Association of Oral Submucous Fibrosis Risk with GSTM1 and GSTT1 Gene Polymorphisms.

OBJECTIVE: To determine the association of GSTM1 and GSTT1 polymorphisms with oral submucous fibrosis (OSF). STUDY DESIGN: A case-control study. Place and Duration of the Study: Department of Human Genetics and Molecular Biology, University of Health Sciences, Lahore and Oral and Maxillofacial Surgery Department, de Montmorency, College of Dentistry/ Punjab Dental Hospital, Lahore, Pakistan, from 1st April 2019 to 31st April 2020. METHODOLOGY: OSF patients were diagnosed with different clinical staging of mouth opening by Vernier caliper with the help of a professional dentist in the Department of Oral and Maxillofacial, de Montmorency, College of Dentistry, Lahore. One hundred and eight blood samples of OSF patients and 108 samples of normal controls were collected. Genomic DNA was obtained from whole-blood extraction. Multiplex PCR amplification using GSTM1, GSTT1, and ß -Globin gene primers was performed. RESULTS: GSTM1 and GSTT1 null genotypes frequencies were found in 43.5% (47/108) and 13.9% (15/108) of controls, whereas 54.6% (59/108) and 25.9% (28/108) of OSF patients, respectively. OSF patients had a greater frequency rate of GSTM1 and GSTT1 null genotypes than controls [OR 1.56, 95% CI 0.91-2.67 (p=0.13)] and [OR 2.17, 95% CI 1.08-4.34 (p=0.04)], respectively. The GSTT1 genotype was found statistically significant with OSF (p=0.05), and risk was also determined. The cumulative effect of null genotypes of GSTM1/GSTT1 did not show any association with the controls and in OSF patients. Proportions of active and null alleles of the patient group were; 86.1%/13.9%; and in control, it was 92.6%/7.4% (OR = 2.01; CI: 0.82-4.97; p=0.18), respectively. CONCLUSION: The study determined a statistically significant association of GSTT1 gene polymorphism with OSF. KEY WORDS: Oral submucous fibrosis, GSTM1, GSTT1, Gene polymorphisms, Genetic risk.

Genetic Alterations, DNA Methylation, Alloantibodies and Phenotypic Heterogeneity in Type III von Willebrand Disease.

Type III von Willebrand disease is present in the Punjab province of Pakistan along with other inherited bleeding disorders like hemophilia. Cousin marriages are very common in Pakistan so genetic studies help to establish protocols for screening, especially at the antenatal level. Factors behind the phenotypic variation of the severity of bleeding in type III vWD are largely unknown. The study was conducted to determine Mutations/genetic alterations in type III von Willebrand disease and also to determine the association of different mutations, methylation status, ITGA2B/B3 mutations and alloimmunization with the severity of type III vWD. After informed consent and detailed history of the patients, routine tests and DNA extraction from blood, mutational analysis was performed by Next Generation Sequencing on Ion Torrent PGM. DNA methylation status was also checked with the help of PCR. In our cohort, 55 cases were detected with pathogenic mutations. A total of 27 different mutations were identified in 55 solved cases; 16 (59.2%) were novel. The mean bleeding score in truncating mutations and essential splice site mutations was relatively higher than weak and strong missense mutations. The mean bleeding score showed insignificant variation for different DNA methylation statuses of the VWF gene at the cg23551979 CpG site. Mutations in exons 7,10, 25, 28, 31, 43, and intron 41 splice site account for 75% of the mutations.

Two homozygous missense mutations in ITGB3 gene as a cause of Glanzmann Thrombasthenia in four consanguineous Pakistani pedigrees.

INTRODUCTION: Glanzmann thrombasthenia (GT) is most common of inherited platelet disorders, resulting from quantitative/qualitative defects in platelet surface integrin αIIbß3, encoded by ITGA2B and ITGB3 genes. Little is known about clinical and molecular characteristics of GT patients from highly consanguineous Pakistani population. METHODS: This study analyzed the clinical and molecular spectrum of six GT patients from four unrelated but consanguineous families. Platelet surface expression of αIIbß3 integrin was determined using flow cytometry analysis. ITGA2B and ITGB3 genes were screened for causative mutations by DNA sequencing. Detected mutations were characterized for their pathogenicity using a variety of in silico tools. RESULTS: Glanzmann thrombasthenia patients in this study generally presented early in life, had a severe course of clinical disease with transfusion dependency for management of bleeding episodes. Molecular analysis revealed 2 homozygous missense mutations in ITGB3 gene, c.422 A˃G (p.Y141C) in three GT patients from a single pedigree with familial segregation and c.1641 C>G (p.C547W) in three unrelated GT patients from three families manifesting type I GT with severe reduction in platelet αIIbß3 levels. In silico pathogenicity predictions, multiple sequence alignment and 3D protein modeling unanimously suggested deleterious nature of the detected mutations, possibly due to aberrant disulfide bonding. Of note, clinical diversity was observed even among GT patients with same mutation in GT1 family. CONCLUSION: This study provides an initial yet important account of clinical and genetic characterization of GT in local patients which may spark further studies to help molecular diagnosis, optimal disease management, and genetic counseling based prevention efforts.

Association of vitamin D receptor gene polymorphisms and risk of urolithiasis: results of a genetic epidemiology study and comprehensive meta-analysis.

Polymorphisms of vitamin D receptor (VDR) gene have been associated with risk of urolithiasis, but, with inconsistent results and lack data from Pakistani population. Therefore, after including our indigenous study data, a comprehensive meta-analysis was performed to provide an evidence-based estimate of any association between VDR polymorphisms and urolithiasis risk. A total of 483 Pakistani subjects, comprising 235 urolithiasis patients and 248 healthy controls, were genotyped for 6 VDR polymorphisms. Additionally, a systematic literature search with subsequent meta-analysis was conducted and pooled odds ratios (ORs) were used to determine the strength of any existent associations. Trial sequential analysis (TSA) was also performed. Results revealed no significant association of any VDR polymorphism and urolithiasis risk in indigenous Pakistani patients. However, meta-analysis of 29 relevant studies indicated that VDR FokI polymorphism significantly increased the risk of urolithiasis in allelic (f vs. F: OR = 1.13; 95% CI = 1.05-1.22; p ≤ 0.01) and recessive (ff vs. FF + Ff: OR = 1.20; 95% CI = 1.05-1.38; p = 0.01) models with no significant heterogeneity. No associations were evident for VDR ApaI, BsmI and TaqI polymorphic variants and urolithiasis risk after correction for multiple testing. Subgroup analysis by ethnicity suggested significant association for FokI variant among Asians. The TSA results demonstrated that the evidence reflecting association of FokI polymorphism and urolithiasis risk was sufficient and conclusive. In conclusion, this meta-analysis suggests that VDR FokI polymorphism is significantly associated with urolithiasis risk, especially in Asians, whereas ApaI, BsmI and TaqI polymorphisms are not associated.

Screening of the LAMB2, WT1, NPHS1, and NPHS2 Genes in Pediatric Nephrotic Syndrome.

Mutations in the NPHS1, NPHS2, LAMB2, and the WT1 genes are responsible for causing nephrotic syndrome (NS) in two third of the early onset cases. This study was carried out to assess the frequencies of mutations in these genes in a cohort of pediatric NS patients. A total of 64 pediatric familial or sporadic SRNS cases were recruited. Among these, 74% had a disease onset of up to 3 years of age. We found one homozygous frameshift mutation in the NPHS1 gene in one CNS case and two homozygous mutations in the NPHS2 gene. Six mutations in four cases in the LAMB2 gene were also identified. No mutation was detected in the WT1 gene in isolated SRNS cases. LAMB2 gene missense mutations were segregating in NS cases with no extra-renal abnormalities. Analysis of the population genomic data (1000 genome and gnomAD databases) for the prevalence estimation revealed that NS is more prevalent than previously determined from clinical cohorts especially in Asian population compared with overall world populations (prevalence worldwide was 1in 189036 and in South-Asian was 1in 56689). Our results reiterated a low prevalence of mutations in the NPHS1, NPHS2, LAMB2, and WT1 genes in the studied population from Pakistan as compared to some European population that showed a high prevalence of mutations in these genes. This is a comprehensive screening of the genes causing early onset NS in sporadic and familial NS cases suggesting a more systematic and robust approach for mutation identification in all the 45 disease-causing genes in NS in our population is required.