Development of a mutation screening service for ARPKD

1. Development of a mutation screening service for ARPKD Wendy Lewis CMGS Spring Conference 2009

2. Polycystic kidney disease • Polycystic kidney disease can be inherited in a dominant (ADPKD) or recessive (ARPKD) manner. • ADPKD is the most commonly inherited kidney disease (incidence of between 1:400 and 1:1,000 live births). • ADPKD is a late onset, chronic, progressive disease. It is incurable and is characterised by numerous fluid-filled cystsin the kidneys and often the liver and pancreas. • Mutations have been identified in the PKD-1 and PKD-2 genes.

3. Autosomal recessive polycystic kidney disease (ARPKD). • Autosomal recessive polycystic kidney disease is an important cause of renal and liver related morbidity and mortality in neonates and infants. • The incidence of ARPKD is estimated to be 1 in 20 000 live births, with a estimated carrier frequency of 1 in 71. • Severely affected neonates display massively enlarged echogenic kidneys with a “Potter” phenotype from oligohydramnios, due to poor foetal renal output.

4. Typical Potter facies • Typical Potter facies phenotype showing: • broad, flat nasal bridge • widely spaced eyes • prominent intraorbital folds • micrognathia • low-set ears.

5. Diagram of a renal medullary ray depicting both normal and abnormal collecting ducts. The left side of the diagram (A) depicts a normal nephron draining into a normal (non-dilated) collecting duct. The right side (B) depicts a normal nephron draining into an ectatic (dilated) collecting duct in ARPKD. • A cut section of a kidney with ARPKD. Note that the cysts are fairly small but uniformly distributed throughout the parenchyma so that the disease is usually symmetrical in appearance, with both kidneys markedly enlarged.

6. PM of infant with ARPKD • This infant died soon after premature birth at 23 weeks gestation from pulmonary hypoplasia as a result of oligohydramnios. • Note the bilaterally enlarged kidneys that nearly fill the abdomen below the liver. • The histological appearance in this case, coupled with the gross appearance, was consistent with autosomal recessive polycystic kidney disease (ARPKD).

7. ARPKD • About 30-50% of affected neonates die shortly after birth from respiratory insufficiency. • Alongside the severe form of ARPKD, there are less severe childhood and adult forms, commonly diagnosed as congenital hepatic fibrosis. • Infants who survive the neonatal period or present later in life express variable disease phenotypes with: • systemic hypertension (60-100%). • abnormalities following portal hypertension (30-75%). • kidney dialysis or transplantation by the age of 10 (~30%). • Chronic lung disease (11%).

8. PKHD1 (Polycystickidney and hepatic disease gene 1) • Despite the variable clinical spectrum, linkage studies indicate that a single locus PKHD1, is responsible for all cases of ARPKD. • PKHD1 is amongst the largest disease genes identified in the human genome and spans approximately 470kb of genomic DNA. • The PKHD1 gene encodes a complicated set of alternatively spliced RNA transcripts ranging from ~8.5-13 kb that are primarily expressed in the kidney. • A 67-exon mRNA transcript of approximately 13 kb encodes the longest predicted ORF which translates to the 4074 amino acid long protein Fibrocystin/Polyductin.

9. Fibrocystin/Polyductin (FPC) • single transmembrane spanning receptor-like protein • an extensive, highly glycosylated N-terminal extracellular region. • a short cytoplasmic tail containing potential phosphorylation sites. • If a significant number of the alternatively spliced products are translated, their exon arrangements predict that both membrane-bound and soluble proteins should be produced.

10. FPC and the cilia. • As the function of FPC is unknown, the pathogenesis of the cystic phenotype in ARPKD is not fully understood. • However FPC has been shown to be localized to primary cilia and concentrated to the basal body area common with many other cystoproteins. • In a mouse model FPC has been shown to associate with the primary cilia of epithelial cells and co-localize with the Pkd2 gene product polycystin-2 (PC2), where the -COOH terminus of FPC physically interacts with the -NH2 terminus of PC2. • This suggests that these two proteins may function in a common molecular pathway which is linked to the dysfunction of primary cilia.

11. The primary cilia

12. Testing of the PKHD1 gene • Traditionally testing for ARPKD was carried out by linkage analysis. • Identification of the gene has enabled other methods of mutation detection, e.g. dHPLC analysis and sequencing. • Patients diagnosed with congenital hepatic fibrosis and Caroli’s disease with minimal or no kidney involvement are thought to be caused by mutations at the same locus. • As the gene is so large an algorithm has been suggested where screening a subset of 27 fragments, will yield an 80% detection rate for known severe PKHD1 mutations.

13. Aims • This project was set up in the hope that genomic analysis of the PKHD1 gene would provide a more comprehensive and reliable result for our families than linkage analysis. • We hoped to achieve a similar detection rate to the published research groups and also develop a full direct screening method that was relatively simple and cost-effective.

14. Method • Primers were designed, ordered and optimised to cover the previously suggested 27 fragments. • A cohort of 16 families referred with a clinical diagnosis of ARPKD or CHF and previously tested by linkage were screened. • Our pick-up rate for two pathogenic mutations from the 27 fragments was 33%. • The screen was extended to cover the whole of the gene. A further 46 fragments were designed, checked and optimised.

15. Results • 16 mutations were identified in all. Of these, 4 were novel. • Two families were homozygous for the same mutation, five families were compound heterozygotes and in two families only one mutation was identified. • Our final pick-up rate for families with two identified pathogenic mutations was 45%.

16. The PKHD1 hotspot!!A heterozygous p.Thr36Met mutation.

17. Sequence of exon 61B showing a heterozygous p.Val3546AlafsX22 mutation.

18. ARPKD mutations detected

19. p.Gln183X p.Gly223Ser p.Ile757Leu p.Arg1775X p.Leu1966ThrfsX4 p.Arg3107X c.2280-1G>A C.5381-12T>C p.Val3546AlafsX22 p.Leu400Ser p.Leu1709Phe p.Gly1971Asp p.Thr36Met Schematic representation of the location and frequency of mutations in PKHD1 from our study.

20. Conclusions • Studies from phenotypically diverse referrals, comparable to our cohort, have a pick-up rate of 47-61%. • This is now set up as a diagnostic service in the Dundee laboratory with a 40 working day turn around time. • The findings have enabled 4 CVS/prenatal tests, one case of PGD and 1 CVS currently on-route.

21. Further work • Testing for large deletions is being investigated by real time analysis using the Rotor-gene 6000. • A MLPA kit is currently being validated MRC Holland. • References • http://medgen.genetics.utah.edu/photographs/pages/potter_phenotype.htm • http://library.med.utah.edu/WebPath/RENAHTML/RENAL039.html • http://radiographics.rsnajnls.org/cgi/content/figsonly/20/3/837

22. Case study • Family suffered peri-natal death of their first born from respiratory arrest (Polycystic kidneys seen on 20wk scan). • PM showed polycystic kidneys, hepatic fibrosis, lung hypoplasia, hypertrophic myocardium and partial malrotation of small and large bowel. Potter’s facies was also noted. • Proband (SC) was initially tested for linkage, which was informative and enabled two subsequent pregnancies to have a prenatal screen, both low-risk with healthy births. • SC included in project and two mutations were identified.

23. Cont. • Both mutations c.[5895dupA] + [9319C>T] previously reported as pathogenic. • Both parents were tested to establish lineage. • Maternal sample heterozygous for c.5895dupA mutation • Paternal sample negative for both mutations!!!!! • From linkage results, no reason to suspect non-paternity. • Second child had inherited the paternal high-risk allele, tested this child again no mutation was detected, therefore gonadal mosaicism less likely. • Concluded that mutation is de novo in proband. • No literature as yet on occurrence rate of de novo mutations in ARPKD.