Development of a mutation Development of a mutation screening service for ARPKDscreening service for ARPKD

Wendy LewisWendy Lewis

CMGS Spring Conference 2009CMGS Spring Conference 2009

Polycystic kidney diseasePolycystic kidney disease

Polycystic kidney disease can be inherited in a dominant Polycystic kidney disease can be inherited in a dominant (ADPKD) or recessive (ARPKD) manner.(ADPKD) or recessive (ARPKD) manner.

ADPKD is the most commonly inherited kidney disease ADPKD is the most commonly inherited kidney disease (incidence of between 1:400 and 1:1,000 live births).(incidence of between 1:400 and 1:1,000 live births).

ADPKD is a late onset, chronic, progressive disease. It ADPKD is a late onset, chronic, progressive disease. It is incurable and is characterised by numerous fluid-filled is incurable and is characterised by numerous fluid-filled cystscysts in the kidneys and often the liver and pancreas.in the kidneys and often the liver and pancreas.

Mutations have been identified in the Mutations have been identified in the PKD-1 and PKD-2PKD-1 and PKD-2 genes.genes.

Autosomal recessive polycystic Autosomal recessive polycystic kidney disease (ARPKD).kidney disease (ARPKD).

Autosomal recessive polycystic kidney disease is an Autosomal recessive polycystic kidney disease is an important cause of renal and liver related morbidity and important cause of renal and liver related morbidity and mortality in neonates and infants.mortality in neonates and infants.

The incidence of ARPKD is estimated to be 1 in 20 000 The incidence of ARPKD is estimated to be 1 in 20 000 live births, with a estimated carrier frequency of 1 in 71.live births, with a estimated carrier frequency of 1 in 71.

Severely affected neonates display massively enlarged Severely affected neonates display massively enlarged echogenic kidneys with a “Potter” phenotype from echogenic kidneys with a “Potter” phenotype from oligohydramnios, due to poor foetal renal output.oligohydramnios, due to poor foetal renal output.

Typical Potter faciesTypical Potter facies

Typical Potter facies Typical Potter facies phenotype showing:phenotype showing:

broad, flat nasal bridgebroad, flat nasal bridge widely spaced eyes widely spaced eyes prominent intraorbital prominent intraorbital

foldsfolds micrognathia micrognathia low-set ears.low-set ears.

Diagram of a renal medullary ray depicting both normal Diagram of a renal medullary ray depicting both normal andand abnormal collecting ducts. The left side of the diagram (abnormal collecting ducts. The left side of the diagram (AA) depicts a ) depicts a normal nephron draining into a normal (non-dilated) collecting duct. normal nephron draining into a normal (non-dilated) collecting duct. The right side (The right side (BB) depicts a normal nephron draining into an ectatic ) depicts a normal nephron draining into an ectatic (dilated) collecting duct in ARPKD. (dilated) collecting duct in ARPKD.

A cut section of a kidney with ARPKD. Note that the cysts are fairly A cut section of a kidney with ARPKD. Note that the cysts are fairly small but uniformly distributed throughout the parenchyma so that small but uniformly distributed throughout the parenchyma so that the disease is usually symmetrical in appearance, with both kidneys the disease is usually symmetrical in appearance, with both kidneys markedly enlarged. markedly enlarged.

PM of infant with ARPKDPM of infant with ARPKD This infant died soon after This infant died soon after

premature birth at 23 weeks premature birth at 23 weeks gestation from pulmonary gestation from pulmonary hypoplasia as a result of hypoplasia as a result of oligohydramnios.oligohydramnios.

Note the bilaterally enlarged Note the bilaterally enlarged kidneys that nearly fill the kidneys that nearly fill the abdomen below the liver. abdomen below the liver.

The histological appearance in The histological appearance in this case, coupled with the this case, coupled with the gross appearance, was gross appearance, was consistent with autosomal consistent with autosomal recessive polycystic kidney recessive polycystic kidney disease (ARPKD).disease (ARPKD).

ARPKDARPKD About 30-50% of affected neonates die shortly after birth About 30-50% of affected neonates die shortly after birth

from respiratory insufficiency.from respiratory insufficiency.

Alongside the severe form of ARPKD, there are less severe Alongside the severe form of ARPKD, there are less severe childhood and adult forms, commonly diagnosed as childhood and adult forms, commonly diagnosed as congenital hepatic fibrosis.congenital hepatic fibrosis.

Infants who survive the neonatal period or present later in life Infants who survive the neonatal period or present later in life express variable disease phenotypes with: express variable disease phenotypes with:

systemic hypertension (60-100%).systemic hypertension (60-100%). abnormalities following portal hypertension (30-75%).abnormalities following portal hypertension (30-75%). kidney dialysis or transplantation by the age of 10 (kidney dialysis or transplantation by the age of 10 (~30%).~30%). Chronic lung disease (11%).Chronic lung disease (11%).

PKHD1 PKHD1 ((PolycysticPolycystic kidney and hepatic kidney and hepatic disease gene 1)disease gene 1) Despite the variable clinical spectrum, linkage studies Despite the variable clinical spectrum, linkage studies

indicate that a single locus indicate that a single locus PKHD1PKHD1, is responsible for all , is responsible for all cases of ARPKD.cases of ARPKD.

PKHD1PKHD1 is amongst the largest disease genes identified is amongst the largest disease genes identified in the human genome and spans approximately 470kb of in the human genome and spans approximately 470kb of genomic DNA.genomic DNA.

The The PKHD1PKHD1 gene encodes a complicated set of gene encodes a complicated set of alternatively spliced RNA transcripts ranging from ~8.5-alternatively spliced RNA transcripts ranging from ~8.5-13 kb that are primarily expressed in the kidney.13 kb that are primarily expressed in the kidney.

A 67-exon mRNA transcript of approximately 13 kb A 67-exon mRNA transcript of approximately 13 kb encodes the longest predicted ORF which translates to encodes the longest predicted ORF which translates to the 4074 amino acid long protein Fibrocystin/Polyductin.the 4074 amino acid long protein Fibrocystin/Polyductin.

Fibrocystin/Polyductin Fibrocystin/Polyductin (FPC)(FPC) single transmembrane single transmembrane

spanning receptor-like proteinspanning receptor-like protein

an extensive, highly an extensive, highly glycosylated N-terminal glycosylated N-terminal extracellular region.extracellular region.

a short cytoplasmic tail a short cytoplasmic tail containing potential containing potential phosphorylation sites.phosphorylation sites.

If a significant number of the If a significant number of the alternatively spliced products alternatively spliced products are translated, their exon are translated, their exon arrangements predict that both arrangements predict that both membrane-bound and soluble membrane-bound and soluble proteins should be producedproteins should be produced..

FPC and the cilia.FPC and the cilia.

As the function of FPC is unknown, the pathogenesis of the As the function of FPC is unknown, the pathogenesis of the cystic phenotype in ARPKD is not fully understood.cystic phenotype in ARPKD is not fully understood.

However FPC has been shown to be localized to primary cilia However FPC has been shown to be localized to primary cilia and concentrated to the basal body area common with many and concentrated to the basal body area common with many other cystoproteins. other cystoproteins.

In a mouse model FPC has been shown to associate with the In a mouse model FPC has been shown to associate with the primary cilia of epithelial cells and co-localize with the Pkd2 primary cilia of epithelial cells and co-localize with the Pkd2 gene product polycystin-2 (PC2), where the -COOH terminus gene product polycystin-2 (PC2), where the -COOH terminus of FPC physically interacts with the -NH2 terminus of PC2.of FPC physically interacts with the -NH2 terminus of PC2.

This suggests that these two proteins may function in a This suggests that these two proteins may function in a common molecular pathway which is linked to the dysfunction common molecular pathway which is linked to the dysfunction of primary cilia.of primary cilia.

The primary ciliaThe primary cilia

Testing of the Testing of the PKHD1 genePKHD1 gene Traditionally testing for ARPKD was carried out by Traditionally testing for ARPKD was carried out by

linkage analysis.linkage analysis.

Identification of the gene has enabled other methods of Identification of the gene has enabled other methods of mutation detection, e.g. dHPLC analysis and mutation detection, e.g. dHPLC analysis and sequencing.sequencing.

Patients diagnosed with congenital hepatic fibrosis and Patients diagnosed with congenital hepatic fibrosis and Caroli’s disease with minimal or no kidney involvement Caroli’s disease with minimal or no kidney involvement are thought to be caused by mutations at the same are thought to be caused by mutations at the same locus.locus.

As the gene is so large an algorithm has been suggested As the gene is so large an algorithm has been suggested where screening a subset of 27 fragments, will yield an where screening a subset of 27 fragments, will yield an 80% detection rate for known severe 80% detection rate for known severe PKHD1PKHD1 mutations. mutations.

AimsAims

This project was set up in the hope that genomic This project was set up in the hope that genomic analysis of theanalysis of the PKHD1 PKHD1 gene would provide a gene would provide a more comprehensive and reliable result for our more comprehensive and reliable result for our families than linkage analysis.families than linkage analysis.

We hoped to achieve a similar detection rate to We hoped to achieve a similar detection rate to the published research groups and also develop the published research groups and also develop a full direct screening method that was relatively a full direct screening method that was relatively simple and cost-effective.simple and cost-effective.

MethodMethod Primers were designed, ordered and optimised to Primers were designed, ordered and optimised to

cover the previously suggested 27 fragments.cover the previously suggested 27 fragments.

A cohort of 16 families referred with a clinical A cohort of 16 families referred with a clinical diagnosis of ARPKD or CHF and previously tested diagnosis of ARPKD or CHF and previously tested by linkage were screened.by linkage were screened.

Our pick-up rate for two pathogenic mutations from Our pick-up rate for two pathogenic mutations from the 27 fragments was 33%.the 27 fragments was 33%.

The screen was extended to cover the whole of the The screen was extended to cover the whole of the gene. A further 46 fragments were designed, gene. A further 46 fragments were designed, checked and optimised.checked and optimised.

ResultsResults

16 mutations were identified in all. Of these, 4 16 mutations were identified in all. Of these, 4 were novel. were novel.

Two families were homozygous for the same Two families were homozygous for the same mutation, five families were compound mutation, five families were compound heterozygotes and in two families only one heterozygotes and in two families only one mutation was identified.mutation was identified.

Our final pick-up rate for families with two Our final pick-up rate for families with two identified pathogenic mutations was 45%.identified pathogenic mutations was 45%.

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

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

ARPKD mutations detectedARPKD mutations detectedFamily Mutations Exon Novel

  Coding Protein    

1 c.5381-12T>C   34 Yes

3 c.5125C>Tc.10637delT

p.Leu1709Phep.Val3546AlafsX22

32e61b

NoNo

4 c.5323C>Tc.5323C>T

p.Arg1775Xp.Arg1775X

3333

NoNo

6 c.2280-1G>Ac.2280-1G>A

  2323

YesYes

7 c.107C>Tc.5912G>A

p.Thr36Metp.Gly1971Asp

337

NoNo

10 c.547C>Tc.667G>A

p.Gln183Xp.Gly223Ser

89

YesNo

11 c.1199T>C p.Leu400Ser 15 Yes

12 c.5895dupAc.9319C>T

p.Leu1966ThrfsX4p.Arg3107X

3658b

NoNo

16 c.2269A>Cc.5895dupA

p.Ile757Leup.Leu1966ThrfsX4

2236

NoNo

Schematic representation of the location and Schematic representation of the location and frequency of mutations in frequency of mutations in PKHD1PKHD1 from our study. from our study.

p.Gln183X p.Gly223Ser

p.Thr36Metp.Leu400Ser

p.Ile757Leu

c.2280-1G>A

p.Leu1709Phe

p.Arg1775X p.Leu1966ThrfsX4

p.Gly1971Asp

p.Arg3107X

p.Val3546AlafsX22C.5381-12T>C

ConclusionsConclusions

Studies from phenotypically diverse referrals, Studies from phenotypically diverse referrals, comparable to our cohort, have a pick-up rate of 47-comparable to our cohort, have a pick-up rate of 47-61%.61%.

This is now set up as a diagnostic service in the This is now set up as a diagnostic service in the Dundee laboratory with a 40 working day turn Dundee laboratory with a 40 working day turn around time.around time.

The findings have enabled 4 CVS/prenatal tests, The findings have enabled 4 CVS/prenatal tests, one case of PGD and 1 CVS currently on-route.one case of PGD and 1 CVS currently on-route.

Further workFurther work

Testing for large deletions is being Testing for large deletions is being investigated by real time analysis using investigated by real time analysis using the Rotor-gene 6000.the Rotor-gene 6000.

A MLPA kit is currently being validated A MLPA kit is currently being validated MRC Holland.MRC Holland.

ReferencesReferences http://medgen.genetics.utah.edu/photographs/pages/potter_phenotype.htmhttp://medgen.genetics.utah.edu/photographs/pages/potter_phenotype.htm http://library.med.utah.edu/WebPath/RENAHTML/RENAL039.htmlhttp://library.med.utah.edu/WebPath/RENAHTML/RENAL039.html http://radiographics.rsnajnls.org/cgi/content/figsonly/20/3/837http://radiographics.rsnajnls.org/cgi/content/figsonly/20/3/837

Case studyCase study Family suffered peri-natal death of their first born from Family suffered peri-natal death of their first born from

respiratory arrest (Polycystic kidneys seen on 20wk respiratory arrest (Polycystic kidneys seen on 20wk scan).scan).

PM showed polycystic kidneys, hepatic fibrosis, lung PM showed polycystic kidneys, hepatic fibrosis, lung hypoplasia, hypertrophic myocardium and partial hypoplasia, hypertrophic myocardium and partial malrotation of small and large bowel. Potter’s facies was malrotation of small and large bowel. Potter’s facies was also noted.also noted.

Proband (SC) was initially tested for linkage, which was Proband (SC) was initially tested for linkage, which was informative and enabled two subsequent pregnancies to informative and enabled two subsequent pregnancies to have a prenatal screen, both low-risk with healthy births.have a prenatal screen, both low-risk with healthy births.

SC included in project and two mutations were identified.SC included in project and two mutations were identified.

Cont.Cont. Both mutations c.[5895dupA] + [9319C>T] previously reported as Both mutations c.[5895dupA] + [9319C>T] previously reported as

pathogenic.pathogenic.

Both parents were tested to establish lineage.Both parents were tested to establish lineage.

Maternal sample heterozygous for c.5895dupA mutationMaternal sample heterozygous for c.5895dupA mutation

Paternal sample negative for both mutations!!!!!Paternal sample negative for both mutations!!!!!

From linkage results, no reason to suspect non-paternity.From linkage results, no reason to suspect non-paternity.

Second child had inherited the paternal high-risk allele, tested this Second child had inherited the paternal high-risk allele, tested this child again no mutation was detected, therefore gonadal mosaicism child again no mutation was detected, therefore gonadal mosaicism less likely.less likely.

Concluded that mutation is Concluded that mutation is de novode novo in proband. in proband.

No literature as yet on occurrence rate of No literature as yet on occurrence rate of de novode novo mutations in mutations in ARPKDARPKD..