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2
*
Diabetes Unit,
Geriatric Unit and
**
Department of Social Medicine, School of Public Health and Community Medicine, Hebrew University Hadassah Medical Center, Jerusalem, Israel
2To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • methylenetetrahydrofolate reductase mutations • type II diabetes • diabetic nephropathy • microalbuminuria • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) is a major complication of
diabetes mellitus (DM) and a primary cause of renal failure.
Microalbuminuria is not only a predictor of diabetic nephropathy, but
also a potent marker of micro- and macrovascular disease. The etiology
of DN is multifactorial and involves both environmental and genetic
factors. A genetic predisposition, based on familial clustering of DN,
has been implicated in the development of DN (Borch-Johnsen et al. 1992
, Pettiti et al. 1990
,
Seaquist et al. 1989
).
Mutations of the methylenetetrahydrofolate reductase (MTHFR) gene
have been shown to be associated with a predisposition to developing DN
in specific populations. MTHFR catalyzes the methylation of
homocysteine to methionine. Elevated levels of total plasma
homocysteine have been linked to increased all-cause mortality
(Kark et al. 1999
), arteriosclerosis (Frosst et al. 1995
) and thromboembolism (Boers et al. 1985
, Clarke et al. 1991
, Genest et al. 1990
, Mayer et al. 1996
, Robinson et al. 1995
, Stampfer et al. 1992
). It has been
reported that a relationship also exists between DN and elevated levels
of plasma homocysteine (Hultberg et al. 1991
). Specific
mutations in the MTHFR gene have been associated with increased total
plasma homocysteine. A commonly occurring mutation, a C 
T
substitution at nucleotide 677 (C677T) of the coding region that
results in the substitution of valine for alanine at position 226 of
the amino acid sequence, is linked with elevated total plasma
homocysteine levels in homozygotes compared with heterozygotes or
normal individuals (D’Angelo and Selhub 1997
,
Engbersen et al. 1995
, Friedman et al. 1999
, Harmon et al. 1996
). Some researchers, but
not others, have found an association between this mutation and DN
(Neugebauer et al. 1998
, Scaglione et al. 1999
, Shcherbak et al. 1999
). Another mutation,
an A C replacement at nucleotide 1298 (A1298C) resulting in a
substitution of alanine for glutamine, results in mild decreases in
MTHFR activity (van der Put et al. 1998
, Weisberg et al. 1998
). Combined heterozygosity of this mutation with
other MTHFR mutations including C677T has been associated with either
increased or decreased total plasma homocysteine levels
(Friedman et al. 1999
, van der Put et al. 1998
). In this study, the allele frequency of both the C677T or
A1298C mutations were measured in the Israeli Jewish diabetic
population, and we examined whether there was any association between
these mutations and DN in this heterogeneous population.
|
SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine the relationship between DN and specific mutations in the MTHFR gene, unrelated Jewish Israeli patients (n = 98) of diverse ethnic backgrounds with type 2 DM were screened for the presence or absence of either the A1298C or C677T mutation. The 98 subjects (41 men and 57 women), aged 45–75 y (mean age 62.4 ± 7.4 y), were recruited from the outpatient diabetes clinic of Hadassah University Hospital in Jerusalem. Trained interviewers administered a structure interview to determine the duration of diabetes, medications, other illnesses and the presence of diabetic complications. The study was approved by the ethics committee of the Israeli Ministry of Health. Informed consent was obtained from all participants.
Biochemical measurements.
Serum creatinine was measured by standard chemical and enzymatic commercial methods in a Kodak 700 XR analyzer C series (Rochester, NY). The reference value for creatinine was 60–106 µmol/L. Urine samples were collected over a 24-h period and urinary microalbumin was measured by immunoturbidimetric method (Roche Reagent, art. 0736872; Basel, Switzerland). DN was defined as follows: persistent micro- or macroalbuminuria (>30 mg/24 h) and no evidence of other nondiabetic renal pathology. Serum vitamin B-12 and serum folate were determined using commercial kits (Vitamin B-12 Elecsys reagent kit, # 1820753 and Folate Elecsys reagent kit, # 1820761, respectively; Roche) using an automated electrochemiluminescence immunoassay (ELICIA); the assays were performed on a Roche Elecsys 2010 immunoassay analyzer. The reference values for serum vitamin B-12 and serum folate were 148–700 pmol/L and 6.8–38.5 nmol/L, respectively.
Genetic analysis.
Genomic DNA was prepared from peripheral blood, as described
(Miller et al. 1988
). The C677T mutation in the MTHFR
gene was analyzed by polymerase chain reaction (PCR) of genomic DNA
using the following primer pairs: 5'-TGAAGGAGAAGGTGTCTGCGGGA-3'
(exonic) and 5'-GGACGGTGCGGTGAGAGTG-3' (intronic), which produced a
fragment that was 198 bp in length. DNA was amplified using a PCR
thermal cycler (Perkin-Elmer, Cetus, MJ Research, MA) using
conditions described previously (Friedman et al. 1999
).
PCR product (10 µL) was digested with the restriction
enzyme HinfI (Gibco BRL, Paisley, Scotland). The C677T mutation
abolishes a HinfI restriction site with the result that digestion of
the mutant allele PCR fragment produces a 198-bp band, whereas
digestion of the wild-type allele produces 175- and 23-bp
fragments. DNA fragments were separated by electrophoresis on a 2%
agarose gel and visualized with ethidium bromide.
The second A1298C mutation was also analyzed by PCR using the following
primer pairs: 5'-CTTTGGGGAGCTGAAGGACTACTAC-3' and 5'-CACTTTGTGACCATTCCG
GTTTG-3' using the conditions described previously (Friedman et al. 1999
). The amplified 163-bp fragment was digested with
MboII (MBI fermentas; Vilna, Lithuania). The A1298C mutation abolishes
an MboII restriction site. Digestion of the 163-bp fragment of the 1298
AA genotype gives five fragments: 56, 31, 30, 28 and 18 bp, whereas the
1298CC genotype results in four fragments: 84, 31, 30 and 18 bp. The
fragments were analyzed by 20% polyacrylamide gel electrophoresis and
visualized with ethidium bromide.
Statistical analyses.
Statistical analyses were performed as described previously
(Friedman et al. 1999
). In brief, allele frequencies
were calculated by allele counting. The Hardy-Weinberg law
(Hardy 1908
) defines a simple relationship between the
frequency of genes in the population and the frequency of genotypes
(i.e., individuals). Under certain assumptions (e.g., random mating, no
migration, no inbreeding, no selective survival among genotypes and
large population sizes), the expected frequencies of genotypes will be
the same in all subsequent generations. It is therefore useful for
estimating allele frequencies from the prevalence of Mendelian traits.
Concordance of genotype frequencies with Hardy-Weinberg equilibrium
was tested by a 
2 goodness-of-fit test. The baseline
values of these groups were compared using the unpaired
t test. Statistical tests were performed using
statistical software (SPSS; Chicago, IL).
|
RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). There were no differences between the normoalbuminuric subjects and
those who had elevated urinary albumin excretion in terms of gender,
age, duration of DM, serum creatinine, serum folate or serum vitamin
B-12.
|
). This was nearly identical to the distribution in DN patients in which
23 (41.8%) were CC, 22 (40%) were CT and 10 (18.2%) were TT. Allele
frequencies for C (wild-type allele) and T allele (mutant allele) were
0.67 and 0.33, respectively, in the normoalbuminuric subjects and 0.62
and 0.38, respectively, in those with evidence of DN. The allele
frequencies of the total study population were 0.64 and 0.36,
respectively. Genotype frequencies between the two groups did not
differ significantly from the frequencies predicted on the basis of the
Hardy-Weinberg law of population genetics.
|
).
In subjects with plasma folate <15.4 nmol/L, plasma homocysteine
levels were 24% higher than those of controls with the same
concentrations of folate who had the normal genotype. When patients in
this study were stratified on the basis of folate concentrations, there
was a relationship between DN and genotype. Among subjects with a serum
folate <15.4 nmol/L, the allele frequency of the mutation was 0.08 in
the normoalbuninuric patients compared with 0.50 in those with DN. The
genotype distribution was also significantly different, with the
proportion of patients who were either heterozygous or homozygous for
the mutation having a greater chance of having DN.
For the A1298 C mutation (Table 3
), the genotype frequencies for the normoalbuminuric group and DN groups
were as follows: 24 (55.8%) were AA, 9 (20.9%) had the heterozygote
AC genotype, and 10 (23.3%) were homozygote for CC in the former group
compared with 26 (47.3%) who were AA, 19 (34.5%) with the AC
genotype, and 10 (18.2%) who had CC in the latter group. Allele
frequencies for A (wild-type allele) and C (mutant allele) were 0.66
and 0.34, respectively, in the normoalbuminuric subjects and 0.65 and
0.35, respectively, in the patients with micro- and macroalbuminuria.
The allele frequencies of the total study population were 0.65 and
0.35, respectively. Similar to the C677T mutation, there were no
significant differences in the genotype distribution frequencies.
However, when this population was also stratified on the basis of serum
folate concentrations, there was evidence that a relationship between
genotype and DN may exist because there was a distinct difference in
the distribution of genotypes (Table 3)
for individuals with plasma
folate <15.4 nmol/L. Individuals homozygous for the mutation appeared
to have a lower incidence of DN because none of the five individuals
with the CC phenotype had DN.
|
, van der Put et al. 1998
). The
population in this study also demonstrated this characteristic, with
the 677T and 1298C allele always being in the trans
configuration. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Chico et al. 1998
,
Smulders et al. 1999a
and 1999b
, Stabler et al. 1999
). The issue is complicated because renal disease
itself might cause elevated levels of homocysteine (Robinson and Dennis 1998
). We examined this issue from a different
perspective. Previous research from our group and others had shown that
specific mutations in the MTHFR gene (C677T) are related to increased
levels of homocysteine. If increased total plasma homocysteine is
related to the development of DN, then it would be reasonable to assume
that there would also be an association between those who were
homozygous for the C677T mutation and an increased frequency of DN. The
results of our study showed that when type 2 diabetics were analyzed as
an aggregate, no difference in genotype frequency among the different
MTHFR mutations could be found between individuals with and without DN.
However, when the analysis was expanded to include stratification by
serum folate levels, individuals with the C677T mutation had a greater
propensity for DN if they have a low serum folate (<15.4 nmol/L).
In a previous study, we found that in subjects drawn from the same
population, those individuals who were homozygous for the C677 T
mutation had higher plasma total homocysteine concentration
(Friedman et al. 1999
), yet no relationship was found
between this mutation and established cardiovascular risk factors.
However, in that study, stratification on the basis of serum folate
levels was not performed. It may be that in a large group of patients
with the C677T mutation, adequate folate supplementation overcomes the
effect of the mutation on MTHFR activity. Both screening studies in
which plasma folate was correlated with total plasma homocysteine
(Jacques et al. 1996
) and prospective studies in which
folate supplementation was provided indicate that high serum levels of
folate restore total plasma homocysteine levels to normal and overcome
the reduction of MTHFR activity associated with the mutant C677T
thermolabile enzyme (Malinow et al. 1997
).
An association between C677T and DN has been shown for both type 1 and
type 2 DM by others (Neugebauer et al. 1998
,
Shcherback et al. 1999
). However, not all researchers
found this association (Scaglione et al. 1999
). Because
these studies were conducted in different populations, it may be that
there are ethnic variations in terms of this relationship. The causes
of DN are multifactorial, and it may be that a given
population may have elements in its genetic makeup that are
protective against the development of DN despite the elevations in
homocysteine levels that are associated with the C677T mutation. Such a
protective effect was found in the Japanese population in which the
C677T mutation was associated with lower than average blood pressure,
which was protective for cerebral vascular disease (Nakata et al. 1998
). The possibility of either protective effects of
C677T or other protective genetic factors related to ethnicity is
suggested by the conflicting results of investigations of this
mutation. Although a majority of studies show that this mutation is
invariably related to elevated total plasma homocysteine, there is no
consistency in terms of an association of this mutation with a variety
of vascular disorders. Alternatively, as our data indicate, serum
folate concentrations may be an intervening variable that must be
considered in genotype-phenotype relationships.
The relationship between the A1298C mutation and DN is more problematic. Our data suggest that individuals homozygous for this mutation may have a lower incidence of DN. However, because all individuals with the 1298 CC genotype also carry the 677 CC wild-type genotype, it may be that the protective affect of 1298 CC is related to the fact that these individuals do not carry the deleterious 677 TT mutation.
In conclusion, our study showed a relationship between mutations in the MTHFR gene and DN in a heterogeneous Israeli population with low serum folate concentrations. It is possible that there are other associations between mutations in the MTHFR gene and DN. For example, individuals with MTHFR gene mutations may have a more rapid decline in renal function than their counterparts who carry the normal alleles. To detect such a relationship, it will be necessary to perform additional and larger long-term, follow-up studies in patients with DN from a variety of ethnic groups. It will also be important to study prospectively whether folate supplementation reduces the incidence of DN in type 2 DM in individuals who carry the C677T allele.
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FOOTNOTES |
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3 Abbreviations used: DM, diabetes mellitus, DN, diabetic nephropathy, MTHFR, methylenetetrahydrofolate reductase, PCR, polymerase chain reaction.
Manuscript received January 31, 2000. Initial review completed March 4, 2000. Revision accepted June 1, 2000.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1. Agardh C. D., Agardh E., Andersson A., Hultberg B. Lack of association between plasma homocysteine levels and microangiopathy in type 1 diabetes mellitus. Scand. J. Clin. Lab. Investig. 1994;54:637-641[Medline]
2. Boers G. H., Smals A. G., Trijbels F. J., Fowler B., Bakkeren J. A., Schoonderwaldt H. C., Kleijer W. J., Kloppenborg P. W. Heterozygosity for homocystinuria in premature peripheral and cerebral occlusive arterial disease. N. Engl. J. Med. 1985;320:1161-1164[Abstract]
3. Borch-Johnsen K., Norgard K., Hommel E. Is diabetic nephropathy an inherited complication?. Kidney Int 1992;41:719-722[Medline]
4. Chico A., Perez A., Cordoba A., Arcelus R., Carreras G., de Levia A., Gonzales-Sastre F., Blanco-Vaca F. Plasma homocysteine is related to albumin excretion rate in patients with diabetes mellitus: a new link between diabetic nephropathy and cardiovascular disease?. Diabetologia 1998;41:684-693[Medline]
5. Clarke R., Daly L., Robinson K., Naughten E., Cahalane S., Fowler B., Graham I. Hyperhomocysteinemia: an independent risk factor for vascular disease. N. Engl. J. Med. 1991;324:1149-1155[Abstract]
6.
D’Angelo A., Selhub J. Homocysteine and thrombotic disease. Blood 1997;90:1-11
7. Engbersen A.M.T., Franken D. G., Boers G.H.J., Stevens E.M.B., Trijbels F.J.M., Blom H. J. Thermolabile 5,10-methylenetetrahydrofolate reductase as a cause of mild hyperhomocysteinemia. Am. J. Hum. Genet. 1995;56:142-150[Medline]
8.
Friedman G., Goldschmidt N., Friedlander Y., Ben-Yehuda A., Selhub J., Babaey S., Mendel M., Kidron M., Bar-On H. A common mutation A1298C in human methylenetetrahydrofolate reductase gene: association with plasma total homocysteine and folate concentration. J. Nutr. 1999;129:1656-1661
9. Frosst P., Blim H. J., Milos R., Goyette P., Sheppard C. A., Matthews R. G., Boers G.J.H., den Heijer M., Kluijtmans L.A.J., van den Heuvel L. P., Rozen R. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat. Genet. 1995;10:111-113[Medline]
10. Genest J. J., Jr, McNamara J. R., Salem D. N., Wilson P. W., Schaefer E. J., Malinow M. R. Plasma homocyst(e)ine levels in men with premature coronary artery disease. J. Am. Coll. Cardiol. 1990;16:1114-1119[Abstract]
11. Hardy G. H. Mendelian proportions in a mixed population. Science (Washington, DC) 1908;28:9-50
12.
Harmon D. L., Woodside J. V., Yarnell J.W.G., McMaster D., Young I. S., McCrum E. E., Gey K. F., Whitehead A. S., Evans A. E. The common "thermolabile" variant of methylenetetrahydrofolate reductase is a major determinant of mild hyperhomocysteinemia. Q. J. Med. 1996;89:571-577
13. Hultberg B., Agardh E., Andersson A., Brattstrom L., Isaksson A., Israelsson B., Agardh C. D. Increased level of plasma homocysteine are associated with nephropathy, but not severe retinopathy in type I diabetes mellitus. Scand. J. Clin. Lab. Investig. 1991;51:277-282[Medline]
14.
Jacques P. F., Bostom A. G., Williams R. G., Ellison R. C., Eckfeldt J. H., Rosenberg I. H., Selhub J., Rozen R. Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation 1996;93:7-9
15.
Kark J. D., Selhub J., Adler B., Gofin J., Abramson J. H., Friedman G., Rosenberg I. H. Nonfasting plasma total homocysteine level and mortality in middle-aged and elderly men and women in Jerusalem. Ann. Intern. Med. 1999;131:321-330
16.
Malinow M. R., Nieto F. J., Kruger W. D., Duell P. B., Hess D. L., Gluckman R. A., Block P. C., Holzgang C. R., Anderson P. H., Seltzer D., Upson B., Lin Q. R. The effects of folic acid supplementation on plasma total homocysteine are modulated by multivitamin use and methylenetetrahydrofoloate reductase genotypes. Arterioscler. Thromb. Vasc. Biol. 1997;17:1157-1162
17. Mayer E. L., Jacobsen D. W., Robinson K. Hyperhomocysteinemia and coronary atherosclerosis. J. Am. Coll. Card. 1996;27:517-527[Abstract]
18.
Miller S. A., Dydes D. D., Polesky H. F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215
19. Nakata Y., Katsuya T., Takami S., Sato N., Fu Y., Ishikawa K., Takiuchi S., Rakugi H., Miki T., Higaki J., Ogihara T. Methylenetetrahydrofolate reductase gene polymorphism: relation to blood pressure and cerebrovascular disease. Am. J. Hypertens. 1998;11:1019-1023[Medline]
20. Neugebauer S., Baba T., Watanabe T. Methylenetetrahydrofolate reductase gene polymorphism as a risk factor for diabetic nephropathy in NIDDM patients [letter]. Lancet 1998;352:454
21. Pettiti D. J., Saad M. F., Bennett P. M., Nelson R. G., Knowler W. C. Familial predisposition to renal disease in two generation of Pima Indians with Type 2 (non-insulin dependent) diabetes mellitus. Diabetologia 1990;33:438-443[Medline]
22. Robinson K., Dennis V. W. From microalbuminuria to hyperhomocysteinemia. Kidney Int 1998;54:281-282[Medline]
23.
Robinson K., Mayer E. L., Miller D. P., Green R., van Lente F., Gupta A, Kottke-Marchant K., Savon S. R., Selhub J., Nissen S. E., Kutner M., Topol E. J., Jacobsen D. W. Hyperhomocysteinemia and low pyridoxal phosphate: common and independent reversible risk factors for coronary artery disease. Circulation 1995;92:2825-2830
24. Scaglione L., Gambino R., Gay M., Cassader M., Pagano G., Cavallo-Perin P. Methylenetetrahydrofolate reductase gene polymorphism is not associated to diabetic nephropathy in type 2 diabetes. Diabetologia 1999;42:A329(abs.)
25. Seaquist E. R., Goetz F. C., Rich S., Barbosa J. Familial clustering of diabetic kidney disease. Evidence for genetic susceptibility to diabetic nephropathy. N. Engl. J. Med. 1989;320:1161-1164
26. Shcherbak N. S., Shutskaya Z. V., Sheidina A. M., Larionova V. I., Schwartz E. I. Methylenetetrahydrofolate reductase gene polymorphism is a risk factor for diabetic nephropathy in IDDM patients. Mol. Genet. Metab. 1999;68:375-378[Medline]
27. Smulders Y. M., Brouwer C. B., Silberbusch J. Is plasma homocysteine related to albumin excretion rate in patients with diabetes mellitus?. Diabetologia 1999a;42:382-383[Medline]
28.
Smulders Y. M., Rakie M., Slaats E. H., Treskes M., Sijbrands E. J., Odekerken D. A., Stehouwer C. D., Silberbusch J. Fasting and post-methionine homocysteine levels in NIDDM. Determinants and correlations with retinopathy, albuminuria, and cardiovascular disease. Diabetes Care 1999b;22:125-132
29. Stabler S. P., Estacio R., Jeffers B. W., Cohen J. A., Allen R. H., Schrier R. W. Total homocysteine is associated with nephropathy in non-insulin-dependent diabetes mellitus. Metabolism 1999;48:1096-1101[Medline]
30.
Stampfer M. J., Malinow R., Willett W. C., Upson B., Ullmann D., Tishler P. V., Hennekens C. H. A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians. J. Am. Med. Assoc. 1992;268:877-881
31. van der Put N.M.J., Gabreëls F., Stevens E.M.B., Smeitink J. A., Trijbels F. J., Eskes T. K., van den Heuvel L. P., Blom H. J. A second common mutation in the methylenetetrahydrofolate reductase: an additional risk factor for neural-tube defects?. Am. J. Hum. Genet. 1998;62:1044-1051[Medline]
32. Weisberg I., Tran P., Christensen B., Sibani S., Rozen R. A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. Mol. Genet. Metab. 1998;64:169-172[Medline]
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