Methylenetetrahydrofolate Reductase gene polymorphism in patients receiving hemodialysis.

Methylenetetrahydrofolate Reductase (MTHFR) is key enzyme in metabolism of homocysteine. Homozygotes for mutation (TT genotype) have hyperhomocysteinemia, risk factor for atherosclerosis development. The aim of the study was to find out distribution of genotype frequencies of C677T MTHFR among patients on maintenance hemodialysis. Possible association of alleles and genotypes of C677T polymorphism of the MTHFR gene with age of onset, duration of dialysis and cause of kidney failure was studied also. Cross-sectional study includes 80 patients from Clinic of Hemodialysis KUCS in Sarajevo. In order to perform genotyping, isolated DNA was analyzed by RFLP-PCR and gel-electrophoresis. From total of 80 patients, 42.5% (n=24) were female, 57.5% (n=46) were male, mean age 54.59+/-1.78 years and duration of dialysis 79.92+/-6.32 months. Genotype distribution was: CC 51.2% (n=41), CT 37.5% (n=30) and TT 11.2% (n=9). Patients with wild-type genotype have longer duration of dialysis in month (87.1 +/- 63.93) comparing to TT genotype patients (67.06 +/- 39.3), with no statistical significance. T allele frequency was significantly higher in group of vascular and congenital cause of kidney failure (Pearson X2 =6.049, P<0.05) comparing to inflammation etiology group. Genotype distribution results are within the results other studies in Europe. Obtained results indicate that C677T polymorphism is not associated with onset, duration and cause of kidney failure in our hemodialysis population. There is an association of T allele of the MTHFR gene and vascular and congenital cause kidney failure.


Introduction
Homocysteine (Hcy) lies at an important metabolic branch point of methionine metabolism, between the remethylation and transsulfuration pathways.These lead to the formation of methionine and cystathionine, respectively.Homocysteine causes endothelial cell dysfunction and damage via production of potent reactive oxygen species during its autooxidation. is metabolite increases tromboxane formation, enhances platelet aggregation and is potent mitogen for vascular smooth muscle cells.Also, Hcy is antagonist of nitric oxide.ose facts may explain why renal impairment patients have a frequent cardiovascular complication and higher overall mortality rate than general population ().

Methylenetetrahydrofolate reductase enzyme
(MTHFR, EC ...) is an enzyme that exists in the cytoplasm of cells.It contains a flavin cofactor and uses NADH as the reducing agent.MTHFR is key enzyme in one-carbone metabolism.The enzyme catalyzes irreversibly reduction of ,-methylenetetrahydrofolate (,-methylTHF) to -methyltetrahydrofolate (-methylTHF), predominate circulating form of folate.Methionine synthase (MS) catalyzes transfer of methyl group from -methylTHF to homocysteine.The product of this B dependent remethylation is methionine and tetrahydrofolate.Methionine serves as a methyl group donor for over  reactions of synthesis, regulation and detoxification.
e most common mutation connected with impaired homocysteine metabolism and hyperhomocysteinemia had been described by Frosst B and coworkers().It is a point mutation on MTHFR gen, located at chromosome , at region p.().MTHFR genetic polymorphism is a cytosine to thymidine transition at nucleotide  in the predicted catalytic domain of MTHFR gene.
is C>T transition results in an alanine to valine substitution and has been associated with a thermolabile enzyme synthesis ().Serum activity of MTHFR in individuals with two copies of this variant (homozygotes with T alleles) is reduced about , while activity in individuals with one mutated allele (heterozygotes with TC alleles) is reduce .About half the general population carries at least one mutated allele and the frequency of the homozygous mutated genotype (TT) ranges from  to  depending on the population ().There is substantial variability in the prevalence of this mutation among different ethnic groups with a prevalence of - in Africans,  in Whites and  in Asians.The TT genotype was particularly common in Australia (,), northern China (), southern Italy (), and Mexico () ().Hyperhomocysteinemia is a major independent risk factor for atherothrombotic diseases and is common in hemodialysis patients.Homozygosity for the CT allele is associated with elevated homocysteine levels, especially in individuals who have a poor folate status ().Many studies have shown that homozygous mutant individuals have a signifi cant higher level of serum tHcy comparing to CT and CC genotype persons.Patient on maintained hemodialysis bearing TT homozygosity have hyperhomocysteinemia in  up to  cases comparing to heterozygous (CT) and wild-type (CC) ().Polymorphism at nucleotide position  CT within MTHFR gene is associated with earlier onset and duration of hemodialysis (,).However this finding was not confirmed in subsequent prospective study at larger scale.Aucella and coworkers claimed that genotypes were not different for time spent on dialysis or age at onset of dialysis ().Aim of our study was to investigate the distribution of allele and genotype frequencies of CT MTHFR among patients on maintenance hemodialysis at Clinic of Hemodialysis, KCUS, Bosnia and Herzegovina.Also, we wanted to investigate is there influence of MTHFR genotype on earlier onset and duration of hemodialysis in patients.

Patients
A total of  patients, both sex, were recruited from Clinic of Hemodialysis KUCS in Sarajevo, Bosnia and Herzegovina and were enrolled in cross-sectional study of MTHFR gene polymorphism.Exclusion criteria included severe malnutrition and patients with CV events.After performing MTH-FR genotyping, patients were separated into three groups: patients with TT genotype, patients with CT genotype, and patients with CC genotype.

Genetic Analysis
Approximately  ml of blood taken by cubital venipunction and stored in EDTA coated vacutainers (BD Biosciences) was used for genetic analysis.DNA was extracted in subsequent salting out procedure using DNA extraction kit (QIAamp blood kit, Qiagen).In order to perform genotyping, isolated DNA was analyzed by RFLP-PCR and gel-electrophoresis.In vitro amplifi cation of MTHFR gene segment that contains polymorphism genomic CT was carried out in  µl PCR reaction under following conditions: X PCR buff er, , mM MgCl, , mM of each of the dNTPs (Fermentas),  pmol of each of the primers (Sigma-Aldrich) forward '-TGAAGGAGAAGGTGTCT-GCGGGA-' and reverse '-AGGACGGTGCGGT-GAGAGTG-',  ng of DNA template and  units of Taq DNA polymerase (Platinum Taq, Invitrogen).Cycling conditions used for amplifi cation of  bp product were as follows:  cycles °C for  seconds, °C for  seconds, °C for  seconds.PCR products were evaluated for successful amplification on  agarose gels stained with ethidium bromide and then digested with Hinf I according to manufacturer's protocol (Pure Extreme, Fermentas).Hinf I recognizes the sequence that contains substitution of T for C nucleotide and is useful for genotype discrimination in this matter.Digested fragments were loaded on  agarose gels stained with ethidium bromide and documented and evaluated as transilluminated image on KODAK Edas software.

Statistical Analysis
Difference in baseline characteristic, possible association of alleles and genotypes of CT polymorphism with age of onset, duration of dialysis and cause of kidney failure was analyzed using SPSS software (version .).Data are presented as mean ± SD.Allelic and genotype frequencies among hemodialysis patients were cal-culated using MedCalc ver ... and tested for statistical signifi cance of allele frequency distribution between comparison groups (Chi square and Fisher's Exact test).A value of p < . was considered statistically signifi cant.

Patient characteristics
Mean age of hemodialysis patients (n=) was . ± . years (Table .)Among them  or . were male and  or . were female.The average duration of dialysis was . ± . months.Age of patients at the initiation of dialysis was . ± . years.No significant difference in the genotype distribution was found between male and female patients (Table .)There were predominant number of male patients in CT and TT genotype groups compare to female patients (. v. . males v. females) but the gender difference was not statistically significant.

D i s t r i b u t i o n o f t h e g e n o t y p e s a m o n g male and female patients on hemodialysis.
Results of gender difference in three diff e r e n t M T H F R g e n o t y p e g r o u p s a r e e xpressed as general number and percentage.
Data in Table  present results about alleles C and T frequencies and allele frequency distribution between three comparison groups, colloquial etiological categories: vascular, congenital and inflammatory (based on primary pathology of end stage renal dis-ease).We observed that minor allele T occurs more frequently in a group of vascular and congenital rather than inflammatory type of end stage renal disease (chi square ., P =.; Fisher's P=.).

Discussion
e results of this study revealed that genotype distribution results for patients receiving hemodialysis are within the results of other studies in Europe.Lim published results of distribution:  for CC, . for CT and . for TT genotype patients.Kimura's results were . for CC, . for CT and . for TT genotype patients.Distribution of MTHFR genotypes obtained for  hemodialysis patients done by Trem-blay was as follows:  had no mutation (CC);  were heterozygotes (CT), and  were homozygotes (TT).If we compare our results of distribution (. for CC, . for CT and .  for TT) with above mentioned ones, no diff erence is observable (P=.).According to the work of Kimura and his associates, we presumed to fi nd a less prevalence of the TT genotype in older age patients on maintained hemodialysis in Bosnia and Herzegovina region.We also, expected that TT genotype patients are signifi cantly younger at initiation of hemodialysis comparing to other two genotype bearing patients.Similar results showed Elizabeth Wrone.She had found that mutant allele frequency and percentage of homozygotes were lower in group of patients with dialysis period longer than one year.We didn't fi nd an association between CT polymorphism and onset and duration of hemodialysis.However, we found that patients with wild-type genotype had a longer duration of dialysis in month (. ± .) comparing to CT (. ± .) and TT genotype patients (. ± .), with no statistical signifi cance (P>.).Possible cause of our fi ndings may be in the diff erence in number of patients included in investigation.Kimura and Wrone had over  patients in their investigation, but our cross-sectional study included  patients and it is obviously less number of examined patients.
Well known fact is that hemodialysis patients have a much higher mortality rate than general popula-

Conclusion
In summary, we have shown in this work that genotype distribution is similar to results of other studies in Europe.Obtained results indicate that CT polymorphism is not associated with onset, duration and cause of kidney failure in our hemodialysis population.But, we found an association of T allele of the MTHFR gene and vascular and congenital cause kidney failure.For precise conclusion about infl uence of MTHFR polymorphism on age of onset, duration of hemodialysis and cardiovascular complication of patients on dialysis in Bosnia and Herzegovina, large prospective study that includes specifi c clinical features and biochemical markers with the application of novel simultaneous genetic risk profi ling is necessary.

TABLE 1 .
Picture .shows an agarose gel that illustrates different genotypes of CT mutation.When the C of the  nucleotide is replaced by T, restriction site for Hinf I is synthesized.Only one fragment of  bp long is presented in lines loaded with wild type C alleles DNA.The  bp fragment is regarded as mutant type T allele and DNA fragments with CT alleles have two  and  bp fragments.Lanes with TT genotype has single  bp fragment.edistribution of the genotypes in the patients was as follows: .  for TT genotype.. of CT genotype and . for CC genotype (Table .).Mean age of patients according to MTHFR genotype was . ± . for TT genotype, . ± . for CT genotype and . ± . for CC genotype.Data on duration of dialysis shows the diff erence between genotype groups: . ± . for TT, . ± . for CT and . ± . months for CC genotype.Wild type genotype Baseline characteristics of patients on dialysis EMINA KISELJAKOVIĆ ET AL.: METHYLENETETRAHYDROFOLATE REDUCTASE GENE POLYMORPHISM IN PATIENTS RECEIVING HEMODIALYSISpatients spend more months on maintained hemodialysis then heterozygous or homozygous patients.but the difference is not significant.No difference in the age at initiation of dialysis was found between three diff erent genotypes groups; . ± ., . ± . and . ±. (TT, CT and CC group respectively).

TABLE 4 .
Allele frequencies distribution among three colloquial cathegories tion.Cardiovascular diseases are major cause of the mortality of these patients.Recently, hyperhomocysteinemia is recognized as a cardiovascular risk factor.MTHFR gene polymorphism determinates level of homocysteine among other factors and may be considered as one of the cardiovascular risk factor.It is important to determine T allele for prediction of Hcy serum concentration and possible prevention or postponing complication and mortality by taking Hcy lowering therapy.Concerning our result that T allele is associated with vascular and congenital cause of kidney failure, we believe that mutation on MTHFR gene may be one of the risk factors for kidney failure or factor that may aggravate pathological changes.