1. Pyridoxine sensitivity in Primary Hyperoxaluria
Christiaan v Woerden, Hans Waterham, Frits Wijburg, Ronald Wanders, Jaap Groothoff Emma Children’s hospital AMC, Amsterdam

2. What has brought them to the top?
What made them the greatest?
genes?
environment?

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

4. 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

5. 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

6. Genotype-phenotype association? Impact B6 sensitivity?
AGT mutation
AGT metabolic activity
level of endogenous oxalate
Clinical severity
oxalate diet, hydration medication
Infection
Renal handling oxalate

7. 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

8. 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

9. Mrs. C, sister of A & B Age 48: ESRD (1 year after diagnosis B)
AGT-activity 9%
Age 49: renal tx
Nephrocalcinosis renal graft

10. 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

11. AGT in liver biopsy specimens
O Immunoreactive AGT -; • immunoreactive AGT +
(Danpure ea J Inher Metab Dis 17: , 1994)

12. AGT deficiency: over 50 mutations
liver biopsy:
immunoreactivity enzyme activity
Protein not synthesized (nonsense m) - -
Protein synthesized OK but inactive + -
Protein synthesized OK but unstable:
Protein rapidly degraded + -
Protein aggregates /-
Protein mistargeting: mitochondrion + +

13. Gly82Glu (Pyr-) mutation abolishes pyridoxine (PLP) binding
(imm+/enz-)
Gly41Arg (Pyr-) abolishes contact 2 monomers: destablilisation aggregation AGT
From Zhang et al, JMB, 2003

14. 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

15. G170R & F152I  activity of P11L-induced mitochondrial mistargeting to 90% by unfolding the AGT
from Danpure et al

16. G170R & F152I  activity of P11L-induced mitochondrial mistargeting to 90% by unfolding the AGT
pyridoxine may increase the activity of 10% peroxisomal AGT
association pyridoxine sensitivity?
from Danpure et al

17. Association B6 sensitivity - outcome 1. the Dutch experience
follow-up PH
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

18. Age at diagnosis

19. End-stage renal disease at diagnosis

20. Outcome: renal function57
at diagnosis 30
preserved renal function 27
renal insufficiency 19
ESRD

21. Outcome: renal function57
at diagnosis 30
preserved renal function 27
renal insufficiency 19
ESRD
2 improved/
2 stabilized
4 ESRD
at follow-up
24 preserved
renal function
5 ESRD/
low GFR 28
ESRD/ low GFR 11
death

22. Clinical & biochemical parameters in relation to renal insufficiency
RR = relative risk, 95%CI = 95% confidence interval

23. 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 ( )
20/33 patients onset < 18th years of age
6/33 patients onset < 1st year of age

24. 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-

25. G170R homozygosity (Pyr+)11
at diagnosis 6
preserved renal function 5
ESRD
1 ESRD (not treated)
5 kidney Tx:
all B6 responsive
at follow-up
5 preserved function
kidney Tx: preserved function
3 preserved function

26. F152I homozygosity (Pyr+)4
at diagnosis 2
preserved renal function 2
ESRD
1 dialysis
1 kidney Tx: B6 responsive
at follow-up
2 preserved function
1 preserved function

27. 33InsC homozygosity (pyr-)
at diagnosis
3 neonatal ESRD
1 deceased
2 liver kidney Tx
at follow-up
1 preserved function
1 deceased
(liver failure)

28. G82R (pyr-) 3 at diagnosis 3 normal GFR 1 preserved 1 decreased GFR
1 ESRD
at follow-up
1 liver kidney-tx
GFR decreasing

29. 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

30. 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

31. The American experience Monico 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

32. The German experience Hoppe 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

33. The Israel experience Frishberg 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

34. 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

36. PH1 group Emma children’s Hospital AMC
Christiaan van Woerden Resident Paediatrics
Simone Denis Technician
Hans Waterham Molecular Geneticist
Ronald Wanders Biochemist
Carla Annink Technician |
Marinus Duran Clinical Chemist
Frits Wijburg Pediatrician Metabolic Diseases
Jaap Groothoff Pediatric Nephrologist

37. 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)

38. a kidney stone