Viktor Kožich
Ústav dědičných metabolických poruch1.LF UK a VFN Praha
Diagnosis of inbornerrors of metabolism
Structure
Diagnosis in generalNewborn screeningSelective screening
clinical approachlaboratory tests
Diagnosis ≈ hypothesis verification
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patient
Successful diagnosis of IEM
+
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knowledgeable physician availability of appropriate test
Diagnostic procedures in IEMs
produkt
substrát
DNA/RNA Enzymes Metabolites
|Clinicsspecific-e.g..
smellurine color
nonspecific-e.g.comaPMRdysmorfic featureshepato/myopathieother
Structure
Diagnosis in general
Newborn screeningSelective screening
clinical approachlaboratory tests
Presymptomatic diagnosis
produkt
substrát
at risk relatives
screening of population segment
newborn screening
single disease or groupof diseases
Newborn screening (NBS)
• active search for disease in the entire population
• presymptomatic diagnosis
• sensu stricto- laboratory analyses of diseases usingdry blood spots
NBS
Classical criteria for NBS
Diseases freqeuncy and severityAsymptomatic latent phaseDisease mechanisms are knownReliable testTest is acceptable by the populationProgram is a continuous processAcceptable treatmetnConditions for dx and rx establishedConsensus on whom and how to treatCost-benefit ratio acceptable by the system
Quality Adjuste Life Yearsusual accepted limit 50.000 USD
QALY for MS-MS (30 IEMs): 6.000-15.000 USD, newborn screening of CF: 13.728 USD
QALY for other situationsdefibrilators in aircrafts/malls 50.000-70.000 USDprostatic ca screening 9.000-23.000 USDmammography 2.000-20.000 (but 232.000 USD under 50 y)MRI screening of breast ca in carriers of BRCA1/2 mutations: 45.000-700.000 USDACE inhibitors in hypertension 5.600 USD
3434
4444
4444 4545
50
5353
3535
51
50
3232
41414747
4646
3737
2929
2929
41414848
4040
DC
51 1331
4545
3535
33
36
2929
50
4141
4848
31
4545
33 4545
4848
52
31
31
3535
54 4949
3232
504949
2424
31
314646
49
51
52
32
5252
31
30
NBS USA-2009
Congenital hypothyroidism
1:4000etiology heterogeneous-peripheral diseaseTSH measurement (also T4) Aim: early substitution with thyroxinprevents irreversible CNS damage(+detection of associated congenitalanomalies)
Congenital Adrenal HyperplasiaDeficient activity of 21-hydroxylase- CYP21 gene mutationsAbnormala steroidogenesis in adrenal cortex –hypokortikalism, hypoaldosteronism andhyperandrogenismClassic (salt wasting or simple virilizing) and lateonset formsClassic form 1:10 000, LO up to 1:100. test: 17-hydroxyprogesteroneAim: early substitution prevents life-threateningcortical insufficency and premature puberty
IEMs of amino acids
accumulation of toxic products: chronic toxicity: e.g. Pheacute toxicity: BCKA, glutarate, isovalerate
clinical featureschronic toxicity: PMR, seizuresacute toxicity: encephalopathy with coma
therapy aim: decrease the load of affectedpathway + supplement other AA
Beta-oxidation defects
common mechanism: decreased production of ketone bodies upon fastingaccumulation of toxic acylCoAs
clinical features:common-hypoglycemia under fastingin some FAO defects-myopathy (especially after prolongedexercise), cardiomyopathy
treatment: prevention of hypoglycemia in catabolic situations, prevention of fasting
Cystic fibrosisAR inheritance1: 2500 – 4000 Mutations in CFTR gen
abnormal chloride channelabnormally thick mucus-bronchi, pancreas, male GU
Severe chronically debilitating disease leading to respiratory failureMedian survival in developed countries: 37yAim ot therapy: slowing down the progression by ATB treatment
Good sampling practice
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correct drying3 hres, no direct heat
Tandem mass spectrometrymodern analytical methodprofile of analyteswide spectrum of compounds: amino acids, acylcarnitines, sugars......enzyme activitiesused for NBS since mid1990s
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MS/MS- verygood identificationof analyteshigh sensitivityfast analysis of many metabolitesin a single assay
suchá krevní kapka
200 µl methanolu
se standardy
odstranění terčíku25 min extrakce
vyražení
3 mm terčíku
odpaření methanolu
butylace 30 min při 60 °Codpaření derivatizačního
činidla
analýza
1 vzorku 2,5 min
100µl
derivatizačního
činidla
NBS for IEMs by MS/MS
200 µl
1:1 acetonitril a voda + 0,02 % kys.
mravenčí
Optimal screening test
•http://www.connectseward.org/shs/da1/review07/Right%20Skewed.jpg
e.g. classical PKU and mild HPA
Low PPV
•http://www.connectseward.org/shs/da1/review07/Right%20Skewed.jpg
e.g. MSUD-Xle, CF-IRT
solution to problem: second tier test
Results from NBS programsNormal
cave!!!!- atypical form of screened disease is not excluded
Borderlineusually non-specificrecall needed-MS/MS on a new sample
Positivevarying PPV (5-100%)diagnostic workup initiated
Structure
Diagnosis in generalNewborn screening
Selective screeninglaboratory testsclinical approach
Diagnostic procedures in IEMs
produkt
substrát
DNA/RNA Enzymes Metabolites
|Clinicsspecific-e.g..
smellurine color
nonspecific-e.g.comaPMRdysmorfic featureshepato/myopathieother
Metabolite analysis- IEMs
specialized testsusually not available in routinely labsmostly profile analysesmostly chromatographic techniques, expensiveequipment neededlaborious methods, lack of kits and controlmaterialcomplex interpretation (e.g. organic acids) by specialist
Small molecules in biochemicalgenetics
definition: < 1500 Dagases, inorganic ionsamino acidsorganic acidssaccharidespolyolssimple lipidspurines, pyrimidinesvitaminsoligomers: peptides up to 5-10 AA, oligosaccharides
cytosol, mitochondrial stromablood, urine
Complex molecules in biochemicalgenetics
definition: > 1500 Daglykolipidssphingolipidsplasmalogensneutral polysaccharides (glycogen)mucopolysaccharides(other polymers: proteins, nucleic acids...)
usually associated with membranesconcentrations in blood/urine rather low, exceptions exist ( x MS/MS technologie)
Sensitivity
Alkaptonuria: 1-5 g homogentisate /dCystinuria: 1-5 g cystine /d
PKU: 0.1 g Phe /l blood
MCAD: C8 acylcarnitin 0.0001 g / l bloodDBS punch 0.003 ml blood
0.2 – 1 ml serum
urine- liters
α L- iduronidase deficiency
(excretion of dermatan sulphate/DS and heparan sulphate/HS )
courtesy Dr.Ledvinová
GC-MS: methylmalonic aciduria
0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0Time [min.]
25
50
75
100
125
150
175
200Vo
ltage
[mV]
3,26
5,54
5,86
6,61
6,64
7,21 7,
77
10,3
610
,63
11,5
112
,08
12,2
8
13,7
213
,80
14,0
0
14,7
014
,81
18,3
90,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0
Time [min.]
50
100
150
200
250
300
350
Vol
tage
[mV
]
3,57
4,24
5,07
5,93
6,63
10,6
4
12,0
812
,29
13,4
713
,72
13,8
1
14,7
014
,80
14,9
6
17,8
0
18,4
0
GC-MS: MCAD deficiency
0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0Time [min.]
50
100
150
200
250
Vol
tage
[mV
]
3,29
5,12
5,94
6,67
10,6
8
12,2
812
,33
13,7
613
,85
14,7
414
,88
18,4
718
,69
0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0Time [min.]
50
100
150
200
250
300
350
Vol
tage
[mV
]
3,57
4,24
5,07
5,93
6,63
10,6
4
12,0
812
,29
13,4
713
,72
13,8
1
14,7
014
,80
14,9
6
17,8
0
18,4
0
Metabolite analysis- IEMs
usually not available in routinely labsmostly profile analyses by chromatographic techniquecomplex interpretation (e.g. organic acids)
Principles of enzyme assays
substrate* product*
cofactor altered cofactor
•separation of substrate from product•quentitation of change
•quentitation of change
Enzyme assays in IEM diagnosisCells are usually needed
leukocytes, fibroblastschorion, amniocytes
Fluorimetry and radiometry (photometry)Measured variable:
substrate/product concentration changecofactor concentration change
ÚDMP: 46 enzyme assays
A GC T T CCAG G G CAC T T C T A T T T G T T T T T G T G G AA A G A CT G G CBases5 10 15 20 25 30 35 40
A GC TT CC AG G G C AC T T CT WT C T R T T T KT KT t GTG AWR G AC WG R C
Bases10 15 20 25 30 35 40 45 50
Heterozygous deletion
Wild type sequence
Structure
Diagnosis in generalNewborn screening
Selective screeninglaboratory tests
clinical approach
Clinical features of IEMs-age
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http://www.hrr.co.uk/acatalog/crocodile_toddler.jpghttp://www.co.shasta.ca.us/html/DSS/images/FosterParentingAdopt/infant.jpg
Clinical features-multisystemicinvolvement
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endogenousexogenousOrigin
N YAcute toxicity
inefficiousefficiousRx-diet, vitamins
tissues (U)blood,urineDx
YNImpact on structure
membranescytosol, ECTLocalization
Y±Chronic progression
> 1500 Da< 1500 Da
Isovaleric aciduria
FTT, vomiting, Kussmaulbreathingconsciousness: comawithin 24-48 h after onsetof symptomsmetabolic acidosis, ketonuriasweaty feet syndrome
http://images.google.com/imgres?imgurl=http://upload.wikimedia.org/wikipedia/commons/8/8b/Isovaleric_acid_structure.png
Diseases of small molecules
usually dependent on exogenous supplymanifestation: (repeated) acute toxicity, usuallywith encephalopathy/comahepatopathy commoncommon disturbances in routine labs-ammonia, Astrup, ketone bodies, glycemia, uric acid...symptoms develop due to specific type of food, fasting, catabolismchronic course possible (if toxicity low)usually good therapeutic response to diet and/orvitamins
Food and IEMs (small molecules)
(sub)acute toxicitymilk (lactose)-hepatopathysaccharose/fructose/sorbitol- hepatopathyand hypoglycemiaexcess protein- vomiting, lethargy, coma(urea cycle disorders, organic acidurias)
Fasting and IEMs
hypoglycemia in GSDhypoglycemia with decreased production of ketone bodies (beta-oxidation defects)acidosis, ketonuria and metabolicencephalopathy in prolonged fasting (organicacidurias)respiratory alkalosis and encephalopathy (urea cycle disorders)
Organic acidurias
several dozens of diseasescommon feature: excretion of carboxylic acids(test-organic acids in urine) orginin usually from carbon skeleton degradationof AAs (or saccharides or lipids)usually acute presentation- „intoxication type“metabolic acidosis common (combination withhyperammonemia frequent)
Mukopolysaccharidosis type I
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Diseases of complex moleculesdisease progresses usually regardless of anyexogenous sources from foodtypical course is progressive (± symptom-freeperiod)dysmorphy at birth possiblefrequent involvement of nervous system andmusculatureorganomegaly due to storage in lysosomalstorage disordersusually untreatable by diet or vitamins
Clinical features: (non)specific signs
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http://gatsome.com/images/iq.gifhttp://www.saratogaschools.org/AcademicServices/MiddleSchool
specific non-specific
e.g. NH3, uric acid
Abnormal urinary smell and colorsmell (small volatile molecules):
sweaty feet-isovaleratemaple syrup-branched ketoacidsboiled cabbage-methionine oxidfish-trimethylamineblackcurrant- organic acidsmouse-phenylacetate
colororange-urateblack upon oxidation-homogentisateblue-indoxyl derivatiesgreen-4-OH-butyrate
Common labs in IEMs
BloodglycemiacholesterolTGuric acidMAchyperammonemia, RAlkALT,ASTCKanemia/pancytopenia
Urineketone bodiesuric acidcrystaluriamyoglobinuria
Selected common situations withhigh risk of IEM
Small moleculesacutelly ill newborn(repeated) atack of long-term uncosciousnessfailure to thrive
Complex moleculesprogressive CNS and musculature involvementfacial dysmorphyorganomegaly (liver, spleen, heart)