Unveiling forensically relevant biogeographic, phenotype and Y-chromosome SNP variation in Pakistani ethnic groups using a customized hybridisation enrichment forensic intelligence panel.

Massively parallel sequencing following hybridisation enrichment provides new opportunities to obtain genetic data for various types of forensic testing and has proven successful on modern as well as degraded and ancient DNA. A customisable forensic intelligence panel that targeted 124 SNP markers (67 ancestry informative markers, 23 phenotype markers from the HIrisplex panel, and 35 Y-chromosome SNPs) was used to examine biogeographic ancestry, phenotype and sex and Y-lineage in samples from different ethnic populations of Pakistan including Pothwari, Gilgit, Baloach, Pathan, Kashmiri and Siraiki. Targeted sequencing and computational data analysis pipeline allowed filtering of variants across the targeted loci. Study samples showed an admixture between East Asian and European ancestry. Eye colour was predicted accurately based on the highest p-value giving overall prediction accuracy of 92.8%. Predictions were consistent with reported hair colour for all samples, using the combined highest p-value approach and step-wise model incorporating probability thresholds for light or dark shade. Y-SNPs were successfully recovered only from male samples which indicates the ability of this method to identify biological sex and allow inference of Y-haplogroup. Our results demonstrate practicality of using hybridisation enrichment and MPS to aid in human intelligence gathering and will open many insights into forensic research in South Asia.

Novel variants underlying autosomal recessive intellectual disability in Pakistani consanguineous families.

BACKGROUND: Intellectual disability (ID) is both a clinically diverse and genetically heterogeneous group of disorder, with an onset of cognitive impairment before the age of 18 years. ID is characterized by significant limitations in intellectual functioning and adaptive behaviour. The identification of genetic variants causing ID and neurodevelopmental disorders using whole-exome sequencing (WES) has proven to be successful. So far more than 1222 primary and 1127 candidate genes are associated with ID. METHODS: To determine pathogenic variants causative of ID in three unrelated consanguineous Pakistani families, we used a combination of WES, homozygosity-by-descent mapping, de-deoxy sequencing and bioinformatics analysis. RESULTS: Rare pathogenic single nucleotide variants identified by WES which passed our filtering strategy were confirmed by traditional Sanger sequencing and segregation analysis. Novel and deleterious variants in VPS53, GLB1, and MLC1, genes previously associated with variable neurodevelopmental anomalies, were found to segregate with the disease in the three families. CONCLUSIONS: This study expands our knowledge on the molecular basis of ID as well as the clinical heterogeneity associated to different rare genetic causes of neurodevelopmental disorders. This genetic study could also provide additional knowledge to help genetic assessment as well as clinical and social management of ID in Pakistani families.

A novel single base pair duplication in WDR62 causes primary microcephaly.

BACKGROUND: Primary microcephaly is a disorder of the brain resulting in a reduced head circumference that can come along with intellectual disability but with hardly any other neurological abnormalities. CASE PRESENTATION: In this study we report on three Pakistani males from a consanguineous family with 2, 4 and 25 years, diagnosed with autosomal recessive primary microcephaly. By genotyping, Sanger sequencing and using bioinformatical approaches the disease causing mutation was identified and evaluated. CONCLUSION: By using a 250K SNP array, we were able to detect an 11Mb large autozygous region in the MCPH2 locus on chromosome 19q13.12. Sequencing of the associated gene, WDR62, revealed the frameshift causing single base pair duplication, c.2527dupG. This mutation is predicted to affect the structural features of WDR62 which in turn changes the conformation and function of the protein. Aspartic acid (D) at position 843 was found to be conserved among various ortholog species. The present findings will be helpful in genetic diagnosis of patients and future studies of WDR62.

Truncation of the E3 ubiquitin ligase component FBXO31 causes non-syndromic autosomal recessive intellectual disability in a Pakistani family.

In this study, we have performed autozygosity mapping on a large consanguineous Pakistani family segregating with intellectual disability. We identified two large regions of homozygosity-by-descent (HBD) on 16q12.2-q21 and 16q24.1-q24.3. Whole exome sequencing (WES) was performed on an affected individual from the family, but initially, no obvious mutation was detected. However, three genes within the HBD regions that were not fully captured during the WES were Sanger sequenced and we identified a five base pair deletion (actually six base pairs deleted plus one base pair inserted) in exon 7 of the gene FBXO31. The variant segregated completely in the family, in recessive fashion giving a LOD score of 3.95. This variant leads to a frameshift and a premature stop codon and truncation of the FBXO31 protein, p.(Cys283Asnfs*81). Quantification of mRNA and protein expression suggests that nonsense-mediated mRNA decay also contributes to the loss of FBXO31 protein in affected individuals. FBXO31 functions as a centrosomal E3 ubiquitin ligase, in association with SKP1 and Cullin-1, involved in ubiquitination of proteins targeted for degradation. The FBXO31/SKP1/Cullin1 complex is important for neuronal morphogenesis and axonal identity. FBXO31 also plays a role in dendrite growth and neuronal migration in developing cerebellar cortex. Our finding adds further evidence of the involvement of disruption of the protein ubiquitination pathway in intellectual disability.

Exploring evolution of brain genes involved in microcephaly through phylogeny and synteny analysis.

BACKGROUND: Human brain development is a complicated process. When normal growth and development of brain or central nervous system is impaired, it leads to neurodevelopemental disorders (NDDs). Autosomal Recessive Primary Microcephaly (MCPH) is one of those, for which seven loci (MCPH1-MCPH7) with the corresponding genes (MCPH1, WDR62, CDK5RAP2, CEP152, ASPM, CENPJ, and STIL) have been reported so far. An important field of study is to find out diversity among organisms due to evolution. How species are related to each other can be inferred through finding evolutionary relationship between organisms in the form of ancestors and descendents. METHODS: MEGA5 was used for phylogenetic tree reconstruction. Pair-wise and multiple alignment was built through ClustalW algorithm. Neighbor joining (NJ) and maximum parsimony (MP) methods were used for tree reconstruction. Bootstrap analysis was done to check the reliability of trees. Synteny analysis was performed using Ensemble synteny view in ensemble database and genome synteny viewer (GSV). RESULTS: Evolutionary time for single gene trees showed that CENPJ (0.02) evolving rapidly while CDK5RAP2 (0.1) evolving with least rate as compare to other genes. All trees were reconciling the species divergence time. Chimpanzee was inferred as closest specie of Human. In MCPH combined tree, five duplications were observed. Four duplications were before and one was after vertebrate and invertebrate divergence. Two genes MCPH1 and WDR62 were closely related with each other. Synteny analysis indicated that maximum conservation of Human was with Chimpanzee. Highly conserved synteny was observed for Human and Chimpanzee in case of CENPJ with no deletion. CONCLUSION: It has been hypothesized that due to having closest relationship, mutations can affect Chimpanzee likewise as these affect Human. Conservation shows that apart from sequence similarity, function of MCPH genes in closely related species is also same and this function disrupts as a result of mutation and hence leads to the diseased state. Huge genomic and proteomic data is available today which enables us to perform In Silico analysis. Our cost and time effective analysis has opened many insights into disease understanding and it will definitely provide a way towards accurate diagnosis.

In silico studies of C 3 metabolic pathway proteins of wheat (Triticum aestivum).

Photosynthesis is essential for plant productivity and critical for plant growth. More than 90% of plants have a C3 metabolic pathway primarily for carbon assimilation. Improving crop yields for food and fuel is a major challenge for plant biology. To enhance the production of wheat there is need to adopt the strategies that can create the change in plants at the molecular level. During the study we have employed computational bioinformatics and interactomics analysis of C3 metabolic pathway proteins in wheat. The three-dimensional protein modeling provided insight into molecular mechanism and enhanced understanding of physiological processes and biological systems. Therefore in our study, initially we constructed models for nine proteins involving C3 metabolic pathway, as these are not determined through wet lab experiment (NMR, X-ray Crystallography) and not available in RCSB Protein Data Bank and UniProt KB. On the basis of docking interaction analysis, we proposed the schematic diagram of C3 metabolic pathway. Accordingly, there also exist vice versa interactions between 3PGK and Rbcl. Future site and directed mutagenesis experiments in C3 plants could be designed on the basis of our findings to confirm the predicted protein interactions.