001 002 003 004 005 006 007 008 009 010 011 012 013 014 015 016 017 018 019 020 021 022 023 024 025 026 027 028 029 030 031 032 033 034 035 036 037 038 039 040 041 042 043 044 045 046 047 048 049 050 051 052 053 054 055 056 057 058 059 060 061 062 063 064 065 066 ORIGINAL RESEARCH published: xx April 2015 doi: 10.3389/fped.2015.00034 Evaluation of gestational diabetes mellitus risk in South Indian women based on MTHFR (C677T) and FVL (G1691A) mutations Imran Ali Khan 1,2,3 , Noor Ahmad Shaik 4 , Vasundhara Kamineni 5 , Parveen Jahan 3 , Qurratulain Hasan 1,2 and Pragna Rao 6* 1 Department of Genetics and Molecular Medicine, Kamineni Hospitals, Hyderabad, India, 2 Department of Genetics, Vasavi Medical and Research Center, Hyderabad, India, 3 Department of Genetics and Biotechnology, Osmania University, Hyderabad, India, 4 Department of Genetic Medicine, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia, 5 Department of Gynecology and Obstetrics, Kamineni Hospitals, Hyderabad, India, 6 Department of Biochemistry, Kasturba Medical College, Manipal University, Manipal, Karnataka, India Edited by: Marijana Pericic Salihovic, Institute for Anthropological Research, Croatia Reviewed by: Suresh Kumar Chitta, Sri Krishnadevaraya University, India Shaik Abdul Nabi, University of Hyderabad, India Tatjana Skaric-Juric, Institute for Anthropological Research, Croatia *Correspondence: Pragna Rao, Manipal, India drpragnarao@gmail.com We aimed to scrutinize the extent to which single amino acid substitutions in the MTHFR and factor V Leiden (FVL) genes affect the risk of gestational diabetes mellitus (GDM) in pregnant women of South Indian descendant. This case–control study was implemented once the ethical approval has been obtained. Overall, 237 women were recruited in this study: 137 had been diagnosed with GDM and the remaining 100 women were used as normal controls or non-GDM. The diagnosis of GDM was confirmed with biochemical analysis, i.e., GCT and oral glucose tolerance tests. Five milliliters of peripheral blood was collected and used for biochemical and molecular analyses. DNA was isolated, and genotyping for MTHFR (C677T) and FVL (G1691A) mutations was performed using PCR–RFLP. FVL (G1691A) locus was not polymorphic in the investigated sample. There was no significant difference in the allele and genotype frequencies of C677T polymorphism between GDM and non-GDM women (p = 0.8892). Keywords: gestational diabetes mellitus, MTHFR, FVL, C677T, G1691A Introduction Specialty section: This article was submitted to Genetic Disorders, a section of the journal Frontiers in Pediatrics Received: 26 November 2014 Accepted: 07 April 2015 Published: xx April 2015 Citation: Khan IA, Shaik NA, Kamineni V, Jahan P, Hasan Q and Rao P (2015) Evaluation of gestational diabetes mellitus risk in South Indian women based on MTHFR (C677T) and FVL (G1691A) mutations. Front. Pediatr. 3:34. doi: 10.3389/fped.2015.00034 Frontiers in Pediatrics | www.frontiersin.org Gestational diabetes mellitus (GDM) is among the most common metabolic disorders affecting pregnant women characterized by glucose intolerance of variable severity, which is usually detected at the onset of or during pregnancy (1). GDM is treated by insulin or dietary modifications. It typically develops during the secondary trimester of pregnancy and resolves after the delivery (2). The risk of GDM women is intensified by advanced maternal age, ancestral disparities, obesity, and a family history of diabetes (3); however, the exact etiology is unknown due to limited knowledge of genetic factors (4). GDM patients are at increased risk of gestational hypertension, pre-eclampsia during pregnancy, and type 2 diabetes (T2DM), as well as metabolic syndromes in later life (5). Pregnant women with a history of GDM are at greater risk of developing T2DM, and a family history of diabetes predisposes pregnant women to GDM (6). Elevated fasting glucose and insulin levels are observed in women who do not have GDM but have a family history of the disease (7). Individuals of Asian descents have two- to sevenfold greater risk of developing GDM than their Caucasian counterparts in developed countries (8). Various epidemiological studies have reported an association between metabolic disorders and risk of developing T2DM or GDM, and numerous 1 April 2015 | Volume 3 | Article 34 067 068 069 070 071 072 073 074 075 076 077 078 079 080 081 082 083 084 085 086 087 088 089 090 091 092 093 094 095 096 097 098 099 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 Khan et al. 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 Mutational studies in gestational diabetes mellitus efforts have been made to identify pathogenic mutations in nuclear and mitochondrial genomes that are linked to both types of diabetes. Various susceptibility loci have been identified by linkage analyses and genome-wide association studies (1). The methylenetetrahydrofolate reductase (MTHFR) gene has been mapped to chromosomal region 1p36.3 and comprises 11 exons encoding 5′ , 10′ -MTHFR (9), a key regulatory enzyme in folate metabolism that converts 5′ ,10′ -MTHF to 5′ -MTHF, the methyl donor for the remethylation of homocysteine to methionine (10). C677T is a common missense mutation in the gene that results in the substitution of alanine for valine at amino acid position 222. The A222V mutation is associated with a 50% reduction in MTHFR enzymatic activity, which increases plasma homocysteine and decreases plasma folic acid concentrations (11, 12). The factor V Leiden (FVL) gene is involved in the blood coagulation; G1691A mutation results in the substitution of guanine with arginine at the amino acid position 506, yielding a defective FVL protein that is unable to interact with the activated protein C, which was associated with increased coagulation activity and susceptibility to thromboembolism in previous studies. This mutation has also been linked to increased thrombosis risk and mortality in women following delivery/pregnancy (13, 14). Numerous studies have found an association between complications during pregnancy and inherited thrombophilias caused by mutations in MTHFR and FVL (15–17). However, there have been no studies to date addressing the potential role of MTHFR and FVL mutations in the development of GDM in the Indian population. The objective of this study was to investigate the extent to which single amino acid substitutions in the MTHFR and FVL genes affect the risk of GDM in pregnant women of South Indian descent. of blood was collected and stored in ethylenediamine tetraacetic acid (EDTA) in order to screen for MTHFR and FVL mutations. Among GDM patients, 58.4% had a family history of T2DM. Screening and management of diabetes during pregnancy were performed by qualified gynecologists according to guidelines set by the American Diabetes Association (18). Clinical and Biochemical Analyses Gestational diabetes mellitus cases were identified using the glucose challenge test between weeks 24 and 28 of gestation; 50 g of glucose was administered to pregnant women with fasting plasma glucose levels exceeding 130 mg/dL. The oral glucose tolerance test (OGTT) was then performed using 100 g of glucose following an overnight fast and 3 days on an unrestricted diet. Fasting plasma samples were drawn 1–3 h after glucose administration. In this study, GDM cases were defined as those whose glucose levels met or exceeded threshold values described by Khan et al. (19). Women diagnosed with T1DM, T2DM, or any other form of diabetes before pregnancy, were excluded from the study. Body mass index (BMI) was calculated as weight/height2 (kg/m2 ). Subjects with BMI >25 kg/m2 were identified as overweight, while a BMI >30 kg/m2 was categorized as obese. Blood samples were collected in order to measure the fasting blood sugar (FBS) early in the morning after overnight fast for more than 10 h and post-prandial blood glucose (PPBG) levels after 2 h of the FBS. DNA Extraction and Genotyping Genomic DNA was extracted from blood samples stored in EDTA using previously described methods (20). The DNA was dissolved in Tris–EDTA buffer at 100 ng/µL and stored at −80°C prior to the molecular analysis. C677T (MTHFR) and G1691A (FVL) mutations were screened using PCR and restriction fragment length polymorphism analysis performed with previously published primers and restriction enzymes (21) (Table 1). PCR amplification conditions were as follows; 35 cycles of denaturation at 95°C for 5 min, followed by annealing at 68°C for 30 s (C677T) or 56°C for 30 s (G1691A), and extension at 72°C for 5 min. PCR products were digested with appropriate restriction enzymes in a total volume of 20 µL for 16 h at 37°C, and analyzed by 2.5% agarose gel electrophoresis with ethidium bromide staining. Materials and Methods Ethics Statement The study protocol was approved by the institutional ethics committee at Kamineni hospitals. Written, informed consent was obtained from all subjects, who were recruited by a clinical research midwife. Study Subjects Statistical Analysis This study was carried out in South Indian population from 2007 to 2011. Study subjects were recruited from Kamineni and Muslim maternity hospitals, Hyderabad. A total of 237 pregnant women participated in the study; of these, 137 had GDM, and 100 had normal glucose levels during pregnancy (i.e., the nonGDM group). A diagnosis of GDM was made by analyzing 3 mL of serum samples obtained from each subject; an additional 2 mL Hardy–Weinberg equilibrium was calculated in the cases and controls as described in our prior study (22). Differences between genotype/allele frequencies of GDM and non-GDM subjects were calculated using T test. The Yates correction was applied prior to analyzing genotype frequencies of MTHFR and FVL mutations. The odds ratio (OR) for genotype/phenotype relations and its 95% TABLE 1 | Details for genotyping MTHFR and FVL genes. Gene SNP location rs no. Amino acid substitution MTHFR Exon 5 rs1801133 Ala 222 Val FVL Exon 10 rs6020 Arg 506 Gln Frontiers in Pediatrics | www.frontiersin.org Forward primer Reverse primer TGAAGGAGAAGGTGT CTGCGGGA TCAGGCAGGAACAA CACCAT GGACGGTGCGGTG AGAGTG GGTTACTTCAAGGACAAAA TACCTGTAAAGCT 2 Fragment Annealing temperature Enzyme 198 bp 68°C HinfI 241 bp 56°C HindIII April 2015 | Volume 3 | Article 34 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 Khan et al. 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 Mutational studies in gestational diabetes mellitus TABLE 2 | Clinical details of GDM cases and controls. S. No. Factors 1 2 3 4 5 6 7 8 Age (years) BMI (kg/m2 ) Weight (kg) Mean gestational age (weeks) FBS (mg/dL) PPBG (mg/dL) Family history Insulin/diet therapy (Rx ) GDM (n = 137) 26.7 ± 5.1 26.8 ± 3.93 69.3 ± 10.18 24.4 ± 5.0 110.6 ± 3.93 158.80 ± 47.76 80 (58.4%) 81 (59.1%)/56 (40.9%) p Valuea Controls (n = 100) 24.6 ± 3.55 24.1 ± 3.55 51.2 ± 6.26 NA 99.24 ± 11.37 112.00 ± 39.70 56 (56%) NA 0.0001 0.28 0.0001 NA <0.0001 0.05 0.66 NA a t-Test applies to calculate the p value between GDM and non-GDM subjects. NA, not analyzed/not applicable, BMI, body mass index; FBS, fasting blood sugar; PPBG, post-prandial blood glucose. confidence interval (CI) was calculated performing chi-square test in the cross-tabs procedure for a 2 × 2 tables using SPSS software. All statistical analyses were performed using SPSS version 19.0 (SPSS Inc., Chicago, IL, USA). Results Characteristics of the Study Population The clinical characteristics of GDM patients and non-GDM subjects are listed in Table 2. GDM cases (n = 137) were aged 22–38 years with a mean age of 26.7 ± 5.1 years, whereas the age and mean age for controls (n = 137) were 17–34 and 24.6 ± 3.55 years. The pre-pregnancy BMI range was 19.8–35.6 kg/m2 (mean: 26.8 ± 3.93 kg/m2 ) for GDM and 19–31.1 kg/m2 (mean: 24.1 ± 3.55 kg/m2 ) for controls (p = 0.28); 40.9% of individuals managed the diabetes with appropriate diet therapy and exercise, while 59.1% of patients required four to eight units of insulin therapy over the entire prenatal period. Age, weight, FBS, and PPBG values differed significantly between GDM cases and controls (p < 0.05). FIGURE 1 | Three percent agarose gel consist of digested products of C677T mutation. It represents the 3% agarose gel picture. Lane 1 consists of 100 bp ladder. Lanes 2–10 consist of homozygous CC genotypes and lane 11 consists of heterozygous CT genotype. TABLE 3 | Allele and genotype frequencies in GDM cases and controls for MTHFR gene. Genotype and allele C677T Allele and Genotype Frequencies Genotype and allele frequencies were in Hardy–Weinberg equilibrium in both groups. The PCR product encompassing the C677T mutation was 198 bp in length; digestion with HinfI (G↓ ANTC) yielded 198 and 175/23 bp fragments representing C and T alleles, respectively (Figure 1). Genotype frequencies for C677T CC, CT, and TT among GDM cases were 81.8, 18.2, and 0%, respectively. The frequencies of the T and C alleles were 9 and 91%. In control subjects, the frequencies of the CC, CT, and TT genotypes were 82, 18, and 0%, respectively, and C and T allele frequencies were 91 and 9%. Table 3 presents just the distribution of genotypes and alleles by GDM status. There was no evidence of a disease association for any of the allelic or genotype classes (OR: -1.015; 95% CI = 0.5378–1.916; p = 0.8892 and OR: -1.011; 95% CI = 0.5211–1.961; p = 0.9746). Controls (n = 100) N (%) N (%) p Valuea MTHFR (rs1801133) CC CT TT C T a 112 (81.8) 25 (18.2) 0 (0) 249 (0.09) 25 (0.09) 82 (82) 18 (18) 0 (0) 182 (91) 18 (9) 0.96a 0.96a Chi-square p value. Discussion This study investigated whether C677T and G1691A mutations are associated with the development of GDM among individuals of South Indian descent by genotyping age-matched cases and pregnant non-GDM subjects. The results did not reveal any association between investigated mutations and GDM risk in this study population. Hyperglycemia is associated with an increased risk of unfavorable outcome in adults (T2DM), children (T1DM), and pregnant G1691A Allele and Genotype Frequencies A 241-bp PCR product encompassing the G1691A mutation was digested with HindIII (A↓ AGCTT), yielding a 209-bp fragment indicating the presence of the A allele (Figure 2). Only the GG (100%) genotype was observed in both cases and controls; therefore, this locus was not further analyzed. Frontiers in Pediatrics | www.frontiersin.org GDM cases (n = 137) 3 April 2015 | Volume 3 | Article 34 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 Khan et al. 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 Mutational studies in gestational diabetes mellitus The prior report from Australian population confirmed that 16.6% women harboring the MTHFR C677T gene mutation developed at least one pregnancy complication; however, the effect was restricted to women with small for gestational age (SGA) infants. There were no differences in genotype distribution among women with intrauterine fetal death, pre-eclampsia, or preterm delivery, and the study concluded that the mutation was a genetic marker for identifying women who are at increased risk of having an SGA infant (30). Diabetes is a risk factor for thrombotic events (31) and atherothrombosis (32). The pathogenesis of diabetes involves hypercoagulability of the blood due to several factors such as the non-enzymatic glycosylation of clotting inhibitors. Patients who are carriers of a thrombophilic gene variant (e.g., factor V A1691G and\or prothrombin A20210G) or other thrombotic risk factors are susceptible to the inherited or acquired thrombophilia. FVL mutations lead to a thrombophilic condition that is heritable both in hetero- and homozygous forms (33). Several previous studies have investigated a potential relationship between FVL gene mutations and diabetes incidence, and there is one report of a possible co-segregation of FVL and T2DM risk alleles (34). A prevalence of 4.6% for the A allele of FVL G1691A (rs6020) has been reported among coronary artery disease patients with T2DM (35). In a later study, these authors reported prevalence rates for this mutation of 3.2, 1.6, and 4.9% among Iranian T2DM patients, T2DM patients with microalbuminuria, and normoalbuminuric T2DM patients, respectively, suggesting that the FVL G1691A mutation is not associated with microalbuminuria in this population (14). This mutation has also been implicated in the pathogenesis of thrombosis (36). No association was found between FVL mutations and T2DM among Lebanese (36), Dutch (37), Japanese (38), and Caucasian patients (39). In conclusion, MTHFR (C677T) and FVL (G1691A) mutations are not likely to be genetic risk factors for the development of GDM in South Indian women. FIGURE 2 | Digested and undigested PCR products appear on 3% agarose gel for (G1691A) mutation. Digested PCR products run on 3% agarose gel. Lane 1 represents 100 bp DNA ladder. Lanes 2–5 represent homozygous GG genotypes. women (GDM) (23). MTHFR and FVL gene mutations are considered risk factors for hereditary thrombophilia and may influence the development of complications during pregnancy (24). We sought to determine whether C677T and G1691A mutations are associated with altered glucose levels during pregnancy, which could indicate GDM risk. Cases and controls differed significantly in terms of age, weight, FBS, and PPBG, but not with respect to genotype frequencies at either locus. These results suggest that the C677T and G1691A mutations are not involved in the development of GDM during pregnancy in Indian population. The frequency of MTHFR gene mutations varies across geographical and ethnic groups. For example, the frequency of C677T in T2DM cases fluctuates between 40 and 49%, depending on the ethnicity of the study cohort (India, 40%; Turkey, 49%; Tunisia, 45%; Brazil, 46%; and China, 44%) (25–29). The C677T genotype frequencies (CC and CT) between north (25) and South Indian populations in T2DM and GDM subjects are varied. Both environmental and genetic factors contribute to the development and progression of GDM during pregnancy; this work showed that MTHFR mutations have no role in GDM in South Indian population. Acknowledgments Gratitude is expressed to all the volunteers who have participated in this study. We are grateful to Indian Council for Medical Research for providing SRF to IAK and funding of this research (Sanction no. 5-3-8-39-2007; RHN). References 4. Khan AI, Jahan P, Hasan Q, Rao P. 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The Review Editor Suresh Kumar Chitta declares that, despite having collaborated on a publication in the last 2 years with author Noor Ahmad Shaik, the review process was handled objectively. Copyright © 2015 Khan, Shaik, Kamineni, Jahan, Hasan and Rao. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 5 April 2015 | Volume 3 | Article 34 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660