indicates pyruvatedehydrogenase (PDH) deficiency and anincreased lactate/pyruvate ratio (>25) is

Fig.1. Causes of metabolic acidosis

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suggestive of pyruvate carboxylase deficiency ormitochondrial respiratory chain abnormalities.3

Secondary lactic acidosis is also a feature oforganic acidemias and fatty acid β-oxidationdisorders.

Hyperammonemia and metabolic acidosiscan occur both in fatty acid oxidation disordersor organic acidemias. Hyperammonemia usuallyoccurs as a result of secondary inhibition of theurea cycle enzymes by the toxic metabolites.Although assays for lactate and ammonia aretechnically straightforward, the concentrations ofboth of these metabolites can be elevated

misleadingly by improper specimen collectionand transportation to the laboratory.

Approach to diagnosis

Clinical features

IEM with metabolic acidosis can presentfrom the newborn period throughout childhood.3,4

The neonatal period is one of the more vulnerableages and many of the IEM with metabolicacidosis present during this time.1,3,4 The neonatehas limited clinical symptoms that are alsocommon to other disorders such as infection,intracranial hemorrhage or other newborn

Fig.2. Approach to lactic acidosis

55


emergencies. These include poor sucking,hypotonia, lethargy, seizures, abnormalmovements, bleeding, hypothermia, apnea andvomiting. The presence of unusual odors suchas the smell of sweaty feet in isovaleric acidemia,maple syrup in maple syrup urine disease andcat urine in 3 methyl-crotonyl carboxylasedeficiency is helpful. In later periods in lifeintermittent periods of ataxia, encephalopathiesand neurological presentations along with“Reye syndrome” like phenotype may beassociated with metabolic acidosis.

A detailed family history is essential toidentify consanguinity as most of the IEMpresenting with metabolic acidosis are inheritedin an autosomal recessive manner. The mostprominent exceptions are the E1-α subunit formof pyruvate dehydrogenase deficiency (primarylactic acidemia) which is inherited in an X-linkedmanner. Another exception is the maternalinheritance pattern seen in mitochondrialdisorders such as MELAS (due to mitochondrialDNA point mutations). Similarly, knowledge ofethnic background is helpful as some of thesedisorders are more common in particular ethnicgroups.5 Enquires should also identify any historyof unexplained neonatal or infant deaths,unexplained mental retardation or children withsimilar disease. Most affected infants are born atfull term with a normal birth weight. A detailedexamination focusing on vital signs, the presenceor absence of facial dysmorphism, organomegalyand detailed neurological examination to look forhypo/hypertonia, abnormal movements, andpresence of petechiae or bleeding should becarried out systematically.

Investigations

Investigative work-up in cases withmetabolic acidosis should include the preliminaryand then the specific tests6 (Table 1). For bestresults, it is helpful to provide adequate clinical

information to the laboratory and to be familiarwith the laboratories reference ranges.

Specific IEM disorders to consider withmetabolic acidosis

Although the list of conditions presentingwith metabolic acidosis is quite long, thedisorders are grouped into the following majorcategories.

1. Organic acidemias

2. Fatty acid β-oxidation disorders

3. Lactic acidoses

1. Organic acidemias

A number of these disorders are due todefects in the catabolic pathways for the branchedchain amino acids or other amino acids afterdeamination. Examples of organic acidemiasinclude methylmalonic acidemia (MMA),propionic acidemia (PA) and isovaleric acidemia(IVA). These disorders may present in theneonatal period with an increased anion gapmetabolic acidosis. The most severe presentationis a healthy newborn infant who becomes veryill within the first 24-48 hours with profoundmetabolic acidosis, hyperammonemia, urineketosis and encephalopathy.7 Features of cerebraledema with a bulging fontanel may be present.Neutropenia and thrombocytopenia withelevations in uric acid are noted. Neurologicalabnormalities worsen with abnormal movements,seizures and alterations in tone. EEG may showburst suppression pattern.

The organic acidemias (IVA, PA and MMA)commonly feature dehydration, moderatehepatomegaly and increased anion gap metabolicacidosis and ketonuria. Presence of hyper-ammonemia can induce respiratory alkalosis ora mixed form of acid base disorder abnormalitywhich can lead to a false diagnosis of urea cycle

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Table 1. Recommended investigations for metabolic disorders

Preliminary Tests Specialized Tests

Blood glucose Urine organic acids by gas chromatography-

Electrolytes mass spectrometry (GC-MS)

Blood gases Plasma amino acids

Calculate anion gap Plasma carnitine (acyl and free)

Ammonia Plasma acylcarnitine profile

Lactate Skin Fibroblasts to measure specific enzyme

Urine for ketones assays for specific disorders

Uric acid DNA based studies for molecular analysis

Complete blood count for Lactate/Pyruvate ratio

pancytopenia, neutropenia CSF lactate, amino acids

thrombocytopenia Muscle biopsy for electron microscopy andrespiratory chain analysis

disorder. Hypo or hyperglycemia with ketosiscan mimic neonatal diabetes. In some casescombination of vomiting, abdominal distensionand constipation may suggest gastro-intestinalobstruction such as pyloric stenosis. Withoutprompt management, these neonates cansuccumb to their illness. The clinical features ofthese diseases tend to be non-specific withconsiderable overlap among the disorders.Definitive diagnosis is dependent on the resultsof laboratory investigations with the key testsbeing plasma amino acids, urine organic acidsand plasma acylcarnitine profile with expertinterpretation.

Maple syrup urine disease (MSUD) presentsas an encephalopathy usually without aprominent metabolic acidosis. The presentationof MSUD in infants overlaps sufficiently withthe organic acidemias with metabolic acidosisthat it is relevant to discuss with the otherdisorders. Maple syrup urine disease ischaracterized by significant increases in

the plasma levels of the branched chain aminoacids, valine, leucine and isoleucine alongwith the pathognomonic alloisoleucine.The corresponding 2- keto acids are present inthe urine and can be identified by2, 4 dinitrophenylhydrazine (DNPH) test as wellas urine organic acid analysis.

Along with the acute infantile presentationsthere can be acute late onset forms where thedisease may present after initial symptom-freeperiod in childhood, adolescence or adulthood.Onset of an acute attack can be precipitated bystress, infection, increased protein intake,constipation or for no overt reason. Most of thelate onset presentations are neurologic (recurrentepisodes of ataxia, coma, seizures, developmentaldelay and hypotonia) or gastro-intestinal(recurrent episodes of vomiting, failure to thriveand pancreatitis-leading to hyperglycemia andhypocalcemia). Other rare presentations are skindisorders resembling staphylococcal scalded skinsyndrome or acrodermatitis enteropathica.

57


Renal complications are commonly seen withMMA and can include renal tubular acidosis ortubulo-interstitial nephritis in older children andadults with MMA. Other organic acidemiasthat can present with metabolic acidosis include3-hydroxy-3 methylglutaryl-CoA lyase(HMG CoA lyase deficiency) that presents withhypoketotic hypoglycemia and shares manyfeatures with fatty acid oxidation disorders suchas medium chain acyl-coA dehydrogenasedeficiency (MCAD deficiency). Other rare causesof metabolic acidosis are ketolytic disorders suchas Succinyl-CoA-3 Oxoacid CoA- Transferasedeficiency (SCOT) and thiolase deficiency andpyroglutamic aciduria (hemolytic anemia,metabolic acidosis, CNS damage and recurrentbacterial infections). All the above disorders areinherited in an autosomal recessive manner.

2. Fatty acid βββββ-oxidation disorders

The disorders include very long chain acyl-CoA dehydrogenase (VLCAD) deficiency,medium chain acyl-CoA dehydrogenase(MCAD) deficiency, carnitine transporterdeficiency and carnitine palmitoyl transferase 1(CPT1A) deficiency among others. Neonatalpresentations can occur with VLCAD andcarnitine transporter deficiency. These twodisorders and other fatty acid β-oxidationdisorders can present in children or adults.The common clinical phenotype is of intermittentmetabolic acidosis and hypoketotichypoglycemia. A low 3-hydroxybutyrate inplasma at the time of hypoglycemia is suggestiveof fatty acid oxidation disorder. Neonatalpresentation can include hyperammonemia andmild lactic acidosis. Cardiomyopathy is a featureof carnitine transporter deficiency and VLCADwhile MCAD and CPT1A deficiency presentwith mild hepatomegaly, hypoglycemiaand encephalopathy (Reye syndrome-likephenotype). There is a nearly 25 % mortalityat the initial presentation of unrecognized

MCAD deficiency thus making a strong case fornewborn screening for these set of disorders.The above disorders are also inherited in anautosomal recessive manner.

3. Lactic acidosis

Patients with IEM presenting with a primarylactic acidosis are divided into the followingtwo categories:

a) Defects in gluconeogenesis (glycogen storagedisease type 1, hereditary fructose intolerance,phosphoenolpyruvate carboxykinase deficiency,fructose -1, 6, diphosphatase deficiency). In thesedisorders, hypoglycemia and lactic acidosis areassociated with hepatomegaly andhyperuricemia.

b) Defects in pyruvate metabolism pyruvatecarboxylase (PC), pyruvate dehydrogenase(PDH) deficiency, mitochondrial electrontransport chain disorders. Lactic acidemia is themost significant metabolic abnormality in thedisorders of pyruvate metabolism. Pyruvatecarboxylase deficiency can be considered as agluconeogenic disorder as well as a disorder inpyruvate metabolism, however it is discussed asa disorder of pyruvate metabolism since lacticacidosis is usually much more prominent thanhypoglycemia.

Pyruvate carboxylase deficiency presentsusually in infancy as a severe and persistent lacticacidosis with significant neurologicalinvolvement. It is inherited in an autosomalrecessive manner and has a poor prognosis evenwith early identification of affected patients.Pyruvate dehydrogenase is a complex enzymethat has four subunits encoded by five genes thatconvert pyruvate to acetyl-CoA for entry into thetricarboxylic acid cycle and ultimately providesenergy for oxidative phosphorylation. The mostcommon type of PDH deficiency isE1α deficiency that is inherited in an X-linked

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manner (as mentioned above) but females areoften affected. The remaining four types of PDHdeficiency are autosomal recessively inherited.Deficiency of PDH is a relatively frequent causeof Leigh’s encephalopathy and neonatal lacticacidosis. Mid-facial hypoplasia (similar to thatseen in fetal alcohol syndrome) and microcephalyare often present. Cranial MRI may showabnormalities in neural development particularlyagenesis of corpus callosum. In PDH deficiencythe lactic acidosis worsens with increasingcarbohydrate intake as pyruvate is converted tolactate instead of acetyl CoA with a normallactate/pyruvate ratio. Fasting hypoglycemia isnot usually seen with PDH deficiency.

Management of infants and childrendiagnosed /suspected of havingmetabolic acidosis caused by IEM

General

Before starting emergency therapyadditional blood and urine samples should beobtained for specific investigations. Blood:4-5 ml in lithium heparin should be centrifugedrapidly and plasma stored frozen at -20° C andUrine: (4-5 ml should be stored at -20° C).Initial management involves stabilization of thesick neonate including use of ventilatory andcardiac support. The goal of the treatment is tominimize catabolism and remove toxicmetabolites. These measures can be started beforethe final diagnosis is established. Promptness indiagnosis and timely initiation of appropriatemanagement are major determinants of theultimate clinical outcome. In the organicacidemias and fatty acid β-oxidation disordersadequate glucose should be provided. Higherrates of glucose infusion require access througha central line and with frequent monitoring.Insulin can be administered if glucose is>12 mmol/L (216 mg/dL) and glycosuria occurs.Acidosis should be corrected by using

bicarbonate 1mEq/kg given gradually. Electrolytedisturbances, dehydration and hypothermiashould be corrected. Abnormal metabolites thatare characteristic of specific organic acidemiasmay accumulate and in some disordershyperammonemia may occur. These may impairneurological functions and should be removedby dialysis. Hemodialysis or hemofiltration arevery useful but availability of hemodialysis andability to place an adequate size catheter in asmall infant may be a challenge. Fluid balanceshould be carefully maintained. The use of ureacycle cocktail is generally not effective in themanagement of hyperammonemia of organicacidemias. In the primary lactic acidemias,hypoglycemia should be identified, corrected andmonitored. However the management principlefor PDH deficiency is to use lipid as the primarysource of energy rather than carbohydrate andbypass PDH to provide acetyl-CoA.

Medications

Carnitine (100 -400 mg/kg) intravenouslydivided into three doses can be institutedprior to the definitive diagnosis (Table 2).Organic acids are excreted in part asacylcarnitines often resulting in low free plasmacarnitine levels in organic acidemias. In isovalericacidemia glycine plays an important role inpromoting metabolite excretion. This can be usedin conjunction with carnitine. Carnitine therapyfor fatty acid oxidation disorders particularlyVLCAD remains controversial in view of risk ofcardiac arrhythmias, however carnitine has beenused in MCAD deficiency and is the mainstay oftreatment of carnitine transporter deficiency.Pharmacological doses of vitamin B

12 for

methylmalonic acidemia are useful in vitaminresponsive methylmalonic acidemias. Similarlybiotin should be given for biotinidase andholocarboxylase synthetase deficiency.Dichloroacetate has been used to reduce lactic

59


acid concentration but does not affect theneurological outcome.8 Use of carbaglu(N-acetyl-glutamate, the natural activator ofcarbomyl phosphate synthetase1 is being exploredin hyperammonemia associated with organicacidemia.9

Nutrition

Dietary treatment of most organic acidemiasrequires protein restriction that should beimplemented as part of a complete nutritionalmanagement program under the supervision of adietitian to avoid nutritional deficiencies andpromote adequate growth. It is important to notethat vitamin responsive IEM are usually lesssevere than the non-vitamin–responsive variants.Ongoing monitoring for trace elementsdeficiency should be carried out in patients onmodified diets. Bone health is also becoming amajor issue with number of metabolic disordersdue to altered mineral status, presence ofmetabolic acidosis and accumulation of toxiccompounds. A regular bone density measurementalong with appropriate supplementation ofcalcium and vitamin D is useful for these patients.

Ongoing management

These emergency measures may causeinadequate nutrition if used for a prolongedperiod and exacerbate catabolism leading to frank

malnutrition. Proper assessment of growth andanthropometry is an essential part ofmanagement. Periodic neurodevelopmentalassessments are recommended as in some of thesepatients hyperammonemia can causeneurological damage. A number of these childrenwith severe metabolic disorders may die despitethe heroic measures. Post mortem metabolicautopsy should be carried out10 (Table 3).

Recent advances

With advent of newborn screening usingtandem mass spectrometry, many of thesedisorders can be diagnosed pre-symptomaticallyin the neonatal period. This requires public healthprograms that include centralized laboratories,efficient communication systems andappropriately trained physicians, nurses anddietitians to identify and retrieve screen positiveinfants to the clinic in a timely manner. However,even with newborn screening, babies at the severeend of the clinical spectrum of some of theorganic acidemias will present before the resultsof newborn screening testing are available.Pediatricians and neonatologists need to considerthese disorders in newborn.11,12 Prenatal diagnosisin future pregnancies can be established morereliably by molecular diagnostic testing thanthrough enzymatic studies for several IEMs. Withimprovements in technology and clinical

Table 2. Suggested management of suspected organic acidemias

High caloric, protein free nutrition (as mentioned above with the help of dietician)

Insulin if required

Hydroxycobalamin (Vitamin B12

1-2 mg/day IV; Cyanocobalamin can be used if hydroxycobalaminis not available. However it may not be as effective) (For MMA)

Biotin 10-20mg/day IV or oral (For biotinidase deficiency, multiple carboxylase deficiency)

Thiamine 10-50 mg/day IV or oral in 1-2 doses (For MSUD)

Riboflavin 20-50 mg/day oral

Carnitine 100-400 mg/kg IV in 3 doses (For propionic and methylmalonic acidemia)

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Table 3. Peri-mortem/post-mortem metabolic autopsy specimens

Plasma 4-5 mls (Lithium Heparin tube) Heparinized, separated and frozen at -20°C

Blood EDTA tube for DNA banking

Filter paper For acylcarnitine analysisblood spots

Urine 4-5 ml frozen and stored at -20° C

Skin Fibroblast culture in Hanks solution stored at 4-8° C (not frozen) forenzyme studies

Liver Frozen for enzymology

Muscle Frozen for histochemistry and enzymology

awareness more babies born with IEM aresurviving than previously. Long term follow upis essential to identify and treat late complicationsthat can occur such as renal complications inMMA. Families affected with these disordersneed support and appropriate genetic counselling.Parent support groups are of great help in thisdirection.

Points to Remember

• Presence of metabolic acidosis particularlywith high anion gap should alert thepediatrician to a possibility of inborn errorsof metabolism.

• Inborn errors of metabolism can mimiccommon neonatal conditions such as sepsisand vice versa.

• Biochemical tests such as ammonia levels,presence or absence of ketones and lactatemeasurements can help with the diagnosisof inborn errors of metabolism.

• Prompt and early diagnosis is essential asmany of these inborn errors of metabolismare treatable.

• Early treatment optimizes theneurocognitive development of the infant.

• Inborn errors of metabolism have a geneticbasis in most instances so establishing adiagnosis will also help in geneticcounselling and family screening.

Acknowledgement

We would like to thank all the patients,families, residents and students who haveinspired us to a better understanding of thesegroups of disorders. Chitra Prasad would like tospecially thank Dr. Mark Korson (Director ofMetabolic Program, Tufts University NewEngland Medical Centre, Boston) for his teachingand approach to metabolic disorders.

References

1. Leonard JV, Morris AA. Diagnosis and earlymanagement of inborn errors of metabolismpresenting around the time of birth. ActaPaediatr 2006;95:6-14.

2. Burton BK. Inborn errors of metabolism:The clinical diagnosis in early infancy. Pediatr1987;79:359-369.

3. Saudubray JM, Nassogne MC, de Lonlay P,Touati G. Clinical approach to inheritedmetabolic disorders in neonates: An overview.Semin Neonatol 2002 ;7:3-15.

4. Garganta CL, Smith WE. Metabolic evaluationof the sick neonate. Semin Perinatol 2005;29:164-172.

61


5. Nyhan WL and Ozand PT. Organic acidemias,propionic acidemia and methylmalonicacidemia. In: Atlas of metabolic diseases.2

nd Edn, Chapman and Hall Medical 1999;

pp1-23.

6. Blau N, Duran M, Blaskovics ME. Organic acidanalysis. In: Physicians guide to the laboratorydiagnosis of metabolic diseases. Hoffman GF,et al, 2

nd Edn, Springer Germany 2002;

pp 31-49.

7. Seashore MR. The Organic Acidemias:An Overview. Gene Reviews (last Revision:July 3, 2007). Available from http://w w w. n c b i . n l m . n i h . g o v / b o o k s h e l f /br.fcgi?book=gene&part=oa-overview. Dateaccessed; May 18

th, 2009.

8. Stacpoole PW, Gilbert LR, Neiberger RE,Carney PR, Valenstein E, Therque DW, et al.Evaluation of long-term treatment of childrenwith congenital lactic acidosis withdichloroacetate. Pediatrics. 2008; 121:1223-1228.

NEWS AND NOTES

9. Levrat V, Forest I, Fouilhoux A, Acquaviva C,Vivaney Saban C, Guffon N. Carglumic acid:An additional therapy in the treatment of organicacidurias with hyperammonemia? Orphanet JRare Dis 2008;30.3:2.

10. Olpin SE. The metabolic investigation of suddeninfant death. Ann Clin Biochem 2004;41:282-293.

11. Saudubray JM, Charpentier C. Clinicalphenotypes: Diagnosis/Algorithms. In: ScriverCR, Beaudet AL, Sly WS, Valle D, Childs B,Kinzler KW. Eds. The Metabolic and MolecularBases of Inherited Diseases. 8

th Edn, McGraw-

Hill, NewYork 2001;pp 1341-1345.

12. Berghe GVD, Fernandes J, Walter JH,Saudubray JM. A clinical approach to inheritedmetabolic diseases. In: Inborn MetabolicDiseases: Diagnosis and Treatment, 4

th Edn.

Saudubray JM, Desguerre I, Sedel F,Charpentier C, (Eds), Springer Germany, 2006;pp 19-22.

FIRST MID TERM NATIONAL CME OF IAP RESPIRATORY CHAPTER

Venue: B.R.Singh Hospital for Medical Education & Research, Sealdah, Kolkata.

at 9. 30 am – 5 pm on 6th June, 2010, Sunday

For details contact

Dr.G.Ghosh

Mobile:98301 71815; E-mail:[email protected]

Dr.K.K.Ghosh

Mobile:98300 34876; E-mail:[email protected]

Dr.S.Roy

Mobile: 98300 36761; E-mail:[email protected]

Website: iapindia@org

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2010; 12(2) : 165

RECURRENT HYPOGLYCEMIAAND INBORN ERRORS OFMETABOLISM

* Madhulika Kabra** Neerja Gupta

Abstract : Many of the inborn errors ofmetabolism, including urea cycle defects,organic acidemias, and certain disorders ofamino acid metabolism, present in the younginfant with symptoms of an acute or chronicmetabolic encephalopathy. Typical symptomsinclude lethargy, poor feeding, apnea ortachypnea, recurrent vomiting, metabolicacidosis and/or hyperammonemia.Hypoglycemia may be the predominant findingin a number of inborn errors of metabolism,including glycogen storage disorders, defects ingluconeogenesis, and fatty acid oxidationdefects.

Keywords : Hypoglycemia, Inborn errors ofmetabolism, Glycogen storage disorders, Fattyacid oxidation defects.

Within a few days or weeks after birth, apreviously healthy neonate may begin to showsigns of an underlying metabolic disorder.Although the clinical picture may vary, infantswith metabolic disorders typically present withlethargy, decreased feeding, vomiting, tachypnea(from acidosis), decreased perfusion, andseizures. As the metabolic illness progresses,


there may be increasing stupor or comaassociated with progressive abnormalities of tone(hypotonia, hypertonia), posture (fisting,opisthotonus), and movements (tongue-thrusting,lip-smacking, myoclonic jerks), and with sleepapnea. Metabolic screening tests should beinitiated. Elevated plasma ammonia levels,hypoglycemia, and metabolic acidosis, if present,are suggestive of inborn errors of metabolism.1

This article will focus primarily on inbornerrors of metabolism causing recurrenthypoglycemia.

Hypoglycemia (blood sugar<45mg/dl at all ages)is a common nonspecific problem in severely illneonates and young infants, regardless of thecause of the illness. Unless the cause of thesymptoms is recognized and treated, this isfollowed by disturbance of consciousness withdrowsiness progressing rapidly to stupor andcoma accompanied by convulsions. In very younginfants, the early signs may be subtle with nothingmore than irritability, sweating and somnolence.A seizure may be the first recognized indicationof the problem. Regardless of the cause,correction of hypoglycemia without delay isatleast as important as making a specificdiagnosis. The differential diagnosis ofhypoglycemia is made easier by someunderstanding of the normal mechanisms formaintaining normal plasma glucoseconcentrations during fasting.

During the intervals between meals, theplasma glucose concentration is maintained bytwo general mechanisms.2

63

* Additional Professor

** Senior Research Officer

Division of Genetics, Department of Pediatrics,AIIMS, New Delhi.


• Mechanisms directed at producing glucose(glycogen breakdown and gluconeogenesis)

• Mechanisms that decrease peripheralglucose utilization by providing alternativeenergy substrate (fatty acid and ketoneoxidation)

Hence, hypoglycemia can occur as a resultof primary or secondary defects in glucoseproduction (deficiency of supply), or as a resultof defects in fatty acid or ketone oxidation (overutilization). Hypoglycemia and its associatedsymptoms occasionally may be seen in infantswith disorders of protein intolerance, but it is seenmore commonly in disorders of carbohydratemetabolism or fatty acid oxidation.

Inborn errors of metabolism causinghypoglycemia

A. Primary defects in glucose production(Gluconeogenesis and glycogenolysisdisorders)

Glucose- 6- phosphatase deficiency (GSD1a)

Glucose 6 phosphate translocase deficiency(GSD1b)

Glycogen synthase deficiency (GSD0)

Amylo 1,6 glucosidase (debranching enzyme)deficiency (GSD3)

Liver phosphorylase deficiency (GSD 6)

Phosphorylase kinase defcicency (GSD 9)

Phosphoenolpyruvate carboxykinase (PEPCK)deficiency (rare)

Fructose 1, 6 diphosphatase deficiency

Pyruvate carboxylase defciceincy

Galactosemia

Hereditary fructose intolerance

B. Over utilization of glucose

(i) Primary or secondary defect in fatty acidoxidation

Carnitine transporter deficiency (primarycarnitine deficiency)

Carnitine palmitoyl transferase 1 deficiency

Carnitine translocase deficiency

Carnitine palmitoly-transferase 2 deficiency

Secondary Carnitine deficiencies

Very long/long/medium/short chain acylCoA dehydrogenase deficiency

(ii)Hyperinsulinism

C. Others

Aminoacid and organic acid disorders

A) Primary defects in glucose production(Gluconeogenesis and glycogenolysisdisorders)

Hypoglycemia with permanenthepatomegaly is mostly due to inborn error ofmetabolism. Acquired or inherited hepatomegalyassociated with severe liver failure in early orlate infancy or childhood can give rise to severehypoglycemia, which appears after 2-3 hour offasting and causes moderate lactic acidosis butno ketosis. Among the best known inborn errorsof metabolism associated with hypoglycemia arethe hepatic glycogen storage diseases (GSD).The hypoglycemia in these disorders is relatedto the inability of the liver to release glucose fromglycogen, and it is most profound during periodsof fasting. Hypoglycemia, hepatomegaly andlactic acidosis are prominent features of thesedisorders. The normal physiologic response todecreased glucose production is increasedmitochondrial fatty acid beta oxidation andproduction of ketones (ketotic hypoglycemia).However, in GSD1, ketogenesis is oftensuppressed and plasma and urinary ketone levels,though elevated, may be inappropriately low forthe degree of hypoglycemia. In gluconeogenesisdefects involving glucose-6-phosphatase or

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fructose diphosphatase, fasting hypoglycemia isclassically associated with ketolactic acidosis.In glycogenosis type III and glycogen synthasedeficiency, fasting hypoglycemia is associatedwith hyperketosis but there is no metabolicacidosis and fasting lactic acidemia is low.However presence of significant postprandialhyperlactic acidemia is highly suggestive of thesediagnoses.

GSD1 (von Gierke disease) : It presents withsevere hypoglycemia at 3-6 months of ageprecipitated when intervals between feeds isprolonged, or there is associated intercurrentillness and is often heralded by a seizure or coma.Some infants may have failure to thrive ortachypnea due to lactic acidosis. Affectedchildren are usually pale and pasty looking withcharacteristic cherubic facies because of the dolllike appearance caused by chubby cheeks.Truncal obesity and marked abdominalprotuberance contrast with typically thinextremities. Recurrent nose bleeds are commonas a result of a secondary defect in plateletfunction.

Enzyme involved : The basic defect in GSD1 isdeficiency of the production of glucose fromglucose 6 phosphate, the final common pathwayfor glycogenolysis and gluconeogenesis.The most common variant is type Ia and is causedby deficiency of glucose 6 phosphatase. It isexpressed in liver and kidney. The nontype Iavariants are caused by deficiency in themicrosomal transport of glucose 6 phosphate(type Ib), phosphate(type Ic) or glucose (typeId).Type Ib and Ic are clinically indistinguishablefrom type Ia and are associated with persistentneutropenia, and affected children typically havehistories of recurrent pyogenic infections andpyorrhea.

Genetics: All GSD except some forms of GSDtype VI (X linked) are autosomal recessive.

Investigations : Blood sugar to look forhypoglycemia. Serial plasma glucose shows thatthe tolerance to fasting is poor often less than3 hours. The hypoglycemia is characteristicallyunresponsive to administration of glucagon.There is a significant rise in plasma lactate inresponse to glucagon .

• Lactate levels for lactic acidosis

• Hypertriglyceridemia, hyperuricemia,Increased transaminases

• Hypophosphatemia

• The kidneys are typically enlarged and mildrenal dysfunction is common

• Liver biopsy shows massive glycogenaccumulation including glycogen within thenucleus of hepatocytes. There is markedaccumulation of macrovesicular fat, buttypically no fibrosis, evidence of biliaryobstruction or inflammation.

• Enzyme studies on fresh liver biopsyspecimen

• DNA analysis for a specific molecular defect

Therapy of all types of GSD I is aimed primarilyat preventing hypoglycemia by administration offrequent (every 2-3 hrs) low fat feeds, containingthe little fructose (less vegetables and fruits) andlactose (soy based milk replacement).This issupplemented by intermittent ingestion ofuncooked cornstarch during the day and tubefeeding with formula during the night.

• Calcium supplementation

• Allopurinol if required

• For Type Ib/Ic granulocyte colonystimulating factor (G-CSF) 2-3 μg/kg/d canbe given for neutropenia

65


Monitor

• Blood sugar (>80 mg/dl) -Aim for bloodlactate <1.5mmol/l,normal triglycerides, uricacid and liver function

• Yearly ultrasound of liver

• Check renal function regularly after 14 yearsof age

GSD III commonly presents as asymptomaticfirm and nontender hepatomegaly discoveredincidentally on routine physical examination.There may be associated splenomegaly. In mostpatients hypoglycemia does not occur or occuronly after prolonged fasting. In a significantminority of patients it may present as severinfantile form like GSD I. However, it is easilydistinguishable by the absence or very mildpresence of lactic acidosis and hyperuricemia.There is ketosis during fasting and moderateincrease in liver AST and ALT, which as a ruledo not occur in GSD I. These patients show risein plasma glucose in response to ingestion ofgalactose, fructose indicating thatgluconeogenesis is intact. They also show asignificant rise in plasma glucose in response toglucagon administration 2-4 hrs after feeding.Liver biopsy shows increased glycogen andvariable interlobular fibrosis, but very little fat.Rarely, the fibrosis progress to frank cirrhosisproducing portal hypertension and liver failure.

Fanconi Bickel syndrome (GSD type XI) ischaracterized by fasting hypoglycemia, markedhepatomegaly associated with early onset renaltubular dysfunction, characterized by polyuria,hypophosphataemic rickets, hyperchloremicmetabolic acidosis and severe growth retardation.This is caused by mutations in GLUT2 geneencoding for the liver type glucose transporter.

The triad of hypoglycemia, markedhepatomegaly and lactic acidosis is alsocharacteristic of other defects of gluconeogenesis

such as hereditary fructose intolerance (HFI),fructose 1,6 diphosphatase deficiency, PEPCKdeficiency and sometimes PC deficiency.Definitive diagnosis requires measurement ofthe enzyme in fresh liver biopsy in formertwo and in the fibroblast predominating inmitochondrial isozyme in latter two. In fructose1,6 diphosphatase deficiency response toglucagon is preserved.

Hypoglycemia may be a prominent featureof both galactosemia and hereditary fructoseintolerance, although symptoms of the latterdisorder occur only after fructose (sucrose) hasbeen introduced in the diet. These patientsdevelop clinical symptoms only after intake oflactose (milk or milk products) or fructose orsucrose respectively, in the diet. If galactosemiaor fructosemia is suspected, the urine should betested simultaneously with Benedict’s reagentand with a glucose oxidase method. The glucoseoxidase method is specific for glucose, andBenedict’s reagent can detect any reducingsubstance. A negative dipstick result for glucosewith a positive Benedict’s reaction means that anonglucose reducing substance is present.With appropriate clinical findings, this is mostlikely to be galactose or fructose. The glycosuriain these conditions typically clears rapidly afterremoval of toxic sugars from diet. Therefore, anegative test does not eliminate the possibilityof one of these disorders, particularly if thepatients have been on intravenous glucose formore than a few hours. If the diagnosis ofgalactosemia is suspected, whether or notreducing substances are found in the urine,galactose-containing feedings should bediscontinued immediately and replaced by soyformula or other lactose-free formula pending theresults of an appropriate enzyme assay onerythrocytes to confirm the diagnosis.

Classical Galactosemia: It is due to deficiencyof the enzyme galactose-1-phosphate uridyl

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transferase results in an accumulation ofgalactose-1-phosphate and other metabolites suchas galactitol that are thought to have a direct toxiceffect on the liver and on other organs. Jaundiceand liver dysfunction in this disorder areprogressive and usually appear at the end of thefirst or during the second week of life withvomiting, diarrhea, poor weight gain, andeventually cataract formation. Progression ofsymptoms occurs after start of milk feeds, usuallystarting on 3rd or 4th day. The disease may presentinitially with indirect hyperbilirubinemiaresulting from hemolysis secondary to high levelsof galactose-1-phosphate in erythrocytes.Alternatively, the effects of acute galactosetoxicity on the brain may rarely cause the CNSsymptoms to predominate. Untreated infants withgalactosemia, if they survive the neonatal period,have persistent liver disease, cataracts, and severemental retardation. Many affected infants die ofEscherichia coli sepsis in the neonatal period,and the early onset of sepsis may alter thepresentation of the disorder.

Diagnosis is confirmed by either enzymeanalysis or specific mutational analysis.

Therapy- Lactose free, galactose restricteddiet through out life is required.

Hereditary fructose intolerance (HFI):The development of symptoms is clearly relatedto the ingestion of fructose or sucrose presentingwith intractable vomiting and symptomatichypoglycemia. More prolonged exposure resultsin failure to thrive, chronic irritability,hepatomegaly, abdominal distension, edema andjaundice. Milder variants of disease are commonand may present with bloating, abdominaldistension and diarrhea.

Investigations shows presence ofhypoglycemia, marked lactic acidosis,hyperuricemia, hypophosphatemia, hepatocelluardysfunction like raised aminotransferases,

prolonged prothrombin and partialthromboplastin time, hypoalbuminemia,hyperbilirubinemia and renal tubular dysfunction(hyperchloremic metabolic acidosis, generalizedaminoaciduria)

Diagnosis is confirmed by demonstration ofdeficiency of aldolase B in fresh liver biopsysample.

B. Over utilization of glucose

Increased glucose utilization (hypoketotichypoglycemia) occurs as a result ofhyperinsulinism or as a result of primary orsecondary defect in fatty acid oxidation and areeasily distinguished by presence of low free fattyacid in the hyperinsulinism. It is important todiscover the timing of hypoglycemia and tosearch for metabolic acidosis and ketosis whenthe patient is hypoglycemic. Unpredictablepostprandial or very short fasting hypoglycemia(2-6hrs) is mostly due to hyperinsulinism andgrowth hormone deficiency. Prolonged fastinghypoglycemia (8-24 hrs) is typically seen in fattyacid oxidation defects or systemic carnitinedeficiency.

Fatty acid oxidation defects: A number ofinherited defects in fatty acid oxidation have beenidentified in infants presenting withhypoglycemia. These disorders are importantbecause of their apparent frequency and becauseof the variability of the initial presentation.Affected infants have an impaired capacity to usestored fat for fuel during periods of fasting andreadily deplete their glycogen stores. Despite thedevelopment of hypoglycemia, acetyl CoAproduction is diminished, and ketone productionis impaired. The hypoglycemia occurring in theseconditions is typically characterized asnonketotic, although small amounts of ketonesmay be produced. Hypoglycemia may occur asan isolated finding or may be accompanied bymany of the other biochemical derangements

67


typically associated with Reye syndrome, suchas hyperammonemia, metabolic acidosis, andelevated transaminases. Hepatomegaly may ormay not be present. Any infant presenting withfindings suggesting Reye syndrome should beevaluated for fatty acid oxidation defects.Because the incidence of true Reye syndrome hasdecreased, most children presenting at any agewith this constellation of findings have aninherited metabolic disorder.

Medium-chain acyl CoA dehydrogenasedeficiency is the most common of the fatty acidoxidation defects and mostly presents as acuteor recurrent Reye like syndrome: vomiting,lethargy, drowsiness, stupor, seizures, hepato-megaly, hypoglycemia, and hyperammonemia.In addition to presenting as nonketotichypoglycemia or a Reye’s-like syndrome, it maypresent as sudden death or an acute life-threatening event. Many reports of infantsdiagnosed as having medium-chain acyl CoAdehydrogenase deficiency have described ahistory of a sibling who died of SIDS.Fat accumulation in the liver or muscle of anyinfant dying unexpectedly should suggeststrongly the possibility of this or a relateddisorder of fatty acid oxidation.

Very long-chain fatty acyl CoA dehydrogenasedeficiency is associated with similar clinicalfindings, although there also may be evidence ofa cardiomyopathy. Infants with this and severalother fatty acid oxidation defects may presentwith cardiac arrhythmias or unexplained cardiacarrest.

The accumulation of fatty acyl CoAs inpatients with fatty acid oxidation defects leadsto a secondary carnitine deficiency, probably asa result of excretion of excess acylcarnitines inthe urine.

Investigations : Urine organic acid analysis,measurement of serum carnitine, and analysis of

the plasma acylcarnitine profile are the mosthelpful laboratory studies in the initial screeningfor defects in fatty acid oxidation. These studiesare sufficient to establish the diagnosis ofmedium chain acyl CoA dehydrogenasedeficiency, which is associated with the presenceof a characteristic metabolite, octanoylcarnitine,on the acylcarnitine profile. Since the organicacid abnormality often disappears when the childis apparently healthy, diagnosis may be difficultif urine and blood samples are not saved fromthe time when the patient was acutely ill.

Enzymatic assays may be necessary for thedefinitive diagnosis of some of the fatty acidoxidation defects.

Treatment: As is true for the defects incarbohydrate metabolism leading tohypoglycemia, treatment of the fatty acidoxidation defects involves avoidance of fastingand provision of adequate glucose. Restrictionof dietary fat intake and supplemental l-carnitinetherapy are recommended.

C. Others 3

When ketoacidosis is present at the time ofhypoglycemia organic acidurias (methylmalonic,propionic, isovaleric, hydroxybutyric,methylglutaconic and methylcrotonic), ketolyticdefects (succinyl co transferase, 3 hetothiolase),late onset Maple syrup urine disease (MSUD),and glycerol kinase deficiency should beconsidered.

In nutshell, approach to the etiology ofhypoglycemia is based on 4 major clinicalcriteria2-4 (Fig.1) and few lab investigations.(Table 1)

• Liver size

• Characteristic of hypoglycemia(unpredictable, permanent, postprandial oronly after fasting)

• Association with lactic acidosis

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• Association with hyperketosis orhypoketosis

An algorithmic approach may also behelpful (Fig.1)

Fig.1. Approach to a child with IEM presenting with hypoglycemia

Basic Essential/Diagnostic

• Blood gases • Acyl carnitines (dried blood spots)

* Blood count * Urine organic acid

* CRP * Plasma aminoacids

* Electrolytes * Serum/Plasma Free fatty acids and plasma ketones

* Phosphate (3-Hydroxybutyrate)

* Liver/Renal function tests * Serum Hormones - Insulin, cortisol, growth hormone

* Creatine kinase

* Uric acid

* Triglycerides

Table 1. Key laboratory investigations during symptomatic hypoglycaemia

69


Points to Remember

• Diagnosis of IEM requires a high index ofclinical suspicion.

• Presence of persistent vomiting, acidosisand seizures with normal sepsis screenpoints towards an IEM.

• The triad of hypoglycemia, markedhepatomegaly and lactic acidosis ischaracteristic of many gluconeogenesisdefects.

• A stepwise correct choice of investigationscan lead to a specific diagnosis and earlymanagement.

References

1. Burton BK. Inborn errors of metabolism ininfancy: a guide to diagnosis.Pediatrics. 1998;102(6):e69.

2. Clarke JTR. Hepatic Syndromes. In: A ClinicalGuide to Inherited Metabolic Diseases, 3rd Edn,Cambridge University Press, New York2006;p126.

3. Ozand PT. Hypoglycemia in association withvarious organic or aminoacid disorders. SeminPerinatol 2000;24:172-193.

4. Diagnostic Approaches. In: The Metabolic andMolecular Bases of Inherited Disease, Eds,Scriver CR, Beaudet AL, Sly WS, Valle D,Childs B, Kinzler KW, Vogelstein B, 8

th Edn,

McGraw-Hill, New York, 2001; p7012.

C Rahm, S Schulz-Juergensen, P Eggert. Effects of desmopressin on the sleep ofchildren suffering from enuresis. Acta Pediatrica, March 2010.(Online Edition)

DDAVP has no effect on the sleep architecture of children with primary monosymptomaticnocturnal enuresis (PME)when analysed by classical, polysomnography which isdetermined by collecting the electric activity of cortical neurons. Taking recent researchfindings into account, this supports the thesis that the disturbances causing PME occurat brain stem level and do not reach consciousness.

CLIPPINGS

Chiappini E, Conti C, Galli L, Maurizio de Martino. Clinical efficacy and tolerabilityof linezolid in pediatric patients: A systematic review. Clinical Therapeutics 2010;32:66-88.

The reviewed pediatric studies in skin and skin–structure infections, bacteremia orpneumonia found that linezolid was associated with high clinical cure rates that did notdiffer significantly from those of vancomycin or cefadroxil. RCTs enrolling childrenwith other types of infection, as well as long–term studies are needed to draw definitiveconclusions about linezolid’s efficacy and tolerability in pediatric patients. Carefulmonitoring for adverse events and possible linezolid resistance continues to be essential.

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INBORN ERRORS OFMETABOLISM PRESENTING ASHYPERAMMONEMIA IN NEONATES

* Lakshmi V** Shanmugasundaram R

Abstract: Neonatal hyperammonemia is amedical emergency requiring prompt recognitionand aggressive therapy. It is apparent thatthe clinical signs of hyperammonemia arenon-specific and could be attributable to manyserious illnesses of the neonate like sepsis,intraventricular hemorrhage, etc. Hyperammo-nemia can be primary or secondary. Urea cycledisorders and other inborn errors of metabolismthough individually uncommon represent animportant cause of hyperammonemia inneonates.Therapy is aimed towards minimizingendogenous production and removal ofammonia.

Keywords: Hyperammonemia, Neonates, Inbornerrors of metabolism

Ammonia is a major waste product ofnitrogen metabolism in all cells. In neonates, asnitrogen turnover is far greater than adults a largeramount of ammonia is produced and excreted.Ammonia is toxic to the nervous system and isexcreted in the nontoxic form by going throughthe urea cycle. A significant exogenous sourceof ammonia comes from decomposition of ureaand other nitrogenous compounds in the intestine

* Consultant Neonatologist,

** Head, Department of Neonatology,

Mehta Children’s Hospital, Chennai

by microorganisms. Primary hyperammonemiaoccurs due to urea cycle defects (UCD) andsecondary in other conditions.There is a directrelationship between plasma ammonia levels andneurological status in patients with UCD and sois the neurological outcome.

Incidence

True incidence in general population is notknown as many die even before diagnosis.The incidence is 1/30,000 births.1

Normal plasma ammonium concentrationin term and preterm babies vary according tothe lab and method from 50-150μg/dL.Values greater than 75 μg/dL in term and150μg/dL in preterm babies are high and need tobe evaluated. Prompt chilling of heparinizedblood and immediate transport to the laboratoryis required to avoid false high levels.

Serum ammonia is measured by any of thefour methods : 1. ammonium sensitive electrode,2. enzymatic method, 3. cation exchange methodand 4. alkaline diffusion method.

Aetiology

Conditions resulting in neonatalhyperammonemia and the various differentialfeatures are shown in Tables 1 and 2 respectively.

1. Urea cycle defects (UCD): Complete absenceof any of the 5 enzymes results in primaryhyperammonemia (Fig.1). With the exception ofarginase deficiency all the other urea cycledefects present in the neonatal period. There isno metabolic acidosis but there is respiratory

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Table 1. Conditions resulting in neonatal hyperammonemia

Urea cycle defects

Carbamyl phosphate synthetase deficiency

Ornithine transcarbamylase deficiency

Arginosuccinate synthetase deficiency ( citrullinemia)

Argino succinate lyase deficiency

Transient hyperammonemia of prematurity

Disorders of branched-chain amino acid metabolism

Beta –ketothiolase deficiency

Isovaleric acidemia

Methylmalonic acidemia

Propionic acidemia

Severe perinatal asphyxia

Total parenteral nutrition

Liver failure

Rare miscellaneous

Congenital lysine intolerance

Syndrome of hyperornithinemia-hyperammonemia-homocitrullinuria

Lysinuric protein intolerance

Syndrome of Rett (hyperammonemia-cerebral atrophy)

Hyperlysinemia with lysine induced crisis

alkalosis.Citrulline, arginosuccinic acid levelsand urinary orotic acid will help in narrowingdown the enzyme defect (Fig.2).

2. Organic acidemia and congenital lacticacidosis

Untoward accumulation of organic acidinhibits ureagenesis and varying degrees ofhyperammonemia have been associated withorganic acidemia though less severe than theprimary form. Presence of wide anion gapmetabolic acidosis, ketosis, hypoglycinemia,neutropenia and thrombocytopenia should arouse

the suspicion of organic acidemias.The mostmarked elevations occur in methylmalonicacidemia and propionic acidemia. The proposedmechanisms are accumulated propionyl coAwhich inhibits NAG synthesis and CPS requiressaturating amount of NAG also and acetyl coAis required for NAG synthesis.

3. Transient hyperammonemia of newborn(THAN)

It is a self-limiting condition seen in stressedpreterm neonates and is usually apparent in thefirst 12 hours of life. Serum ammonia levels have

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Fig. 1. Steps of urea cycle

Fig. 2. Algorithmic approach to diagnosis of UCD

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exceeded 1000μmol/L. Pathogenesis is stillunclear though immature urea cycle has beenproposed.2 Prognosis is good with early treatmentand prompt reduction of ammonia level.

4. Other causes like perinatal asphyxia, livercell failure, TPN etc.

Pathophysiology of central nervoussystem injury

Elevated levels of ammonia is toxic to thenervous system.It causes neuronal injury whichis irreversible when severe and prolonged by thefollowing mechanisms:

1.Interferes with energy metabolism inreticular activating system (RAS).

2.Disturbance of neurotransmittermetabolism and favours release of dopamine andnorepinephrine from nerve endings.

3.Glutamine is a byproduct of ammoniametabolism which favours incorporation oftryptophan into the central nervous system whichis a precursor for neurotonins, serotonin andquinolinic acid

4.Ammonia as a cation has a directinhibitory effect on nerve impulse conduction.

Table 2. Differential diagnosis of neonatal hyperammonemia

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ensues. Further elevation results in respiratoryarrest, fixed-dilated pupils and sustained levelsabove 500μmol/L often cause increasedintracranial pressure which may be irreversible.

Inheritance

Inheritance is autosomal recessive with theexception of ornithine transcarbamylasedeficiency which is x-linked, wherein femalesare carriers and males are affected with disease.Family history for consanguinity and previoussibling neonatal deaths should be elicited.

Fig. 3. Approach to management in hyperammonemia

5.Increased glutamine results in swelling ofastrocytes.

Clinical features

As the serum ammonia exceeds three timesnormal the neonate becomes lethargic, refusesfeeds and repeatedly vomits. Further increaseleads to increased lethargy, hyperventilation andgrunting respirations with respiratory alkalosis,as ammonia is a potent stimulator of therespiratory center. Convulsions and disturbanceof tone are not unknown. When serum ammonialevels are more than 400μmol/L, frank coma

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Investigations

1. Elevated serum ammonia levels(> 500 μmol/L)

2. Elevated glutamine and alanine(both are derived from ammonia)

3. Decreased citrulline (CPS or OTCdefects)

4. Decreased arginine and urea levels

5. Elevated urinary orotic acids in all exceptCPS deficiency (carbamylphosphate when notutilized for urea synthesis results in formationof orotic acid in cytosol)

6. Urinary aminoacids and plasmaaminoacids for citrullinemia andarginosuccinicaciduria

7. Serum electrolytes and pH (wide aniongap metabolic acidosis)

8. Blood gas analysis (respiratory alkalosis)

9. Liver function tests

10.Complete blood counts

11. BUN, glucose, lactate, pyruvate

12.Urine analysis – reducing substance,ketones, aminoacids

Principles of management

Apart from correcting shock, metabolicacidosis, dyselectrolytemia, hypoxia, infectionand maintaining normoglycemia, therapy isaimed towards removal of ammonia and itsimmediate precursors glutamine, glutamate andalanine, by a combination of renal excretion ofhippurate, citrulline and arginosuccinic acid(ASA) and peritoneal dialysis and hemodialysis.

1.Reducing the endogenous production ofammonia by proteolysis by restricting proteinintake to 1.5g/kg/day, substitution of essentialaminoacids and intravenous administration of

10-15% glucose.

2.Stimulation of alternate metabolic pathwayof nitrogen excretion

Administration of sodium benzoate, whichis conjugated in the liver with glycine to formhippurate.

Phenyl acetate conjugated in the liver withglutamine to form phenylacetylglutamine.

Though phenyl acetate promotes moreefficient removal of ammonia, it is not widelyused in view of its offensive odour.These products are not recommended insecondary hyperammonemia like organicacidemia as they may worsen the acidosis.

3. Arginine supplementation

Arginine should be supplemented as theyare insufficient in UCD and are needed fornormal growth. Arginine promotes citrulline andarginosuccinic acid production in citrullinemiaand arginosuccinc aciduria which is excreted inurine and replaces urea as a major nitrogencarrier.

Drugs and dosages

Arginine hydrochloride IV / PO 0.2-0.8g/kg

Sodium benzoate IV / PO 0.25-0.5g/kg

Sodium phenylacetate 0.25g/kg

Essential aminoacids 0.7g/kg

Pyridoxine* 5mg/day

Folic acid * 0.1mg

*Co-factors for glycine synthesis

Megavitamins in congenital lactic acidosisand organic acidemia

Caloric intake 100-120 kcal/kg (MeadJohnson product 80056)

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4. Removal of ammonium

Exchange transfusion: Is a readily availableprocedure and should be the first line procedureto lower serum ammonia levels. Fresh wholeblood is preferred as older blood may contributeto elevated ammonia levels. Two volumeexchanges are recommended although larger uptofour volumes may be necessary in some babies.Repeated exchanges to as many as 10 exchangeshave been necessary in some. It rapidly removesammonia from the circulation. But it is not anongoing procedure as dialysis.

Peritoneal dialysis: Although exchangetransfusion has been recommended, recentstudies show that peritoneal dialysis is moreeffective in removing the accumulatedammonium and improving the survival.3

Peritoneal dialysis is performed using a standarddialysis fluid containing 1.5% added glucose.Ammonia is easily dialyzable. Short 30 minutescycles are used.

Hemodialysis: Hemodialysis may even be moreeffective because access to the circulation maybe easily obtained by umbilical artery and venouscatheterization. Small 30ml extracorporealdialyzers are available.4 Continuous venovenousdiafiltration was also done in the initialmanagement of hyperammonemia.5

Dialysis is continued for 36-60 hours onceserum ammonia levels have come down baby willstart breathing and the sensorium will improve.By this time the cause of hyperammonemiashould be evaluated and appropriate therapyinstituted.

GI drugs: Ammonia is also produced fromintestinal bacteria hence administration of oralunabsorbed antibiotic like neomycin has beenuseful. Lactulose oral or rectal which acidifiesthe gut and promotes poorly absorbable

ammonium has been tried .But above measureshave questionable efficacy in acutely ill neonates.

Prognosis

The neurological and developmentaloutcomes appear to be related to the magnitudeof hyperammonemia, duration of coma and rateof neurologic improvement.6 Although someacute neonatal hyperammonemic syndromes dueto UCD were highly fatal with early aggressivetherapy, Batshaw, et al reported nearly 50% ofsurvivors with normal development and25% with mild retardation.7

Prenatal diagnosis

Prenatal diagnosis is possible in AS andAL deficiency because these enzymes areexpressed in amniocytes.8 Further more ASA maybe detected in the amniotic fluid. In OTCdeficiency where sex determination is done andif male, pregnancy termination may be offered.In CPS deficiency sex determination is not useful.

Points to Remember

• Hyperammonemia incidence is underestimated in view of undiagnosed deaths.

• High index of suspicion in any acutely illneonate especially if there is history ofconsanguinity and sibling death.

• Prompt diagnosis and early agressivetreatment may improve the neurologicaloutcome in survivors.

• Neonatal diagnosis and general counselingshould be offered to the couples.

• Hyperammonemia of newborn has a goodprognosis if treated early and agressively.

• Treatment modalities to remove ammoniafrom the circulation like PD, HD arenecessary during acute episodes.

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References

1. Donn SM, Banagale RC. NeonatalHyperammonemia. Pediatr rev 1984;5:203-208.

2. Ballard RA, Vinocur B, Reynolds JW,Wennhberg RP, Merritt A, Sweetman L, et al.Transient hyperammonemia of a preterm infant.N Eng J Med 1978;299:920-925.

3. Batshaw ML, Brusilow SW. Treatment ofhyperammonemic coma caused by inborn errorsof urea synthesis. J Pediatr 1980;97:893.

4. Donn SM, Swartz RD, Thoene JG.Comparison of exchange transfusion,peritonealdialysis and hemodialysis for the treatment of

hyperammonemia in an anuric newborn infantJ Pediatr 1979;95:67.

5.. Lemley KV, Hintz RS, Enns GM. Continuousrenal replacement therapy in the initialmanagement of Neonatal HyperammonemiaDue to urea cycle defects.NeoReviews 2000;1.

6. Brusilow SW, Batshaw MC, Waber L. Neonatalhyperammonemic coma. Year book, Medicalpublishers, 1982;pp69-101.

7. Batshaw ML,Brusilow SW,Waber.L, etal.Longterm survival in neonatal onset ureacycleenzymopathies.N Eng J Med 1982;306:1387.

8. Goodman SI. Antenatal diagnosis of defects inureagenesis.Pediatr 1981;68:446.

Knuf M, Zepp F, Meyer CU, Habermehl, Maurer L, Burow HM, Behre U. Safety,immunogenicity and immediate pain of intramuscular versus subscutaneos administrationof a measles-mumps-rubella-varicella vaccine to children aged 11-21 months. EuropeanJournal Of Pediatrics,Feb 2010 (Online Edition)

This study compared intramuscular and subcutaneous administration of two doses ofmeasles–mumps–rubella–varicella (MMRV) combination vaccine (Priorix-Tetra™,GlaxoSmithKline Biologicals) in children. Healthy children (N = 328) were randomised toreceive MMRV either intramuscularly or subcutaneously. Reactogenicity was similar betweentreatment groups for immediate vaccination pain, vaccination site pain, redness and incidenceof fever and rashes. Slightly less vaccination site swelling occurred during days 0–3 of thepost-vaccination period after intramuscular administration. Seroconversion rates for allcomponents, 42–56 days post-dose 2, ranged from 99.3% to 100% in the intramuscular groupand from 98.6% to 100% in the subcutaneous. Cell-mediated immunity data supported thehumoral immunogenicity findings. In summary, the MMRV vaccine is well tolerated and highlyimmunogenic when administered either subcutaneously or intramuscularly to children in thesecond year of life.

CLIPPINGS

Federico R. Laham, MD, Amanda A. Trott, MD, Berkeley L. Bennett, MD, MS, ClaudiaA. Kozinetz. LDH concentration in Nasal-Wash Fluid as a Biochemical Predictor ofBronchiolitis Severity. Pediatrics 2010; 125: 225-233.

NW LDH levels in young children with bronchiolitis varied according to viral etiology anddisease severity. Values in the upper quartile were associated with ~80% risk reduction inhospitalization, likely reflecting a robust antiviral response. NW LDH may be a useful biomarkerto assist the clinician in the decision to hospitalize a child with bronchiolitis.

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FATTY ACID OXIDATIONDISORDERS

* Thangavelu S

Abstract: Fatty Acid Oxidation (FAO) disorderis an important group of inborn errors ofmetabolism. Clinical features develop duringperiods of fasting, because fatty acids are themajor source of fuel. When diagnosed and treatedearly, they can be managed with simple dietaryadvice and can live a full life. If the physician isnot aware of the features of FAO disorders, thismay be mistaken as Reye syndrome or sadlyparents may be blamed with the diagnosis ofMunchausen Syndrome by Proxy. Any child withrecurrent hypoglycaemia without ketones or withunexplained neuromuscular disorders orcardiomyopathy needs to be evaluated forFAO disorders.

Keywords : Fatty acid oxidation, SIDS,Hypoketotic hypoglycemia, Reye like syndrome,Cardiomyopathy.

Fatty acid oxidation disorder is an autosomalrecessive inherited condition due to defects inthe enzyme system that metabolizes the fatty acid(FA) by oxidation. Main source of energy forhuman body is glucose. During fasting, whenglucose levels are low, alternate source of energyis ketone body, a metabolic product of FA, whichcomes from the β oxidation of fatty acids. Longchain FAs are a major source of energy for restingskeletal muscle and myocardium. Hence FAOdisorders seriously affect the functioning ofhighly energy dependent tissues like heart,

muscle, brain and liver. Children with Fatty AcidOxidation disorders (FAO) do not get alternateenergy when the serum glucose levels are low.When diagnosed and treated early, these childrencarry a good prognosis.

Pathophysiology

During fasting FAs are released intocirculation from depot stores. They are taken upand oxidised for energy by most tissues exceptcentral nervous system. Because of this there willbe sparing of glucose which will be utilized byCNS. Long chain FAs are transported intomitochondria by carnitine and other threetransporter protein CPT I (Carnitine PalmitoylTransferase), CPT II and Translocase.Once inside mitochondria, they pass throughseries of steps by enzymes of beta oxidation.Based on their chain length specificity, enzymesof beta oxidation are categorized as follows:

VLCAD: Very Long Chain Acyl CoADehydrogenase

LCAD: Long Chain Acyl CoA Dehydrogenase

MCAD: Medium Chain Acyl CoADehydrogenase

SCAD: Short Chain Acyl CoA Dehydrogenase

At the end of the β oxidation acyl coA isconverted into acetyl coA, which is the substratefor energy production in Tricarboxylic Acid(TCA) cycle and for production of ketone bodies.Hence fatty Acid Oxidation (FAO) leads to defectin energy production and accumulation of acylcoA derivatives. Oxidation of FA in mitochondriais one of the major source of energy providingup to 80% of the requirement while fasting.

* Head, Dept. of Pediatrics,Mehta Children’s Hospital, Chennai.

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FAO disorders constitute a group of diseasescaused by deficiency of transporter proteins suchas carnitine, CPT I, Translocase or CPT II andenzymes involved in beta oxidation which arementioned above.

MCAD deficiency is the most common oneamong the FAO disorders. A common pointmutation is 985 A→G of the MCAD cDNA.Second most common disorder is LCADdeficiency and the common mutation is1528 G→C.

Clinical features

There are four groups of clinicalpresentation because of defects in theFA oxidation.

1. Clinical features pertaining to skeletal andcardiac muscles because their main source ofenergy is from fatty acid, resulting in hypotonia,weakness, cardiomyopathy and cardiac failure.

2. Reye like episodes of hypoketotichypoglycemia, hyperammonemia, hepatic failureand encephalopathy, which can occur because offasting associated with illness. A metabolic crisismay be precipitated by simple illnesses like upperrespiratory infection or otitis. Indicators ofpossible crisis are vomiting, diarrhea, excessivesleepiness, seizure and coma. If not properlydiagnosed or suspected child may die withoutdiagnosis or sometimes parents may be blamedwith the diagnosis of Munchausen’s syndromeby proxy.

3. FAO disorders are considered as importantcause of Sudden Infant Death Syndrome (SIDS)though there is no substantiation from molecularstudies.

4. Very rarely mothers of affected fetus maydevelop acute fatty liver of pregnancy or HELLPsyndrome (Hemolysis, Elevated Liver enzymesand Low Platelets)

Diagnosis

Evaluation of FAO disorders includesclinical suspicion and estimation of plasma freeand total carnitine, glucose, ketone bodies,lactate, pyruvate and ammonia. Blood acylcarnitine analysis by TMS, urinary organic acids,measurement of enzymes and mutational analysisare other investigations. Following are thecommonly seen lab findings that lead to thediagnosis.

• Hypoglycemia with inappropriately lowor absent ketones.

• The first line diagnostic test is theanalysis of acyl carnitine esters and its diagnosticmetabolites.

• MCAD deficiency is charecterized byelevated octanoyl carnitine

• Organic acid analyses and and serumcarnitine levels may be useful

• Increased urinary levels of mono and/ordicarboxylic acids, glycin conjucgates andacylated carnitines. This reflects the intracellularaccumulation of acyl-coA derivatives.

• Enzyme assay in fibroblasts andmolecular analysis to confirm the mutation canbe done in specialized laboratories.

Treatment

Objective of management of FAO disorder is toensure that fatty acids are not used as majormetabolic fuel (Table 1).

• Prevention of hypoglycemia is importantby avoiding fasting for more than 8-12 hoursand providing frequent low fat and carbohydraterich meals at bed time. Vigorous treatmentof respiratory infection and gastro-enteritiswill prevent precipitating situations. Acutemanagement includes intravenous glucose at therate of 7-10 mg/kg/min with or without adding

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insulin to maintain serum glucose around100 mg/dl. One should remember the fact thatexcess glucose may increase lactic acidosis.Carnitine supplement is useful in carnitinedeficiency at the dose of 100 mg/kg/day. It shouldbe avoided in disorders with LCAD deficiency.Intravenous lipids are to be avoided but mediumchain triglycerides (MCT) can be given in provendisorders of LCAD deficiency. In an acutesituation, dialysis and exchange transfusion arefound to be useful in disorders of Long ChainFA oxidation and the carnitine cycle. Riboflavincan be tried in the dose of 150 mg/day in ETFdeficiency or SCAD deficiency. Potential use offibrates to increase mutant protein levels andDL-3 Hydroxy Butyrate are the new concepts inthe treatment.

Table 1. Prevention and treatmentof FAO disorders

• Prevention of prolonged fasting.

• Early and vigorous treatment of infection.

• IV glucose infusion 7-10mg/kg/minute.

• Carnitine supplement 100mg/kg/day(except in LCHAD deficiency).

• Medium chain triglycerides

• Dialysis and exchange transfusion

• Riboflavin at 150mg/day.

Prognosis

When diagnosed and treated at birth,outcome in FAO disorders particularly MCADdeficiency is excellent, but more guarded in otherdisorders. But most can adjust to dietary adviceduring fasting and illness and can lead a full life.

Points to Remember

• Fatty acid oxidation disorders are causedby deficiency of the transport protein orβββββ oxidation enzyme system.

• Clincial presentation includes hypoketotichypoglycemia, Reye like syndrome,involvement of skeletal and cardiac muscle.

• Rarely in mothers of affected fetus it cancause acute fatty liver of pregnancy.

• Management mainly involves avoidingfasting and providing frequent low fat andcarbohydrate rich meals at bed time.

Bibliography

1. Ibdah JA, Bennett MJ, Rinaldo P, Zhao V,Gibson B, Sims HP, et al. A fetal fatty acidoxidation disorder as a cause of liver disease inpregnant women. New Engl J Med 1999; 340:1723-1731.

2. Hove JV, Grünewald S, Jaeken J, Demaerel P,Declercq P, Bourdoux P, et al. 3-Hydroxy-butyrate treatment of multiple acyl-CoAdehydrogenase deficiency (MADD) TheLancet, 2003;361:1433-1435.

3. www.fodsupport.org/fods_defined.htm4. Olpin SE. Fatty acid oxidation defects as a cause

of neuromyopathic disease in infants and adults.Clin Lab 2005;51:289-306.

5. Barlett K, Pourfarzam M. Inherited disordersof mitochondrial fatty acid oxidation,Minisyposium: metabolic disease. CurrentPaediatr 1997;7:118-122.

6. Disorders of fatty acid oxidation andketogenesis. In: Manual of metabolic pediatrics.Vademecum metabolicum, 2

nd Edn, Eds,

Iohannes Zschocke, Georg F, Hoffmann,MilupaGmbH, Friedrichsdorf 2004;pp93-97.

7. Sweetman L, Julian C. Williams. Geneticmetabolic disorders. In: Biochemical basis ofpediatric diseases, 3rd Edn. Eds, Steven J.Soldin, Nader Rifai. AACC Press, WashingtonDC, 1998;pp419-455.

8. Thomas JA, Johan LK, Hove V. Inborn errorsof metabolism. In: Current diagnosis andtreatment in pediatrics. 18

th Edn, Eds, William

W.hay Jr, Myron J. Levin, JudithM.Sondheimer, Roin R.Deterding, LangeMedical Books, McGraw-Hill, 2007;pp1003-1004.

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MITOCHONDRIAL DNAAND DIABETES MELLITUS

* Biswajit Mohanty ** Balasubramanian J

Abstract: Diabetes mellitus affectsapproximately 5% of the general population withits prevalence varying between ethnic groups andgeographic regions. The majority of cases areeither type 1 or type 2 diabetes. Although thesedisorders share a common phenotype, fasting andpostprandial hyperglycemia, their etiology isdistinct. A growing body of evidence hasdemonstrated a link between mitochondrialfunctioning and type 2 diabetes. Certainmitochondrial DNA (mtDNA) mutations affectinsulin secretion involving an attenuation ofADP/ATP levels leading to a re-setting of theglucose sensor in the pancreatic ß-cell.Co-morbid conditions include impaired hearing,changes in pigmentation of the retina,gastrointestinal abnormalities, cardiomyopathy,and MELAS syndrome (mitochondrial myopathy,encephalopathy, lactic acidosis and stroke-likeepisodes).

Keywords: Type 2 Diabetes, MitochondrialDNA, Comorbid Conditions.

Diabetes mellitus can be defined as a stateof chronic hyperglycaemia sufficient to causelong-term damage to specific tissues, notably theretina, kidney, nerves and arteries. Maintenance

of normal glucose homeostasis involves theaction of a glucose sensor in the pancreaticß-cell that detects an increase in blood glucoseconcentration and converts that into increasedsecretion of insulin. The ability of pancreaticß-cell to sense ambient glucose levels accuratelyand rapidly depends on the glucose transporterisoform GLUT-2, enzyme glucokinase and thepulsating ratio of ATP/ADP. The ATP helps inexpelling insulin present in the secretory vesiclesinto the extracellular space (exocytosis), fromwhere insulin and C peptide enter the isletcapillaries. The synthesis of ATP takes place inthe inner membrane of mitochondria onrespiratory chain by a process called oxidativephosphorylation. The mutation of mtDNA hasbeen observed to lead on to diabetes.1,2

Mitochondrial DNA

Human mtDNA is a double helical circlecontaining 16,569 base pair. A striking featureof the human mitochondrial genome is itsextreme economy. Nearly every base pairin human mtDNA encodes a protein or aRNA product. This genome encodes 13 proteins,22 tRNAs, 2 rRNAs, NADH reductase, proton–pumping complexes, cytochrome reductase andoxidase subunits and ATP synthase subunitsi.e. components responsible for oxidativephosphorylation or ATP generation in the cell.

mtDNA is vulnerable to mutation comparedto nuclear DNA because it is composed almostexclusively of coding sequences, lacks protectionby histones, has inefficient repair mechanisms.It is also exposed to reactive oxygenspecies (ROS) produced during oxidativephosphorylation while generating ATP.3,4,5

* Professor, Dept. of Biochemistry,

** Professor, Dept. of Pediatrics,

Arupadai Veedu Medical College and Hospital,Puducherry.

GENERAL ARTICLE

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Molecular defect

Mutation of mtDNA occurs by substitutingguanine for adenine (A-to-G) at position 3243 ofleucine transfer RNA (tRNALeu(UUR)).6

Interestingly, tRNALeu(UUR) is a hot spot formutations; 10 disease-related mutations havebeen identified within this gene, 4 of which areassociated with diabetes mellitus. Nearly all thecarriers developed diabetes or IGT before the ageof 70 years; thus, the penetrance of this mutationis nearly 100%. Diabetes can be type 1 or type 2in nature depending on the severity ofinsulinopenia.

Mitochondrial function in beta cell insulinsecretion: Type 2 diabetic patients have animpaired ability of the pancreatic beta-cells tosecrete insulin.7 Glucose-stimulated insulinsecretion (GSIS) has been characterized by itspulsatile nature as generated by oscillations inthe ATP/ ADP ratio.8 A series of steps must becompleted inside and outside the mitochondriabefore insulin can be secreted. This increase inATP/ADP ratio causes the ATP-sensitiveK+ channels to close, causing depolarization ofvoltage-sensitive Ca2+ channels.9,10 The depolari-zation triggers the opening of specific (voltage-gated) Ca2+ channels in the membrane. Ca2+ ionsthen flood into the ß cell from the outside andactivate the contractile proteins which drag thesecretary vesicles containing insulin andC peptide to the cell surface. Here, the vesiclesfuse with the cell membrane and release theircontents into the extracellular space (exocytosis),from where insulin and C peptide enter the isletcapillaries. The processing of proinsulin toinsulin within the secretory granules of the Golgicomplex is dependent on critical levels ofpH (3.5-7.4) as well as ATP.11

Mitochondrial inheritance

Mitochondrial inheritance behavesdifferently from autosomal and sex-linked

inheritance. Nuclear DNA has two copies per cell(except for sperm and egg cells). One copy isinherited from the father and the other from themother. Mitochondria, however, contain theirown DNA, and contain typically from five to tencopies, all inherited from the mother. Mutationsto mtDNA occur frequently, due to the lack ofthe error checking capability that nuclear DNAhas. This means that mitochondrial disordersoccur spontaneously and relatively often.Mitochondrial disease begins to become apparentonce the number of affected mitochondria reacha certain level; this phenomenon is called“threshold expression”.

Clinical presentation

This disorder is associated with diabetesmellitus and deafness. It is inherited maternally(MIDD). A variety of studies have determinedthat of patients with type 1 and type 2 diabetesmellitus, the A/G exchange is present in 0.5–1.5%of patients. If only those patients with a familyhistory of diabetes are considered, the prevalenceof the mutation is two to five times higher. In across sectional study in Netherlands amongdiabetes patients the prevalence of MIDD wasreported to be 1.3%.12 The prevalence of themitochondrial mutation in a Japanese study was4.6% in diabetic patients with maternalinheritance and/or hearing loss.13 Similar findingwere reported in a Chinese study.14 There is apaucity of Indian data on MIDD.

Characterization of affected individuals andtheir siblings demonstrated that 48% presentedwith diabetes and deafness, 21% had diabetesalone, 15% had deafness alone, 3% had deafnessassociated with some neurologic changes, and13% had diabetes, deafness, and findings ofMELAS syndrome (mitochondrial myopathy,encephalopathy, lactic acidosis and stroke-likeepisodes).

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Although patients with mtDNA mutationswere initially diagnosed as having either type 1or type 2 diabetes mellitus, there were differencesbetween these patients and those with type 1 ortype 2 diabetes. Ketosis has been described rarely.Pancreatic antibodies are generally not a featureof MIDD, although islet-cell antibodies havebeen described. Diabetes that is not insulin-requiring at outset can usually be managed withdietary treatment or oral hypoglycemic agents,although there is fairly rapid conversion to insulindependance.

Those initially diagnosed as having type 2diabetes tend to be leaner and more likely to betreated with insulin compared with generalpopulations with type 2 diabetes. Subsequentstudies to define the etiology of diabetes inpatients with mitochondrial mutationsdemonstrated decreased insulin secretion inresponse to an oral glucose tolerance test,whereas measures of glucose utilizationdemonstrated essentially normal insulinsensitivity. The above findings suggest that theprimary defect in patients with mitochondrialmutations is in the pancreatic β cell, oxidativephosphorylation and ATP generation.

Management

In patients with diabetes or IGTaccompanied by hearing loss and maternalhistory of diabetes, a diagnosis of mtDNAmutations as the cause of the diabetes should beconsidered. Family members need to be carefullyscreened for both diabetes and evidence ofsensorineural hearing. Frequently, the hearingloss develops after the onset of diabetes, so thosepatients with diabetes need to be examined overtime for hearing loss. The data suggests thatpatients who replace two percent of the energyfrom trans-fatty acids (TFA) withpolyunsaturated fatty acids could decrease therisk for type 2 diabetes by as much as40 percent.15 TFA contributes to mtDNA

mutation. So patients should be advised to avoidfood containing TFA. Patients with thesemutations are likely to require insulin therapybecause of the insulin deficiency that developssooner or latter. Metformin group of drugs shouldbe avoided as it may lead to lactic acidosis andfurther aggravate the situation.

Conclusion

Type 2 diabetes is not merely a disease ofinsulin insensitivity or lack of insulin release butmay be a global dysfunction of the mitochondrialenergy system. The expression of this phenotypeseems dependent on a critical thresholdassociated with mutations. Although reductionof mtDNA is a critical factor in type 2 diabetespathology, the question as to the nature of theoriginal insult, dysregulated ROS or someexternal protoxins or environmental chemicalsremains to be answered.

Points to Remember

• Type 2 diabetes mellitus may not be mereinsulin insensitivity or release; it maybe part of a global dysfunction ofmitochondrial energy system.

• Mutation of mtDNA as a cause is to beconsidered in diabetes with hearing lossand maternal diabetes.

• Patients with mutation require insulin indue course.

• Metformin group of drugs are to be avoidedas they lead to lactic acidosis.

References

1. Thomas AW, Edwards A, Sherratt EJ, Majid A,Gagg J, Alcolado JC.Molecular scanning ofcandidate mitochondrial tRNA genes in type 2(non- insulin dependent) diabetes mellitus.J Med Genet 1996;33:253-256.

2. Bell GI, Polonsky KS. Diabetes mellitus andgenetically programmed defects in α-cellfunction. Nature. 2001;414:788–791.

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3. Newsholme P, Haber EP, Hirabara SM,Rebelato ELO, Procopio J, Morgan D, et al.Diabetes associated cell stress and dysfunction:role of mitochondrial and non-mitochondrialROS production and activity. J Physiol 2007;583: 9–24.

4. Droge W. Free radicals in the physiologicalcontrol of cell function. Physiol Rev2002;82:47–95.

5. Evans JL, Goldfine ID, Maddux BA, GrodskyGM. Oxidative stress and stress-activatedsignaling pathways: a unifying hypothesis oftype 2 diabetes. Endocr Rev 2002;23:599–622.

6. Gerbitz KD, van den Ouweland JM, MaassenJA, Jaksch M. Mitochondrial diabetes mellitus:a review. Biochim Biophys Acta 1995;1271:253-260.

7. Wollheim CB. Beta-cell mitochondria in theregulation of insulin secretion: a new culprit intype II diabetes. Diabetologia 2000;43:265-277.

8. Deeney JT, Prentki M, Corkey BE. Metaboliccontrol of beta-cell function. Semin Cell DevBiol 2000;11:267-275.

9. Cook DL, Hales CN. Intracellular ATP directlyblocks K+ channels in pancreatic B-cells.Nature 1984;311:271-273.

10. Loussouarn G, Pike LJ, Ashcroft FM,Makhina EN, Nichols CG. Dynamic sensitivityof ATP-sensitive K(+) channels to ATP. J BiolChem 2001;276:29098-29103.

11. Slee DJ, Jones PM, Howell SL. Proinsulinprocessing in electrically permeabilized ratislets of Langerhans. J Mol Endocrinol1990;5:275-280.

12. Maassen JA, Kadowaki T, Maternally inheriteddiabetes and deafness: a new diabetes subtype.Diabetologia 1996; 39: 375-382.

13. Fukui M, Nakano K, Obayashi H, Kitagawa Y,Nakamura N, Mori H, et.al. High prevalence ofmitochondrial diabetes mellitus in Japanesepatients with major risk factors. Metabolism1997; 46: 793-795.

14. LI Ming-zhen, YU De-min, YU Pei, LIU De-min, WANG Kun, TANG Xin-zhi.Mitochondrial gene mutations and type 2diabetes in Chinese families. Chinese MediJournal 2008; 121, 8: 682-686.

15. Salmeron J, Hu FB, Manson JE, Stamper MJ,Colditz GA, Rimm EB, et al. Dietary fat intakeand risk of type 2 diabetes in women. Am J ClinNutr 2001;73:1019-1026.

Alexandra Furwentschesa, Cornelia Bussmanna, Georgia Ramantaniab, FriedrichEbingera, Heike Philippia, Johannes Poschlc, Susanne Schuberta, Dietz Ratinga,Thomas Basta. Levetiracetam in the treatment of neonatal seizures: A pilot study Seizure- European Journal of Epilepsy, Feb 2010(Article in press)

No severe adverse effects were observed. Mild sedation was reported in one infant.All six patients treated with oral LEV became seizure free within 6 days. Five patientsremained seizure free after 3 months with ongoing LEV monotherapy. One infantdeveloped pharmacoresistent epilepsy. Seizures relapsed later in the clinical course oftwo more patients, one of whom was no longer under LEV therapy. Results from thissmall patient group indicate that LEV may be an alternative therapeutic option in neonatalseizures.

CLIPPINGS

85


GENERAL ARTICLE

APPROACH TO ANASARCA

* Maiya PP** Sharanabasavesh M

Abstract: Generalized edema otherwise knownas anasarca, is a common presentation of variousconditions. Common causes include renal,cardiovascular, nutritional and hepatic diseases.Majority of children presenting with anasarcamay have diagnosis referable to any of thesystems mentioned. Occasionally in a child withanasarca there may be difficulty in diagnosis forwhich a systematic approach will help.The approach should include carefully takenhistory, clinical examination and basicinvestigations. An occasional child may requireextensive investigation for the diagnosis.

Keywords: Anasarca, Edema, Hypoalbuminemia,Child.

Anasarca refers to generalized edemacharacterized by puffiness of face, mostnoticeable in the periorbital area and bypersistence of indentation over the medial aspectof ankle following pressure.1 Majority of childrenwith anasarca have straight forward etiologyrelated to cardiovascular, nutritional, renal orhematological condition. Occasionally in somesuch children there may be problem inascertaining the etiology. Hence a systematicapproach will be necessary to diagnose the causeof generalized edema.

Table 1 lists the causes for anasarca, whichwill help to arrive at a diagnosis in a child withanasarca.

Table 1. Causes of anasarca

Cardiac

CCF secondary to congenital heart disease,cardiomyopathy, myocarditis, arrhythmias,rheumatic heart disease, hypertension,cor pulmonale, severe anemia, thyrotoxicosis.

Renal

Nephrotic syndrome, glomerulonephritis,renal failure.

NutritionalKwashiorkor

Hepatic2,3

Fulminant hepatic failure, autoimmunehepatitisDrug induced liver failure: Acetaminophen,Isoniazid, Phenytoin

Metabolic diseases causing liver failure:Wilson disease, glycogen storage diseasetype IV, mitochondrial disorders, Alpha- 1,antitrypsin deficiency, Niemann-Pickdisease.

Hypo perfusion of liver: Budd Chiarisyndrome, Veno-occlusive disease.

Gastro intestinal

Inflammatory bowel disease, gluten sensitiveenteropathy, cystic fibrosis, intestinallymphangiectasia, trypsinogen or enterokinasedeficiency, milk protein sensitivity,eosinophilic gastroenteropathy, short bowelsyndrome.

* Professor and Head,** Lecturer,

Department of Pediatrics,M.S. Ramaiah Medical College andTeaching Hospital, Bangalore.

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Approach to anasarca

Approach to any particular diseasenecessiates a proper history and physicalexamination. This helps in limiting thedifferential diagnosis to specific organ system ina child with anasarca.

History 3-7

Cardiac: Children with anasarca due to cardiacdisease present with the complaints of feedingdifficulties, excessive sweating, failure to thrive,respiratory distress and cyanosis. Older childrenmay also have history of orthopnea, dyspnea andsyncopal symptoms. Edema usually occurs in thedependent area i.e. sacral area in infants and ankleregion in toddlers and older children.

Liver: Ask for history of fever, anorexia,vomiting, pain abdomen, progressive jaundice,fetor hepaticus, bleeding manifestation andabdominal distension. In patients with liverdisorder edema usually starts as ascites andprogresses to develop generalized edema. Infantsmay initially present with irritability, lethargy,poor feeding, and sleep disturbance.

Nutritional: This is the commonest cause ofanasarca in most of the third world countries.Properly taken dietetic history, socioeconomichistory, and growth parameters help in makingthe diagnosis of nutritional cause for anasarca.Early in the disease symptoms include anorexia,lethargy, apathy and irritability. Later theypresent with failure to thrive, increasedsusceptibility to infection and edema.

Renal: Children with renal disorder present withdecreased urine output, hematuria and edema.Edema is in the form of early morning facialpuffiness, which gradually decreases as the dayprogresses in the initial stage of the disease,which later on becomes generalized andpersistent. History of repeated urinary tractinfection, history of sore throat or skin infection

in the recent past and symptoms related tohypertension should be asked for.

In addition history should include historyof chronic diarrhoea, progressive pallor, drugintake, insect bite and burns.

Clinical examination4-7

General Examination should includelooking for peripheral signs of liver cell failure,congestive cardiac failure, rheumatic heartdisease and anemia. One has to also look formanifestations of vitamin and mineraldeficiencies.

CVS: Heart failure occurs when cardiac outputis inadequate to meet the metabolic needs.On examination along with edema other featuresof congestive cardiac failure like tachycardia,tachypnoea, basal rales, tender hepatomegaly andcardiomegaly are looked for. Features of poorperipheral circulation like cold extremitiesprolonged capillary filling time and low bloodpressure can be noticed. Edema is seen in thedependent areas initially which becomesgeneralized. Most common causes of CCF inyoung infants and children are due to congenitalheart defects E.g., VSD, PDA, hypoplastic leftheart syndrome, TGA, etc. Cardiomyopathywhich occurs due to number of causes could bea cause for CCF. Other causes include viralmyocarditis, severe anemia, hyperthyroidism,AV malformation, etc.

Liver disease: On examination of a child withliver disease and anasarca icterus may also be afeature. Increase in liver size initially, whichgradually shrinks without clinical improvementmay be seen in chronic liver disease. Edema startsas ascites which later on becomes generalized asthe disease progress. Mental status changes arenoted with progression of disease. Signs of livercell failure like, fetor hepaticus, spider nevus,asterixis, palmar erythema, testicular atrophy, etcdevelop as the disease progresses.

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Fig 1. Approach to the diagnosis of anasarca

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Renal : Edema occurring in acute phase ofglomerulonephritis is characteristicallyassociated with hematuria, hypertension andoliguria. The edema in this condition is primarilydue to retention of sodium and water by thekidneys. On examination some times pyodermamay be noticed. The age group is generallybetween 5- 15 yrs of age.

Edema in nephrotic syndrome is primarilydue to massive urinary loss of protein leadingonto hypoalbuminemia. Most commonly it isseen in children between the ages of 2 and6 years. Episodes may be precipitated by minorinfections. They usually present with mild edemaaround the eyes and in lower limb that decreasesover the day. As the disease progresses edemabecomes generalized. The child may haveassociated oliguria, anorexia, irritability,abdominal pain and diarrhoea.

Nutritional and Gastrointestinal: Kwashiorkoris a classical example of nutritional deficiencystate leading to anasarca. It is characterized bygrowth retardation, psychomotor changes andedema. Edema starts in the lower extremitiesand later involves upper limb and the face.Associated features like hair changes, skinchanges, hepatomegaly, recurrent infections,vitamin and mineral deficiency signs help inmaking the diagnosis easier.

Gastrointestinal losses of protein either dueto chronic diarrhoea, digestive enzymedeficiency, protein losing enteropathy,inflammatory bowel disease, cystic fibrosis, etclead to anasarca.

Investigations

With proper history and physicalexamination differential diagnosis for anasarcacan be narrowed down and appropriateinvestigation can be asked for. Hence basicinvestigations like hemogram, urine examination,

renal function test, liver function test, chestx-ray may suggest the cause for anasarca.When the basic investigations do not confirm thediagnosis, further investigation may have to bedone. One such investigation would be asciticfluid analysis that helps in differentiating whetherascites is secondary to portal hypertension or dueto non-portal hypertension causes.

Ascitic fluid analysis7,8 : In the past, ascites wasclassified on the basis of protein content eitheras exudative (more than 2.5g/dL) or transudative(less than 2.5 g/dL). It had its own drawbackswith high false positive results. Presently this hasbeen replaced by serum, ascites albumin gradient(SAAG) that is calculated by subtracting asciticfluid albumin from serum albumin. It is classifiedas portal hypertensive (SAAG > 1.1g/dL) or highalbumin gradient ascities which was previouslyknown as transudative ascites and non portalhypertensive (SAAG < 1.1g/dL) or low albumingradient ascites which was previously known asexudative ascites. The accuracy of the SAAGresults is approximately 97% in classifying portalhypertensive and non- portal hypertensive ascites.A high gradient is associated with diffuseparenchymal liver disease and occlusive portaland hepatic venous disease.

Table 2 summarizes the causes for ascitesbased on SAAG values.9

Management

The management depends upon the etiology.Child with renal disease may require fluidrestriction and diuretics, while child with CCFmay require bed rest, digoxin, diuretics andvasodilators. Child with hepatic cause may havegross hypoalbuminemia which requires dietarysupplementation, salt free albumin etc.Occasionally one may come across a child withundiagnosed anasarca. These are the patients whomay require extensive investigations to rule outthe rarer causes like intestinal lymphangiectasia

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and cystic fibrosis where relevant investigationslike sweat chloride test, endoscopy may have tobe carried out.10

Flowchart (Fig.1) summarizes the approachto the diagnosis of anasarca in the child.

Points to Remember

• Edema more in the morning and subsidingby evening is suggestive of renal edema.

• Ascites to start with, followed by edema maysuggest a possibility of hepatic failure.

• Nutritional history combined withanthropometry, vitamin and mineraldeficiency signs, points to the diagnosis ofnutrition deficiency states like kwashiorkor.

• Edema in the dependent part associatedwith tachypnoea and abnormal findings inthe heart suggest the diagnosis ofcardiovascular conditions for anasarca.

References

1. Braunwald E, Loscalzo J. Edema. Harrison’sPrinciples of Internal medicine 17

th Edn 2008

New York. Mc-Graw Hill publishers 2008;pp 231-236.

2. Arora NK, Mathur P, Ahuja A, Oberoi A.Acute liver failure. Indian J Pediatr 2003;70:73-79.

3. Poddar U, Thapa BR, Prasad A, Sharma AK,Singh K. Natural history and risk factors infulminant hepatic failure. Arch Dis Child2002;87:54-56.

4. Pomerant AJ. Pediatric Decision- MakingStrategies to accompany Nelson Textbook ofPediatrics. 1

st Edn, Philadelphi, Saunders,

Harcourt private limited 1996; pp 134-137.

5. Ghai OP. Nutrition and macronutrient disorders.In: Ghai Essential pediatrics 6

th Edn. New Delhi,

CBS Publisher, 2006 ;pp 93-118.

6. Metha M N. Protein energy malnutrition.Eds IAP textbook of pediatrics. 3

rd Edn,

A. Parthasarathy, Jaypee publishers, New Delhi2006;pp120 – 138.

Table 2. Causes of ascites based on SAAG8

High gradient ascities Low gradient ascities

(SAAG > 1.1 gm) (SAAG <1.1 gm)·

• Cirrhosis • Nephrotic syndrome

• Venoocclusive disease, • Tuberculosis

• Fulminant hepatic failure • Nutritional

• Cardiac ascites • Collagen vascular disorder

• Mixed ascites

• Liver metastasis

High SAAG , normal protein Budd chiari syndrome, constrictive pericarditis

High SAAG , low protein Cirrhosis

Low SAAG , low protein Nephrotic syndrome, tuberculosis, nutritional

Low SAAG , normal protein Chylous ascites, pancreatic ascites

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7. Boamah L, William F, Balistreri. Manifestationof liver disease. In: Nelson Textbook ofPediatrics, 18

th Edn, Kliegman RM, Jenson HB,

Stanton BF. Philadelphia, Saunders 2008;pp1661– 1667.

8. Yachha SK, Khanna V. Ascities in childhoodliver disease. Indian J Pediatr 2006:73.816-824.

9. Riyaz A. Ascites .Pediatric gastroenterologyand hepatology. 2

nd Edn, Hyderabad, Paras

Publisher, 2003;pp323 – 343.

10. Maiya PP, Hill PG, Sudarsanam D, Jadhav M.Cystic fibrosis in south India. Trop Geog Med1980; 32: 45–49.

CONTRIBUTOR TO CORPUS FUND OF IJPP

Dr.Suresh K Navandar Aurangabad, Maharashtra. Rs.500/-

CLIPPINGS

Joseph J. Zorc, Caroline Breese Hall, Bronchiolitis: Recent Evidence on Diagnosisand Management . Pediatrics 2010; 125: 342-349.

Viral bronchiolitis is a leading cause of acute illness and hospitalization of young children.Research into the variation in treatment and outcomes for bronchiolitis across differentsettings has led to evidence–based clinical practice guidelines. Ongoing investigationcontinues to expand this body of evidence. Authors of recent surveillance studies havedefined the presence of coinfections with multiple viruses in some cases of bronchiolitis.Underlying comorbidities and young age remain the most important predictors for severebronchiolitis. Pulse oximetry plays an important role in driving use of health careresources. Evidence–based reviews have suggested a limited role for diagnostic laboratoryor radiographic tests in typical cases of bronchiolitis. Several large, recent trials haverevealed a lack of efficacy for routine use of either bronchodilators or corticosteroids fortreatment of bronchiolitis. Preliminary evidence suggests a potential future role for acombination of these therapies and other novel treatments such as nebulized hypertonicsaline.

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MACROLIDES IN CHILDREN

* Jeeson C Unni

Abstract: Macrolides are useful and safeantibiotics for pediatric infections, especiallyif the child is allergic to penicillin and/orcephalosporin. They can be used to treatstreptococcal pharyngitis, other respiratorytract infections, community-acquired pneumoniaand cutaneous infections.

Keywords: Macrolides, Erythromycin,Azithromycin, Clarithromycin, Antibacterialspectrum, Indications, Dosage, Pharma-cokinetics, Drug interactions, Adverse effects.

Macrolides are useful in treating grampositive infections especially when the child isallergic to penicillins and cephalosporins.Currently available macrolides in common usein India are erythromycin and the newermacrolides clarithromycin and azithromycin.Erythromycin, though effective, has a number ofdisadvantages, including a narrow anti-bacterialspectrum, unfavorable pharmacokineticproperties such as a short serum half life, andfrequent GI intolerance.1 Increasing resistance ofgroup A streptococci to erythromycin has beenreported in the West.2 Though isolated studies inIndia show a trend towards this phenomenon.3

others, including the ongoing prospectivehospital surveillance of invasive pneumococcaldisease in seven referral hospitals in India, underIndia CLEN network report very low resistance

to erythromycin.4 Resistance appears to correlatewith the amount of macrolide use within acommunity as evidenced by a decrease inerythromycin resistance among group Astreptococci associated with a nationwidedecrease in macrolide use in Finland.2

The newer macrolides, clarithromycin andazithromycin, are synthesized by altering theerythromycin base to produce compounds thatpossess longer half-life allowing once or twicedaily administration, resistance to gastricdegradation, improved oral bioavailability andpossibly better GI tolerance, excellent tissue andintracellular penetration and an extendedspectrum of activity.5 They can therefore beadministered for children who do not tolerateerythromycin or have infections with organismsresistant to erythromycin and as an alternative toco-amoxiclav or second or third generationcephalosporins.1

Mechanism of action, antibacterialspectrum and clinical uses

Macrolide antibiotics act by binding to theP site of the 50S ribosomal subunit of susceptibleorganisms and by inhibiting bacterialRNA-dependent protein synthesis.6 They may beeither bacteriostatic or bactericidal, dependingon the drug concentration and bacterialsusceptibility. Although generally bacteriostatic,clarithromycin and azithromycin are bactericidalagainst Streptococcus pyogenes, Streptococcuspneumoniae and Haemophilus influenzae.These agents are also useful in managingpertussis and infections caused by Legionella,

DRUG PROFILE

* Editor-in-chief, IAP Drug FormularyDr. Kunhalo’s Nursing Home, Cochin.

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Chlamydia, and Mycoplasma.7,8 Clarithromycinis several-fold more active in vitro thanerythromycin against gram-positive organisms.9

Azithromycin in vitro is considered less potentthan erythromycin against gram-positiveorganisms but this may not be clinicallysignificant as susceptibility concentrations fallwithin the range of blood levels attainable withazithromycin. Further, azithromycin appearsto be more active than erythromycin againstmany Gram-negatives and several otherpathogens, notably Haemophilus influenzae,H. parainfluenzae, Moraxella catarrhalis,Neisseria gonorrhoeae, Urea plasma urealyticumand Borrelia burgdorferi.9,10 Azithromycin hasexcellent in vitro activity against H influenzae,whereas clarithromycin, although less activeagainst H influenzae in vitro, is metabolized intoan active compound, 14-hydroxyclarithromycin,with twice the in vitro activity of the parent drug.Oral azithromycin and clarithromycin are equallyeffective against respiratory tract and soft tissueinfections by S aureus, S pneumoniae,S pyogenes, H influenzae, and M catarrhalis thatare susceptible to these drugs. Thus, these 2 drugsare rational choices for the treatment ofcommunity-acquired pneumonia. However, thelow serum concentrations of azithromycin maybe a problem in patients with bacteremiaassociated with community-acquiredpneumonia.11 Clarithromycin appears more activethan azithromycin and erythromycin againstLegionella pneumophila and Chlamydiapneumoniae, whereas azithromycin demonstratesbetter in vitro activity against M. catarrhalis andMycoplasma pneumoniae.12,13 Clarithromycinappears to be effective for the treatment andprophylaxis of Mycobacterium avium complex(MAC) in patients with AIDS, whileazithromycin appears to be effective forprophylaxis.11

Though azithromycin is the only macrolidewith extended gram negative cover, its only

significant clinical use is its effectiveness againstSalmonella typhi.14 This Cochrane reviewsuggests that azithromycin appears better thanfluoroquinolone drugs in populations thatincluded participants with drug-resistant strainsand that it may perform better than ceftriaxone.Clarithromycin is the macrolide of choice in tripleregimens for the treatment of Helicobacterpylori.15

Parenteral azithromycin has been used as analternate treatment for toxoplasma encephalitisin AIDS patients.16 The newer macrolides are alsoprescribed for the prophylaxis and therapy fordisseminated Mycobacterium avium complexdisease in patients infected with the humanimmunodeficiency virus.17

Topical erythromycin is used in treatmentof acne vulgaris. Oral azithromycin anderythromycin have been used for the treatmentfor acne vulgaris in adolescents but there arerecent reports of resistance to both oral andtopical erythromycin.

The common uses in day-to-daypractice are as follows:

Erythromycin

• Alternative to penicillin in penicillinallergy for secondary prophylaxis in rheumaticfever

• Mild to moderate infections of skin

• Atypical pneumonia

• Neonatal conjunctivitis due toC trachomatis

• Eradication of B pertussis fromnasopharynx/prophylaxis

Clarithromycin

• Eradication regimens for H.pylori

• Atypical pneumonia

Cost and bad taste are disadvantages.

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Azithromycin

• Atypical pneumonia – drug of choice

• Short course therapy for AOM > 2 yr age

• Skin infections

Newer macrolides are prefered toerythromycin in acute otitis media, sinusitis andpneumonia due to higher activity againstH. influenzae.

Pharmacokinetics in children

The bioavailability of oral macrolideantibiotics is poor. At best, only 40% to 50% ofa given dose is absorbed. Food significantlyreduces the absorption of azithromycin ascapsules and the stearate and some base formsof erythromycin. Therefore, they need to be takenat least 1 hour before or 2 hours after food.Clarithromycin; azithromycin suspension; anderythromycin in the estolate, ethylsuccinate, anddelayed-release base (enteric-coated) forms canbe taken without regard to meals.18 In fact, whentaken with meals, the oral bioavailability ofclarithromycin is increased by up to 25%.5

The macrolides penetrate well into therespiratory, genitourinary and GI tracts as wellas into skin, soft tissues, and sinuses. They areonly moderately (40% to 50%) protein-bound.Erythromycin and clarithromycin are mainlymetabolized by the liver. The metabolites oferythromycin are excreted in the bile and urineto a small extent. Clarithromycin is metabolizedto a pharmacologically active compound,14-hydroxy clarithromycin, which undergoesrenal elimination. None of the macrolides crossthe blood brain barrier.

Although serum concentrations ofazithromycin are low following oraladministration, high tissue levels are attained;achieving tissue half-life of approximately three

days. This enables the clinician to use once dailydosing and ensures that a five-day single dailydose regimen for respiratory tract and soft tissueinfections will provide therapeutic tissueconcentrations for at least ten days. It also allowsa single 1 g dose of azithromycin for the effectivetreatment of C.trachomatis genital infections.Clarithromycin has a longer serum half-life andbetter tissue penetration than erythromycin,allowing twice-a-day dosing for most commoninfections.18

Drug interactions

Both erythromycin and clarithromycininhibit the activity of the hepatic cytochromeP-450 enzyme system. As a result, these agentsreduce the metabolism and increase the serumconcentration of other drugs eliminated via theP-450 pathway. Azithromycin, because ofdifferences in its chemical structure, does notcause these interactions. Thus, theophyllineclearance may be decreased by up to 35% whenclarithromycin or erythromycin are prescribedconcurrently; causing clinically importantinteractions especially when theophylline serumconcentrations are already in the high therapeuticrange (i.e., > 15 mcg/ml).19 Clarithromycin anderythromycin may inhibit the metabolism ofcyclosporine via inhibition of theCYP3A4 isoenzyme, thus increasingcyclosporine’s effects and the potential fortoxicity.20 It has been recommended to avoidcyclosporine in combination with thesemacrolide agents or reduce the cyclosporinedosage by 50% when it is necessary to give anyof these macrolides concurrently.

The other medications known to be affectedby erythromycin or clarithromycin areanticoagulants, carbamazepine, cisapride,digoxin and methylprednisolone. Erythromycin,clarithromycin, and azithromycin have beenassociated with the development of ventricular

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arrhythmias, including torsades de pointes andventricular tachycardia, in patients with aprolonged QTc interval.21 In many of the reportedcases, these patients were also receiving amedication that interacts with a macrolide.

Adverse events

Most adverse reactions associated with theuse of macrolide antibiotics are mild and resolveon discontinuing therapy. Because of the lowincidence of toxic effects reported during themore than 55 years of their use, macrolides areconsidered to be the safest antimicrobial agentsavailable,22 so a review of their adverse effectsdeals mostly with rare and unusual events.

In children, the most frequently reportedadverse effects with erythromycin are diarrhea,vomiting (6% each), abdominal pain, rash(3% each), nausea (1% to 2%), and headache(2%). Less frequent but more severe adverseeffects include anaphylaxis, Stevens-Johnsonsyndrome, reversible hearing loss, andhepatocellular cholestatic hepatitis.23

Pseudomembranous colitis, resulting from theovergrowth of Clostridium difficile, has longbeen associated with erythromycin.

Azithromycin and clarithromycin appear tobe well-tolerated. Gastrointestinal intolerance isthe primary adverse side effect of bothantimicrobials but occurs at a significantlyreduced rate when compared to erythromycin.18

Azithromycin causes diarrhea [3.6%], nausea[2.6%], abdominal pain [2.5%], and headache ordizziness [1.3%]. Only 0.7% of patientsdiscontinued azithromycin therapy as comparedto 2.6% of patients receiving comparativemedications.24 The most common adversereactions of clarithromycin are nausea [3.8%],diarrhea [3.0%], abdominal pain [1.9%], andheadache [1.7%].1 Laboratory abnormalities werealso rare and included elevated liver function testsand decreased white blood cell counts. Overall,

fewer than 3% of patients receivingclarithromycin withdrew from studies becauseof adverse effects.25 Though adverse effects havebeen mild, there have been isolated reports ofpseudomembranous colitis (long been associatedwith erythromycin) in patients onclarithromycin.26

Nephrotoxicity caused by macrolides mustbe considered a rare adverse event. Very few dataare available in published case reports, either foradults or children, that show strong evidence fora causal relationship.27,28 No evidence ofnephrotoxicity from clarithromycin has howeverbeen found in clinical trials.

Dosages and administration

Erythromycin

Dosage: General indications: Neonates –Orally/IV < 7 days age 20mg.kg/day 2 timesdaily; > 7 days <1200gm 20mg.kg/day 2 timesdaily; > 7 days >1200gm 30mg.kg/day 3-4 timesdaily

Children: Orally - 30-50 mg/kg/day in4 divided doses max 250mg 4 times daily maybe given. 12-18yrs – 250-500mg 4 times daily.IV – 1mth – 18yr 12.5mg/kg/dose 4 times dailyor as a continuous infusion. Maximum dose -4 g/day. Replace by oral dosage as soon aspossible.

Special indications : Topical (acne vulgaris) :Wash and apply twice daily directly to theaffected area.

Secondary prevention of rheumatic fever –when child is sensitive to penicillin -oral 20mg/kg/day max 500mg twice daily(Contraindicated in liver disorder)

Chlamydia trachomatis pneumonia ininfants and neonates: Oral - 50 mg/kg/day(erythromycin base) in four divided doses for14 days.

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Ophthalmia neonatorum caused by Chlamydiatrachomatis: Neonates – Oral - 50 mg/kg/day infour divided doses for 14 days. If chlamydialconjunctivitis recurs after discontinuing therapy,the erythromycin dosage regimen should berepeated.

A beta-hemolytic streptococcal (GAS)pharyngitis (primary rheumatic fever prophylaxisas an alternative to penicillin in childrenallergic to penicillin)- 40 mg/kg/day in 4 divideddoses x 10 days. Cardiology sub chapter of IAPdoes not recommend using erythromycin for thisindication.

Uncomplicated urethral, endocervical, orrectal gonorrhea, penicillinase-producingNeisseria gonorrhoeae, or for gonorrheaduring pregnancy -Adolescents: 500 mg PO fourtimes per day for 7 days

Treatment and postexposurepertussis prophylaxis – For postexposureprophylaxis, administer to close contacts within3 weeks of exposure, especially in high-riskpatients (e.g., women in 3rd trimester, infants< 12 months).Oral dosage: Infants,Children, andAdolescents: 40-50 mg/kg/day PO (maximum2 g/day) in four divided doses for 14 days.For neonates: Azithromycin is preferred.If azithromycin is unavailable, erythromycin40-50 mg/kg/day PO in 4 divided doses maybe used. Monitor for infantile hypertrophicpyloric stenosis.

Pneumococcal prophylaxis - 1month - 2yr250mg/day, 2-8 yr 500 mg/day, >9 yr 1gm/dayin 2 divided doses.

Gastric stasis – Oral/IV – 1month – 18yr -3mg/kg 4 times daily.

Administration: Oral: total daily dose maybe given in 2 divided doses, but gastrointestinalside-effects can occur.

Patients with hepatic impairment: Use withcaution in patients with impaired hepaticfunction. Although specific dosage guidelines arenot available, a reduced dosage may be necessary.

Patients with renal impairment: No dosageadjustment needed.

Azithromycin

Dosage: For all indications: 6 months -12 years 10mg/kg/day once daily for 3 days uptomaximum of 200 mg (3-7 years), 300 mg(8-11 years, 400 mg (12-14 years) and 500 mg> 14 years. Alternatively, 10mg/kg/day once onfirst day followed by 5mg/kg/day once daily fromday 2 – day 5.

Group A beta-hemolytic streptococcal (GAS)pharyngitis and tonsillitis (primaryprophylaxis of rheumatic fever):12.5 mg/kg/day - single dose for 5 days.(Not recommended for secondary prophylaxis ofrheumatic fever)

Chlamydial infection such as non-gonococcalurethritis (NGU) or cervicitis due to susceptiblestrains of Chlamydia trachomatis: Adolescent:single dose of 1 g orally.

Bacterial endocarditis prophylaxis: 15 mg/kg(single dose max 500mg) 30-60 minutes beforeprocedure in children and adolescents allergic topenicillin.

Treatment and postexposurepertussis prophylaxis – (For postexposureprophylaxis, administer to close contacts within3 weeks of exposure, especially in high-riskpatients (e.g., women in 3rd trimester, infants< 12 months).Oral dosage: Infants > 6 monthsand Children: 10 mg/kg/day (maximum 500 mg)on day 1, then 5 mg/kg/day (maximum 250 mg)on days 2—5. Infants < 6 months: 10 mg/kg/dayfor 5 days. Monitor for infantile hypertrophicpyloric stenosis in infants < 1 month old.

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Uncomplicated typhoid fever: Orally -Adolescent: 8—10 mg/kg/day once dailyfor 7 days (1000 mg on first day, followedby 500 mg once daily for 6 days). Children:10 mg/kg/day once daily for 7 days or 5-dayregimen of 20 mg/kg/day.

Administration: Oral: administer 1 hour beforeor 2 hours after food. If a child vomits within5 minutes of the dose, an additional dose may begiven. If child vomits between 5-60 minutes ofthe dose, alternative therapy should beconsidered. If a child with normal gastricemptying, vomits 60 minutes or thereafter, noadditional dose is warranted.

Clarithromycin

Dosage: Oral 15mg/kg/24hr in 2 divideddoses upto maximum of 500mg twice daily for5-10 days.

H. Pylori - 1-2 yr 125mg, 2-6yr 250mg,6-9yr 375mg, 9-12yr 500mg and 12-18yr1gm/day in 2 divided doses along withamoxycillin and omeprazole or amoxicillin andlansoprazole or metronidazole and omeprazole.

CAP and pharyngitis in children due toChlamydophila pneumoniae, Mycoplasmapneumoniae or Streptococcus pneumoniae givefor 10 days.

Bacterial endocarditis prophylaxis: 15 mg/kg(single dose max 500mg) 30-60 minutes beforeprocedure in children and adolescents allergic topenicillin.

Treatment and postexposurepertussis prophylaxis – (For postexposureprophylaxis, administer to close contacts within3 weeks of exposure, especially in high-riskpatients (e.g., women in 3rd trimester, infants< 12 months). Oral dosage: Infants > 6 monthsand Children: 15 mg/kg/day upto maximum of

500mg twice daily for 7 days. Not used inneonates.

Patients with renal impairment: Creatinineclearance (CrCl) > 60 ml/min: no dosageadjustment needed. CrCl 30-60 ml/min:No dosage adjustment needed except in patientsreceiving concurrent ritonavir. In these patients,reduce the recommended clarithromycin doseby 50%.

CrCl < 30 ml/min: reduce recommendeddose by 50%. In patients receiving ritonavir,decrease the recommended clarithromycin doseby 75%.

Administration: Oral; food does not affect extentof bioavailability but may delay onset ofabsorption.

Conclusion

The use of macrolides in pediatrictherapeutics in India is much less than that ofbeta lactam antibiotics. But it has its place inchildhood infections and it must be usedwherever it is recommended as a first line drug.Since it is not recommended for use in bacteremicillnesses these drugs are used mainly in OPDpractice. The macrolide class as a whole includessome of the safest anti-infective agents availableand therefore they must be used more often whenindicated.

Points to Remember

• Macrolides are the safest group ofantimicrobial drugs available.

• Erythromycin is a good alternative topenicillin in penicillin allergy in mild tomoderate infections by susceptibleorganisms. Also useful in treating atypicalpneumonia and neonatal C.trachomatisconjunctivitis. It eradicates B. pertussis

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from the nasopharynx (reduces period ofinfectivity but does not alter course ofdisease). GI side effects is a drawback.

• Clarithromycin is used in eradicationregimens for H. pylori. Cost andpalatability are drawbacks.

• Azithromycin is highly concentrated intissues allowing short course therapy withonce daily dosing.

• Newer macrolides are preferred toerythromycin in otitis media, sinusitis andnon bacteremic pneumonia due to betterH. influenza and M catarrhalis cover andatypical mycobacteria infections.

References

1. Guay DR. Macrolide antibiotics in paediatricinfectious diseases. Drugs 1996; 51: 515-536.

2. Seppala H, Klaukka T, Vuopio-Varkila J,Muotiala A, Helenius H, Lager K, et al. Theeffect of changes in the consumption ofmacrolide antibiotics on erythromycinresistance in group A streptococci in Finland.N Engl J Med 1997; 337: 441-446.

3. Capoor MR, Nair D, Deb M, Batra K, AgarwalP. Resistance to erythromycin and risingpenicillin MIC in streptococcus pyogenes inIndia. Jpn J Dis. 2006; 59: 334-336.

4. Thomas K, Lalitha MK, Steinhoff MC,Ganesan A. Temporal Trends in AntimicrobialResistance Patterns of Invasive Pneumococciin 7 Hospitals in India - a 10 Year Experience.India Clinical Epidemiology Network(INCLEN); Interscience Conference onAntimicrobial Agents and Chemotherapy. AbstrIntersci Conf Antimicrob Agents ChemotherIntersci Conf Antimicrob Agents Chemother2003 Sep 14-17; 43: abstract no. C2-947.

5. Zuckerman JM. The newer macrolides:azithromycin and clarithromycin. Infect DisClin North Am. 2000; 14: 449-462

6. Sturgill MG, Rapp RP. Clarithromycin: Reviewof a new macrolide antibiotic with improvedmicrobiologic spectrum and favorablepharmacokinetic and adverse effect profiles.Ann Pharmacother 1992; 26:1099-1108.

7. Peters DH, Clissold SP. Clarithromycin: areview of its antimicrobial activity,pharmacokinetic properties and therapeuticpotential. Drugs. 1992; 44: 117-164.

8. Ballow CH, Amsden GW. Azithromycin: thefirst azalide antibiotic. Ann Pharmacother 1992;26:1253-1261.

9. Whitman MS, Tunkel AR. Azithromycin andclarithromycin: overview and comparison witherythromycin. Infect Control Hosp Epidemiol1992; 13: 357-368.

10. Peters DH, Friedel HA, McTavish D.Azithromycin. A review of its antimicrobialactivity, pharmacokinetic properties and clinicalefficacy. Drugs 1992; 44: 750-799.

11. Charles L, Segreti J. Choosing the rightmacrolide antibiotic. A guide to selection.Drugs. 1997; 53: 349-357.

12. Fernandes PB, Bailer R, Swanson R,Hanson CW, McDonald E, Ramer N, et al.In vitro and in vivo evaluation of A-56268 (TE-031), a new macrolide. Antimicrob AgentsChemother 1986; 30: 865-873.

13. Retsema J, Girard A, Schelkly W, ManousosM, Anderson M, Bright G, et al. Spectrum andmode of action of azithromycin (CP-62,993), anew 15-membered-ring macrolide withimproved potency against gram-negativeorganisms. Antimicrob Agents Chemother1987; 31: 1939-1947.

14. Effa EE, Bukirwa H. Azithromycin for treatinguncomplicated typhoid and paratyphoid fever(enteric fever). Cochrane Database Syst Rev.2008; (4): CD006083.

15. Malfertheiner P, Megraud F, O’Morain C,Bazzoli F, El-Omar E, Graham D, etal.The European Helicobacter Study Group(EHSG). Current concepts in the managementof Helicobacter pylori infection: the MaastrichtIII Consensus Report. Gut 2007; 56: 772-781.

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16. Jacobson JM, Hafner R, Remington J,Farthing C, Holden-Wiltse J, Bosler EM, et al.ACTG 156 Study Team. Dose-escalation, phaseI/II study of azithromycin and pyrimethaminefor the treatment of toxoplasmic encephalitisin AIDS. AIDS. 2001; 15: 583-589.

17. Masur H. for the Public Health Service TaskForce on Prophylaxis and Therapy forMycobacterium avium Complex. Recommen-dations on prophylaxis and therapy fordisseminated Mycobacterium avium complexdisease in patients infected with the humanimmunodeficiency virus. N Engl J Med 1996;329: 898-904.

18. Whitman MS. Azithromycin and clarithro-mycin: overview and comparison witherythromycin. Infect Control Hosp Epidemiol1992; 13: 357-368.

19. Kurisu S, Barriere SL. Theophylline-erythromycin interaction. Drug Intel Clin Pharm1984; 18: 390.

20. Spicer ST, Liddle C, Chapman JR, Barclay P,Nankivell BJ, Thomas P, et al. The mechanismof cyclosporine toxicity induced byclarithromycin. Br J Clin Pharmacol 1997;43: 194-196.

21. Yap YG, Camm AJ. Drug induced QTprolongation and torsades de pointes.

Heart 2003; 89: 1363-1372; doi:10.1136/heart.89.11.1363

22. Principi N, Esposito S. Comparative tolerabilityof erythromycin and newer macrolideantibacterials in paediatric patients. Drug Saf1999; 20: 25-41.

23. Periti P, Mazzei T, Mini E, Novelli A. Adverseeffects of macrolide antibacterials. Drug Saf1993; 9: 346-364.

24. Eisenberg E, Barza M: Azithromycin andclarithromycin. Am J Med 1991; 91(suppl 3A):40-45.

25. Hopkins S: Clinical toleration and safety ofazithromycin. Curr Clin Topics Infect Dis 1994;14: 52-79.

26. Braegger CP, Nadal D. Clarithromycin andpseudomembranous enterocolitis. Lancet1994; 343: 241-242.

27. pGómez G, Álvarez ML, P. Errasti P, Lavilla

FJ, García N, Ballester B, et al. Acute tacrolimusnephrotoxicity in renal transplant patientstreated with clarithromycin. TransplantationProceedings 1999; 31: 2250-2251.

28. Viani F, Claris-Appiani A, Rossi LN, Giani M,Romeo A, et al. Severe hepatorenal failure in achild receiving carbamazepine anderythromycin. Eur J Pediatr 1992;151:715-716.

Jan SL, Wu MC, Lin MC, Fu YC, Chan SC, Lin SJ . Pyuria is not always sterile inchildren with Kawasaki disease. Pediatrics International 2010;52:113-117

Pyuria was not always sterile in patients with Kawasaki Disease (KD). Although therewas no different clinical phenotype or coronary outcome in KD patients with or withoutUTI, the authors suggest that UTI should be considered and evaluated in KD patientswith pyuria, a positive nitrite test or a positive result of urine culture. If UTI is definitivelydiagnosed, the patient should be treated for a UTI as well as for KD and complete post–UTI work–up is recommended.

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DERMATOLOGY

DRUG ERUPTIONS –AN OVERVIEW

*Anandan V

Abstract: Adverse drug reaction is a commonproblem in clinical practice posing high degreeof confusion in diagnosis and management.It may lead to fatal results if there is a delay inthe initiation of the appropriate management.A proper knowledge about the variousmorphological patterns of drug eruptions andproper management will reduce the morbidityand mortality.

Keywords: Adverse drug reaction, AGEP, SJS,TEN.

In spite of limited information on theincidence of adverse drug reactions (ADR) inchildren, a meta analysis of prospective studiesrevealed an overall incidence of 9.5% of ADRamong hospitalised children and the rate ofpediatric hospital admissions due to ADR was2.1% of which 39.3% were considered to be lifethreatening reactions. The incidence of ADR inoutpatient setting was reported as 1.5 %.1

Cutaneous eruptions are one of the mostfrequent presentations of ADR and are found tobe present in 40% of the ADR patients whichrange from mild erythematous rashes to lifethreatening toxic epidermal necrolysis (TEN)which could prove fatal.

Approach

The first and foremost in a patient withsuspected ADR is early diagnosis and appropriatemanagement which can reduce the mortality andmorbidity.

Methodical and precise approach towardsthe morphological reaction pattern of ADR willgive the treating physician a clue about theprobable drug, as different drugs are commonlyassociated with specific types of reaction, suchas exanthematous, urticarial, blistering orpustular and among these exanthematous andurticarial with or without angio oedema are thecommon types of eruptions..2 The reactions couldbe simple or complex with the presence of feverand systemic manifestations in the latter.

Drug exposure

ADR could be due to single or multipledrugs and it is essential to inquire about theconcomitant usage of herbal and naturopathicremedies.

Most of the drug reactions occur within1-6 weeks after the drug exposure and sometimesmay take even 3 years as in the case of druginduced lupus.

Morphological approach

1. Exanthematous

Exanthematous drug rash is the commonestform of ADR accounting nearly 95% of the cases3

of which maculopapular is the commonest andthe others being morbilliform and scarletiniform.The reaction can occur from 1-10 days of the

* Associate Professor and HOD,Department of Dermatology,Dharmapuri Medical College, Tamilnadu

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therapy and pruritis is the most commonassociated symptom. Resolution occurs within7-14 days ending with scaling and desquamation.

The common drugs which are implicated forthis type of reactions include penicillin,sulfanomides, barbiturates and anti epileptics.An exanthematous rash could be life threateningif it occurs with fever when one should suspectdrug hypersensitivity syndrome (DHS) which isa triad of fever, skin eruptions and internal organinvolvement. DHS can occur even with the firstexposure of the drug starting from 1-6 weeks ofdrug exposure and these rashes have predilectiontowards the previous radiotherapy sites.

Even though fever and malaise could be thefirst symptoms, pharyngitis and cervicallymphadenopathy also occur early. The initialsigns of eruption are edema and swelling of theface followed by erythema and pruritis of the faceand subsequent caudal spread which can easilyslip into Steven Johnson syndrome (SJS) orTEN. Internal organ involvement results inhepatitis, pneumonitis, pancreatitis, vasculitis,encephalitis, aplastic anemia, SIADH, myositisand thyroiditis.

The drugs associated with this type ofreactions are aromatic anticonvulsants likephenytoin, carbamazepine, phenobarbitol andothers like lamotrigine, sulphanomides,antibiotics, dapsone, minocycline andallopurinol.4

2. Urticarial

Urticarial reactions are characterized by theappearance of erythema and wheals with orwithout pruritis which may last from 1-48 hoursand sometimes even for 5-6 days. These reactionscould be IgE mediated (penicillins andantimicrobials) or non IgE mediated (narcoticanalgesics, radio contrast media, acetyl salicylicacid and NSAIDS).

Latex allergy is a type 1 reaction which canrange from simple urticaria to death, especiallyin children with spina bifida where they areexposed to catheters and latex products morefrequently.5

3. Serum sickness like reaction (SSLR)

SSLR is defined by fever, rash(exanthematous or urticarial), arthralgiaoccurring 1-3 weeks after drug exposure whichcould be associated with lymphadenopathy andeosinophilia; however absence of immunecomplexes, hypocomplementemia, vasculitis andrenal lesions differentiates SSLR from true serumsickness.

Epidemiological evidence in childrensuggests that the risk of SSLR is more withcefaclor than with other antibiotics includingother cephalosporins..6 Recently cefprozil,minocycline, bupropion are reported to causeSSLR.

4. Pustular

a) Acneform eruptions: Acneform eruptionstypically occur over the arms, legs, back and chestwhich are usually monomorphic papules andnodules without comedones sparing prepubertalchildren indicating that prior hormonal primingis a prerequisite.

The drugs that have been reported producingthis type of reaction are steroids, iodides,bromides, ACTH, INH, androgens, lithium andactinomycin-D. This reaction can occur within2 weeks of drug intake and is directlyproportional to dosage and duration of theoffending drug.7

b) Acute generalized exanthematous pustulosis(AGEP): Even though AGEP is rare in children,the estimated incidence is 1-5 cases/million/year.8

The reaction is characterized by an acuteonset with a temperature more than 38OC and a

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cutaneous eruption composed of sterile nonfollicular pustules on an erythematous andedematous base which may be associated withtarget lesions, vasculitis, blisters and erosions ofthe mucus membranes.

Resolution occurs by desquamation within2 weeks after the withdrawl of the offending drug(β lactams, macrolides and calcium channelblockers); Leucocytosis is common anddisappears before desquamation. Eosinophilia,hypocalcemia and renal failure have beenreported with AGEP.

5. Bullous eruptions

a) Pseudo porphyria: A photo toxic disorderwhich is non dose dependent and has neither agenor sex predilection can resemble porphyriacutanea tarda (PCT) or erythropoieticprotoporphyria (EPP) in which normal porphyrinlevels is indicative of pseudo porphyria.Clinically this can present with increased skinfragility, blistering, burning sensation andscarring over the sun exposed areas.

Pseudo porphyria has an estimatedincidence of 12% in patients with juvenilerheumatoid arthritis receiving naproxen,oxaprozin and ketoprofen. Other drugs reportedare furosemide, tetracycline and erythropoietin.Blistering may continue for weeks and skinfragility may persist for months. Resolution isslow and complete except for scarring.9

6. Erythema multiforme major (EMM), SJSand TEN

Even though a strong debate is existing indistinguishing each of these entities by definition,it has been accepted that all these are differentfacets of the same disease process and arbitrarilyit has been accepted that EMM is with 10%,SJS with 10-30% and TEN with more than30% involvement .

Anticonvulsants, antibiotics (penicillins,sulfanomides), allopurinol and NSAIDS(piroxicam) are reported to produce the abovesaid reactions.

The reaction may be acute or insidious withthe onset of fever, malaise and anorexia whichmay last for 1-2 days. Patients with SJS may havethe involvement of mucus membranes witherosions and hemorrhagic crusting ,blisters,ulceration with grey white membrane formation.Patients with TEN initially develop morbilliformor diffuse painful erythema rapidly resulting inblisters and peeling of skin in sheets with positiveNikolsky sign. (skin peeling with lateralpressure). Pseudo membrane formation over thecornea may lead to visual impairment.10

Fluid and electrolyte disturbances andsystemic complications do occur which have tobe tackled cautiously.

7. Miscellaneous drug eruptions

a) Neutrophilic eccrine hidradenitis (NEH):In spite of its rarity, it has been reported to occur11 days after starting the chemotherapy for acutemyelogenous leukemia or lymphoma. The mostcommon clinical presentation is edematouserythema, papules and plaques over face andtrunk associated with neutropenia and fever.Resolution is complete and spontaneous withrecurrences in which case dapsone helps inprevention of recurrences.11

b) Fixed drug eruptions (FDE): FDE withincidence of 22 %,12 is clinically characterizedby oval, pruritic circumscribed, erythematous,plaques occurring over the same sites with there-challenge of offenders. The common sitesinvolved are lips, trunk, legs, arms and genitals.Bullous FDE has been reported and the resolutionoccurs with hyper pigmentation.

The common offenders are sulphonamides,barbiturates, salicylates, acetaminophen,NSAIDS and tetracyclines.

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c) Drug induced lupus (DIL): DIL is defined bythe development of ANA with at least one clinicalsymptom of lupus erythematosis. The drugsreported for DIL are minocycline, procainamideand hydralazine.13 The eruption occurs afterseveral weeks to 2 years which could beurticarial, erythematous or vasculitic witharthritis, hepatitis and with positive ANA.

Differential diagnosis

ADR should be mainly differentiated fromacute viral exanthematous fevers (Drug or Bug),collagen vascular disease, Kawasaki disease andneoplasia which is possible with proper history,high index of suspicion and proper knowledgeof the various morphological patterns of drugreactions produced by specific drugs.

Management

Even though the diagnosis of ADR is basedon the drug history and clinical morphologicalpatterns of reactions by specific drugs,investigations can also serve as corroborativeevidence. CBC may reveal eosinophilia,neutropenia in a case of NEH and leucocytosisin AGEP. ANA will be positive in DIL.Oral provocation test and intra dermal skin testcould be done to confirm the drug hypersen-sitivity and should be done with caution.

Patch testing will be helpful in FDE.Research oriented investigations like radioimmune sorbent assay (RAST), lymphocytetransformation test and lymphocyte cytotoxicityassay could throw some light on betterunderstanding of ADR. Skin biopsy will behelpful in TEN to differentiate it fromStaphylococcal scalded skin syndrome where thesplit will be at the dermo epidermal junction inthe former, where as in the latter it will be moresuperficial. Treatment is mainly avoiding theoffenders. Early initiation of steroids eithertopical or systemic, anti histamines, nutrition,

fluid and electrolyte management will reduce themorbidity and mortality. High dose intravenousgamma globulins (hdIVG) will be life saving inthe case of TEN.

Even though the usage of steroids iscontroversial, in Indian scenario the usage ofsteroids is advocated. Topical steroids of midpotent strength is recommended for milderreactions and systemic steroids like prednisolone1-2mg/kg/day or 3-4 weeks with gradual tapering,injectable dexamethasone 0.2mg/kg/day for5-7 days is life saving. Currently 2.5-3g/kg/dayfor 3 days of intravenous gamma globulins arerecommended to tackle severe reactions likeTEN. Thalidomide was found to be detrimentalin the management of TEN as it causesparadoxical enhancement of tumour necrosisfactor alpha.14

Conclusion

Adverse drug reaction can be properlymanaged by early diagnosis and appropriatemanagement which will reduce the morbidity andmortality.

Points to Remember

• Early diagnosis and management isrewarding.

• Steroids are life saving, especially in TEN.

• Supportive measures are a must.

References

1. Lazarou J, Pomegranz B, Corey P. Incidenceof adverse drug reactions in hospitalisedpatients: a meta analysis of prospectivestudies.JAMA 1998; 279:1200-1205.

2. Roujeau J, Kelly J, Naldi L, Rzany B, Stern RS,Anderson T, et al. Over the counters medicationuse and the risk of Steven-Jhonson syndromeor toxic epidermal necrolysis. N Engl J Med1995; 333:1600-1607.

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3. Bigby M, Jick H, Amdt K Drug inducedcutaneous reactions: A report from the Bostoncollaborative drug surveillance program on15,438 consecutive inpatients, 1975-1986.JAMA 1986;256:3358-3363.

4. Carrol M, Yueng yue K, Esterly N, Dralet B.Drug induced hypersensitivity syndrome inpediatric patients. Pediatrics 2001;108:485-493.

5. Porri F, Pradel M, Lemiere. Associationbetween latex sensitization and reported latexexposure in children. Anesthesiology 1997;86:599-602.

6. Isaacs D Serum sickness like reaction tocefaclor.J Paediatr Child Health 2001;37:298-299.

7. Hurwitz R. Steroid acne . J Am Acad Dermatol1989; 21:1179-1181.

8. Sidaroff A, Halevy S, Barinick J, Vaillant L,Roujean JC. Acute generalised exanthematouspustulosis(AGEP) a clinical reaction pattern.J Cutan Pathol 2001;28:113-119.

9. Levy M, Barron R, Eichenfield A, Haning P.Naproxen induced pseudoporphyria –A distinctive photo dermatitis. J Pediatr1990;117:660-664.

10. Ringheanu M, Laude T. Toxic epidermalnecrolysis in children – an update. Clin Pediatr2000;39:687-694.

11. Sheer N, Knowles S, Shapiro l, Poldre P.Dapsone in prevention of recurrent neutrophilichidradenitis J Am Acad Dermatol 1996;35:819-822.

12. Sharma V, Dhar S. Clinical pattern of cutaneousdrug eruptions among children in north India.Pediatr Dermatol 1995;12:178-183.

13. Bryne P, Williams B, Pritchard M. Minocycline-related lupus. Br J Rheumatol 1994;33:674-678.

14. Wolkenstein P, Latarjet J, Roujeau J,Duguet C, Boudeau S, Vaillant L, et al.Randomised comparison of thalidomideversus placebo in toxic epidermal necrolysis.Lancet 1998;352:1586-1589.

Oduwole O, Meremikwu MM, Oyo-Ita A, Udoh EE . Honey for acute cough in children.Cochrane Database of Systematic Reviews 2010, Issue 1. Art. No.: CD007094. DOI:10.1002/14651858.CD007094.pub2.

Review of one small randomised controlled trial showed that honey was significantlybetter than no treatment for the relief of cough, reducing bothersome cough, improvingchild’s sleep; but no better than ‘no treatment’ in reducing the severity of cough andparent’s sleep. The effects of honey on symptom relief and sleep quality did not differfrom those of dextromethorphan, which is a common ingredient in cough medications.Parents of five children assigned to honey and two assigned to dextromethorphan reportedtheir children suffered from insomnia, hyperactivity and nervousness. However, as withother medications, its benefit should be considered alongside the adverse effects.This review has a limitation in that the results were obtained from a single study involvinga relatively small number of children.

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PHAKOMATOSIS

* Vijayalakshmi G** Elavarasu E

*** Venkatesan MD

Phakomatosis and vascular malformationsalso come under the group of disorders of cellularmigration and neuronal organization.Phakomatoses refer to a group of neurocutaneoussyndromes.

Neurofibromatosis is one of the morecommon types of phakomatosis that is wellknown because of its clinical features.Its hallmark feature is the neurofibroma, whichis a tumor of the Schwann cells surrounding theperipheral nerves. There are two types ofneurofibromatosis (NF). The first type is NF-1,which is the most common type and its clinicalfeatures include cafe au lait spots and neuro-fibromas. The radiological features in NF-1include optic pathway gliomas, sphenoid wingdysplasia and thinning of the cortex of the longbones. The second type, NF-2, only accounts for10% of the cases of neurofibromatosis, and itsclinical feature is bilateral vestibularschwannomas or acoustic neuromas.

Tuberous sclerosis is another phakomatosesthat presents with seizures early in life.These seizures are due to the presence of multiple

hamartomas that are collections of dysplasticcells. They may be seen anywhere in the brain.But the characteristic finding in CT is thecalcified subependymal nodule that is seen in theouter wall of the lateral ventricle (Fig.1).Another peculiar brain lesion in tuberoussclerosis is the giant cell astrocytoma. They aresimilar to the subependymal nodules but with atendency to grow. They usually occur along thecaudo-thalamic groove near the foramen ofMunro and therefore present with hydrocephalus.

Renal involvement occurs in about 80% ofpatients with tuberous sclerosis. The lesionsinclude angiomyolipomas, cysts and renal cellcarcinomas. Angiomyolipomas are localizedproliferations of blood vessels, smooth muscle

RADIOLOGIST TALKS TO YOU

Fig.1. Tuberous sclerosis-Subependymal calcific nodules

* Associate Professor,

** Asst. Professor,

*** Professor,

Department of Radiology,Chengalpet Medical College Hospital,Chengalpet. Tamil Nadu.

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and fat. In ultrasound they appear as multiple,hyperechoic round lesions in the kidneys(Figs.2a and 2b).

Sturge Weber syndrome consists ofcutaneous vascular nevus and leptomeningealangiomatosis. The angiomatosis causes hypoxiaand atrophy of the subjacent brain which givesrise to the characteristic features seen in CT andMRI. Calcification with a tram track appearanceconforming to the gyral pattern is seen in thecortex ipsilateral to the nevus. If sufficientlydense they can be seen in the plain xray of the

skull and this is what was originally describedby Weber. Location of the brain involvement isrelated to the particular area of the trigeminalnerve where the nevus is located. If the nevus isin the area of the ophthalmic division, theoccipital lobe is affected. If the nevus is in themaxillary region, the parietal convexity isaffected. The extent of angiomatoses andtherefore the atrophy correlates to degree ofseizure control and psychomotor development.The child in Fig.3 also had congenital glaucoma,one of the ocular manifestations seen inSturge-Weber syndrome. Note the calcified

Fig 3 Sturge Weber syndrome.Plain CT

Fig 4 Sturge Weber syndrome.contrast CT

Fig 2a and 2b Angiomyolipomas in kidney

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undulating lines posteriorly on the rightcorresponding to calcified gyri. Compare thevolume of the brain parenchyma on either side.The right hemisphere is atrophic and thesubarachnoid spaces are dilated. Contrast CTshows a white blotch in the calcified portioncorresponding to the angiomatosis (Fig.4).Calcification and contrast enhancement are bothwhite in colour and cannot be separated fromeach other in CT. This disadvantage is overcomein MRI where the angiomatoses is brought outclearly with gadolinium enhancement.The enhanced vascular structures are white whilecalcification is black.

A few other phakomatosis are listed herefor completion. Klippel-Trenaunay-WeberSyndrome consists of port-wine hemangiomas,osseous and soft tissue hypertrophy and deepparenchymal vascular malformations.

Osler-Weber-Rendu disease or hereditaryhemorrhagic telangiectasia presents inadolescence with multiple vascularmalformations of the skin, mucosa, lung, brain,spinal cord, and other viscera. MRI is the imagingmodality of choice to identify multiplecavernomas or capillary telangiectasias in thebrain.

Ataxia telengiectasia consists of ocular andcutaneous telangiectasias and cerebellar atrophywith predominant involvement of the anteriorvermis and dentate nuclei.

Though the above disorders are calledneurocutaneous there is a large mesenchymalcomponent in the form of vascular malformationsand hamartomas. Apart from revealing theintracranial involvement, imaging also helps indefining this aspect.

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XXII NATIONAL CONFERENCE OF THEINDIAN SOCIETY OF PEDIATRIC NEPHROLOGY

Venue : Swabhumi, Kolkata, India

Dates : Workshop on Renal Imaging : 12th November 2010

Conference : 13th & 14th November 2010

Organised by

Indian Society of Pediatric Nephrology

West Bengal Academy of Pediatrics

Conference Secretariat

Dr. Jayati SenguptaOrganising Secretary, PENCON 2010WBAP Office , “Oriental Apartment”15C Canal Street, Flat H1, Kolkata - 700 014.

Phone : 033 22654072, email : [email protected] : Dr. Jayati Sengupta 9831174488Dr. Mousumi Nandi Ghosh 9432673388

NEWS AND NOTES


CASE STUDY

INCONTINENTIA PIGMENTIWITH MACROCEPHALY

* Gopal Subramoniam** Prabhu Thanga Marthandan

Abstract : Incontinentia pigmenti (IP) is anX linked dominant disorder of skin which is oftenassociated with ocular, dental and centralnervous system abnormalities. Though lifeexpectancy is normal, quality of life is dependenton the associated abnormalities.

Keywords : Incontinentia pigmenti.

Incontinentia pigmenti is a rare heritable,multi system disorder that is transmitted as anX linked dominant trait, lethal in males.1

Incontinentia pigmenti is characterized by skinlesion, microcephaly, seizure and dentalabnormalities.2 The rarity of incontinentiapigmenti with macrocephaly that too in a malechild was the reason for presenting the case

Case Report

A six-month-old male infant born to thirddegree consanguinous marriage was referred inview of generalized seizures. On examination,he was thriving well with a weight of 7 kgs.There was no anemia or lymphadenopathy.He had hyperpigmented whorly lesion involvingtrunk, back, both upper and lower limbs.There was no lesion over face. Central nervoussystem examination revealed macrocephaly(head circumference-47cms; 97th percentile) with

microphthalmia, wide open anterior fontanellewith sunset sign. The tone in both upper andlower limbs was increased and the deep tendonreflexes were brisk. Fundus examination wassuggestive of severe retinal pigmentary changes.Cardio vascular system and respiratory systemwere essentially within normal limits. There wasno organomegaly. Investigations revealed normaltotal and differential counts. ESR, urinemicroscopy and metabolic screen were normal.Electrolytes and other metabolic parameters werenormal. USG abdomen was normal. EEG showedabnormal epileptiform discharges. MRI of brainshowed right hippocampal atrophy with dilatedright temporal horn, small sized left hippocampuswith patchy T2 hyper density with suspected leftmesial temporal sclerosis. The infant was startedon anticonvulsants. With the features of seizures,hyperpigmented whorly lesion of the skin, retinalpigmentary changes and macrocephaly adiagnosis of incontinentia pigmenti withmacrocephaly was made.

Discussion

Incontinentia pigmenti (IP) is a rareX linked, dominantly inherited disorder of skinpigmentation that is often associated with ocular,dental and central nervous systemabnormalities.3,4 Its incidence is 1 case in 40,000.It is more common in whites than in other races.IP is a lethal syndrome in males. More than 95%of reported cases occur in females. IP may rarelyoccur in males with Klinefeter syndrome (XXY)or as a result of somatic mosaicism or lessdeleterious gene mutation. The male to femaleratio is 1:19-37. Gorrod described the first patientin 1906, Sulzberger described pathological

* Dept. of Pediatrics,Jeyasekaran Hospital and Nursing Home,Nagarcoil, Tamil Nadu.

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changes in1928 and Haber first recognized themulti system nature of disease.5 Happel firstrecognized the skin changes along line ofBlaschko in 1985. Incontinentia pigmenti ischaracterised by loss of melanin from basal cellsin epidermis. Melanin collects in the dermis asaggregates of melanophages. It is caused by agenomic rearrangement of gene for NEMO,Nuclear factor kappa beta essential modulator.6

The defect in the X chromosome is proximal tofactor 7 at Xq28. Two third of mutation originateswith father. NEMO, the ubiquitous proteinbecomes active during embryogenesis and isessential for blood vessel architecture, cellgrowth in stratified epithelium and apoptosis.7

The skin manifestations occur along the line ofBlaschko, which represent the route of embryoniccell migration. Diagnosis is made clinically on

the basis of a history of sequential cutaneouslesions and associated features.3 At least onemajor criterion is necessary for diagnosis ofsporadic incontinentia pigmenti (neonatal rash,hyperpigmentation, linear, atropic, hairlesslesion.) At least one female relative who waspreviously diagnosed with incontinentia pigmentimay be diagnosed with minor criteria (dentalinvolvement, wooly hair, abnormal nail, retinaldisease). Skin change is present in 80% of casesand it involves mostly the sides of torso. Stage 1is vesicular stage with linear vesicles, pustulesand bullae with erythema along lines of Blaschko.Stage 2 is verrucous stage with warty, keratoticpapules and plaque. Stage 3 is hyper pigmentedstage with macular hyper pigmentation in aswirled pattern along the lines of Blaschko.It involves nipple, axilla and groin. Stage 4 is

Fig. 1. Macrocephaly with sunset sign Fig. 2. Hyperpigmented whorly lesionsin back

109


hypo pigmented streaks and patches andcutaneous atrophy. Alopecia and scarring occurin 40% of children.3 In one third of childrendevelopmental delay, seizures, microcephaly,spasticity and paralysis occurs. Ocularimpairment and blindness is seen in 15% ofchildren. Late dentition, conical teeth and partialanodontia are other components. Incontinentiapigmenti must be differentiated from conditionslike microophthalmia, dermal aplasia andsclerocornea (MIDAS) syndrome, linearepidermal nevus, lichen striatus, linear andwhorled nevoid hypermelanosis, dermatopathiapigmentosa reticularis.8 Hypomelanosis of Itois characterised by mirror image lesions of IP,but with swirls of hypopigmentation ordepigmentation. It is not inherited. There is nostage 1 or 2. There can be multisystem

Fig. 3. Typical hyperpigmented whorlylesions of trunk

involvement (eye, skeletal, neurologic) in33-50% of cases. It is associated with Xp11.

No specific treatment is available forincontinentia pigmenti. Stage 1 lesion should beleft intact and kept clean.9,10 Regularconsultations with ophthalmologist, dentist,geneticist and neurologist are advised. The skinabnormalities can improve with age and in someinstances disappear completely. Prognosis in IPdepends on the severity of extra cutaneousmanifestations. Morbidity and mortalityprimarily result from neurologic and ophthalmiccomplications including mental retardation,seizures and vision loss. IP is a geno dermatosisand can be associated with maligancies such asacute myelogenous leukemia, Wilms tumour andretinoblastoma.

References

1. Hegde SK, Bhat SS, Soumya S, Pai D.Incontinentia pigmenti. J Indian Soc Pedod PrevDent 2006;24:24-26

2. Ramalakshmi M, Parthasardhi A. IncontinentiaPigmenti. Indian J Dermatol Venereol Leprol1987;53: 244-245.

3. Joseph G. Morelli.Hyperpigmented lesion .In:Kliegman, Berhman, Jenson, Stanton, NelsonText book of Pediatrics. 18

th Edn, Philadelphia.

Saunders, 2007: pp2681-2682 .

4. Carney RG. lncontinentia pigmenti -.A reportof five cases and review of literature. ArchDermatol 1951; 64: 126-135.

5. Pavithran K, RamChandran P, Zochariah J.Incontinentia pigmenti. Indian J DermatolVenereol Leprol 1984; 50: 274.

6. Mosher DB, Fitzpatrick TB, Ortome JP.Disorders of melanocytes. In: Fitzpatrick TB,Eisen AZ, Wolff K. Eds, Dermatology inGeneral medicine. 3

rd Edn, New York. McGraw-

Hill, 1987:pp856-858.

7. Aradhya S, Courtois G, Rajkovic A, Lewis RA,Levy M, Israel A, et al. Atypical forms of

110

2010; 12(2) : 213

incontinentia pigmenti in male individualsresult from mutations of a cytosine tract in exon10 of NEMO. Am J Hum Genet 2001;68:765-771.

8. Jain VK, Kalla G, Bumb RA. IncontinentiaPigmenti. Indian J Dermatol Venereol Leprol1992;58: 39-40.

9. Gharpuray MB, Joshi MB, Naik SV, et al.Incontinentia pigmenti stage II. Indian JDermatol Venereol Leprol 1987; 53: 122-123.

10. Handa F, Aggarwal RR, Sharma SC, et al.Incontinentia pigmenti - Case Report withreview of literature. Indian J DermatolVenereol 1975; 41 : 63 - 65

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SOUTH NEOCON- 2010

IV SOUTH ZONE & XIII KARNATAKA STATE ANNUAL CONVENTION OF NATIONAL NEONATOLOGY FORUM

Theme: Improving Neonatal Survival - Now or Never

ORGANISED BY:IAP District Branch, Shivamogga, Karnataka.

NNF Karnataka State Chapter,IAP Karnataka State Branch,

IMA District Branches Shivamogga,Shivamogga Institute of Medical Sciences, Shivamogga

PRE CONFERENCE WORKSHOP- On 26th November, 2010

CONFERENCE -On 27th, 28th November 2010.

Venue: Shivamogga Institute of Medical Sciences, Shivamogga 577201

Before Before Before Spot WorkshopREGISTRATION FEES: April 30, Aug .31, Oct, 31-

2010 2010 2010

Reception Committee Members 2500

Delegate 1250 1500 1750 2000 500

Associate delegate 1000 1250 1500 1750

Post Graduate 750 1000 1250 1750 250

Nurses 250 350 500 750 250

HIGHLIGHTS OF THE CONFERENCEPre-conference Work-Shops and CMEs (26th November 2010)1) Certification Neonatal Resuscitation Programme (NRP)*2) Neonatal Ventilation Work-shop*-CPAP3) Continuing Nursing Education (CNE) Programme4) Continuing Medical Education Programme for ObstetriciansAll payments must be made by Demand Draft or Cheque in favour of SOUTH NEOCON-2010 payableat Shivamogga, Karnataka. Add Rs.50/- Outstation Cheques.

CONFERENCE SECRETARIATDr. H V Kotturesha Rasthapuramath Organizing SecretarySOUTH NEOCON-2010 IV SOUTH ZONE &XIII KARNATAKA STATE ANNUAL CONVENTION OF NATIONAL NEONATOLOGY FORUMKottureshwara Hospital Maternal and Child Care, Kamala Nivasa, Achutha Rao Layout 1st CrossShivamogga 577201. Karnataka. Phone: 081825-221432 Mob: Dr. Ganesh s Bidarakoppa- 9845217787Dr. Nagaraj Hommaradi-9448525246 Dr. Chinmaya K S-9448255550Phone: 08182-221432 Mob: 9449400000E-mail: [email protected] website: www.kottureshwarahospital.com.E-mail: [email protected] Website: www.iapshivamogga.org

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2010; 12(2) : 215

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RESPICON 2010XXII National Conference of IAP Respiratory Chapter

Organised byIAP Respiratory Chapter

IAP Respiratory Chapter, Tamil Nadu.IAP - Tamil Nadu State Chapter

IAP – Chennai City Branch

Theme: Theme: Theme: Theme: Theme: “Every Breath Counts”“Every Breath Counts”“Every Breath Counts”“Every Breath Counts”“Every Breath Counts”

Venue: Chennai Trade Centre, Nandambakkam, Chennai.

Date: 13th & 14th November, 2010Dear Colleagues,

Greetings from the Organising Committee of “RESPICON 2010”, Chennai. India.

We welcome you all to Chennai for the XXII National Conference of IAP Respiratory Chapter -RESPICON 2010. We look forward to extending you a warm reception, excellent academic feastand a sumptuous South Indian cuisine with traditional hospitality.

Dr A Balachandran Dr D VijayasekaranOrganising Chairman Organising Secretary

Conference Registration Fee

Up to 30th Up to 30th Up to 31st SpotApr 2010 June 2010 Oct 2010 Registration

IAP member Rs.1750 Rs.2000 Rs.2500 Rs.4000Non-IAP Member Rs.2000 Rs.2250 Rs.2750 Rs.4500*Postgraduates” Rs.1500 Rs.1750 Rs.2000 Rs.3500Foreign Delegates Rs.3500 Rs.4000 Rs.5000 Rs.6000Accompanying Delegates Rs.1250 Rs.1250 Rs.1250 Rs.1500

1. *For PG student, certificate to be produced from Institute signed by HOD 2. Senior Citizens more than 70 years of age – Registration fee is waived. 3. For Senior Citizens, Valid age proof (Driving License / Voters ID / Passport) is required. 4. Pre Conference Workshops – (a) Procedures in Pediatric Pulmonology, (b) Imaging in

Pediatrics Pulmonology, (c) ENT workshop, (d) Workshop on Ventilators (e) Allergy skintests - will be conducted on 11th & 12th November, 2010.

5. Registration fee does not include Pre Conference Workshop Fees.

Conference SecretariatDr D Vijayasekaran, Organising Secretary, RESPICON 20101A, Block II, Krsna Apartment,50, Halls Road, Egmore, Chennai – 600 008. Tamilnadu, - India.Tele: +044-4205900; 28190032Email: [email protected]; Website : www.respicon2010.com


TRYPEDICON 201035th ANNUAL STATE CONFERENCE OF IAP-TNSC

Host : I.A.P. Trichy Chapter - TamilNadu StateVenue : Hotel Sangam, Trichy. Date : August 13th to 15th 2010

Dr. Sunil Srinivasan Dr. Pannerselvam Dr. Suresh ChelliahOrganising Chairman Organising Secretary Treasurer

Registration Fees

Dates I.A.P NON I.A.P P.G. Accompanying(Rs.) (Rs.) (Rs.) Person (Rs.)

01.01.10 - 15.05.10 3000 3500 2500 2750

16.05.10 - 31.07.10 3500 4000 3000 3250

01.08.10 - 15.08.10 4500 5000 4000 4250

1. Mode of Payment : Cash in Person / Cheque / D.D. 2. Senior citizens above 70yrs (as on15.08.2010) Free (Age proof required) 3. Children below 5yrs (as on 15.08.2010) Free 4. Letter fromH.O.D for P.G’S required. 5. Delegate Kit cannot be guaranteed for those registering after01.08.2010 6. Add Rs 50/- extra for outstation cheques. 7. Accomodation and travel details willfollow in first announcement brochure and will also be available in conference website shortly.

Special Features : 1. Thought Provoking informative talks and lively interactive sessions of dayto day interest and recent advances. 2. Topics concering both basic and applied research of practicalimportance by eminent paediatricians. 3. Palate watering south indian, north indian, continental,chinese and tandoori hotcuisines & special low calorie diet. 4. Funfilled activities for theaccompanying spouse and children. 5. Folkarts of Tamilnadu like karagattam, nathaswaram,naiyandimelam, Bharathanatyam 7. Permission from TN medical council for credit hours8. Conveyance arranged from railways, Bus stand and airport to the venue and place of stay.

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Remember this is a conference to make a difference!

Time is running out!

Registration fees to be increased after 01.05.2010.

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For other details see our website www.trypedicon2010.com

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Dr.S.Panner Selvam, Organising Secretary - TRYPEDICON 2010

74/C3, Mullai Salai, Annamalai Nagar, Trichy - 620 018. Cell : 94436 50515

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Suggestions arewelcome toimprove thePerformance ofthe conference

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2010; 12(2) : 217

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2010; 12(2) : 219

PresidentDr.Deepak Ugra

President-2011Dr.T.U.Sukumaran

President-2009Dr.Panna Choudhury

Vice PresidentDr.Santosh T SoansSecretary GeneralDr.Tanmay Amladi

TreasurerDr.Sailesh G Gupta

Editor-in-Chief, IPDr.Piyush Gupta

Editor-in-Chief, IJPPDr.K.Nedunchelian

Joint SecretaryDr.Tarun Patni

Members of the Executive BoardAndhra Pradesh

Dr.Ajoy KumarDr.A.Arumugam

Dr.C.Suresh KumarAssam

Dr.Garima SaikiaBihar

Dr.Shashi BP SinghChhattisgarh

Dr.Ashwani K AgrawalDelhi

Dr.Anju AggarwalDr.A.S.Vasudev

GujaratDr.Digant D ShastriDr.Satish V Pandya

HaryanaDr.H.S.Sabharwal

Jammu & KashmirDr.Rekha Harish

JharkhandDr.Brij Bhushan Sahni

KarnatakaDr.L.H.Bidari

Dr.A.Prahalad KumarDr.Santosh T Soans

KeralaDr.T.M.Ananda Kesavan

Dr.George F MoolayilDr.Shaji Thomas John

Madhya PradeshDr.Suhas B Dhonde

Dr.J.S.TutejaMaharashtra

Dr.Anand K ShandilyaDr.Bakul Jayant Parekh

Dr.Manoj T RathiDr.Rhishikesh P ThakreDr.Vasant M Khalatkar

OrissaDr.Arabinda Mohanty

PunjabDr.Rajinder Gulati

RajasthanDr.Ashok Gupta

Dr.Raj Kumar GuptaTamilnadu

Dr.S.BalasubramanianDr.Major K Nagaraju

Dr.S.ThangaveluTripura

Dr.C.M.ChhajerUttarkhand

Dr.Geeta D KhannaUttar Pradesh

Dr.Arun Kumar AgrawalDr.Girish Chandra Agrawal

Dr.B.D.BhatiaWest Bengal

Dr.Amaresh DeDr.Arup Roy

ServicesBrig.P.L.Prasad

IAP’s Spl. RepresentativeDr.Shyam Kukreja

A.A.A.Dr.Pravin J Mehta

Indian Academyof Pediatrics

Kailash Darshan, Kennedy Bridge,Mumbai - 400 007.IAP Team - 2010


IJPP Team - 2010

JOURNAL COMMITTEE

Editor-in-Chief

Dr.K.Nedunchelian

Executive Editor

Dr.S.Thangavelu

Managing Editor

Dr.N.C.Gowrishankar

Associate Editors

Dr.Janani Sankar

Dr.P.Ramachandran

Dr.T.L.Ratnakumari

Dr.C.V.Ravisekar

Dr.S.Shanthi

Executive Members

Dr.G.Durai Arasan

Dr.S.Lakshmi

Dr.V.Lakshmi

Dr.E.Mahender

Dr.V.Poovazhagi

Dr.T.Ravikumar

Dr.Shanthi Ramesh

Dr.So.Shivbalan

Dr.Tanmay Amladi(Ex-officio)

NATIONAL ADVISORY BOARD

President 2010, IAP

Dr.Deepak Ugra

President 2011, IAP

Dr.T.U.Sukumaran

Editor-in-Chief, IP

Dr.Piyush Gupta

Members

Dr.Arvind Bagga

Dr..K.E.Elizabeth

Dr.Mamta V Manglani

Dr.N.C.Mathur

Dr..D.Narayanappa

Dr.Rajiv Kumar Bansal

Dr.Sandhya Bhatnagar

Dr.Shyama Saikia

Emeritus Editors

Dr.A.Parthasarathy

Dr.B.R.Nammalwar

Dr.M.Vijayakumar

Dr.A.Balachandran

Indian Journal ofPractical Pediatrics

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2010; 12(2) : 221

INBORN ERRORS OF

META

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SM ISSN 0972-9607

VOL.12 NO.2

APR.-JUN. 2010

IJPP

2010; 12(2):103-218, INB

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Indian Journal

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Indian Academy of Pediatrics

www.ijpp.in

IJPP

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