Comparison of TCN-2 (776C>G) Gene Polymorphism and Vitamin B12 Status with Different Body Mass Index among Saudi Adults.

BACKGROUND: Overweight and obesity (OO) are significant public health issues, and many elements, including genetics, epigenetics, sedentary lifestyle, comorbid conditions, psychological and environmental pressures, have been linked to OO. More than 2 billion people are presently impacted by the global obesity epidemic, which is still advancing relentlessly. It is a significant public health concern and a major contributor to healthcare costs, because it increases the chance of developing conditions such as heart disease, stroke, type 2 diabetes, and chronic kidney disease (CKD). Using the ranges of 18.5-25 for normality, 25-30 for overweight, and 30 for obesity, BMI (in kg/m2) is used to identify obesity. Vitamin deficiency is one of the causative factors associated with the increasing trend of obesity. Altered vitamin B12 status is a multifactorial trait; changes in B12 status are produced by several single nucleotide polymorphisms (SNPs) in various genes that interact with the environment. They also support coordinated efforts to alter the built environment that is causing the obesity pandemic. Therefore, the present study aimed to evaluate the TCN-2 (776C>G) gene alteration and vitamin B12 levels with respect to different body mass index, as well as associating BMI with other biochemical parameters. METHODS: 250 individuals were involved in the study; among them, 100 were in the healthy weight range category (BMI: 18.5 to <25 kg/m2), 100 were overweight (BMI: 25.0 to <30 kg/m2), and 50 were obese (BMI: >30 kg/m2). Participants visited during the screening program were subjected to blood pressure measurement, and further peripheral blood samples were drawn from all the participants in plain as well as in EDTA vials for biochemical (lipid profile and vitamin B12 level) analysis and single nucleotide polymorphism studies. Extracted DNA from whole blood collected in EDTA vials using kit protocol was used for genotyping by PCR-RFLP. RESULTS: The levels of systolic (p < 0.0001) and diastolic blood pressures (p < 0.0001), HDL (p < 0.0001), LDL (p = 0.04), TG (p < 0.0001), cholesterol (p < 0.0001), and VLDL (p < 0.0001) showed significant differences between healthy controls, overweight, and obese groups. The healthy control TCN-2 (776C>G) genotypes were compared with those of overweight and obese participants, and compared to the healthy controls it was observed that overweight (p = 0.01) and obese (p = 0.002) subjects had significant differences in TCN-2 (776C>G) genotypes. For genotypes CG and GG, the odds ratio was 1.61 (0.87-2.95; p = 0.12), and 3.81 (1.47-9.88; p = 0.005) for overweight participants, respectively, and obese participants' calculated odds ratios were 2.49 (1.16-5.36; p = 0.01) and 5.79 (1.93-17.35; p = 0.001), respectively. The relative risk for genotypes CG and GG, was 1.25 (0.93-1.68; p = 0.12), 2.17 (1.12-4.17; p = 0.02) for overweight participants, while the obese participants' calculated relative risks were 1.31 (1.03-1.68; p = 0.01) and 2.02 (1.12-3.65; p = 0.001), respectively. Vitamin B12 levels were analyzed, and it was observed that a significant difference existed among overweight (305.5 pmol/L, p < 0.0001) and obese patients (229 pmol/L, p < 0.0001), respectively, as compared to healthy controls (385.5 pmol/L). Correlation analysis showed a significant association of vitamin B12 level with TG, cholesterol and VLDL; it showed a negative correlation, suggesting that decreases in B12 levels may impact the lipid profile. CONCLUSION: The study concluded that a predisposition to the GG genotype of TCN-2 gene polymorphism (776C>G) may increase susceptibility to obesity and the related complications, and higher odds and relative risk for the GG genotype may increase the risk of having obesity and further related complications. Lower vitamin B12 levels were linked with obesity and overweight, and impaired lipid parameters suggested that lower vitamin B12 may impact the altered lipid profile.

Vitamin B12 binding to mutated human transcobalamin, in-silico study of TCN2 alanine scanning and ClinVar missense mutations/SNPs.

Many missense mutations/SNPs of the TCN2 gene (which yield Transcobalamin (TC)) were reported in the literature but no study is available about their effect on binding to vitamin B12(B12) at the structural level experimentally nor computationally. Predict the effect of TC missense mutations/SNPs on binding affinity to B12 and characterize their contacts to B12 at the structural level. TC-B12 binding energy difference from the wildtype (ΔΔGmut) was calculated for 378 alanine scanning mutations and 76 ClinVar missense mutations, repeated on two distinct X-ray structures of holoTC namely 2BB5 and 4ZRP. Destabilizing mutations then went through 100 ns molecular dynamics simulation to study their effect on TC-B12 binding at the structural level employing 2BB5 structure. Out of the studied 454 mutations (378 alanine mutations + 76 ClinVar mutations), 19 were destabilizing representing 17 amino acid locations. Mutation energy results show a neutral effect on B12 binding of several missense SNPs reported in the literature including I23V, G94S, R215W, P259R, S348F, L376S, and R399Q. Compared to the wildtype, all the destabilizing mutations have higher average RMSD-Ligand in the last 25% of the MD simulation trajectories and lower average hydrogen bond count while the other parameters vary. Previously reported TCN2 SNPs with an unknown effect on TC-B12 binding were found to have a neutral effect in the current study based on mutation energy calculations. Also, we reported 17 possible amino acids that destabilize TC-B12 binding upon mutation (four listed in ClinVar) and studied their structural effect computationally.

Case report: Novel compound-heterozygous mutations in the TCN2 gene identified in a chinese girl with transcobalamin deficiency.

Transcobalamin (TC) deficiency is a rare autosomal recessive disease characterized by megaloblastic anemia. It is caused by cellular vitamin B12 depletion, which subsequently results in elevated levels of homocysteine and methylmalonic acid. This disease is usually diagnosed by genetic analysis of the TCN2 gene. Here, we described a 2.2-month-old Chinese girl with TC deficiency presenting with diarrhea, fever and poor feeding. Whole-exome sequencing detected a pair of compound-heterozygous mutations in TCN2 gene, c.754-12C>G and c.1031_1032delGA (p.R344Tfs*20). To our knowledge, it is the first time that they were identified and reported in TC deficiency. This study contributes to a better understanding of the TC deficiency, expanding the spectrum of TCN2 mutations in this disorder and also supporting the early diagnosis and proper treatment of similar cases in the future.

Whole exome sequencing, a hypothesis-free approach to investigate recurrent early miscarriage.

RESEARCH QUESTION: Are there genetic determinants shared by unrelated women with unexplained recurrent early miscarriage (REM)? DESIGN: Thirty REM cases and 30 controls were selected with extreme phenotype among women from Eastern Brittany (France), previously enrolled in an incident case-control study on thrombophilic mutations. Cases and controls were selected based on the number of early miscarriages or live births, respectively. Peripheral blood was collected for DNA extraction at initial visit. The burden of low-frequency variants in the coding part of the genes was compared using whole exome sequencing (WES). RESULTS: Cases had 3 to 17 early miscarriages (20 cases: ≥5 previous losses). Controls had 1 to 4 live births (20 controls: ≥3 previous live births) and no miscarriages. WES data were available for 29 cases and 30 controls. A total of 209,387 variants were found (mean variant per patient: 59,073.05) with no difference between groups (P = 0.68). The top five most significantly associated genes were ABCA4, NFAM1, TCN2, AL078585.1 and EPS15. Previous studies suggest the involvement of vitamin B12 deficiency in REM. TCN2 encodes for vitamin B12 transporter into cells. Therefore, holotranscobalamin (active vitamin B12) was measured for both cases and controls (81.2 ± 32.1 versus 92.9 ± 34.3 pmol/l, respectively, P = 0.186). Five cases but no controls were below 50 pmol/l (P = 0.052). CONCLUSIONS: This study highlights four new genes of interest in REM, some of which belong to known networks of genes involved in embryonic development (clathrin-mediated endocytosis and ciliary pathway). The study also confirms the involvement of TCN2 (vitamin B12 pathway) in the early first trimester of pregnancy.

Transcobalamin II deficiency in twins with a novel variant in the TCN2 gene: case report and review of literature.

Objectives Transcobalamin II (TC) is an essential plasma protein for the absorption, transportation, and cellular uptake of cobalamin. TC deficiency presents in the first year of life with failure to thrive, hypotonia, lethargy, diarrhea, pallor, mucosal ulceration, anemia, pancytopenia, and agammaglobulinemia. Herein, we present TC deficiency diagnosed in two cases (twin siblings) with a novel variant in the TCN2 gene. Case presentation 4-month-old twins were admitted with fever, respiratory distress, vomiting, diarrhea, and failure to thrive. Physical examination findings revealed developmental delay and hypotonia with no head control, and laboratory findings were severe anemia, neutropenia, and hypogammaglobulinemia. Despite normal vitamin B12 and folate levels, homocysteine and urine methylmalonic acid levels were elevated in both patients. Bone marrow examinations revealed hypocellular bone marrow in both cases. The patients had novel pathogenic homozygous c.241C>T (p.Gln81Ter) variant in the TCN2 gene. In both cases, with intramuscular hydroxycobalamin therapy, laboratory parameters improved, and a successful clinical response was achieved. Conclusions In infants with pancytopenia, growth retardation, gastrointestinal manifestations, and immunodeficiency, the inborn error of cobalamin metabolism should be kept in mind. Early diagnosis and treatment are crucial for better clinical outcomes. What is new? In literature, to date, less than 50 cases with TC deficiency were identified. In this report, we presented twins with TCN2 gene mutation. Both patients emphasized that early and aggressive treatment is crucial for achieving optimal outcomes. In this report, we identified a novel variation in TCN2 gene.

Genetic Variants in Folate and Cobalamin Metabolism-Related Genes in Nonsyndromic Cleft Lip and/or Palate

The aim of this study was to evaluate the association of the polymorphisms in TCN2 (rs1801198) gene and in MTRR (rs1801394) gene with nonsyndromic cleft lip and/or palate (NSCL/P) in a Brazilian population. Genomic DNA was extracted from buccal cells. The polymorphisms in TCN2 (rs1801198) and MTRR (rs1801394) genes were genotyped by carrying out real-time PCR and Taqman assay. Chi-square test was used to determine the association between genotype and allele frequencies with NSCL/P and NSCL/P subgroups (cleft lip only, cleft lip and palate, and cleft palate only). Eight hundred and sixty seven unrelated individuals (401 cases with NSCL/P and 466 individuals without cleft) were evaluated. Genotype distributions of TCN2 and MTRR polymorphisms were in Hardy-Weinberg equilibrium. The TCN2 polymorphic genotype GG was identified in 16.7% of the NSCL/P group and in 14.1% of the non-cleft group (p>0.05). Similarly, the frequency of MTRR genotype (GG) was similar in NSCL/P group (15.5%) and control group (17.8%) (p>0.05). Multivariate analysis showed an association between MTRR and the subgroup that the mother smoked during pregnancy (p=0.039). Our findings did not demonstrate an association between TCN2 polymorphisms and NSCL/P, however suggests an association between MTRR and NSCL/P etiology.

Resumo O objetivo desse estudo foi avaliar a associação entre os polimorfismos no gene TCN2 (rs1801198) e no gene MTRR (rs1801394) com fissura de lábio e/ou palato não sindrômica (NSFL/P) em uma população brasileira. DNA genômico foi extraído de células bucais. Os polimorfismos nos genes TCN2 (rs1801198) e MTRR (rs1801394) foram genotipados através do PCR em tempo real pelo método Taqman. O teste do qui-quadrado foi utilizado para determinar a associação entre a frequência alélica e genotípica e NSFL/P e nos subtipos (fissura de lábio, fissura de lábio com palato e fissura de palato). Oitocentos e sessenta e sete indivíduos não aparentados (401 casos com NSFL/P e 466 indivíduos sem fissura) foram avaliados. A distribuição dos genótipos dos polimorfismos de TCN2 e MTRR estavam em equilíbrio de Hardy-Weinberg. O genótipo polimórfico GG do gene TCN2 foi identificado em 16,7% do grupo com NSFL/P e em 14,1% do grupo sem fissura (p>0,05). Da mesma forma, a freqüência do genótipo GG do gene MTRR foi bastante semelhante entre o grupo com NSFL/P (15,5%) e o grupo controle (17,8%). A análise multivariada mostrou associação entre o gene MTRR e o subgrupo que apresentou tabagismo materno durante a gestação (p=0,039). Nossos resultados mostraram que não há associação entre os polimorfismos nos genes TCN2 e NSFL/P, entretanto sugerem uma associação entre MTRR e a etiologia de NSFL/P.