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

1. Novel Mutations in the Connexin 43(GJA1) may contribute to Nonsyndromic Hearing Loss. By Asha Kiran Akula Master of Research

2. 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.

3. 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

5. 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.

6. 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.

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

12. 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.

13. 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.

15. 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.

17. General mechanism of hearing Bones maleus, incus and stapes (middle ear) Tympanic membrane (Outer ear) Sound waves stimulate the auditory nerve Movement of stapes Pressure waves (fluid filled inner ear) hairs to move in the inner ear

18. Organ of Corti Endolymph Stria Vascularis Tectorial membrane Outer hair cells Inner hair cells Spiral Limbus Supporting cells Basilar membrane Epithelial tissue

19. CX43 • 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.

20. 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.

21. Plasmid construction with the mutations Permanent transfected HeLa-CX43 cell line Immunostaining Dye Transfer

22. Plasmidwas 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

23. Electrophoresis -Results Expression analysis of GJA1 mRNA in four stable transfected HeLa cells by RT-PCR. RT-PCR analysis of total RNA from HeLa cells expressing CX43 WT, CX43 S69P, CX43 932delC and CX43 T326I confirms expression of the corresponding mRNAs in stably transfected 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

24. Western Blot-Results • confirm the expressed CX43 mutant proteins • Primary antibody- monoclonal anti-CX43 antibody • Secondary antibody- HRP-conjugated anti-mouse IgG • GAPDH-Internal Control • Mock HeLa cells- Negative control

25. 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

26. 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

28. Dye Transfer • Functionality of gap junction formed. • Transfected HeLa cells-microinjected with Lucifer Yellow.

29. Lucifer Yellow transfer stably expressed with WT or mutant Cx43 HeLa cells 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 30 100 HeLa-Cx43 S69P 0 30 0 HeLa-Cx43 T326I 0 30 0 HeLa-Cx43 A311V 0.54 ±1.471 50 86 HeLa 0 30 0

30. Discussion 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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