cx26
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Screening for
CONNEXIN 26 MUTATION in hearing impaired families
Balaji. A
External guide:Dr. C.R. SRIKUMARI SRISAILATHYUGC Research Scientist – BDept of GeneticsInstitute of Basic Medical Sciences (IBMS)Chennai
Internal guide:Mr. R.Balachander M.PhilDept. of biotechnologyPrathyusha Engg. College.
▪ Objective
Introduction
Introduction
Hearing loss is a common sensory disorder in the human population. The incidence of congenital hearing loss is estimated at 1 in 1000 births.
Of which appropriately equal numbers of case are attributed to environmental and genetic factors
The hearing disorder attributed to genetic causes, approximately 70% are classified as nonsyndromic and remaining 30% as syndromic.
Mutations in Connexin26 (encoded by GJB2 gene) have been established as a major cause(50%) of inherited non syndromic deafness in different populations.
In India population, W24X is the major mutation(87% ) found in the GJB2 gene.
To screen for W24X, W77X, Q124X and 35delG mutations in connexin26 ( GJB2 gene) in hearing impaired families of Thiruvallur Dist.
To compare with the general agrees these mutation with the trend in India.
To analyze the results for genetic counseling.
IntroductionIntroduction
LiteratureLiterature
Result & Discussion Result & Discussion
Materials & Methods Materials & Methods
Conclusion Conclusion
▪ Structure , location & function
Literature
Literature
IntroductionIntroduction
LiteratureLiterature
Deafness
connexin26»»Result & Discussion Result & Discussion
Materials & Methods Materials & Methods
Conclusion Conclusion
1. Gap junctions contain channels that connect neighboring cells.
2. They are relatively nonspecific, and the molecular movement through the channels occurs by passive diffusion.
3. 26 in connexin26 represents its molecular weight.
Molecular Models for Connexin26 Topology
▪ Structure , location & function
▪ Mechanism of Hearing & Role of Cx26 in it
Literature
IntroductionIntroduction
LiteratureLiterature
Result & Discussion Result & Discussion
Materials & Methods Materials & Methods
Conclusion Conclusion
Deafness
connexin26»»
Expression of Cx26 in the epithelial network of cochlear cells involved in recycling of K+ ions between the fluids of inner ear (organ of corit).
Any mutation in GJB2 gene (location 13q11) will interfere the recycling of K+ ions, which results in deafness.
▪ Mechanism of Hearing & Role of Cx26 in it
▪ Hair Cells
Literature
The most likely model for hair cell function proposes that deflection of the sterocilia pulls on fine links that join adjacent sterocilia at their tips.
The tips link acts as a gating spring to open one or more transduction channels, allowing cations ( k+, Ca 2+ ) to flood into the cell and depolarize it.
IntroductionIntroduction
LiteratureLiterature
Result & Discussion Result & Discussion
Materials & Methods Materials & Methods
Conclusion Conclusion
Deafness
connexin26»»
▪ Recycling of k+ ions
▪ Classification of Etiologies
Literature
<1%
Hearing Loss
~50%
~50%
Environmental
Genetic
Ototoxic drugsAcoustic traumaBacterial infectionsViral infections
30%
70%
Non-syndromic
Autosomal Dominant(DFNA1-DFNA54)
Autosomal Recessive(DFNB1-DFNA67)
X-Linked (DFN1-DFN8)
Mitochondrial~1%
Alport NorriePendred UsherWaardenburgBranchio-Oto- RenalJervell and Lange-Nielsen
Syndromic
~22%
~77%
IntroductionIntroduction
LiteratureLiterature
Result & Discussion Result & Discussion
Materials & Methods Materials & Methods
Conclusion Conclusion
Deafness
connexin26
»»
▪ Classification of Etiologies
▪ Non-syndromic hearing loss
Literature
Connexin26 ( GJB2 gene ) contribute to both autosomal dominant (Locus: DFNA3) and recessive ( Locus: DFNB1) nonsyndromic hearing loss.
IntroductionIntroduction
LiteratureLiterature
Result & Discussion Result & Discussion
Materials & Methods Materials & Methods
Conclusion Conclusion
Deafness
connexin26
»»
Mutations in the Connexin 26 gene. Highlighted mutations are focused in this present study Mutation name Nucleotide change Codon Amino acid change Domain
-3170G -3170G>A ---- Splice site None
M1V 1AG 1 MetVal IC1
31del14 del of 14 nt at 31 11-15 Frameshift IC1
31del38 del of 38 nt at 31 11-23 Frameshift IC1
G12V 35GT 12 GlyVal IC1
35delG del of G at 30-35 10-12 Frameshift IC1
35insG ins of G at 30-35 10-12 Frameshift IC1
51del 12insA del of 12 nt at 52 17-21 Frameshift IC1
S19T 56GC 19 SerThr IC1
W24X 71GA 24 TrpStop TM1
M34Ta 101TC 34 MetThr TM1
V37Ib 109GA 37 ValIle TM1
W44C 132GC 44 TrpCys EC1
W44X 132GA 44 TrpStop EC1
G45E 134GA 45 GlyGlu EC1
E47X 139GT 47 GluStop EC1
167del T del of T at 167 56 Frameshift EC1
Q57X 169CT 57 GlnStop EC1
G59A 176G 59 GlyAla EC1
176-191del 16 del of 16 nt at 176 59-64 Frameshift EC1
Y65X 195CG 65 TyrStop EC1
D66H 196GC 66 AspHis EC1
R75W 223TG 75 ArgTrp EC1
W77R 229TC 77 TrpArg TM2
W77X 231GA 77 TrpStop TM2
235del C del of C at 233-235 78-79 Frameshift TM2
Mutation name Nucleotide change Codon Amino acid change Domain
V84L 250GC 84 ValLeu TM2
L90P 269TC 90 LeuPro TM2
269ins T Ins of T at 269 90 Frameshift TM2
V95M 283GA 95 ValMet IC2
R98Q 293GA 98 ArgGln IC2
H100Yc 298CT 100 HisTyr IC2
299-300del AT del of AT at 299 100 Frameshift IC2
314del 14d del of 14 nt at 314 104-110 Frameshift IC2
333-334del AA del of AA at 333-335 111-112 Frameshift IC2
S113R 339G 113 SerArg IC2
358-360del GAGe del of GAG at 358 120 Del of Glu 120 IC2
K122I 339TG 122 LysIle IC2]
Q124X 370CT 124 GlnStop IC2
R127H 380GA 127 ArgHis IC2
Y136X 408CA 136 TyrStop IC2
R143W 427CT 143 ArgTrp IC2
509insA ins of A at 509 170 Frameshift TM3
P175T 523CT 175 ProThr EC2
R184P 551GC 184 ArgPro EC2
S199F 596T 199 SerPhe EC2
631-632del GT del of GT at 631-632 210 Frameshift IC3
Diagrammatic representation of the Connexin 26 protein traversing the membrane. Mutations of Cx26 are also showed. Mutations in Red Colour are focused in this study
▪ Pedigrees
Materials & Methods
Materials & Methods
Samples
Total no. of families : 7
Total no. individuals : 30(blood collected)
Total no. of affected: 16
134-1 134-2
134-3
FAMILY CODE: ZTVR 134
(36)(30)
(5)
135-3
135-2135-1
135-5
FAMILY CODE: ZTVR 135
(32) (27)
(6) (8) (1/2)
136-3 136-4 136-5
136-2136-1
FAMILY CODE: ZTVR 136
(38)(28)
(12) (10) (8)
138-2 138-3
138-1
FAMILY CODE: ZTVR 138
(42) (38)
(8)(6)
137-6
137-5
137-4
137-3
137-2
FAMILY CODE: ZTVR 137
32 23
5 2 2137-1
(83)
(57) (44)
(24) (22) (20) (17)
139-1
139-2
139-3
139-4
139-5
FAMILY CODE: ZTVR 139
(50)
(22) (27) (23) (20) (25)
FAMILY CODE: ZPON 84
84-1 84-2
84-3 84-4 84-5
(52) (40)
(15)(17)(20)
connexin26
IntroductionIntroduction
LiteratureLiterature
Materials & Methods Materials & Methods
Samples
Isolation of DNA
Dissolving of DNA
Screening W24X
Screening W77X,Q124X & 35delG
»»Result & Discussion Result & Discussion
Conclusion Conclusion
1. 5-10 ml of peripheral blood was collected in a vacutainer tube containing liquid EDTA and was centrifuged for 25 min at 3000 rpm.
2. The supernatant was discarded and the buffy coat was transferred into a sterile 50 ml conical centrifuge tube. Final volume was brought to 50 ml using RBC lysis buffer.
3. Blood with RBC lysis buffer was placed on ice (4°C) for 30 mins and was inverted for every 10min.
4. This was spinned down at 3500 rpm for 10 min at 4°C and the supernatant 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 mechanically sheared to break the clumps. This was done until the DNA released from WBC which was indicated by viscosity of the solution.
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.
7. This was centrifuged at 3500 rpm for 10 min at 4°C and the supernatant was carefully transferred to 15 ml conical tube containing 5 ml of isopropanol.
8. The tube was gently inverted, until the solution losses its high viscosity, to precipitate DNA. The pellet was then transferred to a 1.5 ml micro centrifuge tube containing 70% ethanol, spinned down and dissolved in TE
buffer.
▪ Protocol
▪ Pedigrees
▪ Reagents
Materials & Methods
1 RBC lysis buffer : Ammonium Chloride 7 g/l , Ammonium bicarbonate 70 mg/l
2 Cell Lysis buffer : 1M Tris, 0.5M EDTA, and 10% SDS, pH 8.2.
3 5M Ammonium acetate
: 19. 27 mg/50 ml.
4 TE buffer : 10 mM Tris and 1 mM EDTA, pH 8.0.
IntroductionIntroduction
LiteratureLiterature
Isolation of DNA
Dissolving of DNA
Screening W24X
Screening W77X,Q124X & 35delG
Materials & Methods Materials & Methods
Samples
»»
Result & Discussion Result & Discussion
Conclusion Conclusion
▪ Protocol
Materials & Methods
Cottony mass of DNA stored in 70 % EtOH at the end of DNA isolation↓
Vortex↓
Centrifuged at 13000 rpm for 2 min↓
Supernatant discarded↓
To the pellet 70% of alcohol ( 200 µl) added↓
Vortex↓
Cf. at 13000 rpm for 2 min↓
Discard the supernatant↓
To the pellet, add 200 µl of 100% EtOH↓
↓Vortex
↓Cf. at 13000 rpm for 2 min
↓Discard the supernatant
↓Dry the pellet
↓Add TE*
↓Incubate 1 hr at 65oC ( water bath)
↓Incubate at 37oC2 for 2-3 hrs
IntroductionIntroduction
LiteratureLiterature
Isolation of DNA
Dissolving of DNA
Screening W24X
Screening W77X,Q124X & 35delG
Materials & Methods Materials & Methods
Samples
»»
Result & Discussion Result & Discussion
Conclusion Conclusion
Genomic DNA
Amplification of Exon2 of GJB2 gene ( chr. 13q11 ) using PCR
Exon2 (286 bp)
Alu I restriction enzyme
Wild type W24X mutant
( 286 bp)
( 184 & 102bp)
▪ Protocol
▪ PCR conditions
▪ Overview
Materials & Methods
10X PCR buffer : 2.0 µlMgCl2 : 2 mMdNTPs : 200 µMPrimer forward :2.5 pmol Reverse : 2.5 pmolTaq polymerase : 0.5 UTemplate DNA : 50-100 ng
Mutation Initial Denaturation
Denaturation Annealing Extension Final Extension
W24X 95°C-5min 95°C-40 sec 65°C-40sec 72°C-30sec 72°C-2min
AG CTTC GA
GG CTCC GA
Primer sequences:
Primer type
Primer Sequence Amplicon length
PCR Cycles
Forward Reverse
5’-TCT TTT CCA GAG CAA ACC GC-3’5’-GAC ACG AAG ATC AGC TGC AGG-3’
286 30
IntroductionIntroduction
LiteratureLiterature
Isolation of DNA
Dissolving of DNA
Screening W24X
Screening W77X,Q124X & 35delG
Materials & Methods Materials & Methods
Samples
»»
Result & Discussion Result & Discussion
Conclusion Conclusion
▪ RFLP
▪ PCR conditions
Materials & Methods
IntroductionIntroduction
LiteratureLiterature
Isolation of DNA
Dissolving of DNA
Screening W24X
Screening W77X,Q124X & 35delG
Materials & Methods Materials & Methods
Samples
»»
Result & Discussion Result & Discussion
Conclusion Conclusion
The PCR product was digested in a 10µl reaction volume which
consist of 2.0µl of 10X buffer, 2.5 U of Alu I restriction enzyme.
The reaction volume was incubated at 37oC for 16 hours.
▪ RFLP
▪ Primer sequences
▪ Overview
▪ PCR conditions
Materials & Methods
IntroductionIntroduction
LiteratureLiterature
Isolation of DNA
Dissolving of DNA
Screening W24X
Screening W77X,Q124X & 35delG
Materials & Methods Materials & Methods
Samples
»»
Result & Discussion Result & Discussion
Conclusion Conclusion
Genomic DNA
Amplification of Exon1 of GJB2 gene ( chr. 13q12 ) using ASO-PCR
Mutant + common primer
Normal + common primer
Amplification for wild type only
Amplification if the mutation is present
Allele Specific Oligonucleotide - Polymerase Chain Reaction [ASO-PCR]10X PCR buffer : 2.0 µlMgCl2 : 2.5 mMdNTPs : 200 µMPrimer forward :0.4 µmol Reverse : 0.4 µmolTaq polymerase : 0.5 UTemplate DNA : 50-100 ng
Mutation Effect Primer Sequence Amplicon length
PCR Cycles
G-to-Abp 231
W77X Nor 5’- TACTTCCCCATCTCCCACATCCGGCTATTG-3’Mut 5’-TACTTCCCCATCTCCCACATCCGGCTATTA-3’Com 5’- GATGACCCGGAAGAAGATGCTGCTTGTGTA- 3’
234 bp 30
G deletionbp 35
35delG Nor 5’-TTGGGGCACGCTGCAGACGATCCTGGGGAG-3’ Mut 5’- TTGGGGCACGCTGCAGACGATCCTGGGGAT -3’Com 5’- GAAGTAGTGATCGTAGCACACGTTCTTGCA-3’
202 bp 30
C-to-Tbp 370
Q124X Nor 5’-GAATTTAAGGACATCGAGGAGATCAAAACAC-3’Mut 5’-GAATTTAAGGACATCGAGGAGATCAAAACAT-3’Com 5’ GACACAAAGCAGTCCACAGTGTTGGGACAA–3’
210 bp 30Mutation Initial Denaturation
Denaturation Annealing Extension Final Extension
W77X 95°C-5min 95°C-40sec 66°C-30sec 72°C-30sec 72°C-3min
Q124X 95°C-5min 95°C-40sec 66°C-30sec 72°C-30sec 72°C-3min
35delG 95°C-5min 95°C-40sec 66°C-30sec 72°C-30sec 72°C-3min
▪ Gel Photos
Result & Discussion
IntroductionIntroduction
LiteratureLiterature
Result & Discussion Result & Discussion
Materials & Methods Materials & Methods
Conclusion Conclusion
Result & Discussion
ZPON84-1 ZPON84-2
ZPON84-3 ZPON84-4 ZPON84-5
HET HET L HOMO HOMO NOR
L - 100 bp LADDER
HET -HETEROZYGOUS
HOMO -HOMOZYGOUS
NOR - NORMAL
286 bp184 bp102 bp
Gel photograph showing mutational status of individuals belonging to family ZPON84.
Conclusion
IntroductionIntroduction
LiteratureLiterature
Result & Discussion Result & Discussion
Materials & Methods Materials & Methods
Conclusion Conclusion
Conclusion
S.No.
Family ID Age/gender Phenotype
Genotype
W24X W77X 35delG Q124X
1 ZPON84-1 52/M Normal Heterozygous - - -
2 ZPON84-2 40/F Normal Heterozygous - - -
3 ZPON84-3 20/MBilateral profound
HomozygousMutant
- - -
4 ZPON84-4 17/MBilateral Profound
Homozygous Mutant
- - -
5 ZPON84-5 15/F Normal Normal - - -
Phenotype – Genotype Correlation of ZPON84 Family
Among the remaining six families (ZTVR134, ZTVR135, ZTVR136, ZTVR137, ZTVR138 and ZTVR139) all the affected screened in the first phase tested negative for all the four common mutations screened. Figure 4.5 shows absence of W24X in these families. Hence the family members were not further tested. Screening for other known mutations would explain their etiology.
THANK YOU I Profusely Thank All The Probands And Family Members
Who Participated In This Study And Made This Work A Reality
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