Folic acid is essential for the de novo synthesis of nucleotide precursors for normal DNA synthesis and for normal cellular methylation reactions. The methyltetrahydrofolate reductase enzyme catalyzes the synthesis of 5-methyltetrahydrofolate, the methyl donor for the B12-dependent remethylation of homocysteine to methionine. Methionine is the precursor for the synthesis of S-adenosylmethionine (SAM), the major cellular methyl donor for DNA, RNA, protein, and phospholipids methylation.
The human MTHFR gene is composed of 11 exons; the exon sizes, location of intronic boundaries, and intron sizes are quite similar those in the mouse mthfr gene. In addition, the promoter region of the MTHFR gene does not have a TATA box but contains CpG islands, multiple potential SP1-binding sites, and binding sites for several other transcription factors.
The MTHFR 677 C-T polymorphism has been implicated in the etiology of neural tube defects. The polymorphism probably acts by decreasing enzyme activity and raising the dietary requirement for folic acid to maintain normal remethylation of homocysteine to methionine. Chronic elevation in intracellular homocysteine can lead to an increase in SAH and potent product inhibition of the DNA methyltransferase leading to DNA hypomethylation. DNA hypomethylation during embryogenesis has been proposed to negatively affect chromatin structure and critical regulatory genes required for normal development. There is also evidence that chronic folate/methyl deficiency can lead to chromosome instability, and aberrant chromosome segregation.
Studies to examine folate metabolism and polymorphisms in the MTHFR gene and the methionine synthase reductase (MTRR) gene in mothers of children with Down syndrome found a higher frequency, relative to control mothers, of both heterozygous and homozygous T mutant allele in MTHFR gene and homozygous G mutant allele in MTRR gene in Down syndrome mothers. In addition, plasma homocysteine were higher in these mothers. It is possible that increased frequency of MTHFR and MTRR polymorphisms and altered folate status in mothers of children with Down syndrome could promote DNA hypomethylation and meiotic non-disjunction.
Al-Habboubi et al. (2003) studied the prevalence of the MTHFR C677T mutation among healthy Bahrainis by performing PCR- restriction fragment length polymorphism analysis on 152 subjects. Their ages were (33.44 ± 11.12) and the male:female ratio was 170:238. All subjects had neither thrombotic problems nor a family history of thrombosis. The C677T mutation was present in 30 participants giving an overall carrier frequency of 19.7%. There were 26 heterozygous carriers (17.11%). On the other hand, only four carriers (2.63%) were in the homozygous state. No difference in the MTHFR C677T mutation frequency was seen with respect to gender [See also: Lebanon > Al-Habboubi et al., 2003]. One year later, Al-Habboubi et al. (2004) studied the prevalance of C677T and A1298C single nucleotide polymophisms in a set of 155 healthy Bahraini nationals (103 males, 52 females). Genotype analysis was peformed by PCR followed by RFLP using HinfI for the C677T mutation, and MboII for the A1298C mutation. Of the subjects analyzed, 21.94% were found to have the C677T mutation, with 19.4% being heterozygous (C/T), and 2.6% being homozygous (T/T) carriers. This frequency was comparable to values reported earlier for other East Asian countries, and lower than that for southern European and South East Asian countries. The distribution of this mutation was seen to be in accordance with Hardy-Weinberg equilibrium. However, gender differences were seen in the distribution of the mutations, with females showing more heterozygous and carriers (25.5% C/T and no T/T), compared to males (16.3% C/T, 3.8% T/T). With respect to the A1298C mutation, 60.67% of the subjects were shown to have the mutation (53.3% heterozygous A/C, and 7.3% homozygous C/C). The C/C frequency was found to be comparable to the frequencies for US and UK, lower than those for Jews, and higher than the frequencies reported from Asians. The distribution of this mutation was not according to Hardy Weinberg equilibrium, and no gender difference was seen in the distribution.
Al Jishi et al. (2005) described, for the first time, a 41-year-old married woman with a combination of Takayasu arteritis (TA) and primary antiphospholipid antibodies (aPL) syndrome who underwent carotid stenting. She had single nucleotide polymorphism (SNP) double homozygosity for methylenetetrahydrofolate reductase (MTHFR); C677T (T/T genotype) and A1298C (C/C genotype). Serum homocysteine was elevated and that might be attributed to C677T SNP. It was thought that MTHFR 1298C/C genotype would be an independent risk factor for ischemic stroke.
In a study on 118 patients and 120 control subjects, Almawi et al. (2009) found a positive association between MTHFR C677T and A1298C genotypes and stroke. The C677T mutation was also found to be associated with elevated homocysteine levels in both controls and patients.
Nemr et al. (2010) investigated the association of the c.C677T and c.A1298C mutations and diabetic nephropathy in patients with Type 2 DM. The study consisted of 252 Lebanese and 225 Bahraini subjects with Type 2 DM related nephropathy, and a control group of 309 Lebanese and 328 Bahraini patients with Type 2 DM in the absence of nephropathy. The Lebanese patients with nephropathy were found to have significantly higher frequencies of the 677T allele and C/C and T/T genotypes, when compared to Lebanese control patients. This did not hold for the Bahraini patients. The 1298C allele frequencies were comparable in the control and study groups. 677T/T carriers in both populations were found to have a higher homocysteine concentration. This increased concentration was linked with nephropathy in Lebanese, but not Bahraini patients. Regression analysis revealed the C677T mutation to be associated with nephropathy only among Lebanese patients. Nemr et al. (2010) stressed that the relationship between MTHFR mutations and diabetic nephropathy is dependent on the racial/ethnic background.
Gharaibeh et al. (2014) studied 50 consecutive patients with retinitis pigmentosa and 50 controls matched by age and gender to determine the prevalence of thrombophilic factors. Among patients/controls they found the MTHFR 677 TT heterozygous mutation (48/52). Gharaibeh et al. (2014) concluded that the difference between patients with retinitis pigmentosa and the control group was not statistically significant for the prevalence of this studied factor.
[See also: Saudi Arabia > Bu et al., 2004].
Adekile et al. (2001) screened a random cohort of 41 Kuwaiti sickle cell disease (SCD) patients attending the Hematology Clinic of Kuwait University Teaching Hospital for the 677C>T mutation of the methylenetetrahydrofolate reductase (MTHFR) gene. The cohort consisted of 33 SS and 8 S/beta (0)-thal patients aged 2-41 years with a mean of age of 12.8+/- 8.6 years. PCR/RFLP was employed for the screening of MTHFR mutation and revealed 15 patients heterozygous for the mutation and one patient as homozygous, resulting in an allele frequency of 20.7%. In addition, 21 subjects (14 SS and 7 S/beta (0)-thal) were screened for osteonecrosis using MRI of the hip which showed 7 subjects with diverging degrees of osteonecrosis resulting in a frequency of 21.4% for the 677C>T allele. Adekile et al. (2001) stated that the obtained allele frequencies do not suggest an association with osteonecrosis.
Al Tawari et al. (2002) reported a family from Kuwait with severe MTHFR deficiency showing unique dermatological and brain imaging features. Three siblings in this family, two of them boys and a girl, died before the age of 3-months due to the deficiency. The fourth child, a boy, was treated with betaine, which resulted in clinical and biochemical improvement.
Al-Habboubi et al. (2003) demonstrated the prevalence of MTHFR C677T mutation among healthy Lebanese individuals by performing PCR- restriction fragment length polymorphism analysis on 408 subjects. Their ages were (32.94 ± 10.94) and the male:female ratio was 108:44. All subjects had neither thrombotic problems nor a family history of thrombosis. The C677T mutation was present in 169 participants giving an overall carrier frequency of 41.4%. There were 159 heterozygous carriers (38.97%). On the other hand, only ten carriers (2.45%) were in the homozygous state. No difference in the MTHFR C677T mutation frequency was seen with respect to gender.
Almawi et al. (2005) undertook a case control study to understand the prevalence of the prothrombotic mutations G1691A FV-Leiden, factor II G20210A, and MTHFR C677T mutations. One hundred and ninety eight Lebanese patients (84 males, 114 females), diagnosed with venous thromboembolism (VTE) were compared to 697 healthy age and gender matched controls. Diagnosis of VTE was performed using Doppler ultrasound, duplex scan, D-dimer levels, and phlebogram. Mutation analysis of the subjects was performed by PCR followed by RFLP using restriction enzyme HinfI for MTHFR. Multiple triple regression model analysis was also performed. The MTHFR C677T mutation did not show a significant difference between the patients (0.3838) and the control (0.3092) groups. The MTHFR C/T mutation was equally prevalent in both the groups. However, the T/T mutation showed a higher prevalence in the patients (20.7% vs. 11.0% in controls). Analysis of combined inherited risk factors showed that though the FV-Leiden allele and the G20210A allele by themselves were clearly associated with VTE (odds ratio2.71 and 3.590 respectively), they synergized with the MTHFR 677 T/C allele in increasing the odds of developing the disease. Logistic regression analysis showed that individuals below 45 years of age were 5.5 times more prone to developing VTE.
[See also: Bahrain > Nemr et al., 2010; Saudi Arabia > Bu et al., 2004].
Pathare et al. (2002, 2004) reported a 27-year old Omani man who presented with multiple episodes of venous thrombosis and was found to have elevated levels of homocysteine. This 27 year old male had his first episode of venous thrombosis as deep venous thrombosis (DVT) seven years before reporting his case and he was appropriately anticoagulated with heparin then by warfarin for one year, but as his thrombophilia workup (Proteins C and S, antithrombin III, lupus anticoagulant, APCR and plasminogen assay) was normal his anticoagulant therapy was stopped. Three years later, he presented with a second episode of DVT along with pain in the right lumbar and hypochondria which when investigated by abdominal ultrasound, thrombosis of the distal inferior vena cava and the whole of common iliac vein was detected. There was no family history of thrombophilia, and his systemic examination was unremarkable. Repeating the thrombophilia workup had only yielded normal results. He was managed by heparin and later by warfarin (to maintain INR between 2 and 2.5). The patient was admitted three years later because of a third episode of DVT as he had stopped his anticoagulant for three months. On examination, his left lower limb had dilated veins but with no ulcerations or edema, but the right lower limb was edematous at the ankle. Doppler scan revealed a persistent DVT along with complete blockage of the common femoral vein, and superficial femoral vein with partial blockage of the popliteal vein on the right side. He was anticoagulated and investigated with thrombophilia workup which was normal, as well as with specific molecular diagnostic tests recommended for thrombophilia evaluation (mutations in MTHFR gene, CBS 844ins68, prothrombin gene G20210A mutation, and factor V Leiden gene). His routine blood investigations revealed normal blood counts with normal reticulocyte counts with unremarkable peripheral blood film, ESR of 12 mm/hr, and negative ANA, Anti-DNA, autoantibody screen, lupus anticoagulant and anticardiolipin antibodies. He was found to be HbE trait on hemoglobin electrophoresis (HbE was 23% and HbA was 76.4%), and was found to have elevated levels of homocysteine of 23 micromol/l with normal levels of serum folate (4.5ng/l) and B12 (208pg/l). Genetic analysis was carried out by extracting the DNA from which the MTHFR gene was amplified by PCR with specific primers and the PCR products was then digested by specific enzyme for MTHFR gene mutation (endonuclease Hinfl). A heterozygous MTHFR C677T mutation was identified in the patient. Four years later, Pathare et al. (2006) studied the prothrombotic risk factors in Omani patients who presented with their first thrombophilic event. All Omani patients (39 patients with 24 females and 15 males) who were admitted between 2001 and 2003 with a first episode of deep venous thrombosis (DVT) with or without pulmonary embolism (PE) or with thrombosis in an unusual site were included in the study. MTHFR polymorphism (667C T) was identified using PCR amplification with appropriate primers, followed by digestion with HinfI. The MTHFR mutation (C677T) was detected in 14 patients (35.89%). It was found that seven normal Omani control subjects (8.75%) were heterozygous, while two (2.5%) were homozygous for the mutation. Out of the patients 43.58% had either one or both of the hyperhomocysteinemic mutations (MTHFR C677T and CBS 844ins68). One patient had three risk factors which were a combination of the two mutations (MTHFR C677T and CBS 844ins68) with protein C deficiency, while two patients had both mutations as risk factors, and 11 patients had only the MTHFR C677T. Pathare et al. (2006) pointed that the population prevalence of the hyperhomocysteinemic mutations in their study was significantly lower than that of the Caucasian population.
In 2001, Al-Gazali et al. described a male child of a first-cousin couple of Palestinian origin who had Down syndrome and neural tube defect. Al-Gazali et al. (2001) evaluated the 677 C-T MTHFR polymorphism and found that both the mother and child were homozygous for the TT mutation. In contrast, both mother and child were wild type for the A-C MTRR mutation. Al-Gazali et al. (2001) suggested that altered folate status plus homozygous mutation in the MTHFR gene in the mother could promote chromosomal instability and meiotic non-disjunction resulting in trisomy 21.
Bu et al. (2004) studied the frequencies of alleles and genotypes for CYP1A1, NAT2, GSTs, MTHFR, MTR (MS), and NQO*1 genes among Arabs. Of the studied group, 95% were from Saudi Arabia, and 5% were from Jordan, Syria, Lebanon, and Yemen. The mean age was 37 years, and male to female ratio was (25:1). Allele frequencies for the MTHFR C677T and MTHFR A1298C variants were 15%, and 32%, respectively in Arabs.
Fawaz et al. (2004) carried out a molecular study to assess the prevalence of the MTHFR C677T mutation in sickle cell disease (SCD) patients and healthy controls. Study subjects comprised 87 SCD patients (38 males and 49 females; mean age 23.10 ± 14.15 years) and 105 controls (65 males and 40 females; mean age 32.28 ± 15.00 years). All patients were Saudi nationals from Eastern Saudi Arabia. PCR-RFLP analysis was used to detect the MTHFR C677T mutation. A significant increase in MTHFR C677T carrier rate was seen in SCD patients (36/87, 42%) compared to healthy subjects (26/105; 25%; P = 0.022). While the prevalence of C/T (32% and 21%; P = 0.109) and T/T (9% and 4%; P = 0.232) genotypes was higher among patients than controls, respectively. However, this was not statistically significant (P = 0.217; OR = 2.56) due to small sample size. Thus, Fawaz et al. (2004) suggested that a larger study is needed to confirm or alternatively rule out involvement of MTHFR C677T in SCD pathogenesis.
Al-Ali et al. (2005) studied the prevalence of the C677T mutation in MTHFR gene among Saudi patients with cardiovascular disease (CAD). Restriction fragment length polymorphism and allele specific hybridization were performed in 83 CAD patients and 40 age and gender-matched controls over a period of two years (2003-20004). The frequencies of the T allele and homozygous TT allele were 20% and 11% among CAD patients and 15% and 0% among controls. Al-Ali et al. (2005) concluded that the MTHFR C677T variant is a risk factor for CAD.
Mammo et al. (2007) reported the incidences of five prothrombotic gene polymorphisms among Saudi Arabians. The data for this study came from the Saudi Thrombosis and Familial Thrombophilia Registry. The subjects were healthy consenting donors from different regions, tribes, and origins in Saudi Arabia. Between 2001 and 2005, DNA samples from 902 Saudis (793 men and 109 women) were collected and genotyped. The MTHFR 677C>T mutation was tested in 884 of these subjects. Heterozygous carrier rate for this mutation was found to be 25%, while 2% of the subjects were homozygous rare allele carriers. The incidence of the rare allele was 27% (frequency: 15%).
Siraj et al. (2007) conducted a study among Saudi diffuse large B lymphoma (DLBCL) patients. One hundred and sixty DLBCL patients and 511 Saudi controls were included in the study. All samples were analyzed for MTHFR C677T and A1298C functional polymorphisms using restriction fragment length polymorphism in association with MGMT and FHIT genes promoter hypermethylation. In the DLBCL patients the frequency of MTHFR A1298C genotype CC was 31%, AC was 35% and the frequency of C allele was 66%, whereas in the controls the frequencies were 10, 43, and 53% respectively. It was found that Saudi individuals who carry MTHFR 1298CC and the 1298C allele had 4.23 and 1.73-fold higher risk of developing DLBCL, respectively. Also CCC genotype was associated with 3.489, and CTCC was related to 9.515-fold higher risk, compared with full MTHFR enzyme activity. The results suggested an association between MTHFR polymorphisms and increased susceptibility to develop DLBCL among Saudi population.
The C677T polymorphism in the MTHFR gene is associated with hyperhomocysteinemia. The latter is one of the factors that account for the high prevalence of cardiovascular death in patients with progressive renal disease. This triggered Al-Muhann et al. (2008) to determine the prevalence of this polymorphism in Saudi patients with end-stage renal disease (ESRD) on hemodialysis. The control group only included people with no past, present or family history of renal disease. The T allele frequency and homozygous genotype of MTHFR in ESRD patients were 14% and 2%, respectively compared to 13% and 0%, respectively in the control group. The results showed that the homozygotes T allele genotype was overrepresented in the hemodialysis group compared to the control one.
Alghasham et al. (2012) performed a common-variant association study, investigating the association of MTHFR C677T and A1298C polymorphisms with hypertension in Saudi subjects. Genotypic and allelic frequencies were obtained through a PCR-based reaction assay using hybridization probes specific to the corresponding polymorphic variant. All cases showed a higher carrier frequency of the mutant 677T allele than controls (41% vs 26%, p=0.004) and significant association was described using the odds ratio (OR=1.9, 95% CI 1.2-3.1). Filtering hypertensive cases with obesity, a significantly higher carrier frequency (48% vs 26%, p=0.004) and higher odds ratio (OR=2.6, 95% CI 1.3-5.01) was found. Hypertensive cases with obesity and diabetes, and cases with hypertension alone, both showed a higher carrier frequency than controls, albeit without significance (p=0.06, p=0.6 respectively). All cases showed a higher carrier frequency of the mutant 1298C allele than controls (59% vs 42%, p=0.003), and significant association was described (OR=1.98, 95% CI 1.3-3.1). Filtering hypertensive cases with obesity, a significantly higher carrier frequency (61% vs 42%, p=0.02) and higher odds ratio (OR=2.2, 95% CI 1.1-4.2) was found. Hypertensive cases with obesity and diabetes showed a significantly higher carrier frequency (66% vs 42%, p=0.002) and odds ratio (OR=2.6, 95% CI 1.4-4.9). Cases with hypertension alone showed no significant increase in 1298C carriage (p=1). Genotype data was further filtered through gender, age, parental consanguinity, and smoking, each of which showed no significant association with susceptibility to hypertension (p>0.05), suggesting that these polymorphisms predispose to hypertension regardless of the listed risk factors.
AlShahrani et al. (2016) studied 490 Saudi subjects to find the frequency of allele and genotype of MTHFR C677T polymorphism in patients with primary glaucoma. The study included 210 Saudi patients and 280 Saudi controls. Primary open angle glaucoma was diagnosed in 144 of these patients. The frequencies of genotype CT and allele T were significantly higher in POAG, whereas the frequencies of genotype CC and allele C were significantly lower as compared to controls, indicating that the genotype CT and allele T are associated with susceptibility to primary glaucoma. Such an association was not seen for Primary Angle Closure Glaucoma.
Lajin et al. (2012) investigated the role of three common polymorphisms of the folate-homocysteine metabolic pathway in an Arab population from Syria consisting of 85 schizophrenic patients and 126 healthy controls. Results indicated a strong association between MTHFR c.A1298C and schizophrenia. The variant C allele frequency was significantly higher in the patients group [40% vs 29%, OR=1.6, 95% CI (1.06-2.41), p=0.023]. A statistically significant association was found for MTHFR c.677TT genotype under the recessive model in the male patients subgroup [OR=2.6, 95% CI (1.04-6.5), p=0.036], and MTHFR c.677CT genotype under the overdominant model in the total patients group [OR=0.52 95% CI (0.29-0.92), p=0.024].
El Hajj Chehadeh et al. (2016) aimed to determine the association between MTHFR SNPs and T2DM in the Emirati population. Genomic DNA was obtained from 209 diabetics and 169 healthy controls. Genotyping was carried out for the MTHFR variants C677T and A1298C. No significant association was found between the SNPs and T2DM for the Emirati cohort. However, when the patients were stratified based on complications, it was found that the C677T SNP was associated with a history of cerebrovascular accidents (CVA) (p=0.0330), history of nephropathy (p=0.0280) and levels of LDL cholesterol (p=0.0409), while the A1298C polymorphism was associated with hypertriglyceridemia (p=0.0305). The authors concluded that while the MTHFR SNPS were not associated with T2DM in the Emirati population, they could be used as risk markers for CVA, nephropathy, high LDL cholesterol and triglycerides in T2DM patients, thereby allowing for targeted risk assessment and initiation of early preventive measures.
[See: Palestine > Al-Gazali et al., 2001].
Nakai et al. (2004) applied a chip-based MALDI-TOF mass spectrometry to screen for 15 single nucleotide polymorphisms (SNPs) from 14 candidate genes involved in the renin-angiotensin system, lipid metabolism, cytokines and adhesion molecules, and growth factors, and the coagulation-fibrinolysis system. The study included the analysis of 47 healthy Yemenite Jews, a population known for its low incidence of coronary artery disease (CAD). The incidence of the C/T (p.A222V) allele in the MTHFR gene was 0.479 vs 0.181.
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