Transcript Genetic Study of Polycystic Kidney Disease

Molecular Pathology of
Kidney Diseases
Dr. K.W. Chan
1. Hereditary Kidney
Diseases
•Adult
polycystic disease
•Infantile polycystic disease
•Alport syndrome
1. Hereditary Kidney
Diseases
•[Adult
polycystic disease]
•Infantile polycystic disease
•Alport syndrome
Characteristics of APKD
•
•
•
•
•
•
•
Gene frequency 1/1000.
Autosomal dominant.
Symptoms onset in middle age.
Large polycystic kidneys.
>50% end stage renal failure.
A disorder affecting multiple organ systems.
Genetically heterogeneous.
Localization of APKD genes
•
1986 (Reeders et al)
– A gene locus of APKD (now called PKD1) was
shown to be closely linked to the -globin locus
on 16p.
•
1988 (Kimberling)
– Genetic heterogeniety of APKD was discovered.
•
1992 (Peters et al)
– The PKD2 locus was localized to 4q21-23.
•
1994 (EPKDC)
– PKD1 identified to be a gene encoding a 14-kb
transcript
– encoding a 4,302 a.a. protein called polycystin-1
•
1996 (Mochizuki et al)
– PKD2 was cloned and polycystin-2 characterized
•
1997 (Ariza et al)
– described a 2-generation Spanish family with
PKD in which linkage to the PKD1 and PKD2 loci
was excluded -> evidence of PKD3
Localization of PKD1
•
A Portuguese family with both APKD
and TSC
– Father normal karyotype
– Mother 46,XX t(16;22)(p13.3;q11.21), suffers
APKD
– Son 45,XY 16pter-p13.3 and 22pter-q11.21,
suffers TSC and APKD
– The breakpoint at 16p13.3 has disrupted
the PKD1
Genetic Heterogeneity of APKD
Gene Involved Location Proportion
PKD1
16p13.3
80-90%
PKD2
4q21-23
10-15%
PKD3
not mapped
rare
Characteristics of PKD1
•
•
•
•
Located on 16p13.3
~52kb genomic DNA, 14kb mRNA, 46 exons,
4,302 a.a.
Encodes for polycystin 1, an integral
membrane glycoprotein
70% duplication on 16p13.1 - the HG area
– HG-A 21 kb
– HG-B 17 kb
– HG-C 8.5 kb
Characteristics of PKD2
•
•
•
•
Located on 4q21-23.
Encodes a 4 kb mRNA, 968 a.a. product.
Encodes for polycystin 2, an integral
membrane glycoprotein.
Polycystin 1 and 2 function together as
part of a multi-component membranespanning complex involved in cell-cell
or cell-matrix interactions.
Polycystin 2
•
•
•
Polycystin 2 has six transmembrane
spans with intracellular amino- and
carboxyl-termini.
It has amino acid similarity with PKD1,
and the family of voltage-activated
calcium (and sodium) channels
It contains a calcium-binding domain.
Aims of genetic study
•
•
•
•
Early diagnosis, including prenatal and
presymptomatic diagnosis.
Select embyro by “test tube baby”
Correlate between phenotype and
genotype.
Understand mechanisms involved in
cyst formation and other associated
lesions of APKD at the molecular level.
Problems in the study of PKD1
•
•
•
Only about 2.5 kb out of the 14 kb
transcript is not duplicated.
Mutations affecting the duplicated part
are difficult to determine.
No “hot spot” mutations.
Diagnosis by Imaging
•
•
•
•
Ultrasonography
Intravenous urography
Urogram with bolus intravenous
nephrotomography
Computerized tomography
Diagnostic Criteria of APKD
Age
No. of cysts
Remarks
< 30
2
Unilateral /
Bilateral
30 – 59
2
In each kidney
> 60
>3
In each kidney
Strategies for genetic study
• Genetic linkage study for early diagnosis
•
– Microsatellite studies in APKD kindreds
Mutation analysis
– Sequencing
• Only about 2.5 kb out of the 14 kb
transcript is not duplicated.
• Mutations affecting the duplicated part
are difficult to determine.
• No “hot spot” mutations.
Mutation Identified
Mutation Type Mutation
Deletion
Insertion
Missense
Nonsense
Splicing Error
Translocation
Method Paper Published
Exon 31-34, 35-46 PCR
Exon 39, 44
SSCP
Novel aa
SSCP
Eur PKD Consortium 94
Peral et al. 96
Peral et al. 96
GluAsp
2 CT
GlnStop
CysStop
Arg, GluStop
CT
CT
Exon 44
Intron 43
Exon 15
Peral et al. 96
Rossetti et al. 96
Turco et al. 95
Neophytou et al. 96
Peral et al. 96
Rossetti et al. 96
Turco et al. 96
Eur PKD Consortium 94
Peral et al. 95
Eur PKD Consortium 94
SSCP
HA
HA
SSCP
SSCP
HA
HA
HA
CA
Microsatellite Polymorphism
•
Variation in the number of
dinucleotides within (AC)n or other
simple sequence repeats
Microsatellite Markers for
PKD1
Marker
Locus
Repeat Type
Heterozygosity
SM7
D16S283
(AC)n
76.8%
CW2
D16S663
(AC)n
83%
AC2.5
D16S291
GGG(GT)nGAT
79%
SM6
D16S665
(TG)10C(CG)10TGC(GT)3
69%
(AC)n
56%
(AC)n
76.8%
KG8
D16S521
D16S521
Microsatellite Markers for
PKD2
Locus
Repeat Type
Heterozygosity
D4S231
(AC)n
87%
D4S1563
(AC)n
81%
D4S414
(AC)n
86%
Scope of work
•
Characterization of markers in HK
Population (a sample of 63 unrelated
adults)
– 6 PKD1 markers, 3 PKD2 markers
•
Microsatellite haplotyping in 5 APKD
families with a total of 42 members.
Method
•
•
•
•
20 ml of peripheral blood
DNA extracted and purified
PCR amplification of each of the 9
microsatellite markers
Southern blot
Microsatellite
Characterization
Marker
No. of subjects
Type of Allele
HET
PIC
SM7
57
9
0.639
0.609
CW2
46
7
0.789
0.760
AC2.5
54
9
0.754
0.720
SM6
54
14
0.652
0.635
KG8
56
4
0.246
0.229
D16S521
51
11
0.639
0.609
D4S231
52
10
0.840
0.807
D4S1563
57
7
0.775
0.741
D4S414
54
9
0.823
0.793
Family A
Ia
Ib
Ic
55
49
48
IIa
IIb
IIc
21
20
18
PKD1 Markers in Family A
PKD2 Markers in Family A
Family B
Ia
Ib
Ic
Id
Ie
50
45
43
41
34
IIa
IIb
IIc
20
17
16
PKD1 Markers in Family D
Family D
Ia
Z+2
Z+2
Z
Z
Z-8
Z+6
(SM7)
(SM6)
(CW2)
(KG8)
(AC2.5)
(D16S521)
Ib
Z+6
Z-1
Z
Z
Z+2
Z+5
Z+4 Z
Z Z+1
Z+4 Z
Z+4 Z
Z Z
Z Z
SM7
CW2
AC2.5
SM6
KG8
D16S521
IIa
IIb
Z+2 Z+4
Z+2 Z
Z Z+4
Z Z+4
Z-8 Z
Z+6 Z
Z+6Z+4
Z-1Z
Z Z+4
Z Z+4
Z+2Z
Z+5Z
IIc
Z+6 Z
Z-1 Z+1
Z Z
Z Z
Z+2 Z
Z+5 Z
PKD2 Markers in Family D
PKD1 Markers in family E
Family E
Ia
Z+6
Z
Z
Z-9
Z-2
Z
Z-4
Z-3
Z+6
Z
Z
Z+6
Ib
Z
Z-2
Z
Z
Z
Z
(SM7)
(SM6)
(CW2)
(KG8)
(AC2.5)
(D16S521)
Ic
SM7
CW2
AC2.5
SM6
KG8
D16S521
Z+4
Z-1
Z+2
Z-5
Z-2
Z
IIa
Z+6
Z
Z
Z-9
Z-2
Z
Z
Z-2
Z
Z
Z
Z
IIb
Z-4
Z-3
Z+6
Z
Z
Z+6
Z
Z-2
Z
Z
Z
Z
Z+2
Z+1
Z+7
Z
Z
Z+7
Z-4
Z-3
Z+6
Z
Z
Z+6
Results of Genetic Diagnosis
Family
Generation
No. of
Member
ADPKD1
ADPKD2
A
2
6
+
-
B
2
8
++
--
C
3
18
+++
--
D
2
5
-
-
E
2
5
ND
ND
PKD3 - Is there one?
•
2001 (Pei et al)
– evidence of bilineal disease and transheterozygotes in a large family of ADPKD
– 28/48 members affected
– SSCA screened for and found a PKD2
mutation (2152delA; L736X) in 12 affected
pedigree members
– linkage analysis with markers at the PKD1
locus, found significant LOD scores (13.0).
PKD3 - Is there one?
•
2001 (Pei et al)
– evidence of bilineal disease and transheterozygotes in a large family of ADPKD
– 28/48 members affected
– in 2/48, who had severe disease, evidence
of trans-heterozygotes
1. Hereditary Kidney
Diseases
•Adult
Polycystic Disease
•[Infantile Polycystic Disease]
•Alport Syndrome
Infantile Polycystic Kidney Disease
•
•
•
Autosomal recessive.
Usually incompatible
with life.
Early antenatal
diagnosis for
termination of
pregnancy is desirable.
Infantile Polycystic Disease
•
•
In infantile PKD, the liver is always
affected.
The abnormal bile ducts in the liver are
accompanied by periductal fibrosis.
Hence called congenital hepatic fibrosis.
Localization of PKHD1
•
1994
– Gene locus in 6p12.1-p21.
•
2002 (Ward et al)
– PKHD1 cloned
– 16kb transcript, 4,074 a.a. receptor-like
protein called fibrocystin
– Missense and truncating mutations
identified in 14 probands
Genetics of PKHD
•
•
•
Compound heterozygosity.
Double truncating mutations – severe
disease.
Some families with mild disease show
compound heterozygosity for a
missense and a truncating mutation.
1. Hereditary Kidney
Diseases
•Adult
Polycystic Disease
•Infantile Polycystic Disease
•[Alport Syndrome]
Alport Syndrome
•
A hereditary disorder of basement
membrane collagen characterized
clinically by hematuria, progressive
renal failure, and, frequently,
neurosensory hearing loss and ocular
abnormalities.
Alport Syndrome
•
•
Genetically heterogeneous.
In the majority of cases, the disease is
inherited as an X-linked trait, but an
autosomal recessive form and also an
autosomal dominant form exist.
Alport Syndrome
•
•
•
•
X-link recessive: mutations of COL4A5 on
Xq22.
X-link recessive associated with diffuse
leiomyomatosis: mutations of COL4A5 and
COL4A6.
Autosomal recessive: mutations of COL4A3
and COL4A4 on 2q.
Autosomal dominant : no mutation in any of
the COL4 genes.
Prenatal Diagnosis of AS
•
•
X-link recessive: by genetic linkage
analysis using polymorphic markers in
and around COL4A5.
Autosomal recessive: by genetic linkage
analysis using polymorphic markers in
and around COL4A3 and COL4A4.
Fabry disease
Fabry disease
The story begins…
Fabry disease
•
•
X-linked recessive inborn error of
glycosphingolipid catabolism that results
from the deficient activity of the lysosomal
enzyme α-galactosidase A (EC 3.2.1.22).
Accumulation of glycosphingolipid
substrates in the vascular endothelium
causing occlusive microvascular diseases
mainly affecting the kidney, the heart,
peripheral nerves and the brain.
F/50 Southern Chinese
•
•
•
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Asymptomatic proteinuria
24 hour urine protein 0.6 g
No skin or corneal lesions
Normal renal function tests
Family history
•
•
Mother died of kidney disease
4 siblings
– one younger brother (age 44) in end stage
renal failure since age of 35
– one younger brother (age 41) has
cardiomyopathy since age of 33, not in
renal failure
Renal Biopsy Diagnosis
•
Consistent with heterozygous Fabry
disease
Diagnosis Confirmed
•
•
Consistent with
heterozygous Fabry disease
Low serum α-galactosidase A activity at
5.2nmol/ml.hr
– (normal range: 8.8-14.5nmol/ml.hr)
•
Low serum α-galactosidase A activity in
both brothers (both at <0.5nmol/ml.hr)
Genetic studies of 2 affected
brothers
•
•
Direct DNA sequencing of all 9 exons of
α-galactosidase gene
New primers are designed for this
study
Genetic studies of 2 affected
brothers
•
•
Direct DNA sequencing of all 9 exons of αgalactosidase gene
Results
– single T-to-C transition in codon 14 of exon 1
– missense mutation predicting a leucine to proline
substitution (L14P)
– same mutation in both brothers
– other exon sequences are normal
DNA Sequence of Exon 1
Brother A
Normal subject
Complementary strand, exon
1
Brother A
Normal subject
Significance of the study
•
•
•
Successful design of new primers for
direct sequencing studies of the Fabry
disease gene
A novel mutation is documented
The two male patients have the same
genetic mutations despite different
phenotypic manifestations
2. Tumors of the Kidney
•Renal
Cell Carcinoma
•Wilms’ tumor (nephroblastoma)
2. Tumors of the Kidney
•[Renal
Cell Carcinoma]
•Wilms’ tumor (nephroblastoma)
Renal Cell Carcinoma
•
Types (histological with a genetic
basis):
–
–
–
–
clear cell
papillary
chromophobe
collecting duct carcinoma
Renal Cell Carcinoma
•
Types:
– clear cell (VHL gene on 3p
inactivated)
– papillary (trisomies 7, 16, 17; loss of
Y; t(X;1))
– chromophobe (monosomy of
different chromosomes)
Nephroblastoma (Wilms’
tumor)
•
•
•
•
20% of malignant childhood
tumors
highest incidence at age 3
abdomincal mass
hematuria, pain, fever
Nephroblastoma
•
•
Deletions or mutations of WT1 or
WT2 genes
Both WT1 and WT2 genes are on
the short arm of chromosome 11,
but are distinctly different genes.
WT1
•
•
•
•
WT1 gene is encoded by 10 exons, resulting in
messenger RNA subject to a complex pattern of
alternative splicing.
WT1 gene encodes a zinc finger transcription factor,
which binds to GC-rich sequences and functions as a
transcriptional activator or repressor for many
growth factor genes.
WT 1 protein is mainly expressed in developing
kidney, testis, and ovary, indicating that it is
involved in the differentiation of genitourinary
tissues, all thought to be the sites of origin of Wilms’
tumor.
The point mutation of WT1 results in Denys-Drash
syndrome.
WT1
High level WT1 expression in leukemia
cells is linked to a poor prognosis.
 A correlated expression between WT1 and
mdr-1 in vincristine resistant cells indicates
a close relation with multi-drug resistance
and is a promising diagnostic marker for
chemoresistance in hematologic
malignancies.

Cystinuria
•
Background
– A hereditary disorder of cystine and
dibasic amino acid transport across the
luminal membrane of renal proximal
tubule and small intestine.
– Cystine is of low solubility and forms
urinary stones in sufferers of cystinuria.
Localization of Cystinuria Genes
•
1992 (Bertran et al)
– rBAT (SLC3A1) on 2p (type I cystinuria).
•
1999 (Feliubadalo et al)
– SLC7A9 on 19q (non-type I).
•
•
•
SLC7A9 is the transmembrane channel
mediating the uptake of these a.a.
rBAT is a smaller protein.
rBAT forms a heterodimeric complex with the
channel and is critical for its targeting to the
luminal membrane.
•
Hereditary Kidney Diseases
–
–
–
–
–
•
Adult polycystic disease
Infantile polycystic disease
Alport syndrome
Fabry disease
Tubular transport - Cystinuria
Tumours of the Kidney
– Renal Cell Carcinoma
– Wilms’ tumor (nephroblastoma)
~ the end ~