Exome sequencing identifies homozygous variants in MBOAT7 associated with neurodevelopmental disorder.

Intellectual disability (ID) is a large group of neurodevelopmental disorders characterized by a congenital limitation in intellectual functioning (reasoning, learning, and problem solving), adaptive behavior (conceptual, social, and practical skills), originated at birth and manifested before the age of 18. By whole exome sequencing of five consanguineous Pakistani families presenting hallmark features of ID, global developmental delay, aggressive and self-injurious behaviors, microcephaly, febrile seizures and facial dysmorphic features, we identified three novel homozygous missense variants (NM_024298.5: c.588G > T; p.Trp196Cys, c.736 T > C; p.Tyr246His and c.524A > C; p. Asp175Ala) and one rare homozygous in-frame deletion variant (c.758_778del;p.Glu253_Ala259del) in membrane-bound O-acyltransferase family member 7 (MBOAT7) gene previously associated with autosomal recessive neurodevelopmental disorder. The segregation of the variants was validated by Sanger sequencing in all family members. In silico homology modeling of wild-type and mutated proteins revealed substantial changes in the structure of both proteins, indicating a possible effect on function. The identification and validation of new pathogenic MBOAT7 variants in five cases of autosomal recessive ID further highlight the importance of this genes in proper brain function and development.

Molecular identification and cross-immunogenic study on two field isolates of Mycoplasma synoviae isolated from broilers in five districts of Khyber Pakhtunkhwa.

Background: Mycoplasma synoviae (MS) is an important poultry pathogen causing heavy economic losses Worldwide. Subclinical persistence of this pathogen is the major issue to control its prevalence. Aim: This study aimed to determine the molecular and cross-immunogenicity of MS among broilers in five Districts of Khyber Pakhtunkhwa (KP). Methods: This study was conducted by collecting 434 specimen samples from 40 broiler farms and desi poultry in five districts of KP. Specimen samples from the broiler birds (n = 150), broiler farm environment (n = 264), and desi poultry birds (n = 20) were aseptically collected and serially passaged in Modified Frey's broth. The homologous and heterologous antibody reactions were studied in rabbits. Before inoculation into rabbits, the MS isolates were inactivated by formalin and adjuvanted with Montanide. Results: The overall turbidity prevalence in Frey's broth was observed as 109/434 (25.11%) samples, and these turbidity-positive samples were shifted on Frey's agar. After the appearance of classic fried egg colonies, the Biochemical confirmation was supported by the production of catalase and phosphatase, reduction of tetrazolium, film and spot assay, and fermentation of glucose for species differentiation in avian mycoplasma. The MS prevalence percentage was recorded as 2% (9/434) through biochemical tests. The PCR results showed 0.5% MS prevalence with two field isolates (named MS-1 and MS-2). Both MS-1 and MS-2 field isolates showed similar values (42.2) of homologous geometric mean titer (GMT). While the heterologous GMT for MS-1 serum against MS-2 isolate was lower (27.9) as compared to MS-2 serum against MS1 isolate (38.9). No titer was detected in the control group (Group-III). Conclusion: In conclusion, the results indicated the existence of MS in broiler birds and high homologous titers recorded between field isolates, which is a perpetual menace to poultry.

Frequency of urinary tract infection and antibiotic sensitivity of uropathogens in patients with diabetes.

OBJECTIVE: To determine the frequency of urinary tract infections and antibiotic sensitivity among patients with diabetes. METHODS: This observational study was carried out in Microbiology Department of Baqai Institute of Diabetology and Endocrinology (BIDE), Baqai Medical University from April 2015 to June 2016. All patients with diabetes having symptoms of UTI attending out patients department of BIDE were analyzed. All samples received in the laboratory were processed according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Antimicrobial susceptibility pattern was determined by disc diffusion method. RESULTS: A total number of 199 urine specimens, frequency of UTI were 24 (12.06%) in male and 175 (87.94%) in female. UTIs were highly found in (age group 51-60) 70 (35.18%). Escherichia coli was the most frequent pathogen (71%), followed by Klebsiellapneumoniae (7.48%), Proteus mirabilis (1.87%), Staphylococcus aureus (9.35%), Candida (5.61%) and Candidaalbicans were (2.80%). Majority of gram negative uropathogens were shown high sensitivity towards Imipenem and Piperacillin / Tazobactam followed by Nitrofurantion, Ceftriaxone, Levofloxacin, Ofloxacine, Ciprofloxacin, Norfloxacin, Cefixime, Nalidixic acid and Cephradine. Gram positive was most sensitive to Nitrofurantionand Vancomycin followed by Piperacillin / Tazobactam, Imipenem, Cephradine, Ceftriaxone, Norfloxacin and Cefixime. CONCLUSION: We observed the higher frequency of UTIs in female as compared to male participants due to poor hygiene. E.coli was the most frequent pathogen responsible for UTI in patients with diabetes, followed by Staphylococcus aureus.

Genetic studies of multiple consanguineous Pakistani families segregating oculocutaneous albinism identified novel and reported mutations.

Oculocutaneous albinism (OCA) is an autosomal-recessive disorder of a defective melanin pathway. The condition is characterized by hypopigmentation of hair, dermis, and ocular tissue. Genetic studies have reported seven nonsyndromic OCA genes, among which Pakistani OCA families mostly segregate TYR and OCA2 gene mutations. Here in the present study, we investigate the genetic factors of eight consanguineous OCA families from Pakistan. Genetic analysis was performed through single-nucleotide polymorphism (SNP) genotyping (for homozygosity mapping), whole exome sequencing (for mutation identification), Sanger sequencing (for validation and segregation analysis), and quantitative PCR (qPCR) (for copy number variant [CNV] validation). Genetic mapping in one family identified a novel homozygous deletion mutation of the entire TYRP1 gene, and a novel deletion of exon 19 in the OCA2 gene in two apparently unrelated families. In three further families, we identified homozygous mutations in TYR (NM_000372.4:c.1424G > A; p.Trp475*), NM_000372.4:c.895C > T; p.Arg299Cys), and SLC45A2 (NM_016180:c.1532C > T; p.Ala511Val). For the remaining two families, G and H, compound heterozygous TYR variants NM_000372.4:c.1037-7T > A, NM_000372.4:c.1255G > A (p.Gly419Arg), and NM_000372.4:c.1255G > A (p.Gly419Arg) and novel variant NM_000372.4:c.248T > G; (p.Val83Gly), respectively, were found. Our study further extends the evidence of TYR and OCA2 as genetic mutation hot spots in Pakistani families. Genetic screening of additional OCA cases may also contribute toward the development of Pakistani specific molecular diagnostic tests, genetic counseling, and personalized healthcare.

Characterization of gut bacterial flora of Apis mellifera from north-west Pakistan.

Gut microbiota has been recognized to play a beneficial role in honey bees (Apis mellifera). Present study was designed to characterize the gut bacterial flora of honey bees in north-west Pakistan. Total 150 aerobic and facultative anaerobic bacteria from guts of 45 worker bees were characterized using biochemical assays and 16S rDNA sequencing followed by bioinformatics analysis. The gut isolates were classified into three bacterial phyla of Firmicutes (60%), Proteobacteria (26%) and Actinobacteria (14%). Most of the isolates belonged to genera and families of Staphylococcus, Bacillus, Enterococcus, Ochrobactrum, Sphingomonas, Ralstonia, Enterobacteriaceae, Corynebacterium and Micrococcineae. Many of these bacteria were tolerant to acidic environments and fermented sugars, hence considered beneficial gut inhabitants and involved the maintenance of a healthy microbiota. However, several opportunistic commensals that proliferate in the hive environment including members Staphylococcus haemolyticus group and Sphingomonas paucimobilis were also identified. This is the first report on bee gut microbiota from north-west Pakistan geographically situated at the crossroads of Indian subcontinent and central Asia.

Ophthalmo-genetic analysis of Pakistani patients with nonsyndromic oculocutaneous albinism through whole exome sequencing.

Oculocutaneous albinism (OCA) is a disorder of defective melanin biosynthesis that is characterized by hypo-pigmentation of skin, hair and retinal pigment epithelium. Phenotypically, OCA patients exhibit white milky skin, whitish to golden hair and deterioration of retinal cells. Until recently, genetic studies have reported seven causative genes (TYR, TYRP1, OCA2, SLC45A2, SLC24A2, C10ORF11 and MCIR) and an uncharacterized OCA5 locus. Herein we present the medico-genetic study of three Pakistani patients inheriting autosomal recessive OCA. Whole exome sequencing, followed by Sanger DNA sequencing for segregation analysis, revealed recurrent mutations c.346C>T (p.Arg116*) and c.1255G>A (p.Gly419Arg) (family A and B respectively) in TYR gene, while the patient from family C did not reveal any known gene mutation, which suggests the involvement of some novel genetic factor. It is the first report of mapping c.346C>T mutation in a Pakistani patient. Our study further extends the evidence of genetic hotspots regions in TYR gene causing OCA in Pakistani population.

PCR/RFLP-based analysis of genetically distinct Plasmodium vivax population of Pvmsp-3α and Pvmsp-3ß genes in Pakistan.

BACKGROUND: Plasmodium vivax is one of the widespread human malarial parasites accounting for 75% of malaria epidemics. However, there is no baseline information about the status and nature of genetic variation of Plasmodium species circulating in various parts of Pakistan. The present study was aimed at observing the molecular epidemiology and genetic variation of Plasmodium vivax by analysing its merozoite surface protein-3α (msp-3α) and merozoite surface protein-3ß (msp-3ß) genes, by using suballele, species-specific, combined nested PCR/RFLP detection techniques. METHODS: A total of 230 blood samples from suspected subjects tested slide positive for vivax malaria were collected from Punjab, Sindh, Khyber Pakhtunkhwa, and Balochistan during the period May 2012 to December 2013. Combined nested PCR/RFLP technique was conducted using Pvmsp-3α and Pvmsp-3ß genetic markers to detect extent of genetic variation in clinical isolates of P. vivax in the studied areas of Pakistan. RESULTS: By PCR, P. vivax, 202/230 (87.82%), was found to be widely distributed in the studied areas. PCR/RFLP analysis showed a high range of allelic variations for both msp-3α and msp-3ß genetic markers of P. vivax, i.e., 21 alleles for msp-3α and 19 for msp-3ß. Statistically a significant difference (p ≤ 0.05) was observed in the genetic diversity of the suballelic variants of msp-3α and msp-3ß genes of P. vivax. CONCLUSION: It is concluded that P. vivax populations are highly polymorphic and diverse allelic variants of Pvmsp-3α and Pvmsp-3ß are present in Pakistan.

Fermentative production of L-lysine: fungal fermentation and mutagenesis-II: a review.

Lysine has been recognized as one of the most deficient essential amino acids. It is an economically important food and feed supplement. It has also got various pharmaceutical applications in the formulation of diets with balanced amino acid concentration and in amino acid infusions. Chemical, enzymatic and fermentation processes have been used to synthesize lysine. The objective of this review is to outline the research work of various scientists which provide important and beneficial information regarding the production of lysine through fungal fermentation and mutagenesis. Moreover, methods to improve the total yield and quality of lysine are also presented.