Novel Mutations in the Connexin 43(GJA1) may contribute to Nonsyndromic Hearing Loss. - PowerPoint Presentation

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Novel Mutations in the Connexin 43(GJA1) may contribute to Nonsyndromic Hearing Loss.

By

Asha

Kiran Akula

Master of Research

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Gap Junctions

Intercellular communication channels.

Gap junctions allow the selective permeability to ions and small molecules.

Movement through these channels is passive and non specific.

Gap junctions are made up of clusters of closely packed connexons

The structural unit of gap junction is Connexon.

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Connexon

Consist of pairs of transmembrane channels.

The connexon hemi channel in one cell membrane docks with a connexon hemi channel in an adjacent cell.

Hexameric: they consist of arrays of 6 connexin protein subunits

Different connexin isoforms have been identified.

Homomeric or heteromeric

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Functions

Gap junctions involve in regulation of

Tissue homeostasis

Regulation of cell growth

Embryonic development

Electrical and metabolic coupling

The loss of connexins, or the existence of mutations affecting their normal functions, has been implicated in a variety of diseases and disorders, including cancers.

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Gap Junctions play a major role in intercellular calcium

signalling

.

Gap

junctional

opening is controlled by the intracellular concentration of calcium.

The low intracellular calcium concentration enables the gap junction channels to open and vice versa.

Controlled gating of gap junction channels may be responsible for the normal functioning of the cell.

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Connexins

Show overlapping tissue expression patterns, most tissues expressing more than one connexin type.Each connexin contains 4 TM domains, with two extracellular and three cytoplasmic regions.

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Both N- and C-termini –face the cytoplasm

The third TM domain -

amphipathic

in nature

forms the lining of the formed channel.

Amino acid sequence identity between the isoforms is ~50-80%, with the TM domains being well conserved.

Both extracellular loops – contain conserved

cysteine

residues, which likely form

intramolecular

disulphide bonds.

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Single putative intracellular loop (between TM domains 2 and 3) and the

cytoplasmic

C terminus are highly variable among the family members.

Six connexins associate to form a hemi-channel, or connexon. Two connexons then interact (likely via the extracellular loops of their connexins) to form the complete gap junction channel.

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Connexin Gene

Multigene

family comprising 20 in mouse and 21 genes in human genome (Cardiovascular Research, 2010).

α and

β

gene families.

The "

Gja

/

Gjb

" nomenclature-adopted by the NCBI data base.

Cells express multiple types of connexin-potentially associate to form gap junction channels containing more than one type of connexin.

 

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Mutations

Alterations in the gap junction,

hemichannel

, or general functions of the connexins.

Cause various human diseases like skin diseases,

nonsyndormic

and

syndromic

deafness, cataracts,

Oculodentodigital

Dysplasia (ODDD), cancers etc.

Mutations in Cx32 - Charcot-Marie-Tooth disease. Cx26 -deafness and skin disease.

Cx30, Cx30.3, and Cx31- hearing loss and skin disorders.

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Hearing Loss

Affects 1 in 1000 newborns.

Syndromic and nonsyndromic

70% of genetically related hearing loss-nonsyndromic.

Two types-

Conductive hearing loss

Sensorineural Hearing Loss

Mutations in Cx26- common cause of congenital bilateral non-syndromic sensorineural hearing loss.

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Hearing

The ear is made up of three different sections:

1.the outer ear

External auditory canal

Tympanic

membrabe

2.the middle ear-bones

3.the inner ear

Cochlea

Vestibular system.

 

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Cochlea

Main auditory portion of the inner ear.

Core component-organ of

corti

Organ of

corti

- sensory organ of hearing.

Organ of

corti

comprises - hair cells

supporting cells

Endolymph

Hair cells-two types

Inner hair cells

Outer hair cells

Connexins are present in supporting cells.

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Sound waves

Tympanic membrane(Outer ear)

Bones maleus, incus and stapes(middle ear)

Movement of stapes

Pressure waves (fluid filled inner ear)

hairs to move in the inner ear

stimulate the auditory nerve

General mechanism of hearing

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Organ of Corti

Outer hair cells

Inner hair cells

Tectorial

membrane

Basilar membrane

Supporting cells

Spiral

Limbus

Stria

Vascularis

Endolymph

Epithelial tissue

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CX43- Four predicted membrane-spanning segments (M1–M4) linked by two extracellular (E1–E2)

and one cytoplasmic loop. Amino and carboxyl tails faced intracellularly.Expressed in non sensory epithelial cells of the inner ear.

CX43

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Three novel missense mutations have been identified in the

GJA1 gene -

related to hearing loss.

The three missense mutations

c.205T>C (p.S69P) - Extracellular loop

c.932delC - C-terminal region

c.977C>T (p.T326I) - C-terminal

cytoplasmic

domain.

Studied the

intracellular distribution,

assembly and

the effects of the three Cx43 mutants with the wild type Cx43.

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Plasmid construction with the mutationsPermanent transfected HeLa-CX43 cell lineImmunostainingDye Transfer

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Plasmid

was constructed using CX43WT

cDNA

and cloned into pCDNA3.1 vector

Constructed plasmid-expression in HeLa cells via transfection-

lipofectamine

method.

Addition of G418- isolation of stable

transfectants

RT-PCR

– success of transfection and expression of transfected genes.

PCR products- Gel electrophoresis

CX43WT-Positive control.

HeLa and water-Negative control

β

-

actin

-internal control

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Expression analysis of

GJA1 mRNA in four stable transfectedHeLa cells by RT-PCR. RT-PCR analysis of total RNA from HeLacells expressing CX43 WT, CX43 S69P, CX43 932delC and CX43T326I confirms expression of the corresponding mRNAs in stablytransfected HeLa cell lines (upper panel). -actin served as a reference for the loading amount of total RNA for each sample (lower panel).Mock HeLa and water were used as negative controls

Electrophoresis -Results

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confirm the expressed CX43 mutant proteinsPrimary antibody- monoclonal anti-CX43 antibodySecondary antibody- HRP-conjugated anti-mouse IgGGAPDH-Internal ControlMock HeLa cells- Negative control

Western Blot-Results

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Immunostaining

Transfected cells - washed and fixed.

Primary antibodies

Mouse anti-pan-

cadherin

antibody (anti-CH19) – cell membrane

Mouse anti-CX43-epitope of CX43 protein

Secondary Antibodies

Alexa

Fluor 488 and

Alexa

Flour 594

Nuclei-stained with DAPI

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Immunostaining -Results

Localization analysis of CX43 WT in stably transfected HeLa cells by

immunocytochemistry

using anti-CX43 and pan-

cadherin

antibody. Analysis of fluorescence microscopy on HeLa cells expressing CX43 WT reveals localization of the CX43 protein in the

plasmamembranes

. CX43 proteins are indicated by

arrows. The cells were

counterstained with 4-6-diamidino-2-phenylindole, DAPI, to highlight the nuclei.

Scale bars 10 m

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Dye Transfer

Functionality of gap junction formed.

Transfected HeLa cells-microinjected with Lucifer Yellow.

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Cell line Dye-filled Number of Total number neighbor injections of cell (n)showing cell number (n) dye transfer (mean ± SE)HeLa-Cx43 WT 4.17 ±1.621 30 100HeLa-Cx43 S69P 0 30 0HeLa-Cx43 T326I 0 30 0HeLa-Cx43 A311V 0.54 ±1.471 50 86HeLa 0 30 0

Lucifer Yellow transfer stably expressed with WT or mutant

Cx43 HeLa cells

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D

iscussion

With the above results, these three mutations-risk factor for the development of hearing.

Three mutations-loss of function of CX43 –hearing

loss.

CX43WT- found localized to the cell membranes at the point of contact between adjacent expressing cells.

Membrane localization-confirmed by

colocalization

with pan-

cadherin

.

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