Development of a mutation screening service for ARPKD

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Development of a mutation screening service for
ARPKD

• Wendy Lewis
• SGLC Meeting 2008

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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 1800 and 11,000
  live births).
• ADPKD is a late onset chronic, progressive
  disease. It is incurable and is characterised by
  numerous fluid-filled cysts in the kidneys and
  often the liver and pancreas.
• Mutations have been identified in the PKD-1 and
  PKD-2 genes.

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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 of live births, with a estimated carrier
  frequency of 1 in 71.
• Severely affected neonates display
• massively enlarged echogenic kidneys
• and Potter phenotype from oligohydramnios
• due to poor foetal renal output.

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PM of infant with ARPKD

• This infant died soon after premature birth at 23
  weeks gestation from pulmonary hypoplasia as a
  result of oligohydramnios. The oligohydramnios
  resulted from markedly diminished foetal urine
  output as a consequence of polycystic kidney
  disease. 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).

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ARPKD

• About 30-50 of affected neonates die shortly
  after birth from respiratory insufficiency.
• Infants who survive the neonatal period or
  present later in life express variable disease
  phenotypes with systemic hypertension, end stage
  renal disease and abnormalities following portal
  hypertension.
• Patients diagnosed with congenital hepatic
  fibrosis and Carolis disease with minimal or no
  kidney involvement are thought to be caused by
  mutations at the same locus.

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ARPKD

• Despite the variable clinical spectrum of ARPKD,
  linkage studies indicate mutations at 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.
• An estimated minimum of 86 exons (12,222bp) are
  assembled into a variety of alternatively spliced
  transcripts ranging from 9-16 kb of the
  fibrocystin/polyductin protein (FPC) .

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Fibrocystin/polyductin (FPC)

• The longest PKHD1 transcript includes 67 exons
  with an ORF composed of 66 exons and encodes the
  4074 amino acid protein FPC.
• Northern blot analysis of human tissue revealed a
  broad, smeared signal consistant with the
  existence of multiple different-sized
  transcripts.
• 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.

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FPC protein structure

• FPC is a
• single transmembrane spanning receptor-like
  protein
• with an extensive, highly glycosylated N-terminal
  extracellular region
• a short cytoplasmic tail containing potential
  phosphorylation sites
• And is highly expressed kidney, with lower levels
  in liver and pancreas.

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PKHD1 and the cilia.

• FPC has been shown to be localized to primary
  cilia and concentrated to the basal body area
  common with many other cystoproteins.
• The pathogenesis of the cystic phenotype in ARPKD
  is not fully understood, as the function of FPC
  in normal tissue is not yet known.
• However the genetic and molecular data suggest
  that the genesis of cysts in all polycystic
  diseases is linked to the dysfunction of primary
  cilia.

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The primary cilia
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Testing for PKHD1

• 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.
• As the gene is so large an algorithm had been
  suggested where screening a subset of 27
  fragments, would yield an 80 detection rate for
  known severe PKHD1 mutations.

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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.

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• Primers were designed, ordered and optimised to
  cover the suggested 27 fragments.
• These were then split into three smaller groups
  of between 8-13 fragments each .
• 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.

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Results

• The screen was extended to cover the whole of
• the gene. A further 46 fragments were designed,
  checked and optimised.
• 15 mutations were identified in all. Out of
  these, 4 were novel.
• One family was homozygous, five families were
  compound heterozygotes and in three families only
  one mutation was identified.
• Our final pick-up rate for families with two
  identified pathogenic mutations was 40

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ARPKD mutations detected
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Conclusions

• Studies from phenotypically diverse referrals,
  comparable to our cohort, have a pick-up rate of
  47-61.
• We feel that this is beneficial to our patients
  to offer this as a diagnostic test.
• We aim to submit a gene dossier to UKGTN to
  notify our intention to set up mutation screening
  for ARPKD as a service in the Dundee laboratory.

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Further work

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