WT1 GENE MUTATION-A CAUSE FOR

NEPHROTIC SYNDROME IN CHILDREN

Presenting by:-
Praveen Kr Choudhary
Soumya Ranjan Swain
Venkatesh Tripathi
Co-ordinator
External:-Dr.V.Vettriselvi
Asst.Prof ,S.R.M.C
Internal:- Mani megalai
Dr.M.G.Runiversity

PROJECT VENUE-
SRI RAMACHANDRA
UNIVERSITY,CHENNAI
Index
Introduction
Aim of the project
Materials & reagents
Methodology
Remaining work
Expected result
Conclusion
 Introduction
WT1 gene:- wilm’s Tumour gene
* This gene encodes a transcription factor that contains four
zinc-finger motifs at the C-terminus and a
proline/glutamine-rich DNA-binding domain at the N-
terminus. It has an essential role in the normal development
of the urogenital system.
• present on the chromosome no.-11p13
• Description10 exons spanning 48 kb of genomic DNA.
• Transcription3 kb mRNA; four alternative splice forms: +/-
exon 5 and alternative splice donor sites at exon 9.
 Nephrotic syndrome
Nephrotic syndrome is a nonspecific disorder in which
the kidneys are damaged, causing them to leak large
amounts of protein (proteinuria )from the blood into
the urine.
Kidneys affected by nephrotic syndrome have small pores in
the podocytes, large enough to permit proteinuria and
subsequently hypoalbuminemia,hyperlipidemia,edema.
 View of project
Aim of the project Importance of project
WT1 gene mutation a In Indian
cause for Nephrotic population,mutational
analysis in WT1 gene on
syndrome.
nephrotic syndrome has not
Type of mutation & been performed in patients.
response to the Nature & type of mutation &
treatment. its association with the
nephrotic syndrome is
unclear in Indian
population.
Apparatus & Reagents Reagents
Apparatus DNA extraction(KIT method)
Eppendroff -red cell lysing buffer(RCLB)
-tritonX
MicroPippete -nucleated lysing buffer(NLB)
-sodium dodecyl sulphate(SDS),Nacl
Centrifuge -70% ethanol
-RNAse free water
Gel electrophoretic Gel electrophoresis
-agarose
tank -1x TAE buffer
PCR -EtBr(ethidium bromide)
-bromophenol blue
Seqencer 3730 model PCR
-mastermix
Laminar air flow -forward & reverse primer
-buffer,water
Vortex machine -template DNA
Seqenncing
-ready reaction mix
-dilution buffer,water
-template DNA
-primer
 METHODOLOGY
DNA EXTRACTION

Blood sample from the 10 different patients are collected,3ml of blood from each sample
has been taken in centrifuge tube.Add RCLB.make upto 10ml.

Add Triton X(100µl).incubate at 37˚C for 5mins,spin at 2000 rpm for 15 mins.
Discard the supernatant and add RCLB(10 ml).Vortex and mixing.spin at 2000 rpm for 15
mins(step is repeated until it turns white pellete).add 1 ml NLB and 20µl SDS.incubate in
water bath at 55˚C for 1 hr.

Transfer the whole sample into the eppendroff tube (2ml).add 400µl Nacl.spin at
10,000rpm for 10-15 mins.take the supernatant and add to 15 ml tube.Add absolute Alcohol
double the volume of supernatant.

Scoop the DNA,put it in a eppendroff and add 500µl of 70% Ethanol.spin at

10,000rpm.Discard the supernatant .Dry the pellet for 2-3 hrs.Add 100µl of RNAse free
water.
GEL electrophoresis

0.2 gm of Agarose in 25ml 1x TAE buffer.boil for 2min.Add 4µl of EtBr .

Cast the gel

Add Bromophenol blue(1µl) & 10µl of DNA. Mix well & load the molecular ladder by using
micropipette. Run the gel at 50 /100 volts for 30/15 mins.Observe the result in UV
transilluminator.
PCR

Take micro centrifuge tube.& take 6µl of mastermix,add 0.2µl of forward & reverse
primer.add 1.6µl of nuclease free water. At the last add 2µl of template DNA,in each tube.

Set the PCR programe in PCR mechine prior to use.keep the centrifuge tube in PCR
machine & run for the 30 cycles.

Check for the PCR amplification

Perform the gel electrophoresis at 150 volts for 15min,for checking the purity of the PCR
amplification.observe the result in UV transilluminator.
Cycle sequencing reaction
Take micro centrifuge tube.& take 1µl of Ready Reaction mix (2.5X). Add dilution
buffer(5X), add 2µl of forward & reverse primer .Add 4.5µl of nuclease free water. At the last
,add 1µl of template DNA ,in each tube.

Set the programe for Cycle sequencing reaction,run the PCR for 30 cycle.& hold the sample
at 4˚c after the amplification process.

Post sequencing purification method

Add 12µl of master mix 1 to each reaction. Add 52µl of master mix 2 to each reaction.brief
spin.incubate at room temperature for 15mins.spin at 10,000 rpm for 30mins.

Discard the supernatant.add 100µl of 70% ethanol to each sample.spin at 10,000 rpm for 5
mins ,discard the supernatant.
Remaining work & expected result
Results obtained
DNA has been extracted successfully from the nephrotic syndrome patients.
Quality & quantity of the DNA has been checked.it reveals a good quality DNA.
PCR work is done and amplified DNA has been obtained.

Remaining works

Mutational analysis of the DNA sample for WT1 gene.

Expected result

Nature & type of mutation in WT1 gene.

WT1 gene mutation & response to the treatment.
Mutation frequency
THANK YOU