RESEARCH GROUP

calendargrumpyBiotechnology

Dec 14, 2012 (4 years and 10 months ago)

251 views

Muhammad Ajmal, Muhammad Nasir and

Abdul Hameed


Institute of Biomedical and Genetic Engineering

24
-
Mauve Area, G
-
9/1

Islamabad

Association of a 3
-
Amino Acid Deletion Mutation of
NPHS2 gene with Congenital Nephrotic Syndrome
(CNS) in a consanguineous Pakistani family



Autosomal

recessive

nephrotic

syndrome

(NS)

belongs

to

the

heterogeneous

group

of

familial

nephrotic

syndrome
.


The

disease

is

characterized

by

childhood

onset

of

proteinuria
,

progression

to

end
-
stage

renal

disease,

and

minimal

change

nephrotic

syndrome
.


NS

results

from

alterations

of

the

glomerular

capillary

wall
.


Proteinuria

and

hypoalbuminemia
,

often

associated

with

edema

and

generalized

hyperlipidemia


Introduction

Genetic of NPHS


Majority

of

nephrotic

syndrome

(NS)

occurs

as

a

sporadic

form,

the

incidence

of

familial

cases

are

from

3

to

5
%
.


Mutations

of

seven

genes,

NPHS
1

(
19
q
13
.
1
),

NPHS
2

(
1
q
25
-
31
),

ACTN
4

(
19
q
13
),

CD
2
AP,

WT
1
,

TRPC
6

(
11
q
21
-
22
),

and

LAMB
2

have

been

recognized

to

date

are

responsible

for

various

forms

of

NS

are

summarised

in

the

table
.


NPHS: Known Loci

Disease

Gene

Locus

Protein

Mode of
Inheritance

Age of onset

CNS

NPHS1

19q13.1

Nephrin

AR

Prenatal

SRNS

NPHS2

1q25
-
31

Podocin

AR

Early childhood

FSGS1

ACTN4

19q13

α4actinin

AD

childhood

FSGS2

TRPC6

11q21
-
22

TRPC6

AD

Early childhood

FSGS3

CD2AP

6p12

CD2AP

AD

Early childhood

Syndrome
Fraiser

Syndrome
Densy
-
Drash

WT1

11p13

Transcription factor

AD

Adulthood

Syndrome Pierson

LAMB2

3p14
-
22

Lamininβ2 chain

AR

?

Diffuse Mesengial Slerosis

NPHS3

AR

Early childhood

NPHS Family: Sample collection

A

three

generation

consanguineous

Pakistani

family,

in

which

congenital

nephrotic

syndrome

was

segregating

as

an

autosomal

recessive

trait

was

ascertained

through

nephrology

clinic

in

Peshawar,

Pakistan


Three

living

members

of

the

family

were

found

to

be

affected

with

NPHS


Detailed

clinical

examination

was

performed

on

the

available

family

members

including

3

affected

individuals

A pedigree of Pakistani NPHS Family

Janat Sher
NPHS007
Jamila Bibi
NPHS008
Rehana Bibi
NPHS004
Munir
NPHS009
Imamuddin
NPHS010
Amana Bibi
NPHS011
Rahib
NPHS012
Metab-uddin
NPHS013
Junaid
NPHS014
Rema
NPHS015
Mohibullah
NPHS003
Mobashir
NPHS005
Abdur-Rehman
NPHS006
Haji Baghisher
NPHS001
Banarasa Bibi
NPHS002
1NPHS (NEPHROTIC SYNDROME FAMILY)
Legend

Male Normal

Female Normal

Male affected

Female affected

Marriage

Cousin
Marriage

Aim of the study

The aim of present study was


1.
To

locate

and

study

Pakistani

families

affected

with

autosomal

recessive

congenital

nephrothic

syndrome
.

2.
To

identify

the

genes

responsible

for

producing

specific

phenotypes

in

Pakistani

family

with

CNS
.

3.
To

establish

the

prenatal

diagnosis

and

carrier

screening

tests

of

the

disease

in

Pakistan
.

4.

Genetic Counseling
Avoid cousin marriages and /or
avoid children

NPHS Family
: sample collection & DNA
extraction


Blood

samples

from

affected

individuals,

their

parents

and

clinically

normal

siblings

of

the

family

members

were

collected

with

informed

consent


Blood

samples

were

also

collected

from

100

ethnically

matched

unrelated

normal

Pakistani

individuals

and

were

used

as

controls

for

allele

frequencies

and

confirmation

of

disease

associated

mutation


Genomic

DNA

for

linkage

analysis

was

extracted

from

peripheral

blood

by

the

standard

phenol

chloroform

extraction

procedure

Genotyping and Linkage Analysis



To

identify

the

locus

responsible

for

the

disease

in

this

family,

genomic

DNA

from

each

individual

was

genotyped

using

microsatellite

marker

for

the

known

NS

loci



Microsatellite

markers

in

the

disease

region

were

amplified

by

polymerase

chain

reaction

(PCR)


PCR Conditions


PCR

reactions

were

performed

in

10


l

volume,

each

containing

1
.
5
mM

MgCl
2
,

0
.
6

M

of

each

primer,

0
.
2
mM

dNTPs
,

1
U

Taq

DNA

polymerase

and

PCR

buffer

[
16
mM

(NH
4
)

2
SO
4
,

67
mMTris
-
HCI

(pH

8
.
8
),

and

0
.
01
%

of

the

nonionic

detergent

Tween
-
20
]



Amplification

was

performed

with

an

initial

denaturation

for

4

min

at

94
o
C,

followed

by

35

cycles

of

denaturation

at

94
o
C

for

45
sec,

annealing

at

55
o
C

for

45

sec,

extension

at

72
o
C

for

45

sec

and

a

final

extension

at

72
o
C

for

5

min

PAGE Electrophoresis



The

PCR

products

were

separated

on

10
-
12
%

non
-
denaturing

polyacrylamide

gels



The

gel

was

stained

with

ethidium

bromide,

visualized

under

UV

illumination

and

photographed
.



Alleles

were

assigned

to

each

individuals

and

genotyping

was

carried

out
.


NPHS2 Gene: Mutation Screening


NPHS

family

was

mapped

to

NPHS
2

locus

on

chromosome

1
q
25
.
2



NPHS
2

gene

was

the

candidate

gene

in

this

region,

which

comprises

of

8

exons
.



Polymerase chain reaction (PCR) amplification of the
NPHS2

gene was performed with eight pairs of primers
spanning all 8 exons by using
intronic

forward and
reverse primers

NPHS2 Gene: PCR Amplification Conditions


PCR

amplification

was

performed

in

50

µl

reaction

volume

containing

250

ng

of

genomic

DNA,

amplification

buffer

containing

600

nM

of

each

primer,

1
.
5

mM

MgCl
2
,

200

mM

of

dNTPs

and

2
.
5

U

Taq

polymerase

in

an

PxE

thermal

cycler
.



The

amplification

conditions

were

95
o
C

for

5

min,

followed

by

35

cycles

of

95
o
C

for

45

s,

primer

specific

annealing

temperature

for

45

s,

extension

on

72

o
C

for

45

s

and

final

extension

at

72

o
C

for

7

min
.


NPHS2 Gene: Sequencing


Aliquots

(
5

µl)

of

the

PCR

products

were

analyzed

by

2
.
5
%

agarose

gel

electrophoresis
.


PCR

products

were

then

purified

using

QIAquick

PCR

Purification

Kit
.


Sequenced

directly

using

Big

Dye®

Terminator

v
3
.
1

cycle

sequencing

kit

in

an

ABI

3130

genetic

analyzer
.


Potential

mutations

were

confirmed

by

bi
-
directional

sequencing

and

assessing

100

control

samples

having

ethnic

backgrounds

matching

to

patients
.


NPHS2:

Mutation Identified in Pakistani family

Patient’s

electrophergram

showing

homozygous

9
bp

deletion

(arrow)

in

exon

5

of

NPHS
2

gene
.

An

electrophergram

of

a

normal

carrier

showing

double

peaks

at

the

point

of

deletion

(arrow)

in

exon

5

of

NPHS
2

gene
.


Patient

Carrier

PCR
-
RFLP

Analysis

to

Confirm

Mutation


Restriction

fragment

length

polymorphism

(RFLP)

analysis

and

confirmation

of

the

mutation

identified

by

sequencing

within

the

exon

5

of

the

NPHS
2

gene

from

the

patient

and

the

parents


The

sequencing

indicated

the

9
-
bp

homozygous

deletion

(
704
del
9
)

in

exon

5

of

the

NPHS
2

in

the

patient
.


The

restriction

enzyme

map

of

this

gene

region

was

analyzed

by

NEBcutter
,

version

2
.
0
,

software
.

PCR
-
RFLP


To

determine

the

presence

of

the

mutation

identified

in

NPHS
2

and

confirm

its

disease
-
association
,

we

amplified

exon

5

of

this

gene

in

all

the

CNS

family

members

and

a

panel

of

100

Pakistani

control

individuals
.


The

PCR

derived

293

bp

fragment

was

then

digested

with

the

restriction

enzyme

XbaI,

and

resolved

on

an

agarose

(
2
.
0
%
)

gel

and

visualized

on

a

UV

transilluminator
.

PCR
-
RFLP for mutation confirmation

(A)
Pedigree of NPHS
family shows
consanguineous
family with three male
patients (filled boxes).

(B) Agarose gel showing
RFLP analysis .

The

293
bp

PCR

product

of

exon

5

for

all

family

members

was

digested

with

XbaI

restriction

enzyme
.

Fragments

were

resolved

on

2
.
0
%

agarose

gel
.

Individuals

with

a

single

fragment

of

284
bp

were

identified

as

homozygous

mutant

since

they

did

not

contain

the

restriction

site

for

XbaI
.

Those

with

two

fragments

(
225
bp

and

68

bp)

were

heterozygous

normal/carriers

for

the

mutation
.

M
:

DNA

size

standard,

UC
:

Undigested

control

PCR

product

Results


While

testing

known

loci

for

NPHS,

strong

linkage

was

obtained

between

the

phenotype

of

NPHS
2

family

and

the

markers

D
1
S
1677
,

D
1
S
1589

and

D
1
S
518

at

chromosome

1
q
25
-
31


This

chromosomal

region

harbors

a

gene

podocin

that

has

previously

been

reported

to

be

associated

with

NPHS
2


On

mutation

screening

of

NPHS
2

gene,

a

homozygous

9
bp

deletion

(
704
del
9
)

in

exon

5

of

NPHS
2

gene

(Figure

1
)

was

found

to

be

segregating

with

CNS

phenotype

in

Pakistani

family

Results
-

Continued


The

704
del
9

deletion

result

in

a

3
-
amino

acid

deletion

(residues

235
-
237
)

of

podocin

protein

product
.


In

exon

5
,

704
del
9

abolishes

the

digestion

for

XbaI

restriction

enzyme
.


To

confirm

mutation

and

screening

of

100

controls,

exon

5

PCR

products

of

NPHS
2

gene

were

digested

with

XbaI
.

It

confirmed

the

presence

of

homozygous

deletion

in

only

the

affected

individuals

(Figure

2
B)
.

Discussion


Mutations

in

the

podocin

gene,

NPHS
2
,

have

been

shown

in

familial

and

sporadic

forms

of

steroid
-
resistant

nephrotic

syndrome

(SRNS)
.



Podocin

is

an

integral

membrane

protein

located

at

the

slit

diaphragm

of

the

glomerular

permeability

barrier



The

genetic

and

clinical

features

of

familial

SRNS

have

not

been

fully

studied

in

Asian

patients


Discussion
-

continued


More

than

60

unique

NPHS
1

mutations

have

been

reported

in

Caucasian

countries

and

NPHS
2

is

mutated

in

26

38
%

of

the

familial

and

10
.
5

23
%

of

the

sporadic

SRNS

in

a

large

European

survey


In

a

study

on

36

Japanese

sporadic

SRNS

cases,

no

NPHS
2

mutation

were

identified,

suggesting

a

significant

ethnic

difference

in

disease

genes



These

observations

point

to

a

need

for

worldwide

study

to

investigate

the

nature

of

disease

genes

in

SRNS

families

outside

Caucasian

countries
.



Identification

of

9
bp

deletion

and

its

association

with

CNS

in

Pakistani

family

is

the

first

of

NPHS
2

gene

involvement

in

any

country

of

Asian

origin


In

Pakistan,

consanguineous

marriages

for

hundreds

of

years

have

resulted

into

the

segregation

of

recessively

inherited

disease

in

large

inbred

families


The

identification

of

pathogenic

NPHS
2

gene

mutation

in

Pakistani

family

may

help

us

in

further

understanding

the

functional

role

of

NPHS
2

gene,

establishing

genotype
-
phenotype

correlation,

more

accurate

disease

diagnosis,

improved

genetic

counseling

and

carrier

screening
.