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.
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.
To acknowledge above data, we screen GJA8 gene of 27 cataract patients with no family history of cataract, a subtle 1104G>C (pE368Q) (GenBank KY556641) transversion that substitutes glutamic acid to glutamine was identified at exon 2 of GJA8 gene in one of the patient, which revealed that glutamic acid at position 368 in normal GJA8 protein was changed to glutamine, which is highlighted in Figure 6.
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.
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.