Pyridoxine sensitivity in Primary

Hyperoxaluria

Christiaan v Woerden, Hans Waterham, Frits Wijburg, Ronald Wanders, Jaap GroothoffEmma Children’s hospital AMC, Amsterdam

What has brought them to the top?

environment?

genes?What made them the

greatest?

Primary Hyperoxaluria I (PHI): peroxisomal enzyme (AGT) deficiency in the liver

Role of pyridoxine (B6)

• Essential co-factor of AGT

• mutation Gly82Glu: inhibits B6 binding no AGT activity

• Reduction of oxalate excretion by B6 in B6 deficiency

• Reduction oxalate excretion pharmacological dosages B6 in 30% of PH1 patients W Europe

PH1: extreme heterogenous phenotypical expression

No symptoms, sole kidney stones, nephrocalcinosis, UTI

or

Interstitial nephritis & fibrosis, ESRD systemic oxalosis: retinopathy , blunted vision, bone pain, fractures, growth, arthopathy

peripheral neuropathy, heartblock, myocarditis, skin calcification, peripheral,

gangreen, pancytopenia, splenomegaly, vascular calcification, arterial wall stiffening

AGT mutation

AGT metabolic activity

level of endogenous oxalate

oxalate diet, hydration medicationInfectionRenal handling oxalate

Clinical severity

Genotype-phenotype association? Impact B6 sensitivity?

Mrs. A

• Age 22: kidney stone

Hyperoxaluria (5x normal) & hyperglycoluria

• Liver biopsy: AGT residual activity of 48%

• Reduction of hyperoxaluria to “high normal” (0.057 mmol/mmol

kreat) under pyridoxine 50 mg

• Age 38: good health, 1 new stone removed, US small

calcifications

Mrs. B, sister of Mrs. A

• Age 6: kidney stones, surgical removal

• Age 30: diagnosis PH1, lost to follow-up

• Age 50: kreat 200 μmol/l, nephrocalcinosis

• Liver biopsy: 15% AGT-activity

• Age 51: ESRD

Mrs. C, sister of A & B

• Age 48: ESRD (1 year after diagnosis B)

• AGT-activity 9%

• Age 49: renal tx

• Nephrocalcinosis renal graft

Mrs. C, sister of A & B

• Age 48: ESRD (1 year after diagnosis B)

• AGT-activity 9%

• Age 49: renal tx

• Nephrocalcinosis renal graft

• All 3 sisters homozygous G170R mutation

OO Immunoreactive AGT -; Immunoreactive AGT -; •• immunoreactive AGT + immunoreactive AGT + (Danpure ea J Inher Metab Dis 17: 487-499, 1994)(Danpure ea J Inher Metab Dis 17: 487-499, 1994)

AGT in liver biopsy specimens

AGT deficiency: over 50 mutations

liver biopsy:immunoreactivity enzyme

activity1. Protein not synthesized (nonsense m) - -

2. Protein synthesized OK but inactive + -

3. Protein synthesized OK but unstable:– Protein rapidly degraded + -– Protein aggregates + +/-– Protein mistargeting: mitochondrion + +

Gly82Glu (Pyr-) mutation abolishes pyridoxine (PLP) binding

(imm+/enz-)

Gly41Arg (Pyr-) abolishes contact 2 monomers: destablilisation aggregation AGT

From Zhang et al, JMB, 2003

2 polymorphic variants: a “major” & “minor” allele

• Minor allele: 4% population Europe/USA

• Normal AGT: peroxisomal localisation by way of Peroxisomal Targeting Sequence 1 as folded dimer

• Minor allele: P11L aa replacement: catalytic act AGT to 30% dimerisation AGT in vitro at 37°– 5% mitochondrial location AGT by a weak Mitochondrial

Targeting Sequence at N-terminus– Mitochondrial AGT import only as an unfolded monomer

G170R & F152I activity of P11L-induced

mitochondrial mistargeting to 90% by unfolding the AGT

from Danpure et al

G170R & F152I activity of P11L-induced

mitochondrial mistargeting to 90% by unfolding the AGT

from Danpure et al

pyridoxine may increase the activity of 10% peroxisomal AGT

association pyridoxine sensitivity?

Association B6 sensitivity - outcome 1. the Dutch experience

• follow-up PH 1972-2002• search for patients:

– Dutch Registration Renal Replacement Therapy (RENINE)– Dutch Society of Pediatric Nephrology– Dutch Society of Nephrology

• if no answer: contact by phone• review of all available medical charts

• Total number of patients: 62 • PH1: 57 PH2: 1• PH-unidentified: 4

• Prevalence PH1 = 2.9 per 106

• Incidence PH1 = 0.15 per 106 per year

v Woerden et al, NDT 18, 2003 & Kidney Int 66, 2004

Age at diagnosis

70

30%

pediatric age(n=40)

adult age(n=17)

End-stage renal disease at diagnosis

pediatric age (n=10)

77%

23%ESRD

adult age (n=9)

41%

59%ESRD

Outcome: renal function

57

30preserved renal function

27renal insufficiency

at diagnosis

19 ESRD

Outcome: renal function

57

30preserved renal function

27renal insufficiency

at diagnosis

19 ESRD

at follow-up

24 preserved renal function

11death

28ESRD/ low GFR

5 ESRD/low GFR

4 ESRD2 improved/ 2 stabilized

Clinical & biochemical parameters in relation to renal insufficiency

parameter n RR 95%CI

UOx > median 18/37 1.1 0.5-2.2

AGT<15% 19/29 0.45 0.3-1.8

nephro- calcinosis

33/57 1.8 1.0-3.4

Pyridoxine unresponsive

12/36 2.2 1.1-4.2

RR = relative risk, 95%CI = 95% confidence interval

Mutation analysis: patients

• 33/57 patients of 26 families

• Median age onset of symptoms/diagnosis 5.7/6.6 (0.1-50/57)

• Mean follow up after diagnosis 12.5 years (0.1- 49)

• 20/33 patients onset < 18th years of age

• 6/33 patients onset < 1st year of age

Mutations

• 11 patients homozygous for G170R - pyr+

• 4 patients homozygous for P152I - pyr+

• 3 patients homozygous for 33InsC - pyr-

• 3 patients homozygous for G82R - pyr-

• 1 patient homozygous for G170R & V336D

mutation - pyr-

• 11 patients compound heterozygous - pyr-

G170R homozygosity (Pyr+)

11

6preserved renal function

5ESRD

at diagnosis

5 kidney Tx: all B6 responsive

at follow-up

5 preserved function 3 preserved function

1 ESRD (not treated)

kidney Tx: preserved function

F152I homozygosity (Pyr+)

4

2preserved renal function

2ESRD

at diagnosis

1 kidney Tx: B6 responsive

at follow-up

2 preserved function 1 preserved function

1 dialysis

33InsC homozygosity (pyr-)

3

3 neonatal ESRDat diagnosis

1 deceased(liver failure)

at follow-up 1 deceased

1 preserved function

2 liver kidney Tx

G82R (pyr-)

3

3 normal GFRat diagnosis

at follow-up 1 preserved

1 liver kidney-tx

1 decreased GFR 1 ESRD

GFR decreasing

Mrs. B, sister of Mrs. A

• Age 6: kidney stones, surgical removal• Age 30: diagnosis PH1, lost to follow-up• Age 50: kreat 200, nephroclacinosis• Liverbiopsy: 15% AGT-activity• Age 51: ESRD

Follow-up (8 years):• Same year renal Tx, calcification Tx kidney, GFR 46 at 5 years

follow up• Normalisation oxalate excretion under pyridoxine

Mrs. C, sister of A & B

• Age 48: ESRD (1 year after diagnosis B)• AGT-activity 9%• Age 49: renal tx • Nephrocalcinosis graft

Follow-up (7 years):• Normalisation oxalate excretion under B6• GFR graft 56 after 5 years of follow-up

• All 3 sisters homozygous G170R mutation

The American experienceMonico et al Am J Nephrol 2005

• 23 PH1 patients

• 6 homozygotes G170R

• 1 homozygous F152I

• Homozygotes G170R & F152I B6 responsive and high AGT residual act (19 vs.10 heterozygotes G170R & 8 non-G170R)

• No follow up

• Conclusion: association B6 and G170R & F152I

The German experienceHoppe et al, Am J Nephrol 2005

• Patients: 65 PH; 42 PH1; 12 unclassified

• 7 B6 full response - no mutation found - AGT 7.2 (1 patient)

• 9 B6 partial response (25-50%)- 4 heterozygous G170R - AGT 4.7

• Time interval symptoms - diagnosis: 1-31 year

• 17 no B6 response - AGT 5.2

• 25 (38%) ESRD - 2 homozygous G170R

• 6 isolated kidney tx - 1 successful, 3 recurrences, 2 failed

The Israel experienceFrishberg et al Am J Nephrol 2005

• 56 PH1 patients

• 21 families

• 15 mutations, 1 nonsense, 13 missense mutations

• No B6 responsiveness, AGT-activity near to 0

• Prevalent phenotype; early onset CRF– 20 ESRD childhood (18†), 15 at infancy– Clinical presentation 43 < age 5– 12 asymptomatic at diagnosis

Conclusions pyridoxine sensitive PH1

• Homozygosity G170R and F152I & minor allele, others?

• 20-30% PH1 patients Western Europe/USA

• Relatively late onset: adult patients!!

• Diagnosis often delayed

• Good outcome if early diagnosed

• no indication for liver Tx

PH1 group Emma children’s Hospital AMC

Christiaan van Woerden Resident PaediatricsSimone Denis TechnicianHans Waterham Molecular GeneticistRonald Wanders BiochemistCarla Annink Technician |Marinus Duran Clinical ChemistFrits Wijburg Pediatrician Metabolic DiseasesJaap Groothoff Pediatric Nephrologist

Participating centres

AN Bosschaart (Enschede)

WT v Dorp (Haarlem)

MAGL ten Dam (Nijmegen)

CFM Franssen (Groningen)

IH Go (Nijmegen)

JJ Homan vd Heide (Groningen)

JP v Hooff (Maastricht)

F Th Huysmans (Leiden)

JE Kist-van Holthe tot Echten (Leiden)

W Koning-Mulder (Enschede)

G Kolsters (Zwolle)

MR Liliën (Utrecht)

S Lobatto (Hilversum)

LAH Monnens (Nijmegen)

J Le Noble (Schiedam)

C Ramaker (Amsterdam)

EMA vd Veer (Amsterdam)

ED Wolff (Rotterdam)

R Zietse (Rotterdam)

a kidney stone