Primary microcephaly families mapped with different microcephalic genes by using whole exome sequencing; Insilco 3D Model's prediction of STIL, CENPJ, and CEP135 protein

Abstract

BackgroundPrimary microcephaly (MCPH- OMIM 251200) is inherited from an autosomal recessive, heterogeneous, non-progressive, and neurodevelopmental disorder. It is considered by small occipital-frontal circumference (OFC) for a person's age and gender at birth. MethodsPrimary microcephaly affected consanguineous families (A, B, C, D) in various remote villages of Punjab, Pakistan were identified. Blood samples were collected followed by we used salting-out method for DNA extraction. WES (whole exome sequencing) was used to analyze the mutations and filtered variants that were confirmed by sanger sequencing. ResultsWhole-exome sequencing revealed a sequence variant c.453 + 5G > A in intron 5 of the STIL gene that generated a frameshift and truncated protein (p. Asp89Glyfs*8) by introducing a premature stop codon in family A. We have mapped a mutation in exon 2 (c.18delC) of the CENPJ gene in family B. This variant stopped the transcript synthesis by the premature introduction of the stop codon (p. Ser7Profs*2). The sequence variant in exon 23 (9557C > G) of the ASPM gene in family C, causing a sudden halted the translation process (p. Ser3186X). In family D revealed a variant in intron 11 (c.1473 + 1G > A) of the CEP135 gene caused a frameshift mutation (p. Glu417Glyfs*2) and terminated the protein synthesis, these variants were previously reported and our study supports them. However, no three-dimensional structure models of STIL, CENPJ, and CEP135 protein have been solved in the protein database. As a result, we predicted 3D protein models for the first time in this study by using Insilco technologies such as I TASSER, Swiss model, and phyre2. The validation of these protein models was done by using Ramachandran plot and SAVES server while refinement of the selected models was done by using Galaxy WEB server. In the Protein Model Database, the 3D model of STIL protein predicted and refined was submitted with the entry number PM0084074, whereas the 3D model of CENPJ protein was deposited with the accession number PM0084076. The 3D model of CEP135 protein was deposited in the Protein Model Database (PMDB – https://bioinformatics.cineca.it/PMDB/) with the accession number PM0084077. ConclusionsWe exposed DNA variants in primary microcephaly-affected individuals which impaired the expression of genes that are necessary to build a proper functioning of the brain. This study provides supporting evidence to the pathogenicity of previously reported mutations. Inslico protein models prediction provides functional exploration for clinicians and geneticists for establishing reliable diagnostic strategies for MCPH families.