Zubair Anwar

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.

Identification of Missense Mutation (I12T) in the BSND Gene and Bioinformatics Analysis

Nonsyndromic hearing loss is a paradigm of genetic heterogeneity with 85 loci and 39 nuclear disease genes reported so far. Mutations of BSND have been shown to cause Bartter syndrome type IV, characterized by significant renal abnormalities and deafness and nonsyndromic nearing loss. We studied a Pakistani consanguineous family. Clinical examinations of affected individuals did not reveal the presence of any associated signs, which are hallmarks of the Bartter syndrome type IV. Linkage analysis identified an area of 18.36 Mb shared by all affected individuals between markers D1S2706 and D1S1596. A maximum two-point LOD score of 2.55 with markers D1S2700 and multipoint LOD score of 3.42 with marker D1S1661 were obtained. BSND mutation, that is, p.I12T, cosegregated in all extant members of our pedigree. BSND mutations can cause nonsyndromic hearing loss, and it is a second report for this mutation. The respected protein, that is, BSND, was first modeled, and then, the identified mutation was further analyzed by using different bioinformatics tools; finally, this protein and its mutant was docked with CLCNKB and REN, interactions of BSND, respectively.

Physico-chemical growth requirements and molecular characterization of indigenous Spirulina

The present study was designed to explore the indigenous Spirulina and its mass cultivation by optimizing the physico-chemical growth requirements. One hundred and twenty samples were collected from different soils and water from three districts (Sargodha, Lahore and Faisalabad) of Punjab. Collected samples were shifted to laboratory immediately under sterile conditions to avoid contamination and were kept under dim light at 30°C. Then Spirulina was isolated from collected samples and cultivated under different nutrient, temperature and light regimens to get its maximum biomass in our laboratory. Our results showed that maximum growth of indigenous Spirulina was obtained at 30°C and at 1500 lux light intensity. Nitrogen concentrations of 0.625, 1.25 and 1.875 g/L had no effect on the growth, while phosphate concentrations of 0.5, 1.0 and 1.5 g/L had a minimal and gradual effect on growth as the concentrations were increased. For the confirmation and molecular characterization of indigenous Spirulina, DNA was isolated by chloroform-isoamyl alcohol extraction method and its polymerase chain reaction (PCR) was carried out by using specific primer of 16s rDNA gene and PCR products were run on gel giving an amplicon size of 700 bp. Our study shows that Spirulina can be grown in lab conditions by optimizing the physico-chemical growth requirements.