{"id":56369,"date":"2024-03-20T10:28:53","date_gmt":"2024-03-20T10:28:53","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=56369"},"modified":"2024-04-02T04:23:36","modified_gmt":"2024-04-02T04:23:36","slug":"association-of-mthfd1-g1958a-mthfd1-t401c-and-cbs-844ins68bp-with-breast-cancer-in-jordan","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no1\/association-of-mthfd1-g1958a-mthfd1-t401c-and-cbs-844ins68bp-with-breast-cancer-in-jordan\/","title":{"rendered":"Association of MTHFD1 G1958A, MTHFD1 T401C and CBS 844ins68bp with Breast Cancer in Jordan"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Female breast cancer (BC) is the most commonly worldwide diagnosed cancer, with a recorded 2.3 (11.7%) million new cases in 2020 1<\/sup>. In Jordan, BC is the most common cancer, accounting for 38.4% of all detected cancers in females (National Cancer Registry (JNCR., 2021). Despite the high prevalence of BC and the identification of various risk factors, the causes of BC are not completely clear. A critical step for interventions and good management of BC is to identify risk factors for its development. There are several known risk factors for breast cancer including genetic factors 2’3<\/sup> which are highly heterogeneous 3’4<\/sup>. While only a minor fraction of these genetic factors arise from mutations in established high-penetrance susceptibility genes, the predominant share is believed to stem from common genetic variants, including single nucleotide polymorphisms (SNPs). Identification of the genetic risk factors for any type of cancer in any population is essential for these factors can be changed and adjusted to reduce the risk of cancers. Among those that can allow protective effect against cancers in particular are different common genetic variants, for example, single nucleotide polymorphisms (SNPs) in the genes encoding functional enzymes and coenzymes in the folate\/ one-carbon metabolism and lifestyle risk factors 5’6’7’8<\/sup>. SNPs in genes related to the folate\/ one-carbon metabolism alter gene function or regulation by changing the structure and the catalytic activities of the affected enzymes, and increasing DNA methylation in promoters of many genes and methylation reduction in promoters of other genes. These changes lead to activation of proto-oncogenes and inactivation of tumor suppressor genes 9’10<\/sup> and alter cell division leading to aberrant chromosomal segregation and tumorigenesis 11’12’13’14<\/sup>. Different studies reported the characteristics and risk factors for BC in Jordanian women; these included lifestyle risk factors such as alcohol consumption, cigarette smoking 6’15<\/sup> high-fat nutrient intake pattern, and insufficient exercise 7<\/sup>. Other risk factors reported for BC in Jordan were age,  obesity, body mass index (BMI), high level of estrogen, age at menarche and menopause, reproduction history, exposure to ionizing radiation, and hereditary background 16’17<\/sup>. Unlike the epidemiological factors, few and limited reports were published on the genetic factors and different gene polymorphisms (SNPs) associated with the risk of BC in Jordan. These included the known germline, high penetrance susceptibility genes, which include the germline BRCA1<\/em> and BRCA2<\/em> mutations 18’19<\/sup> and few studies examined the association of SNPs in genes related to the folate\/one-carbon metabolism with BC 20’21<\/sup><\/p>\n\n\n\n

MTHFD1<\/em> and CBS<\/em> are genes encoding the enzymes methylenetetrahydrofolate dehydrogenase (MTHFD1<\/em>) and cystathionine \ud835\udefd-Synthase (CBS<\/em>) respectively have central roles in folate and homocysteine metabolism through the folate \/one-carbon metabolism and are candidate genes for cancer susceptibility 22’23’24’25’26<\/sup>. MTHFD1 enzyme catalyzes the irreversible conversion of 5,10-methylenetetrahydrofolate to the primary form of circulatory folate 5-methyltetrahydrofolate 27<\/sup>, and is essential for the synthesis of purine and pyrimidine bases 28<\/sup> and regeneration of SAM 29<\/sup>. CBS is a major enzyme in the transsulfuration pathway, at the homocysteine (Hcy) junction of preserving methionine or converting it to cysteine. CBS<\/em> eliminates Hcy by catalyzing the condensation of serine and Hcy to form cystathionine 30<\/sup>, which is then hydrolyzed to cysteine, a precursor of the potent antioxidant glutathione 28’31<\/sup>. <\/p>\n\n\n\n

Different studies showed an association of\nMTHFD1<\/em> G1958A with different diseases including cardiovascular diseases 27<\/sup>‘<\/sup>32<\/sup>\nmaternal risk for fetal loss 33<\/sup>\nand neural tube defects 34<\/sup>‘<\/sup>24<\/sup>‘<\/sup>35<\/sup>\nand psychiatric disorders 36<\/sup>.\nSeveral clinical studies showed over-expression of CBS<\/em> and, increased\nproduction of H2S in many cancer types including colon, ovarian, gastric,\ncolorectal, prostate, and gastroesophageal cancer 35<\/sup>‘<\/sup>37<\/sup>‘<\/sup>38<\/sup>‘<\/sup>39<\/sup>‘<\/sup>40<\/sup>.\nThe main CBS-derived metabolites are the anti-inflammatory (H2<\/sub>S) and\nHcy 31<\/sup>.\nH2S is associated with signaling and protective effects on antioxidant defenses\ninhibits the production of hydrogen peroxide (H2O2) and other reactive oxygen\nspecies (ROS) and preserves the activity of key antioxidant enzymes including\ncatalase and superoxide dismutase, glutathione peroxidase, and\nglutathione-S-transferase 41<\/sup>‘<\/sup>42<\/sup>‘<\/sup>43<\/sup>.\nFunctional SNPs in CBS<\/em> gene can promote carcinogenesis 11<\/sup>‘<\/sup>40<\/sup>‘<\/sup>44<\/sup>‘<\/sup>45<\/sup>‘<\/sup>46<\/sup>.<\/p>\n\n\n\n

The association between\neach of MTHFD1<\/em> and CBS<\/em> genes with BC are still controversial and\nindefinite. To our knowledge, this is the first study to examine associations\nof MTHFD1<\/em> and CBS<\/em> genes with breast cancer in Jordanians. Thus in\nthis case-control study, we aimed to examine the possible association of MTHFD1<\/em>\nG1958A (R653Q), MTHFD1<\/em> T401C (R134K), and the insertion mutation CBS<\/em>\n844ins68bp with BC among Jordanian women.<\/p>\n\n\n\n

Materials and methods<\/strong><\/p>\n\n\n\n

Subjects<\/strong><\/p>\n\n\n\n

A group of two hundred\nwomen with confirmed diagnoses of breast cancer (cases) as well as a group of\n200 age-matched unaffected women (controls) were recruited from two major\nreferral hospitals for cancer, King Abdullah Hospital in Irbid, (northern part\nof Jordan) and Al-Basheer Hospital in Amman (central part of Jordan). Each of\nthe participants in the study provided informed consent to donate samples of\ntheir blood and use clinical data for research. All procedures used were in\nstrict compliance with the principles of the Helsinki II Declaration. All\nprocedures used were in strict compliance with the principles of the Helsinki\nII Declaration. The study was ethically approved by the Yarmouk University\n(Irbid, Jordan) IRB committee (YU IRB DSR 2023\/190).<\/p>\n\n\n\n

Blood sampling, DNA isolation and Genotyping<\/strong><\/p>\n\n\n\n

Blood samples (3ml) were withdrawn\nfrom subjects into EDTA vacationers and stored at 4\u00b0C until DNA extraction.\nGenomic DNA was extracted from the collected blood samples according to the\nmanufacturer’s instructions using a commercial kit (OMEGA Biokit). Genotyping\nof both MTHFD1<\/em> G1958A (rs2236225) and MTHFD1<\/em> T401C (rs1950902)\nwas achieved by specific PCR amplification of the genomic DNA, followed by\nsubsequent digestion with the proper restriction enzyme according to 33<\/sup> and\n47<\/sup>\nrespectively. At the same time, the detection of CBS<\/em>844ins68bp was\naccomplished according to 48<\/sup>\nby direct PCR.<\/p>\n\n\n\n

Table 1 summarizes the\ngenotyping conditions including the sequences of the primers, amplification\nconditions, restriction endonucleases and sizes of the DNA fragments produced.\nAll PCR reactions were carried out in a total volume of 25\u03bcl, containing 1\u03bcl of\n(5pmole\/\u03bcl) of the proper forward and reverse primers specific for each SNP,\n12.5\u03bcl  of 2X master mix (Promega, USA),\nand 1\u03bcl of DNA sample in nuclease-free water up to 25 \u03bcl. Amplification\nproducts were visualized by electrophoresis on 2% agarose gel (Agarose A; Biobasic)\nfollowing staining with 0.5\u03bcg\/ml ethidium bromide (Sigma, USA).<\/p>\n\n\n\n

Statistical analyses<\/strong><\/p>\n\n\n\n

Comparisons between\ngroups and the different allele frequencies were evaluated using Pearson\nchi-square and the goodness of fit test (P>0.05). The allele and genotype\nfrequencies of the variants in the case and control groups were calculated. Logistic\nregression analyses were recruited to estimate the associations between the\nrisk of breast cancer and each examined variant, determining odds ratios (ORs)\nand their corresponding 95% confidence intervals (CIs). The statistical\nanalyses were conducted using SPSS version 22.0 (SPSS, Chicago, IL).<\/p>\n\n\n\n

Table 1: Primers sequences and PCR amplification conditions for the genotyping of the targeted polymorphisms MTHFD1<\/em> G1958A, MTHFD1<\/em> T401C, and CBS<\/em> 844ins 68<\/strong><\/p>\n\n\n\n\n\n\n\n\n
\n

Polymorphism<\/strong><\/p>\n<\/td>\n

\n

Primer sequence (5\u2019\u2192 3\u2019)<\/strong><\/p>\n<\/td>\n

\n

PCR  conditions: denaturation, annealing, and extension <\/strong><\/p>\n<\/td>\n

\n

Restriction enzyme and  incubation conditions<\/strong><\/p>\n<\/td>\n

\n

Fragment length produced in base pairs <\/strong>(bp)<\/p>\n<\/td>\n

\n

Primers reference<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

MTHFD1 G1958A<\/em><\/p>\n

 (Arg653Gln)<\/p>\n<\/td>\n

\n

F:5\u2019CACTCCAGTGTTTGTCCATG-3\u2019<\/p>\n

R:5\u2019GCATCTTGAGAGCCCTGAC-3\u2019<\/p>\n<\/td>\n

\n

Total of 35 cycles: denaturation at 94\u02daC for 30 sec, annealing at 58\u02daC for 1 min., and extension at 72\u02daC for 1 min.<\/p>\n<\/td>\n

\n

*Msp<\/em>I incubated at 37\u00b0C, 3h<\/p>\n<\/td>\n

\n

G allele: 196bp, 71bp and 63bp<\/p>\n

 <\/p>\n

A allele: 267 and 63bp<\/p>\n<\/td>\n

\n

33<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

MTHFD1 T401C<\/em><\/p>\n<\/td>\n

\n

F: 5\u2019-GGCGTACAAGGAATGAAAC3\u2019<\/p>\n

R: 5\u2019-GGATGTGGATGGGTAAGTG3\u2019<\/p>\n<\/td>\n

\n

35 cycles: 95\u02daC for 30 sec, 48\u02daC for 40 sec, 72\u02daC for 40 sec<\/p>\n<\/td>\n

\n

*BsmA<\/em>I<\/p>\n

Incubated at 37 \u00b0C,<\/p>\n

16 h<\/p>\n<\/td>\n

\n

T allele: 180 and 45 bp<\/p>\n

C allele: 131, 49, and 45 bp<\/p>\n<\/td>\n

\n

47<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

CBS 844ins 68<\/em><\/p>\n

 <\/em><\/p>\n<\/td>\n

\n

F: 5\u2019-CGCCCTCTGCAGATCATTGG3\u2019<\/p>\n

R: 5\u2019-CCTTCCACCTCGTAGGTTGTC3\u2019<\/p>\n<\/td>\n

\n

32 cycles: 95\u02daC for 30 sec, 61\u02daC for 40 sec, 72\u02daC for 40 sec<\/p>\n<\/td>\n

\n

–<\/em>–<\/p>\n<\/td>\n

\n

Wild type: 100bp, Homozygous mutant:168bp<\/p>\n

Heterozygous: 100bp and 168bp.<\/p>\n<\/td>\n

\n

48<\/sup><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

*Source: (New England Biolabs, Ipswich, MA, USA).<\/p>\n\n\n

Results <\/strong><\/p>\n\n\n\n

The mean ages of the\nstudied BC patients (50.22 \u00b1 10.8 years) and the unaffected controls (49.03\u00b1\n10.4 years) were statistically not different (P= 0.919). Figures 1 and 2 show\nrepresentative results of the PCR \u2013 RFLP genotyping of MTHFD1<\/em> G1958A and\nMTHFD1<\/em> T401C respectively, while Figure 3 shows representative results\nof CBS<\/em> 844ins68bp by direct PCR. Table 2 shows the observed frequencies\nof both alleles and genotypes in BC patients and the unaffected controls.  The alleles and genotypes of each of the three\nexamined SNP were in Hardy Weinberg equilibrium (P> 0.05). Table 2 shows\nthat the distribution of the wild type and mutant alleles of the individual\npolymorphisms MTHFD1<\/em> G1958A, MTHFD1<\/em> T401C and CBS<\/em>\n844ins68bp as well as the frequencies of the different genotypes of each\nexamined polymorphism in the BC group were not significantly different from\ntheir frequencies in the controls. In addition, the sums of the mutant\ngenotypes of each of these three polymorphisms were not significantly different\nbetween the patients and the control groups. The odd ratios of the sums of\nmutant genotypes MTHFD1<\/em> G1958A (GA+AA), MTHFD1T401C (TC+CC) and CBS\n844ins68 (w\/Ins + Ins\/Ins) were 0.918 (95% CI=0.601-1.401; p = 0.67), 1.042\n(95% CI=0.328-3.306; P=1.00) and 1.197 (95%CI = 0.701-2.044; p=0.498)\nrespectively. <\/p>\n\n\n\n

However, the frequencies\nof the double compound mutant genotypes GA\/CC and AA\/CC of the two\nnon-synonymous MTHFD1<\/em> polymorphisms G1958A and T401C were higher in BC\npatients group compared to the unaffected controls. The double compound\ngenotypes MTHFD<\/em> GA\/CC and AA\/CC seemed to increase the risk for BC by\n3.4 and 5.1 respectively (Table 3) but their confidence intervals  were wide possibly due to the small and\nlimited numbers of the observed individuals carrying double or triple compound\ngenotypes.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1:<\/strong> Gel image shows PCR-RFLP pattern for MTHFD1<\/em> G1958A and MTHFD1<\/em> T401C resolved on 3% agarose gel stained with ethedium bromide.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 2:<\/strong> Gel image shows the PCR analysis of CBS<\/em> 844ins68 resolved on 3% agarose gel stained with ethedium bromide. <\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 2: Allele and genotype frequencies of MTHFD1 <\/em>G1958A, MTHFD1 <\/em>T401C, and CBS <\/em>844ins68polymorphisms in BC and the control groups.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

 <\/strong><\/p>\n

 Polymorphisms<\/strong><\/p>\n<\/td>\n

\n

%  Controls<\/strong><\/p>\n

 ( n) <\/strong><\/p>\n<\/td>\n

\n

%  Cases  <\/strong><\/p>\n

(n)  <\/strong><\/p>\n<\/td>\n

\n

OR (95% CI)<\/strong><\/p>\n<\/td>\n

\n

P<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

MTHFD1 <\/em><\/strong>G1958A<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

G (ref)<\/strong><\/p>\n<\/td>\n

\n

0.59% (235)<\/p>\n

 <\/p>\n<\/td>\n

\n

0.58% (231)<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

A<\/strong><\/p>\n<\/td>\n

\n

0.41% (165)<\/p>\n<\/td>\n

\n

 0.42% (169)<\/p>\n<\/td>\n

\n

0.960 (0.725-1.271)<\/p>\n<\/td>\n

\n

0.774<\/p>\n<\/td>\n<\/tr>\n

\n

GG (ref)<\/strong><\/p>\n<\/td>\n

\n

0.30 (61)<\/p>\n<\/td>\n

\n

0.32 (65)<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

GA<\/strong><\/p>\n<\/td>\n

\n

0.57 (113)<\/p>\n<\/td>\n

\n

0.51 (101)<\/p>\n<\/td>\n

\n

0.847 (0.544-1.317)<\/p>\n<\/td>\n

\n

0.229<\/p>\n<\/td>\n<\/tr>\n

\n

AA<\/strong><\/p>\n<\/td>\n

\n

0.13 (26)<\/p>\n<\/td>\n

\n

0.17 (34)<\/p>\n<\/td>\n

\n

1.270 (0.677-2.375)<\/p>\n<\/td>\n

\n

0.263<\/p>\n<\/td>\n<\/tr>\n

\n

GA and AA<\/strong><\/p>\n<\/td>\n

\n

0.70 (139)<\/p>\n<\/td>\n

\n

0.68 (135)<\/p>\n<\/td>\n

\n

0.918 (0.601-1.401)<\/p>\n<\/td>\n

\n

0.667<\/p>\n<\/td>\n<\/tr>\n

\n

MTHFD1 <\/em><\/strong>T401C<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

T (ref)<\/strong><\/p>\n<\/td>\n

\n

0.14% (56)<\/p>\n<\/td>\n

\n

0.13% (52)<\/p>\n<\/td>\n

\n

0.918  (0. 612-1.377)<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

C<\/strong><\/p>\n<\/td>\n

\n

0.86% (344)<\/p>\n<\/td>\n

\n

 0.87% (348)<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

0.679<\/p>\n<\/td>\n<\/tr>\n

\n

TT (ref)                 <\/strong><\/p>\n<\/td>\n

\n

0.03 (6)<\/p>\n<\/td>\n

\n

0.03 (6)<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

TC<\/strong><\/p>\n<\/td>\n

\n

0.22 (44)<\/p>\n<\/td>\n

\n

0.2 (40)<\/p>\n<\/td>\n

\n

0.955 (0.282-3.233)<\/p>\n<\/td>\n

\n

0.623<\/p>\n<\/td>\n<\/tr>\n

\n

CC<\/strong><\/p>\n<\/td>\n

\n

0.75 (150)<\/p>\n<\/td>\n

\n

0.77 (154)<\/p>\n<\/td>\n

\n

1.054 (0.329-3.375)<\/p>\n<\/td>\n

\n

0.623<\/p>\n<\/td>\n<\/tr>\n

\n

TC and CC<\/strong><\/p>\n<\/td>\n

\n

0.97 (194)<\/p>\n<\/td>\n

\n

0.97 (194)<\/p>\n<\/td>\n

\n

1.042 (0.328-3.306)<\/p>\n<\/td>\n

\n

1.000<\/p>\n<\/td>\n<\/tr>\n

\n

CBS <\/em><\/strong>844ins68<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

w  (ref.)<\/strong><\/p>\n<\/td>\n

\n

0.92% (369)<\/p>\n<\/td>\n

\n

0.91% (365)<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Ins<\/strong><\/p>\n<\/td>\n

\n

0.078% (31)<\/p>\n<\/td>\n

\n

0.086% (35)<\/p>\n<\/td>\n

\n

0.876 (0.529-1.451)<\/p>\n<\/td>\n

\n

0.607<\/p>\n<\/td>\n<\/tr>\n

\n

w\/w  (ref.)<\/strong><\/p>\n<\/td>\n

\n

0.85 (170)<\/p>\n<\/td>\n

\n

0.83 (165)<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

w\/Ins<\/strong><\/p>\n<\/td>\n

\n

0.15 (29)<\/p>\n<\/td>\n

\n

0.18 (35)<\/p>\n<\/td>\n

\n

1.222 (0.711-2.100)<\/p>\n<\/td>\n

\n

0.413<\/p>\n<\/td>\n<\/tr>\n

\n

Ins \/Ins<\/strong><\/p>\n<\/td>\n

\n

0.005 (1)<\/p>\n<\/td>\n

\n

0.0 (0)<\/p>\n<\/td>\n

\n

0.0<\/p>\n<\/td>\n

\n

0.0<\/p>\n<\/td>\n<\/tr>\n

\n

w\/ Ins  +  Ins\/ Ins<\/strong><\/p>\n<\/td>\n

\n

0.15 (30)<\/p>\n<\/td>\n

\n

0.18 (35)<\/p>\n<\/td>\n

\n

1.197 (0.701-2.044)<\/p>\n<\/td>\n

\n

0.498<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Abbreviations: Ref – Reference category (wild-type allele\/genotype); n – number of subjects; OR – Odds Ratio; CI – confidence interval of OR based on multinomial logistic regression; P > 0.05; w – wild-type allele of CBS 844 (100bp); Ins – CBS 844ins68bp.”<\/p>\n\n\n

Table 3: Frequencies and numbers of the observed double compound genotypes among BC patients and the control groups.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Compound polymorphisms<\/strong><\/p>\n<\/td>\n

\n

Controls (n)<\/strong><\/p>\n<\/td>\n

\n

Cases (n)<\/strong><\/p>\n<\/td>\n

\n

OR (95% CI)<\/strong><\/p>\n<\/td>\n

\n

P<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

MTHFD1 G1958A\/MTHFD1 T401C<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

GG\/TT (Ref.)<\/p>\n<\/td>\n

\n

0.02 (4)<\/p>\n<\/td>\n

\n

0.01 (1)<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

GG\/TC<\/p>\n<\/td>\n

\n

0.08 (15)<\/p>\n<\/td>\n

\n

0.07 (13)<\/p>\n<\/td>\n

\n

1.467 (0.343 – 35.056)<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

GG\/CC<\/p>\n<\/td>\n

\n

0.21 (42)<\/p>\n<\/td>\n

\n

0.26 (51)<\/p>\n<\/td>\n

\n

1.857 (0.523 – 45.127)<\/p>\n<\/td>\n

\n

0.695<\/p>\n<\/td>\n<\/tr>\n

\n

GA\/TT<\/p>\n<\/td>\n

\n

0.01 (2)<\/p>\n<\/td>\n

\n

0.02 (4)<\/p>\n<\/td>\n

\n

1.000 (0.500 – 127.900)<\/p>\n<\/td>\n

\n

0.287<\/p>\n<\/td>\n<\/tr>\n

\n

GA\/TC<\/p>\n<\/td>\n

\n

0.13 (26)<\/p>\n<\/td>\n

\n

0.12 (24)<\/p>\n<\/td>\n

\n

1.69 (0.385 – 35.400)<\/p>\n<\/td>\n

\n

0.411<\/p>\n<\/td>\n<\/tr>\n

\n

GA\/CC<\/p>\n<\/td>\n

\n

0.43 (85)<\/p>\n<\/td>\n

\n

0.37 (73)<\/p>\n<\/td>\n

\n

3.435 (0.376 – 31.425)<\/p>\n<\/td>\n

\n

0.762<\/p>\n<\/td>\n<\/tr>\n

\n

AA\/TT<\/p>\n<\/td>\n

\n

0.00 (0)<\/p>\n<\/td>\n

\n

0.01 (1)<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

0.220<\/p>\n<\/td>\n<\/tr>\n

\n

AA\/TC<\/p>\n<\/td>\n

\n

0.02 (4)<\/p>\n<\/td>\n

\n

0.03 (5)<\/p>\n<\/td>\n

\n

1.000 (0.388 – 64.387)<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

AA\/CC<\/p>\n<\/td>\n

\n

0.11 (22)<\/p>\n<\/td>\n

\n

0.14 (28)<\/p>\n<\/td>\n

\n

5.091 (0.531- 48.852)<\/p>\n<\/td>\n

\n

0.736<\/p>\n<\/td>\n<\/tr>\n

\n

MTHFD1 G1958A\/CBS ins68<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

GG\/ ww (Ref.)<\/p>\n<\/td>\n

\n

0.26 (51)<\/p>\n<\/td>\n

\n

0.26 (51)<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

GG\/ wIns<\/p>\n<\/td>\n

\n

0.05 (10)<\/p>\n<\/td>\n

\n

0.07 (14)<\/p>\n<\/td>\n

\n

1.400  (0.569 -3.442)<\/p>\n<\/td>\n

\n

0.400<\/p>\n<\/td>\n<\/tr>\n

\n

GA\/ ww<\/p>\n<\/td>\n

\n

0.48 (96)<\/p>\n<\/td>\n

\n

0.44 (87)<\/p>\n<\/td>\n

\n

0.906 (0.906 -1.471)<\/p>\n<\/td>\n

\n

0.366<\/p>\n<\/td>\n<\/tr>\n

\n

GA\/ wIns<\/p>\n<\/td>\n

\n

0.09 (17)<\/p>\n<\/td>\n

\n

0.07 (13)<\/p>\n<\/td>\n

\n

0.765  (0.337\u20131.736)<\/p>\n<\/td>\n

\n

0.448<\/p>\n<\/td>\n<\/tr>\n

\n

AA\/ ww<\/p>\n<\/td>\n

\n

0.12 (23)<\/p>\n<\/td>\n

\n

0.14 (27)<\/p>\n<\/td>\n

\n

1.174  (0.596-2.313<\/p>\n<\/td>\n

\n

0.545<\/p>\n<\/td>\n<\/tr>\n

\n

MTHFD1 T401C\/ CBS ins68<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

TT\/ w\/w (Ref.)<\/p>\n<\/td>\n

\n

0.02 (4)<\/p>\n<\/td>\n

\n

0.02 (4)<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

TT\/ wIns<\/p>\n<\/td>\n

\n

0.01 (2)<\/p>\n<\/td>\n

\n

0.01 (2)<\/p>\n<\/td>\n

\n

1.000 (0.091-11.028)<\/p>\n<\/td>\n

\n

1.000<\/p>\n<\/td>\n<\/tr>\n

\n

TC\/ ww<\/p>\n<\/td>\n

\n

0.18 (35)<\/p>\n<\/td>\n

\n

0.19 (37)<\/p>\n<\/td>\n

\n

1.057 (0.245\u2013 4.556)<\/p>\n<\/td>\n

\n

0.795<\/p>\n<\/td>\n<\/tr>\n

\n

TC\/ wIns<\/p>\n<\/td>\n

\n

0.04 (8)<\/p>\n<\/td>\n

\n

0.02 (3)<\/p>\n<\/td>\n

\n

0.375 (0.055\u2013 2.555)<\/p>\n<\/td>\n

\n

0.126<\/p>\n<\/td>\n<\/tr>\n

\n

CC\/ ww<\/p>\n<\/td>\n

\n

0.66 (131)<\/p>\n<\/td>\n

\n

0.62 (124)<\/p>\n<\/td>\n

\n

0.947 (0.232-3.867)<\/p>\n<\/td>\n

\n

0.467<\/p>\n<\/td>\n<\/tr>\n

\n

CC\/ wIns<\/p>\n<\/td>\n

\n

0.10 (19)<\/p>\n<\/td>\n

\n

0.15 (30)<\/p>\n<\/td>\n

\n

1.579 (0.352-7.079)<\/p>\n<\/td>\n

\n

0.093<\/p>\n<\/td>\n<\/tr>\n

\n

CC\/ InsIns<\/p>\n<\/td>\n

\n

0.01 (1)<\/p>\n<\/td>\n

\n

0 (0)<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

OR: odd ratios; CI: confidence interval of OR according to multinomial logistic regression; P > 0.05; Ref: reference ; w: wild type (100 bp); Ins: mutant CBS 844ins68bp (168 bp).<\/p>\n\n\n

In order to find out the effect of the observed triple compounds of the examined polymorphisms, MTHFD1<\/em>G1958A\/ MTHFD1<\/em>T401C\/ CBS<\/em> 844ins68 genotypes were examined in the cases and the controls. The results are shown in Table 4, which shows that there are no significant differences between the observed frequencies of the triple compound genotypes between the cases and the controls expect for the triple compound genotype MTHFD1<\/em> 1958AA\/MTHFD1<\/em> 40CC\/CBS<\/em> 844ins68 (w\/ins68) which have increased odd ratio to 5. This suggested that the presence of these three polymorphisms together in the same individual have a synergistic effect on increasing the risk for BC.<\/p>\n\n\n\n

Table 4: Numbers of the observed triple compound genotypesof MTHFD1<\/em> G1958A, MTHFD1<\/em> T401C and CBS<\/em> 844ins68 among the cases and the control groups.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Variants of MHFD1<\/em> G1958A\/ MTHFD1<\/em> T401C\/ CBS<\/em> 844ins68<\/strong><\/p>\n<\/td>\n

\n

Number of Controls<\/strong><\/p>\n

 <\/strong><\/p>\n<\/td>\n

\n

Number of Patients<\/strong><\/p>\n

 <\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

OR<\/strong><\/p>\n

 <\/strong><\/p>\n<\/td>\n

\n

 <\/strong><\/p>\n

95% C.I<\/strong><\/p>\n

 <\/strong><\/p>\n<\/td>\n

\n

 <\/strong>P value<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

GG\/TT\/ ++ (Ref.)<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

GG\/ TT\/+ins<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

GG\/ TC\/ ++<\/p>\n<\/td>\n

\n

13<\/p>\n<\/td>\n

\n

11<\/p>\n<\/td>\n

\n

1.692<\/p>\n<\/td>\n

\n

0.135- 21.270<\/p>\n<\/td>\n

\n

0.674<\/p>\n<\/td>\n<\/tr>\n

\n

GG\/ TC\/ +ins<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

1.000<\/p>\n<\/td>\n

\n

0.090- 44.350<\/p>\n<\/td>\n

\n

1.000<\/p>\n<\/td>\n<\/tr>\n

\n

GG\/CC\/++<\/p>\n<\/td>\n

\n

36<\/p>\n<\/td>\n

\n

39<\/p>\n<\/td>\n

\n

2.167<\/p>\n<\/td>\n

\n

0.188- 24.929<\/p>\n<\/td>\n

\n

0.701<\/p>\n<\/td>\n<\/tr>\n

\n

GG\/CC\/+ ins<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

12<\/p>\n<\/td>\n

\n

2.000<\/p>\n<\/td>\n

\n

0.299- 3.468<\/p>\n<\/td>\n

\n

0.148<\/p>\n<\/td>\n<\/tr>\n

\n

GA\/TT\/++<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

2.000<\/p>\n<\/td>\n

\n

0.150 – 59.890<\/p>\n<\/td>\n

\n

0.653<\/p>\n<\/td>\n<\/tr>\n

\n

GA\/TC\/++<\/p>\n<\/td>\n

\n

20<\/p>\n<\/td>\n

\n

22<\/p>\n<\/td>\n

\n

2.000<\/p>\n<\/td>\n

\n

0.185- 26.157<\/p>\n<\/td>\n

\n

0.744<\/p>\n<\/td>\n<\/tr>\n

\n

GA\/TC\/+ins<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

0.333<\/p>\n<\/td>\n

\n

0.014- 8.182<\/p>\n<\/td>\n

\n

0.057<\/p>\n<\/td>\n<\/tr>\n

\n

GA\/CC\/++<\/p>\n<\/td>\n

\n

74<\/p>\n<\/td>\n

\n

63<\/p>\n<\/td>\n

\n

1.703<\/p>\n<\/td>\n

\n

0.151- 19.222<\/p>\n<\/td>\n

\n

0.246<\/p>\n<\/td>\n<\/tr>\n

\n

GA\/CC\/+ins<\/p>\n<\/td>\n

\n

11<\/p>\n<\/td>\n

\n

11<\/p>\n<\/td>\n

\n

1.000<\/p>\n<\/td>\n

\n

0.157- 25.404<\/p>\n<\/td>\n

\n

1.000<\/p>\n<\/td>\n<\/tr>\n

\n

AA\/TT\/ ins,ins<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

AA\/TC\/++<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

5<\/p>\n<\/td>\n

\n

1.333<\/p>\n<\/td>\n

\n

0.204- 54.532<\/p>\n<\/td>\n

\n

0.475<\/p>\n<\/td>\n<\/tr>\n

\n

AA\/CC\/++<\/p>\n<\/td>\n

\n

20<\/p>\n<\/td>\n

\n

21<\/p>\n<\/td>\n

\n

1.100<\/p>\n<\/td>\n

\n

0.176- 25.010<\/p>\n<\/td>\n

\n

0.869<\/p>\n<\/td>\n<\/tr>\n

\n

AA\/CC\/+ins<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

7<\/p>\n<\/td>\n

\n

5.000<\/p>\n<\/td>\n

\n

0.397- 25.37<\/p>\n<\/td>\n

\n

0.092<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

OR: odd ratios; CI: confidence interval of OR according to multinomial logistic regression; P > 0.05; +: wild type CBS<\/em> (100 bp); ins: mutant CBS<\/em> 844ins68 (168 bp); Ref: reference.<\/p>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

In this current\npopulation-based case-control study, we investigated the association between MTHFD<\/em>\n1958A, MTHFD1<\/em> 401C variants and the insertion CBS<\/em>844ins68bp with\nthe risk of BC. MTHFD1<\/em> gene, mapped to chromosome 14 (14q24), codes for\nthe enzyme MTHFD1<\/em>, which is a core enzyme in the folate\/ one-carbon\nmetabolism 49<\/sup>.\nMTHFD1<\/em> is a trifunctional enzyme consists of two major domains, an\nN-terminal containing the dehydrogenase and cyclohydrolase activities and a\nsynthetase domain in the C terminus 50<\/sup>,\nand is associated with epigenetic events and DNA methylation that can influence\nsusceptibility to cancers 13<\/sup>.\nThe SNP MTHFD1<\/em> G1958A is a G to A transition at position 1958 located\nwithin the 10-formyl-THF synthetase domain 28<\/sup>,\nresulting in the substitution of glutamine with arginine residue at position\n653 (R653Q). MTHFD1 G1958A causes reduction in the activity and stability of\nthe enzyme 40<\/sup>‘<\/sup>51<\/sup> and\nfacilitates different developmental diseases and different types of cancers,\nincluding breast cancer 14<\/sup>,\ncolorectal 52<\/sup>,\ngastric 47<\/sup>,\nmethotrexate sensitivity in acute lymphoblastic leukemia 53<\/sup>‘<\/sup>54<\/sup>.\nHowever, other different studies including a meta-analysis of Asians showed\nlack of association between MTHFD1<\/em> G1958A and different types of cancers\nincluding lung cancer, and head and neck cancer 55<\/sup>‘<\/sup>56<\/sup>.\nFurthermore, MTHFD1<\/em> G1958A decreased the risk for acute lymphoblastic leukemia\n57<\/sup>.\nThe less common SNP MTHFD1<\/em> T401C in which the arginine at position 134\nis substituted by lysine (R134K), lies within the dehydrogenase\/cyclohydrolase\ndomain of the enzyme 50<\/sup>,\nwhich may result in disturbance of the folate-mediated homocysteine pathway\nthat is associated with cancer 34<\/sup>‘<\/sup>14<\/sup>‘<\/sup>40<\/sup>.\nThe activity of MTHFD1<\/em> G1958A was correlated to reduction in the\nsynthase activity, which is essential for the remethylation of homocysteine\n(Hcy) to methionine, which is a precursor for the synthesis of SAM 29<\/sup>‘<\/sup>40<\/sup>.\nReduced levels of SAM lead to low methyl supply, which can result in global DNA\nhypomethylation, and very little conversion of dUMP to dTMP, leading to uracil\nmisincorporation into DNA 58<\/sup>,\nDNA strand breaks, chromosomal instability, alteration of gene expression and\nconsequently promoting carcinogenesis 58<\/sup>‘<\/sup>59<\/sup>.\nHowever, current results showed that neither the  individual polymorphism MTHFD1 G1958A\nnor  MTHFD1 T401C per se were associated\nwith BC, which supported the reported lack of association between MTHFD1 401C\nand human cancers 34<\/sup> as\nwell as the lack of association between MTHFD 1958A and BC in west Siberian\nregion of Russia 60<\/sup>.\nHowever, our results were contrary to those obtained from studying a mix of\nWhite, African American, Hispanic, Asian, and unknown postmenopausal females,\nwhich reported association of MTHFD1 T401C with risk of BC 14<\/sup>.\nIn addition, results of our current study were also different from those\nreported association of MTHFD1<\/em> gene with other different types of cancer\nincluding gastric, colon and head and neck cancers 47<\/sup>‘<\/sup>47<\/sup>.\nDifferent studies also showed that the homozygous mutant genotype MTHFD1<\/em>\n1958AA induced significant reduction in the overall cancer risk, and the risk\nof primary liver and colon cancers 61<\/sup>.\nIn addition, homozygous patients with MTHFD1<\/em> 1958AA genotype had\nsignificantly higher frequency of tumor CpG island hypermethylation compared to\nwild-type MTHFD1 1958GG homozygotes, which were significantly associated with\nDNA hypomethylation 13<\/sup>,\nsuggesting that the G allele may exert a protective effect for cancer risk by\nprotecting from DNA hypomethylation 61<\/sup>.<\/p>\n\n\n\n

Cystathionine \ud835\udefd-synthase\n(CBS<\/em>) is another core enzyme in the folate\/ one-carbon related\nmetabolism, specifically at the reverse transsulfuration pathway, which\ntransfers sulfur from the cytotoxic metabolite homocysteine to cysteine. The\nCBS gene located in the subtelomeric region q.22.3, of chromosome 21 62<\/sup> encodes\nfor the 63-kDa CBS subunits of the tetramer active enzyme 63<\/sup>.\nEach subunit of CBS<\/em> consists of N- and C- terminal domains. The\nN-terminal domain binds to the cofactor heme and is essential for proper\nfolding and assembly of the protein, but is not essential for its catalytic\nactivity 64<\/sup>.\nThe C-terminal regulatory domain contains the binding sites for the allosteric\nactivator SAM and is responsible for CBS<\/em> subunit tetramerization 63<\/sup>.\nThe variant CBS ins68, which initially detected in a heterozygous patient with\nhomocystinuria due to CBS<\/em> deficiency 65<\/sup>\nhad been associated with lowered plasma homocysteine levels, consists of an\nidentical insertion of 68bp DNA repeat within exon 8 of the CBS gene 66<\/sup>.\n<\/p>\n\n\n\n

The observed results\ndemonstrated that CBS<\/em> 488ins68 polymorphism is not a risk factor for BC,\nwhich supported previous reports that considered CBS<\/em> 844ins68 as a\nneutral variant 67<\/sup>,\nwhich generates an alternative splice site, which allows elimination of the\nentire insertion to form a normal CBS<\/em> mRNA transcript 68<\/sup>.\nCurrent results were also in harmony with the lack of association between CBS<\/em>\n844ins68 polymorphism with different types of cancer including colorectal cancer\n47<\/sup> carcinomas\nof the upper gastrointestinal tract 69<\/sup>\nand prostatic carcinoma 70<\/sup>.\nFurthermore, our results were in harmony with the unaltered expression of CBS<\/em>\nenzyme in BC and the majority of cancer types where the role of CBS has been\nexamined (with the exception of liver cancer and glioma) 42<\/sup>.\nHowever, our current results were contrary to the reported significant\nassociation of CBS<\/em> 844ins68 with risk of BC in Mexicans. In Mexicans,\nboth homozygous and heterozygous genotypes of CBS 844ins68 were associated with\nthe risk of BC 45<\/sup>.\nSuch controversial results could be due to ethnic differences as well as\ndifferent lifestyles, diet and uptake levels of folate in the Jordanian and\nMexican populations. Furthermore, current results are also different from the\nreported expression of CBS<\/em> in breast cancer-affected tissue, compared to\nthe normal control unaffected breast tissue in mastectomy samples of BC\npatients 42<\/sup>‘<\/sup>\n71<\/sup>.<\/p>\n\n\n\n

The conflicting and\ncontroversial reports in the literature concerning the association of MTHFD1<\/em>\nG1958A, MTHFD1<\/em> T401C and CBS 844ins68bp with the risk of BC could be\nreflection to the complexity of the regulation of CBS<\/em> and MTHFD1<\/em>\ngenes, as well as the complexity of the carcinogenesis process itself. <\/p>\n\n\n\n

Both CBS<\/em> and MTHFD1<\/em>\nare involved in methyl group metabolism 70<\/sup>.\nCBS<\/em> catalyzes different but interrelated cellular biochemical pathways,\nincluding availability of SAM and DNA methylation, while MTHFD1<\/em> G1958A\nis associated in breast cancer with hormone receptor content and DNA\nmethylation frequency 13<\/sup>.\nBC patients homozygous for the MTHFD1<\/em> 1958AA genotype had a\nsignificantly higher frequency of tumor CpG island hypermethylation compared to\nthe wild-type homozygotes MTHFD1<\/em> 1958 GG 13<\/sup>.\nCBS<\/em> transcription is regulated by different mechanisms including the\nmethylation status of the CpG islands in its two principal GC-rich promoters 72<\/sup>‘<\/sup>73<\/sup> and\nby several hormones and transcription factors 57<\/sup>‘<\/sup>74<\/sup>‘<\/sup>75<\/sup>‘<\/sup>76<\/sup>.\nCBS<\/em> also regulates the production of both ROS, which is triggered by\nglutathione abundance 65<\/sup>\nand the intrinsic cellular regulator H2S 77<\/sup>‘<\/sup>78<\/sup>.\nIn addition, the process of carcinogenesis involves altered methylation cycle\naccompanied with promoter hypermethylation, which leads to inactivation of\ngenes in almost all pathways protective of carcinogenesis such as DNA repair, cell\ncycle control and apoptosis 31<\/sup>.\nAll these factors including ethnicity, which is a possible cause for\ndifferences in genetic variants in MTHFD1<\/em>, CBS<\/em> and other genes in\nthe folate\/one-carbon metabolism in different ethnic groups, could contribute\nto differential risks of developing breast cancer between different populations\n79<\/sup>.\nIn addition, other factors specific for different populations such as\nenvironmental, lifestyles, nutrition and uptake levels of folate may act upon\nthese SNPs to generate a gradient of intermediates in the folate\/ one-carbon\nmetabolism and associated transsulfuration pathway.<\/p>\n\n\n\n

Various studies showed that different individual polymorphisms in different genes of the folate\/ one-carbon metabolism were not per se associated with breast cancer. These include the polymorphisms MTHFR<\/em> C677T and MTHFR<\/em> A1298C in the methylenetetrahydrofolate reductase, TYMS 1494 ins\/del 6 in the thymidylate synthase and MTRR<\/em> A66G in 5-methytetrahydrofolate homocysteine methyltransferase reductase (MTRR<\/em>). However, the presence of mutant alleles for two polymorphisms of these genes increased the risk of BC or were associated with increasing the risk of developing more BC aggressive phenotypes 80’81<\/sup>. We examined the effect of double compound genotypes of MTHFD1 T401C, MTHFD1 1958A and CBS<\/em> 488ins68 CBS<\/em> 488ins68 on the risk of BC. Results showed that the double compound states of both MTHFD1 T401C and MTHFD1<\/em> 1958A (1958GA\/ 401CC) and (1958AA\/401CC) seemed to increase the risk of BC by 3.4 and 5.1 folds respectively (Table 3), but such increase was accompanied by large confidence intervals possibly due to the observed small numbers of these compound genotypes. Further investigations are required to explore the effect of double compound genotypes of MTHFD1<\/em> T401C, MTHFD1<\/em> 1958A and CBS<\/em> 488ins68 on the risk of BC. In conclusion, CBS<\/em> 844ins68bp, MTHFD1<\/em> G1958A and MTHFD1 T401C polymorphisms per se are not risk factors of BC in Jordan females. For better BC management, further studies can be considered for understanding the influence of double compound genotypes of these polymorphisms on the risk of BC in Jordan.<\/p>\n\n\n\n

Conclusion <\/strong><\/p>\n\n\n\n

This study revealed that each of MTHFD1 G1958A, T401C and CBS 844ins68 polymorphisms alone had no direct risk for BC in the Jordanian women, compared to the wild-type genotypes. <\/p>\n\n\n\n

Acknowledgment<\/strong><\/p>\n\n\n\n

We would like to extend\nour gratitude to all women who participated in this study and to the Ministry\nof Health in Jordan. Yarmouk University completely supported this work.<\/p>\n\n\n\n

Conflict of interest <\/strong><\/strong><\/p>\n\n\n\n

The authors have no\nconflict of interest to declare.<\/p>\n\n\n\n

Funding\nSource<\/strong><\/p>\n\n\n\n

The Deanship of Scientific Research and Graduate Studies at Yarmouk University, Irbid, Jordan (Grant # 20\/2014).<\/p>\n\n\n\n

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