Abu Saeed Hashmi

Identification of two novel ALS2 mutations in infantile-onset ascending hereditary spastic paraplegia

ABSTRACT Biallelic mutations of ALS2 cause a clinical spectrum of overlapping autosomal recessive neurodegenerative disorders: infantile-onset ascending hereditary spastic paralysis (IAHSP), juvenile primary lateral sclerosis (JPLS), and juvenile amyotrophic lateral sclerosis (ALS2). We report on eleven individuals affected with IAHSP from two consanguineous Pakistani families. A combination of linkage analysis with homozygosity mapping and targeted sequencing identified two novel ALS2 mutations, a c.194T > C (p.Phe65Ser) missense substitution located in the first RCC-like domain of ALS2/alsin and a c.2998delA (p.Ile1000*) nonsense mutation. This study of extended families including a total of eleven affected individuals suggests that a given ALS2 mutation may lead to a phenotype with remarkable intrafamilial clinical homogeneity.

Identification of functional consequence of a novel selection signature in CYP11b1 gene for milk fat content in Bubalus bubalis.

Genomic selection for traits of economic importance is an emerging approach carrying tremendous potentials. Many of polygenic traits as milk fat, protein and yield have been characterize at genomic level and important selection signatures have been identified. Cytochrome P450 enzymes are potential loci for affecting many of dairy capabilities. Present study was conducted for genomic dissection of CYP11b1 gene in riverine buffaloes and seven genetic variations were identified. Out of these, one novel polymorphism (p.A313T) was found well associated with milk fat %age. AB genotyped buffaloes were found to have higher milk fat %age (8.9%) for this loci. p.A313T was further validated at larger data set by restriction digestion using CviAII enzyme. Functional consequences of this locus were also predicted by studying three dimensional structure of CYP11b1 protein. For this purpose, 3D protein model was predicted by homology modeling, secondary structural attributes were determined, signal peptide was predicted and a transmembrane helix was also identified. One of polymorphism (p.Y205L) was found in the vicinity of functionally significant F-G loop region, which is the part of protein gets attached to the inner mitochondrial membrane. But this variation could not be associated and needs further investigation. p.A30V, a popular selection marker in cattle, was found in buffaloes as well but could not be associated and might need further confirmation on larger data set. Results of this study illustrate the impending potential of this gene in determining dairy capabilities of buffaloes and might have a role in selection of superior dairy buffaloes.

Cloning and expression of NS3 gene of Pakistani isolate type 2 dengue virus

Abstract Introduction Dengue is one of the major emerging viral diseases in the world, with dramatic increases in reported cases in the last few decades and annual worldwide occurrence of approximately 390 million infections. It is a highly important mosquito-vectored disease and is a problem in tropical and subtropical areas of the world. The major aim of this study was to clone and express the dengue NS3 gene, in service to its therapeutic importance for the development of stable cell lines. Material and Methods Blood samples from dengue fever (DF) patients were collected and subjected to PCR amplification of the NS3 gene of dengue virus serotype-2 (DENV-2). The NS3 gene was amplified using gene specific primers and cloned in the TA cloning vectors. Results The gene was successfully expressed in mammalian expression vector pcDNA3.1. The current finding was different from a previously reported DENV-2 strain replicon constructed in different cells, in which the whole genetic material of the virus was used instead of an active protease gene, and which gave a low yield of replicon expressing cells. Conclusion Recombinant NS3 could be used to produce an antibody that is possibly helpful for developing a single step diagnostic assay to detect the dengue virus NS3 antigen in sera of dengue patients.

In-Silico Analysis of Gene Als2 Genetic Variants Identified in the Affected Horses and Humans with Motor Neuron Disease

ABSTRACT Motor neuron disease (MND) is a spontaneous neurologic disorder of humans and adult horses, which results from the degeneration of motor neurons in the spinal cord and brain stem. The etiology of MND disease in the South Asian region is not well understood. To achieve the objective of the study, blood samples were collected from both affected humans and horses. DNA was purified by inorganic method; subsequently, ALS2 gene was sequenced both in humans and horses of Pakistani origin diagnosed with MND. In case of affected individuals, by targeted sequencing, we identified two novel ALS2 mutations, the missense substitution c.194T>C (p.Phe65Ser) located in the first RCC1 domain of ALS2 and the nonsense mutation c.2998delA (p.Ile1000*) located in the PH domain of ALS2. In case of affected horses, we identified two missense substitution mutations c.247G>A (p.Ile83Val) and c.914T>G (p.Leu305Arg) in the ALS2 gene. In‐silico analysis was performed by PolyPhen‐2 and SIFT of identified genetic variants to predict whether a substitution of specific amino acid affects protein function and to understand the pathogenicity of identified genetic variants. Our results suggest that genetic variants identified in human families were found to be pathogenic, whereas missense substitutions identified in horse ALS2 gene were not causing MND in affected horses. HighlightsDegeneration of motor neurons in the spinal cord and brain stem causes motor neuron disease (MND) in adult horses.In this study, ALS2 gene was sequenced both in humans and horses of Pakistani origin diagnosed with MND.In humans, two novel ALS2 mutations, the missense substitution located in the first RCC1 domain of ALS2 and the nonsense mutation located in the PH domain of ALS2 were recorded; whereas in horses, two missense substitution mutations in the ALS2 gene were found.Genetic variants were found to be pathogenic, whereas missense substitutions identified in horse ALS2 gene were not causing MND in affected horses.