Transcript Development of a mutation screening service for ARPKD

Development of a mutation
screening service for ARPKD
Wendy Lewis
CMGS Spring Conference 2009
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
cysts in the kidneys and often the liver and pancreas.

Mutations have been identified in the PKD-1 and PKD-2
genes.
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.
Typical Potter facies

Typical Potter facies
phenotype showing:

broad, flat nasal bridge
widely spaced eyes
prominent intraorbital
folds
micrognathia
low-set ears.
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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.
PM of infant with ARPKD
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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.
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The histological appearance in
this case, coupled with the
gross appearance, was
consistent with autosomal
recessive polycystic kidney
disease (ARPKD).
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:
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systemic hypertension (60-100%).
abnormalities following portal hypertension (30-75%).
kidney dialysis or transplantation by the age of 10 (~30%).
Chronic lung disease (11%).
PKHD1 (Polycystic kidney 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 ~8.513 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.
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.
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.
The primary cilia
Testing of the PKHD1 gene
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Traditionally testing for ARPKD was carried out by
linkage analysis.
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Identification of the gene has enabled other methods of
mutation detection, e.g. dHPLC analysis and
sequencing.
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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.
Aims
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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.
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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.
Method
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Primers were designed, ordered and optimised to
cover the previously suggested 27 fragments.
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A cohort of 16 families referred with a clinical
diagnosis of ARPKD or CHF and previously tested
by linkage were screened.
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Our pick-up rate for two pathogenic mutations from
the 27 fragments was 33%.
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The screen was extended to cover the whole of the
gene. A further 46 fragments were designed,
checked and optimised.
Results
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16 mutations were identified in all. Of
these, 4 were novel.
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Two families were homozygous for the
same mutation, five families were
compound heterozygotes and in two
families only one mutation was identified.
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Our final pick-up rate for families with two
identified pathogenic mutations was 45%.
The PKHD1 hotspot!!
A heterozygous p.Thr36Met mutation.
Sequence of exon 61B showing a
heterozygous p.Val3546AlafsX22 mutation.
ARPKD mutations detected
Family
Mutations
Coding
Exon
Novel
34
Yes
Protein
1
c.5381-12T>C
3
c.5125C>T
c.10637delT
p.Leu1709Phe
p.Val3546AlafsX22
32e
61b
No
No
4
c.5323C>T
c.5323C>T
p.Arg1775X
p.Arg1775X
33
33
No
No
6
c.2280-1G>A
c.2280-1G>A
23
23
Yes
Yes
7
c.107C>T
c.5912G>A
p.Thr36Met
p.Gly1971Asp
3
37
No
No
10
c.547C>T
c.667G>A
p.Gln183X
p.Gly223Ser
8
9
Yes
No
11
c.1199T>C
p.Leu400Ser
15
Yes
12
c.5895dupA
c.9319C>T
p.Leu1966ThrfsX4
p.Arg3107X
36
58b
No
No
16
c.2269A>C
c.5895dupA
p.Ile757Leu
p.Leu1966ThrfsX4
22
36
No
No
Schematic representation of the location and
frequency of mutations in PKHD1 from our study.
p.Gln183X
p.Gly223Ser
p.Ile757Leu
p.Arg1775X
p.Leu1966ThrfsX4
p.Arg3107X
c.2280-1G>A
C.5381-12T>C
p.Thr36Met
p.Val3546AlafsX22
p.Leu400Ser
p.Leu1709Phe
p.Gly1971Asp
Conclusions
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Studies from phenotypically diverse referrals,
comparable to our cohort, have a pick-up rate of 4761%.
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This is now set up as a diagnostic service in the
Dundee laboratory with a 40 working day turn
around time.
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The findings have enabled 4 CVS/prenatal tests,
one case of PGD and 1 CVS currently on-route.
Further work
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Testing for large deletions is being
investigated by real time analysis using
the Rotor-gene 6000.
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A MLPA kit is currently being validated
MRC Holland.
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References
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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
Case study
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Family suffered peri-natal death of their first born from
respiratory arrest (Polycystic kidneys seen on 20wk
scan).
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PM showed polycystic kidneys, hepatic fibrosis, lung
hypoplasia, hypertrophic myocardium and partial
malrotation of small and large bowel. Potter’s facies was
also noted.
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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.
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SC included in project and two mutations were identified.
Cont.
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Both mutations c.[5895dupA] + [9319C>T] previously reported as
pathogenic.
Both parents were tested to establish lineage.
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Maternal sample heterozygous for c.5895dupA mutation
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Paternal sample negative for both mutations!!!!!
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From linkage results, no reason to suspect non-paternity.
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Second child had inherited the paternal high-risk allele, tested this
child again no mutation was detected, therefore gonadal mosaicism
less likely.
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Concluded that mutation is de novo in proband.
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No literature as yet on occurrence rate of de novo mutations in
ARPKD.