Congenital septal defects in Karachi, Pakistan: an update of mutational screening by high-resolution melting (HRM) analysis of MTHFR C677T.

BACKGROUND: Congenital heart defects (CHDs) are the heart structural malformations present at birth. Septal defects account for 40% of CHD, including atrial, ventricular and atrioventricular septal defects. In Pakistan, the prevalence of CHD is 3.4 in 1000, and a study estimated that 60,000 babies are born with CHD annually. Methylenetetrahydrofolate reductase (MTHFR), a chief enzyme, involved in the folate metabolism. The missense mutation, C677T (rs1801133), exists in MTHFR gene, results in a MTHFR thermolabile variant having low enzymatic activity. The study is aim to identify the MTHFR C677T variant association with septal defects. METHODS: Samples of 194 CHD patients (age [Formula: see text]= 5.8 ± 5.1) and 50 normal echo controls (age [Formula: see text]= 6.0 ± 4.9), confirmed by pediatric consultant, were collected. Extracted DNA, quantified by agarose gel electrophoresis and nanodrop, was screened for SNP by high-resolution melting (HRM). Further, HRM results were confirmed using restriction analysis and sequencing. HRM was simply and precisely genotyped the samples within 3 h at low cost. RESULTS: Genotypic data suggested that heterozygous mutant (CT) was frequent in congenital septal defect patients (0.26) which was higher than controls (0.143), p > 0.05. Mutant (TT) genotype was not found in this study. CONCLUSIONS: rs1801133 has lack of significant association with congenital septal defects. The absence of TT genotype in this study suggesting the role of natural selection in targeted population. HRM is an easy, fast and next generation of PCR, which may be used for applied genomics.

Missense mutations in the CITED2 gene may contribute to congenital heart disease.

BACKGROUND: Congenital heart disease (CHD) is a lifelong abnormality present from birth. Multiple studies have shown that mutations in genes involved in heart development could cause congenital heart disease. The CITED2 gene works as a transcription factor in the hypoxic pathway for the development of the heart. Therefore, five CHD types, ventricular septal defect, atrial septal defect, atrioventricular septal defect, tetralogy of fallot, and patent ductus arteriosus, were evaluated by conducting a targeted single nucleotide polymorphism (SNP) analysis of the CITED2 gene variant rs375393125 (T > C). This study aimed to identify the association of CITED2 gene mutations in CHD patients. METHODS: Three hundred fifty samples, 250 from patients and 100 from controls, were collected for this genetic analysis. Allele-specific PCR and gel electrophoresis were used to identify the target missense mutations. The genotypic results of the CHDs were further validated through Sanger sequencing. RESULTS: The frequency of the homozygous mutant (CC) in CHD patients was 48.4%, and of the heterozygous mutant (TC) genotype was 11.4%; these percentages are higher than controls (1%). The control samples had only one heterozygous TC and no homozygous CC genotype. The chi-square value was obtained at 103.9 with a probability of 0.05, more significant than the significance value of 21.03. The odds ratio was 43.7, which is > 1. The calculated value of ANOVA was 11.6, which was more significant than the F critical value of 3.7. As a result of sequencing, the mutant sample of each selected CHD type was found heterozygous or homozygous, and the results were like those obtained through conventional PCR. CONCLUSION: The samples of CHD patients showed mutations. Therefore, the CITED2 gene SNP might be associated with CHD.

Identification of bacterial community in a rapid composting method using 16SrDNA genes sequencing.

Composting is a process of microbial degradation of organic waste and is commonly applied for waste management. This is a slow process and requires a lot of land and human resources. The present study investigated mechanical augmentation with required microbial culture for composting municipal solid waste (MSW). Thirty isolates were subjected to 16S rDNA PCR amplification and gene sequencing. The isolates' sequencing from the compost samples was processed on BLASTn. Fourteen strains were identified for further experiments. The results divulge that Empedobacter (04), Bacillus (02), Proteus (02), Lactiplantibacillus (01), Klebsiella (01), Citrobacter (01), Brevibacillus (01), E. coli (01) and one unidentified strain were growing during composting. Eleven combinations of bacterial consortium and respective additives were applied for the organic waste decomposition in the next stage, resulting in varied completion periods ranging from 3 to 14 days. Two combinations were completed within 3 days, which are considered ideal combinations for composting. The microbial consortium was significantly diverse, which is a reason for rapid biodegradation. The present study reveals that the technology will be highly feasible for municipal solid waste management in tropical/subtropical countries.

Cytogenetic investigation of couples with recurrent spontaneous miscarriages.

BACKGROUND & OBJECTIVE: Spontaneous pregnancy loss has always been the frustrating experience for the couples and concern clinician. Chromosomal abnormality in either of the parent is considered to be the one of the leading cause of recurrent spontaneous miscarriages. This study was designed to evaluate the possible chromosomal etiology of miscarriage and the subsequent intimacy of maternal or paternal genetic abnormality. METHODS: This case-control study was conducted between January 2016 and October 2016 at a tertiary care hospital in Karachi. A total of thirty-two couples were selected who had suffered with recurrent spontaneous miscarriages (RSM). Using conventional cytogenetic technique karyotyping was performed on all of the subjects. For the control twenty couples were also selected with no history of pregnancy loss. All the karyotypes were recorded on the standard method. Data was analyzed through SPSS version 22. RESULTS: Among thirty-two cases nine cases were found to have abnormal karyotype. In which sex chromosomal trisomy=02 (46,XY/47,XXY), marker chromosome=01 (47,XX,+mar), Robertsonian translocation=01 (45,XY,der,(14:21),(q10;q10)), reciprocal translocation=01 (46,XX,t(11;22)(q23;q11.2)), inversion=02 (46,XX,inv(9)(p11q13)) and minor structural abnormalities=02 (46,XX,15PS+) were found. Approximately equal ratio with 1:1.25 was observed among male and female carrier respectively. Non-significant difference was found between the ages of both carriers (p=0.34). Though a significant different value was calculated in the case of number of miscarriage (p=0.004*). Moreover, no significant association was found among spontaneous miscarriage (SM) and recurrent spontaneous miscarriage (RSM) with respect to maternal age (p= 0.157). CONCLUSION: In the recent study possible chromosomal abnormalities suggested the evaluation of the patient with the history of recurrent spontaneous miscarriage must include conventional cytogenetic. Moreover, probe development and extended investigation can ease the prognosis among pregnancy related complication.

TNF gene promoter region polymorphisms and association with young-onset rheumatoid arthritis.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that shares a major global economic burden due to disabilities and mortality risk. It affects all age groups with a female predominance. Tumor Necrosis Factor (TNF) a proinflammatory cytokine is one of the key players in etiology of autoimmune diseases such as RA. TNF gene promoter polymorphisms predict disease susceptibility, severity and therapeutic response. Therefore, the current case-control study was designed to evaluate the possible association of TNF gene promoter polymorphisms (-238 and -308) with susceptibility to young-onset RA. The study involves 102 individuals (50 young-onset RA patients, 52 healthy individuals). Genomic DNA was extracted using a standard phenol-chloroform method followed by PCR-RFLP for the screening of TNF gene promoter polymorphisms (-238 and -308). The study resulted in the association of TNF -238G/A polymorphism with susceptibility to young-onset RA in the homozygous form GG (Odds Ratio = 3.23, p-value= <0.05), though no significant difference was observed for -308G/A polymorphism among young-onset RA patients and controls. Thus concludes; TNF -238/G/A contributes to the risk of susceptibility to young-onset RA, conversely, TNF -308 G/A protects against the disease. Consequently, the study has demonstrated a possible relationship of studied TNF polymorphism with young-onset RA.

Prospects of Microfluidic Technology in Nucleic Acid Detection Approaches.

Conventional diagnostic techniques are based on the utilization of analyte sampling, sensing and signaling on separate platforms for detection purposes, which must be integrated to a single step procedure in point of care (POC) testing devices. Due to the expeditious nature of microfluidic platforms, the trend has been shifted toward the implementation of these systems for the detection of analytes in biochemical, clinical and food technology. Microfluidic systems molded with substances such as polymers or glass offer the specific and sensitive detection of infectious and noninfectious diseases by providing innumerable benefits, including less cost, good biological affinity, strong capillary action and simple process of fabrication. In the case of nanosensors for nucleic acid detection, some challenges need to be addressed, such as cellular lysis, isolation and amplification of nucleic acid before its detection. To avoid the utilization of laborious steps for executing these processes, advances have been deployed in this perspective for on-chip sample preparation, amplification and detection by the introduction of an emerging field of modular microfluidics that has multiple advantages over integrated microfluidics. This review emphasizes the significance of microfluidic technology for the nucleic acid detection of infectious and non-infectious diseases. The implementation of isothermal amplification in conjunction with the lateral flow assay greatly increases the binding efficiency of nanoparticles and biomolecules and improves the limit of detection and sensitivity. Most importantly, the deployment of paper-based material made of cellulose reduces the overall cost. Microfluidic technology in nucleic acid testing has been discussed by explicating its applications in different fields. Next-generation diagnostic methods can be improved by using CRISPR/Cas technology in microfluidic systems. This review concludes with the comparison and future prospects of various microfluidic systems, detection methods and plasma separation techniques used in microfluidic devices.

Biotechnological approaches to the production of plant-derived promising anticancer agents: An update and overview.

The plant kingdom is a rich source of bioactive compounds, many of which have been used since pre-history for their therapeutic properties to treat a range of illnesses. These metabolites have recently attracted attention to their antineoplastic activities to treat various cancers relying on different mechanisms. Some of these molecules are glycosides, which have proven useful as anti-cancer agents, namely podophyllotoxin (PPT) anaryltetralin lignan or alkaloids. There are three primary forms of alkaloids, such as indole alkaloids (vincristine and vinblastine from Catharanthus roseus), quinoline alkaloid (camptothecin from Camptotheca acuminata), and diterpenoid alkaloid (taxol and it's analogous from Taxus and Corylus species). This review considers various plant biotechnology approaches used to enhance the production of these anticancer molecules in different species. In this regard, many in vitro culture techniques such as stimulation of suspension culture and hairy roots are being used to investigate the effects of plant growth regulators and elicitors on various explants.