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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.

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1 hereditary kidney diseases

1. Hereditary Kidney Diseases

Adult polycystic disease

Infantile polycystic disease

Alport syndrome

1 hereditary kidney diseases1

1. Hereditary Kidney Diseases

[Adult polycystic disease]

Infantile polycystic disease

Alport syndrome

characteristics of apkd
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
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.
slide6
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
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
characteristics of pkd1
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
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 membrane-spanning complex involved in cell-cell or cell-matrix interactions.
polycystin 2
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
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
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
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

Diagnostic Criteria of APKD
strategies for genetic study
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.
microsatellite polymorphism
Microsatellite Polymorphism
  • Variation in the number of dinucleotides within (AC)n or other simple sequence repeats
scope of work
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
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

Microsatellite Characterization
pkd1 markers in family d
PKD1 Markers in Family D

(SM7)

(SM6)

IIa

IIa

IIb

IIb

IIc

IIc

Ia

Ia

Ib

Ib

Z,Z

Z,Z

Z,Z+4

Z,Z+4

Z,Z+4

Z,Z+6

Z,Z+4

Z+2,Z+4

Z+4,Z+6

Z+2,Z+6

(CW2)

(KG8)

Z,Z

Z,Z

Z,Z

Z-1,Z

Z,Z+1

Z,Z+2

Z,Z+2

Z,Z+2

Z-1,Z+2

Z-1,Z+1

(AC2.5)

(D16S521)

Z,Z

Z,Z

Z,Z

Z,Z+6

Z,Z+5

Z,Z+4

Z,Z+4

Z,Z+4

Z,Z+5

Z+5,Z+6

pkd1 markers in family e
PKD1 Markers in family E

(SM7)

(SM6)

Ic

Ic

IIb

IIb

IIa

IIa

Ib

Ib

Ia

Ia

Z,Z

Z,Z

Z-4,Z

Z-9,Z

Z,Z+4

Z-5,Z

Z-9,Z

Z-4,Z+2

Z-4,Z+6

Z,Z+6

(CW2)

(KG8)

Z,Z

Z,Z

Z-3,Z

Z-2,Z

Z-2,Z

Z-2,Z

Z-2,Z

Z-2,Z-1

Z-3,Z-2

Z-3,Z+1

(AC2.5)

(D16S521)

Z,Z

Z,Z

Z,Z

Z,Z+2

Z,Z+6

Z,Z+6

Z,Z+6

Z,Z+6

Z,Z+6

Z+6,Z+7

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

Results of Genetic Diagnosis
pkd3 is there one
PKD3 - Is there one?
  • 2001 (Pei et al)
    • evidence of bilineal disease and trans-heterozygotes 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 one1
PKD3 - Is there one?
  • 2001 (Pei et al)
    • evidence of bilineal disease and trans-heterozygotes in a large family of ADPKD
    • 28/48 members affected
    • in 2/48, who had severe disease, evidence of trans-heterozygotes
1 hereditary kidney diseases2

1. Hereditary Kidney Diseases

Adult Polycystic Disease

[Infantile Polycystic Disease]

Alport Syndrome

infantile polycystic kidney disease
Infantile Polycystic Kidney Disease
  • Autosomal recessive.
  • Usually incompatible with life.
  • Early antenatal diagnosis for termination of pregnancy is desirable.
infantile polycystic disease
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
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 mutationsidentified in 14 probands
genetics of pkhd
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 diseases3

1. Hereditary Kidney Diseases

Adult Polycystic Disease

Infantile Polycystic Disease

[Alport Syndrome]

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 syndrome1
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 syndrome2
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
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 disease1

Fabry disease

The story begins…

fabry disease2
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
F/50 Southern Chinese
  • Asymptomatic proteinuria
  • 24 hour urine protein 0.6 g
  • No skin or corneal lesions
  • Normal renal function tests
family history
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
Renal Biopsy Diagnosis
  • Consistent with heterozygous Fabry disease
diagnosis confirmed
Diagnosis Confirmed
  • Consistent withheterozygous 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
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 brothers1
Genetic studies of 2 affected brothers
  • Direct DNA sequencing of all 9 exons of α-galactosidase 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
DNA Sequence of Exon 1

Brother A

Normal subject

complementary strand exon 1
Complementary strand, exon 1

Brother A

Normal subject

significance of the study
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

2. Tumors of the Kidney

Renal Cell Carcinoma

Wilms’ tumor (nephroblastoma)

2 tumors of the kidney1

2. Tumors of the Kidney

[Renal Cell Carcinoma]

Wilms’ tumor (nephroblastoma)

renal cell carcinoma
Renal Cell Carcinoma
  • Types (histological with a genetic basis):
    • clear cell
    • papillary
    • chromophobe
    • collecting duct carcinoma
renal cell carcinoma1
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
Nephroblastoma (Wilms’ tumor)
  • 20% of malignant childhood tumors
  • highest incidence at age 3
  • abdomincal mass
  • hematuria, pain, fever
nephroblastoma
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.
slide62
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.
slide63
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
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
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.
slide66
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)