A Comprehensive Functional And Computational Study Of Sequence Variants In ASS1Gene And Marker Chromosome Inv Dup (15) Associated Mental Retardation

DNA Sequence variants may be either benign variations or functional mutations. Identifying these mutations may provide useful information concerning severity of a defect and likely prognosis. Sequencing the respective gene as part of the confirmatory process can be critical in establishing the presence of a disorder. Beyond sequencing, interpretation of the results is critical. Apart from nucleotide changes in the genome that lead to genetic disorders, Chromosomal imbalances of variable size resulting in the gain or loss of chromosomal material (copy number variants; CNVs) have been recognized as another cause of human disorders. Microdeletions and microduplications are believed to be among the most frequent genetic causes of mental retardation (MR).Part I:Mutations in the ASS1gene associated with Citrullinemia type1(CTLN1) patients as well as ASS1nonsynonymous nucleotide variants (nsSNVs) were analysed, for ASS1gene mutations. Classical citrullinemia type I (CTLN1) is an inborn error of the urea cycle caused by deficiency of argininosuccinate synthetase. Its marked by elevation of citrulline and ammonia levels in plasma and depletion of arginine. Patient1exhibited a mild phenotype characterized by an elevated biochemical profile. Sequencing revealed that he carries novel donor site (c.174+1G>A) and missence (p.V141G) mutations in exon7, the second base. Mutation (c.174+1G>A) caused abnormal splicing, resulting in deletion of exon4and consequently a truncated protein. Mutation (p.V141G) causes partial abnormal splicing of exon7as the exon splicing enhancers (ESE) are not completely abolished. Patient2presented with early onset neonatal citrullinemia marked by an elevated biochemical profile and a clinical phenotype four days after birth. ASS1gene sequencing detected (c.773+1G>A) and (R265C) compound heterozygous mutation. The (c.773+1G>A) mutation in vitro pSPL3exon trapping strategy experiments showed that the mutations caused an abnormal splicing. This resulted in a transcript lacking exon11and another having retention of79bp from intron11. Both transcripts would result in premature termination of the protein. In addition, the tools SIFT, PolyPhen-2, I-Mutant2.0and CUPSAT mutant locus protein prediction analysis, illustrate the effect of the mutations in the protein structure. Missing exon4due to (c.174+1G>A) leads to loss of23aminoacids locating in the ATP binding site. Valine at position141locates in a hydrophobic pocket, substitution with Glycine according to structure prediction analysis, indicate this may interfere with ATP binding. These two mutations may reduce ATP binding. R265C found in an earlier study introduces abnormality in the citrulline binding site. This study showed that missense mutations and splice site mutations caused by different transcripts mechanism induced disease. Data mining of55ASS1nsSNVs data from dbSNP database (NCBI) was performed with retrival of the SNV ID as well as the amino acid change. Analysis of sequence variants detected in this era of next generation sequencing is very critical in understanding their pathological significance. The impact on the molecular mechanisms and structural effects would provide understanding of their disease relatedness. As such mutations damaging on analysis, if present in individuals would put them at risk of citrullinemia. In the analysis, a three step approach was exploited using SIFT, PolyPhen-2and I-Mutant2.0as initial filtering step. PANTHER, SNP&GO and Mutpred tools were used for functional prediction. This resulted in a total of20nsSNVs being found damaging at step1and2. Out of this, the seven most deleterious were evaluated for structural impact on modeled mutant and native3D structures based on RMSD, total energy value and atomic contact. These four variants SNV ID:TMP ESP9133364846, rs121908643,rs121908637and, TMP ESP9133355834had destabilising increase in total energy value. Structural analysis on the3D structure for mechanistic insight, predicted some variants disturb hydrogen bonding networks, others decrease hydrophobicity as well as introduction of steric constraints. On the overall our findings may provide useful information for prioritization of these nsSNPs for future molecular experimental studies and in new born screening.Part II:In the study a six year old patient of Chinese descent with severe mental retardation was investigated for presence of chromosomal aberrations. He exhibited learning disabilities, hypotonia, ataxia, cognitive defects, and speech delay but was devoid of dysmorphic features. Karyotype analysis using conventional Chromosomal analysis revealed a Supernumerary marker chromosome (SMC),47, XY,+mar. Parental karyotypes were observed to be normal indicative the supernumerary marker chromosome arisen denovo. Further characterization by Array comparative genome hybridization (array-CGH) indicated a7.7Mb duplication extending from BP1to BP3segments. This encompass the Prader-Willi/Angelman Critical region (PWACR) important to expression of clinical phenotype. The chromosome region15q11-q13is prone to cytogenetic abnormalities including; deletions, interstitial duplications, translocations, inversions, and marker chromosomes. Maternally-derived duplications that include the imprinted region on the proximal long arm of chromosome15underlie a complex neurobehavioral disorder characterized by cognitive impairment, seizures and a substantial risk for autism spectrum disorders. The duplications most often take the form of a supernumerary pseudodicentric derivative chromosome, the isodicentric15. The phenotypic expression associated with this duplications is marked with clinical variability with symptoms ranging from autism, developmental delay, seizures, speech delay, learning disabilities, various levels of mental retardation as well as poor motor coordination.Copy number increase for genes in this region was confirmed by Quantitative PCR (qPCR), Ct method in which genes important for neurodevelopment had their copy number duplicated. The duplication arisen de novo was as well confimed by parents’ qPCR results. Methylation analysis of the SNRPN locus revealed the duplication to be maternally derived. This SMC is postulated to be the probable pathogenic cause of the clinical phenotype observed in the patient. Multiple genetics tests are shown to present a synergetic approach in molecular diagnostics in unexplained developmental delays.