Cx26

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



Deafness

connexin26»»Result & Discussion Result & Discussion



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






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.






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%






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.






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


(32) (27)

(6) (8) (1/2)

136-3 136-4 136-5

136-2136-1


(38)(28)

(12) (10) (8)

138-2 138-3

138-1


(42) (38)

(8)(6)

137-6

137-5

137-4

137-3

137-2


32 23

5 2 2137-1

(83)

(57) (44)

(24) (22) (20) (17)

139-1

139-2

139-3

139-4

139-5


(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




Samples

Isolation of DNA

Dissolving of DNA

Screening W24X

Screening W77X,Q124X & 35delG

»»Result & Discussion Result & Discussion


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.



Isolation of DNA

Dissolving of DNA

Screening W24X



Samples

»»



▪ 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



Isolation of DNA

Dissolving of DNA

Screening W24X



Samples

»»



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



Isolation of DNA

Dissolving of DNA

Screening W24X



Samples

»»



▪ RFLP

▪ PCR conditions

Materials & Methods



Isolation of DNA

Dissolving of DNA

Screening W24X



Samples

»»



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



Isolation of DNA

Dissolving of DNA

Screening W24X



Samples

»»



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






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






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

Cx26

Documents

rbc lysis

methods samples

hearing loss

family code

hearing amp

dna dissolving

family members

restriction