Identification of Hearing Loss-Associated Variants of PTPRQ, MYO15A, and SERPINB6 in Pakistani Families.

The inner ear is an essential part of a well-developed and well-coordinated hearing system. However, hearing loss can make communication and interaction more difficult. Inherited hearing loss (HL) can occur from pathogenic genetic variants that negatively alter the intricate inner ear sensory mechanism. Recessively inherited forms of HL are highly heterogeneous and account for a majority of prelingual deafness. The current study is designed to investigate genetic causes of HL in three consanguineous Pakistani families. After IRB approval, the clinical history and pure tone audiometric data was obtained for the clinical diagnosis of HL segregating in these three Pakistani families. We performed whole exome sequencing (WES) followed by Sanger sequencing in order to identify and validate the HL-associated pathogenic variants, respectively. The 3-D molecular modeling and the Ramachandran analysis of the identified missense variants were compiled to evaluate the impact of the variants on the encoded proteins. Clinical evaluation revealed prelingual severe to profound sensorineural HL segregating among the affected individuals in all three families. Genetic analysis revealed segregation of several novel variants associated with HL, including a canonical splice-site variant (c.55-2A>G) of PTPRQ in family GCFHL-01, a missense variant [c.1079G>A; p.(Arg360Gln)] of SERPINB6 in family LUHL-01, and an insertion variant (c.10208-10211insCCACCAGGCCCGTGCCTC) within MYO15A in family LUHL-011. All the identified variants had very low frequencies in the control databases. The molecular modeling of p.Arg360Gln missense variant also predicted impaired folding of SERPINB6 protein. This study reports the identification of novel disease-causing variants in three known deafness genes and further highlights the genetic heterogeneity of HL in Pakistani population.

Identification and computational analysis of USH1C, and SLC26A4 variants in Pakistani families with prelingual hearing loss.

Hearing loss (HL) is clinically and genetically heterogeneous disorder and is the most frequent occurring sensory deficit in humans. This study was conducted to decipher the genetic cause of HL occurring in two large consanguineous Pakistani families (GCNF-01, GCNF-03). Family history and pure tone audiometry of both families suggested prelingual HL, while the affected individuals of GCNF-01 also had low vision and balance problems, consistent with cardinal features of Usher syndrome type I (USH1). Exome sequencing followed by segregating analysis revealed a novel splice site variant (c.877-1G > A) of USH1C occurring with USH1 phenotype in family GCNF01. While the affected individual of family GCNF-03 were homozygous for the c.716 T > A, p.(Val239Asp) previously reported pathogenic variant of SLC26A4. Both variants have very low frequencies in control database. In silico mutagenesis and 3-dimensional simulation analyses revealed that both variants have deleterious impact on the proteins folding and secondary structures. Our study expands the mutation spectrum of the HL genes and emphasizes the utility of exome sequencing coupled with bioinformatics tools for clinical genetic diagnosis, prognosis, and family counseling.

Delineation of Homozygous Variants Associated with Prelingual Sensorineural Hearing Loss in Pakistani Families.

Hearing loss is a genetically heterogeneous disorder affecting approximately 360 million people worldwide and is among the most common sensorineural disorders. Here, we report a genetic analysis of seven large consanguineous families segregating prelingual sensorineural hearing loss. Whole-exome sequencing (WES) revealed seven different pathogenic variants segregating with hearing loss in these families, three novel variants (c.1204G>A, c.322G>T, and c.5587C>T) in TMPRSS3, ESRRB, and OTOF, and four previously reported variants (c.208C>T, c.6371G>A, c.226G>A, and c.494C>T) in LRTOMT, MYO15A, KCNE1, and LHFPL5, respectively. All identified variants had very low frequencies in the control databases and were predicted to have pathogenic effects on the encoded proteins. In addition to being familial, we also found intersibship locus heterogeneity in the evaluated families. The known pathogenic c.226C>T variant identified in KCNE1 only segregates with the hearing loss phenotype in a subset of affected members of the family GCNF21. This study further highlights the challenges of identifying disease-causing variants for highly heterogeneous disorders and reports the identification of three novel and four previously reported variants in seven known deafness genes.

Phosphoglycerate mutase 1 in cancer: A promising target for diagnosis and therapy.

Altered enzymatic machineries are a substantial biochemical characteristic of tumor cell metabolism that switch metabolic profile from oxidative phosphorylation to amplified glycolysis as well as increased lactate production under hypoxia conditions. Reprogrammed metabolic profile is an emerging hallmark of cancer. Overexpression of several glycolytic enzymes and glucose transporters has been reported in 24 different types of cancers that represent approximately 70% of all the cancer cases around the globe. Thus, targeting glycolytic enzymes could serve as tempting avenue for drug design against cancer. Phosphoglycerate mutase 1 (PGAM1) is an important glycolytic enzyme that catalyzes the conversion of 3-phosphoglycerate to 2-phosphoglycerate. Recent investigations have revealed the overexpression of PGAM1 in several human cancers that is linked with tumor growth, survival, and invasion. The aim of this review is to update scientific research network with cancer-specific role of PGAM1 to elucidate its capability as bonafide therapeutic target for cancer therapy. Moreover, we have also summarized the reported genetic and pharmacological inhibitors of PGAM1. This study suggests that further investigations on PGAM1 should focus on the exploration of molecular mechanisms of PGAM1 overexpression in development of cancer, assessment of biosafety profiles of known inhibitors of PGAM1, and utilization of PGAM1 inhibitors in combinatorial therapies. These future studies will surely support the unbiased strategies for the development of novel PGAM1 inhibitors for cancer therapies.

Identities and frequencies of variants in CYP1B1 causing primary congenital glaucoma in Pakistan.

Purpose: Primary congenital glaucoma (PCG) is a clinically and genetically heterogeneous disease. The present study was undertaken to find the genetic causes of PCG segregating in 36 large consanguineous Pakistani families. Methods: Ophthalmic examination including fundoscopy, or slit-lamp microscopy was performed to clinically characterize the PCG phenotype. Genomic nucleotide sequences of the CYP1B1 and LTBP2 genes were analyzed with either Sanger or whole exome sequencing. In silico prediction programs were used to assess the pathogenicity of identified alleles. ClustalW alignments were performed to determine evolutionary conservation, and three-dimensional (3D) modeling was performed using HOPE and Phyre2 software. Results: Among the known loci, mutations in CYP1B1 and LTBP2 are the common causes of PCG. Therefore, we analyzed the genomic nucleotide sequences of CYP1B1 and LTBP2, and detected probable pathogenic variants cosegregating with PCG in 14 families. These included the three novel (c.542T>A, c.1436A>G, and c.1325delC) and five known (c.868dupC, c.1168C>T, c.1169G>A, c.1209InsTCATGCCACC, and c.1310C>T) variants in CYP1B1. Two of the novel variants are missense substitutions [p.(Leu181Gln), p.(Gln479Arg)], which replaced evolutionary conserved amino acids, and are predicted to be pathogenic by various in silico programs, while the third variant (c.1325delC) is predicted to cause reading frameshift and premature truncation of the protein. A single mutation, p.(Arg390His), causes PCG in six (~43%) of the 14 CYP1B1 mutations harboring families, and thus, is the most common variant in this cohort. Surprisingly, we did not find any LTBP2 pathogenic variants in the families, which further supports the genetic heterogeneity of PCG in the Pakistani population. Conclusions: In conclusion, results of the present study enhance our understanding of the genetic basis of PCG, support the notion of a genetic modifier of CYP1B1, and contribute to the development of genetic testing protocols and genetic counseling for PCG in Pakistani families.

Estimation of Antifungal Activity of Mevastatin Produced by Aspergillus terreus GCBL-03 on pretreated substrate in solid state fermentation.

This project was planned to study the production of cholesterol lowering drug mevastatin by Aspergillus terreus GCBL-03 on pretreated substrate bagasse in solid state fermentation. Bagasse was pretreated by potassium hydroxide to delignify the substrate to readily become available to microorganism. It was noticed that pretreated bagasse exhibited 13.69±0.64 mg/100mL yield of mevastatin and 6.63±0.48 g dry cell mass as compared to nontreated substrate that showed 7.04±0.81 mg/100 mL and 6.04±0.24 g dry cell mass. The fermentation profile like pH (5.5), temperature (30 °C), moisture contents (60%) inoculum size (2 mL), and incubation time (72 h) showed the optimum production (30.63±1.24 mg/100 mL) of mevastatin. Furthermore, fermented drug showed noteworthy antifungal activity against battery of selected fungal strains assessed by disc diffusion and measurement of minimum inhibitory concentration by micro dilution method. It was concluded from the study that fermented mevastatin was effective against fungal strains.

Screening of indigenously isolated fungi for lovastatin production and its in vivo evaluation.

Seven indigenously isolated fungal strains (Aspergillus niger, Aspergillus flavus, Aspergillus terreus, Penicillium citrinum, Penicillium notatum, Pleurotus ostreatus and Trichoderma viradae) were tested for their potential to produce cholesterol lowering drug lovastatin by using different agro-industrial wastes (Corn cobs, corn stover, banana stalk, wheat straw, wheat bran, bagasse) in submerged as well as solid state fermentation. Aspergillus terreus showed maximum production of 18.74 mg/100 mL by wheat bran in solid state fermentation. The fermentation parameters (pH, temperature, Inoculum size, moisture contents and fermentation time) were also optimized for optimum production of lovastatin. It was found that Aspergillus terreus could produce 27.14 mg/100 mL lovastatin under optimum condition of pH (6), temperature (30°C), Inoculum size (2 mL), moisture contents (60%) and fermentation time (120 hrs) in solid state fermentation. The optimized lovastatin was extracted from fermented broth and orally administered to rats. The hypocholesterolemic effect of fermented lovastatin was evaluated on serum ALT, AST, HDL-C, LDL-C, TG and TC level of rats. It was concluded from the study, fermented lovastatin effectively lower the cholesterol level of rats.

Oxidative stress elevated DNA damage and homocysteine level in normal pregnant women in a segment of Pakistani population.

Maternal oxidative stress during pregnancy may impair fetal growth and help in the development of diseases in adulthood. The aim of current study was to assess total oxidation status (TOS), related parameters and their relationship to DNA damage (%) and homocysteine level in normal pregnant women in low-income participants. In a cross-sectional study healthy women were grouped as normal, while age matched nulliparous and singleton pregnancies were included for first, second and third trimester groups. TOS (P<0.01), melanodialdehyde (MDA) (P<0.001), aspartate aminotransferase (AST) (P<0.01), triiodothyronine (T3) (P<0.01), thyroxine (T4) (P<0.01), and homocysteine (P<0.001), in pregnant women were significantly higher as compared to normal healthy women. While serum total proteins (P<0.01), albumin (P<0.01) and total antioxidant status (TAS) (P<0.001) decreased significantly as compared to normal healthy women. Women in third trimester showed a significantly high level of body temperature (P<0.01), triglyceride (P<0.01), LDL-cholesterol (P<0.05), AST (P<0.01), T3 (P<0.01), homocysteine (P<0.001), TOS (P<0.01) and MDA (P<0.001) but a lower concentration of serum proteins, albumin and TAS at the end of the pregnancy. Pearson correlation indicated a positive relationship of homocysteine with triglycerides (P<0.027), TOS (P<0.01), MDA (P<0.035) and had a negative relationship with total protein (P<0.026). DNA damage was strongly related with T3 (P<0.008), TOS (P<0.02), MDA (P<0.037) and MBI (P<0.048) profiles of pregnant women. These changes were considered normal for pregnant women having optimum blood pressure and normal child birth. Hormonal influences and hemodilution may contribute towards the observed changes in this study.

Plasma homocysteine and DNA damage profiles in normal and obese subjects in the Pakistani population.

Dependence of plasma total homocysteine (tHcy) and DNA damage profiles on melanodialdehyde (MDA), oxidative stress, liver function tests (LFT), and lipids was studied in non-obese and obese subjects in the Pakistani population. Development of obesity is influenced by both genetic, biochemical and environmental factors. Plasma homocysteine (Hcy) and DNA damage profiles play a pivotal role in its progression. We studied 160 obesity patients and 160 lean subjects. Leukocytes were evaluated for DNA damage by comet assay and blood plasma for biochemical properties using commercial kits. Plasma Hcy level and DNA damage were strongly correlated with triglycerides (P < 0.000), LDL-cholesterol (P < 0.001), systolic blood pressure (P < 0.001), cholesterol (P < 0.004), MDA (P < 0.004) and total oxygen stress (P < 0.004) in obese individuals. Both Hyc and DNA damage were negatively associated with total anti-oxidant response and globulin. Both Hcy profile and DNA damage may have a role in the endothelium damage even in the normal range and are related to triglycerides, ALT, MDA, TOS, HDL- and LDL-cholesterol in the Pakistani population.