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CONNEXIN 26
MUTATION
in hearing impaired
families
External
Internalguide:
guide:
Dr.
Mr.
C.R.
R.Balachander
SRIKUMARI SRISAILATHY
M.Phil
UGC
Dept.
Research
of biotechnology
Scientist – B
Dept
Prathyusha
of Genetics
Engg. College.
Institute of Basic Medical Sciences (IBM
Balaji. A
Chennai
Introduction
▪ Objective
Introduction
Literature
Hearing loss is a common sensory disorder in the human population.
The incidence of congenital hearing loss is estimated at 1 in 1000
aterials & Methods To births.
screen for W24X, W77X, Q124X and 35delG mutations in
esult & Discussion connexin26 ( GJB2 gene) in hearing impaired families of Thiruvallur
Dist.
Of which appropriately equal numbers of case are attributed to
Conclusion
environmental and genetic factors
To compare with the general agrees these mutation with the trend in
India.
The hearing disorder attributed to genetic causes, approximately
70% are classified as nonsyndromic and remaining 30% as
To syndromic.
analyze the results for genetic counseling.
Literature
Literature
» connexin26
Deafness
Any mutation in GJB2 gene (location 13q11) will interfere the recycling of
K+ ions, which results in deafness.
Literature
▪ Mechanism
Hair Cells of Hearing & Role of Cx26 in it
Introduction
Literature
» connexin26
Deafness
Conclusion
» Deafness
Syndromic
Hearing Loss
aterials & Methods Alport Norrie
Pendred Usher
esult & Discussion Waardenburg
Branchio-Oto- Renal
Conclusion Jervell and Lange-Nielsen
~50% 30%
Non-syndromic
Genetic ~22% Autosomal Dominant
(DFNA1-DFNA54)
Literature
connexin26
» Deafness
»
esult & Discussion
Samples 5 (83) (36)
136-1
2
136-2 (30)
2
134-1 134-2
Conclusion Total137-1
no. of families : 7
Isolation of DNA
(22) (27) (23) (20) (25)
Samples Total no.(12)
individuals
139- (5) : 30 139-
139-
(10) 139-
(8)
Dissolving of DNA 2 3 134-3 4 5
(blood
2
collected)
3 136-3 136-4 136-53 2
FAMILY
FAMILY CODE:
CODE: ZTVR
ZPON 84 135
Screening W24X
Total no. of affected: 16
FAMILY
(57) CODE: ZTVR 138
(44)
Screening W77X, 137-2 137-
Q124X & 35delG (32) 3 (27)
(52) 135-1 (40)
135-2
1
(24) (22) (20) (17)
137- 137- 137-
(20) (17) (15)
(6) 4 (8) 5 (6) 6
(8) 84-3 84-4 84-5 (1/2)
135-3 135-5
138-2 138-3
Materials & Methods
▪ Protocol
Pedigrees
Reagents
Introduction 1. 5-10 ml of peripheral blood was collected in a vacutainer tube containing
Literature 1 liquid EDTA
RBC lysis and was
buffer centrifugedChloride
: Ammonium for 25 min
7 g/lat ,3000 rpm.
Ammonium
2. The supernatant was bicarbonate
discarded 70 andmg/l
the buffy coat was transferred into a
aterials & Methods
2 sterile 50 ml
Cell Lysis conical
buffer : 1M centrifuge tube.
Tris, 0.5M Finalandvolume
EDTA, waspH
10% SDS, brought to 50 ml
Samples using RBC lysis buffer. 8.2.
3.3 Blood with RBC lysis
5M Ammonium : 19.buffer was placed
27 mg/50 ml. on ice (4°C) for 30 mins and was
» Isolation of DNA acetate for every 10min.
inverted
4.4 This
TE buffer : 10 mM
was spinned down Tris rpm
at 3500 and 1for
mM 10EDTA,
min atpH4°C8.0.and the supernatant
Dissolving of DNA
was discarded. The step was repeated until WBC’s pelleted without RBC’s.
5. The WBC pellet was then suspended in 5 ml of cell lysis buffer and was
Screening W24X
mechanically sheared to break the clumps. This was done until the DNA
Screening W77X, released from WBC which was indicated by viscosity of the solution.
Q124X & 35delG 6. 2.5 ml of 5M ammonium acetate was added to the solution and the tube was
inverted for 5 min to precipitate proteins out of the solution.
esult & Discussion 7. This was centrifuged at 3500 rpm for 10 min at 4°C and the supernatant was
» Screening W24X
Primer Primer Sequence
Alu I restriction
Amplico PCR
type enzyme n length Cycles
Screening W77X,
Q124X & 35delG GG CT AG CT
Wild type W24X mutant
Forward
CC GA 5’-TCTTTT CCA GAG CAA ACC GC-3’ 286 30 TC GA
Reverse 5’-GAC ACG AAG ATC AGC TGC AGG-3’
esult & Discussion
( 286 bp)
Conclusion
Mutation Initial Denaturation Annealing Extension Final Extension
Denaturation
( 184 & 102bp)
W24X 95°C-5min 95°C-40 sec 65°C-40sec 72°C-30sec 72°C-2min
Materials & Methods
▪▪ PCR
RFLPconditions
Introduction
Literature
aterials & Methods The PCR product was digested in a 10µl reaction volume which
Samples consist of 2.0µl of 10X buffer, 2.5 U of Alu I restriction enzyme.
Isolation of DNA
The reaction volume was incubated at 37oC for 16 hours.
Dissolving of DNA
» Screening W24X
Screening W77X,
Q124X & 35delG
Conclusion
Materials & Methods
▪▪▪PCR
Primer
Overview
RFLP sequences
conditions
Introduction
Literature
10X PCR
Allele buffer
Specific Oligonucleotide :- Polymerase
2.0 µl Chain Reaction [ASO-PCR]
MgCl2 : 2.5 mM
aterials & Methods dNTPs : 200 µM
Primer
Samples Genomic DNA
Mutation Effect Primer Sequence Amplicon PCR
forward :0.4 µmol length Cycles
Isolation of DNA
Reverse
G-to-A W77X Nor : 0.4 µmol
5’- TACTTCCCCATCTCCCACATCCGGCTATTG-3’ 234 bp 30
Taq
bp 231 polymerase Mut : 0.5 U
5’-TACTTCCCCATCTCCCACATCCGGCTATTA-3’
Com 5’- GATGACCCGGAAGAAGATGCTGCTTGTGTA- 3’
Dissolving of DNA
Template DNA : 50-100
Amplificationng
of Exon1 of GJB2 gene ( chr. 13q12 )
G 35delG Nor 5’-TTGGGGCACGCTGCAGACGATCCTGGGGAG-3’
using ASO-PCR 202 bp 30
deletion Mut 5’- TTGGGGCACGCTGCAGACGATCCTGGGGAT -3’
bp 35 Com 5’- GAAGTAGTGATCGTAGCACACGTTCTTGCA-3’
Screening W24X
C-to-T
MutationQ124X
bp 370
InitialNor 5’-GAATTTAAGGACATCGAGGAGATCAAAACAC-3’
Denaturation Annealing Extension
Mut 5’-GAATTTAAGGACATCGAGGAGATCAAAACAT-3’
210 bp
Final 30
»
Mutant + common
Screening W77X, Denaturation
Com 5’ GACACAAAGCAGTCCACAGTGTTGGGACAA–3’
primer
Extension
Normal + common
Q124X & 35delG W77X
primer
95°C-5min 95°C-40sec 66°C-30sec 72°C-30sec 72°C-3min
Literature
Conclusion
ZPON84-3 ZPON84-4 ZPON84-5
286 bp
184 bp
102 bp
HET HET
Result & Discussion
L HOMO HOMO NOR
L - 100 bp LADDER
HET -HETEROZYGOUS
HOMO -HOMOZYGOUS
NOR - NORMAL
Conclusion
Introduction
Phenotype – Genotype Correlation of ZPON84 Family
Literature
Genotype
aterials & Methods S
Family ID Age/gender Phenotype
.No.
esult & Discussion W24X W77X 35delG Q124X
1 ZPON84-1 52/M Normal Heterozygous - - -
Conclusion
2 ZPON84-2 40/F Normal Heterozygous - - -
Bilateral Homozygous
3 ZPON84-3 20/M - - -
profound Mutant
Bilateral Homozygous
4 ZPON84-4 17/M - - -
Profound Mutant
5 ZPON84-5 15/F Normal Normal - - -