I-TASSER anticipated secondary structure of the wild type GJA8 protein.
Five 3-D models of wild type GJA8 protein on the basis of energy and functional annotation were projected by I-TASSER.
Impact of mutation on the function of GJA8 protein was determined by online tool HOPE.
GJA8 gene code for connexin-50, its expression is exceedingly high in fiber cells, and crucial for maintenance of lens appropriate structure and function (Rong et al., 2002).
Recently, GJA8 gene was knocked down in a rabbit model by aid of CRISPR/Cas9 system at zygote level which revealed the significance of GJA8 in perpetuation of lens normal phenotype (Yuan et al., 2016).
Polymerase chain reaction (PCR) primer sets were designed to sequence the first exons of MAF, EPHA2 , and NHS genes not covered in targeted sequencing, as well as to analyze the segregation of the GJA8 deletion in the family [Supplementary Table 1].
Of the remaining variants with potential function importance (including ten nonsynonymous, 3 splice acceptor and donor site mutations, and 1 coding InDels), only the heterozygous in-frame coding on GJA8 was novel meeting the criteria as nonpolymorphic with an allele frequency <5% in any of the single nucleotide polymorphism database, HapMap, or 1000 genome project database.
This novel heterozygous deletion was concerned with a sequence deletion of 5 amino acids (or 15 bp) at the intracellular loop domain (cytoplasmic loop [CL]) of the Cx50 protein (or GJA8 ) [Figure 1]d, which connects two transmembrane domains and was suspected to serving as binding sites of Ca [sup]2+/CaM in gap junction channel function.[sup], Mutations on GJA8 have caused multiplex ADCCs (Cataract 1, multiple types, OMIM#116200) including nuclear cataracts.
Cx50 ( GJA8 ) and Cx46 ( GJA3 ) are the major components of mammalian lens fiber cells, and mutations of these two genes account for approximately 20% of nonsyndromic familial cataract cases.[sup] The typical structure of Cx includes a cytoplasmic N-terminal domain (NT), four transmembrane domains (TM1 to TM4), two extracellular loops (E1 and E2), a CL between TM2 and TM3, and a C-terminal domain (CT).
Third, the cataract morphology of the affected members and inheritance pattern of the cataracts in the family were compatible with those of cataract caused by GJA8 mutations.