Spectrum of MTHFR gene SNPs C677T and A1298C: a study among 23 population groups of India.

Abstract

Elevated homocysteine is a risk factor for many complex disorders. The role of methylenetetrahydrofolate reductase (MTHFR) gene in methylation of homocysteine makes it one of the most important candidate genes for these disorders. Considering the heterogeneity in its distribution in world populations, we screened MTHFR C677T and A1298C single nucleotide polymorphisms in a total of 23 Indian caste, tribal and religious population groups from five geographical regions of India and belonging to four major linguistic groups. The frequencies of MTHFR 677T and 1298C alleles were found to be 10.08 and 20.66%, respectively. MTHFR homozygous genotype 677TT was absent in eight population groups and homozygous 1298CC was absent in two population groups. 677T allele was found to be highest among north Indian populations with Indo-European tongue and 1298C was high among Dravidian-speaking tribes of east India and south India. The less common mutant haplotype 677T-1298C was observed among seven population groups and overall the frequency of this haplotype was 0.008, which is similar to that of African populations. cis configuration of 677T and 1298C was 0.94%. However, we could not find any individual with four mutant alleles which supports the earlier observation that presence of more than two mutant alleles may decrease the viability of foetus and possibly be a selective disadvantage in the population.

Full text

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Spectrum of MTHFR gene SNPs C677T and A1298C: a study
among 23 population groups of India
Kallur Nava Saraswathy•Mohammad Asghar•
Ratika Samtani•Benrithung Murry•Prakash Ranjan Mondal•
Pradeep Kumar Ghosh•Mohinder Pal Sachdeva
Received: 8 April 2011/Accepted: 30 November 2011
? Springer Science+Business Media B.V. 2011
Abstract
complex disorders. The role of methylenetetrahydrofolate
reductase (MTHFR) gene in methylation of homocysteine
makes it one of the most important candidate genes for
these disorders. Considering the heterogeneity in its dis-
tribution in world populations, we screened MTHFR
C677T and A1298C single nucleotide polymorphisms in a
total of 23 Indian caste, tribal and religious population
groups from five geographical regions of India and
belonging to four major linguistic groups. The frequencies
of MTHFR 677T and 1298C alleles were found to be 10.08
and 20.66%, respectively. MTHFR homozygous genotype
677TT was absent in eight population groups and homo-
zygous 1298CC was absent in two population groups. 677T
allele was found to be highest among north Indian popu-
lations with Indo-European tongue and 1298C was high
among Dravidian-speaking tribes of east India and south
India. The less common mutant haplotype 677T-1298C
was observed among seven population groups and overall
the frequency of this haplotype was 0.008, which is similar
to that of African populations. cis configuration of 677T
and 1298C was 0.94%. However, we could not find any
individual with four mutant alleles which supports the
earlier observation that presence of more than two mutant
alleles may decrease the viability of foetus and possibly be
a selective disadvantage in the population.
Elevated homocysteine is a risk factor for many
Keywords
Haplotypes ? Indian populations
MTHFR C677T ? MTHFR A1298C ?
Introduction
Methylenetetrahydrofolate reductase (MTHFR) catalyzes
irreversiblythereductionof5,10-methylenetetrahydrofolate
to 5-methyltetrahydrofolate, thereby playing an important
role in the methylation of homocysteine to methionine by
providing methyl group. Molecular defects in the gene
that encodes the enzyme reduce its activity which results
in increased plasma homocysteine [1], and supplementa-
tion of folate being an important co-factor in the con-
version of homocysteine to methionine, may compensate
for this effect [2]. Hyperhomocysteinemia is a risk factor
for many complex disorders like coronary artery disease
[3, 4], neural tube defects [5] and recurrent pregnancy
loss [6]. MTHFR gene is mapped on human chromosome
1p36.3 [7].
C677T (rs1801133) and A1298C (rs1801131) are the
two most common MTHFR gene single nucleotide poly-
morphisms (SNPs) that have confirmed effect on the
enzyme activity [1, 8]. The former SNP is result of a point
mutation at the position 677 in exon 4 leading to the sub-
stitution of alanine by valine [1]. This mutation is associ-
ated with the reduced enzyme activity by about 70 and 40%
in homozygotes and heterogzygotes, respectively resulting
in elevation of plasma homocysteine [8] and diminished
DNA methylation [9]. The latter SNP is characterized by a
point mutation at position 1298 in exon 7 of the gene that
leads to the replacement of glutamine by alanine [10]. This
mutation also results in the reduction of MTHFR activity
but to a lesser extent than in the case of C677T [8].
Many studies have reported the association of MTHFR
C677T and A1298C mutations with cardiovascular dis-
eases [11], neural-tube defects [10], various types of cancer
[12–16], male infertility [17], pregnancy complications
[18–20] and other complex diseases where the role of
K. N. Saraswathy ? M. Asghar ? R. Samtani ? B. Murry ?
P. R. Mondal ? P. K. Ghosh ? M. P. Sachdeva (&)
Biochemical and Molecular Lab, Department of Anthropology,
University of Delhi, Delhi 110007, India
e-mail: mpsachdeva@rediffmail.com
123
Mol Biol Rep
DOI 10.1007/s11033-011-1299-8

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folate is largely documented [21]. The frequency distri-
bution of both these mutations especially of C677T shows
a high heterogeneity among different ethnic and regional
groups [22–25]. Moreover, C677T and A1298C mutations
are known to interact in vivo [8, 10] and a combined effect
of these two is likely to carry a selective disadvantage and
contribute to decreased foetal viability [26].
Because of the changing life styles and dietary habits,
like in any developing country the number of patients
suffering from complex disorders is increasing in India and
there is a need of knowing the distribution pattern of var-
ious candidate genes for these disorders in general popu-
lation. The Indian Genome Variation Consortium had
included MTHFR C677T as one of its SNPs but it failed to
demonstrate clear cut population variation [27]. Consider-
ing the implication of MTHFR gene in various complex
disorders coupled with its high heterogeneity among dif-
ferent ethnic and regional population of the world, the
present study was planned to investigate the heterogeneity
of this gene in different population groups of India,
including various castes and tribes and a religious com-
munity (the Muslim) inhabiting different geographical
regions of the country and representing its major linguistic
groups [28].
Materials and methods
Subjects and material
A total of 1,069 random blood samples was collected from
healthy individuals within the age group of 20–55 years,
belonging to 23 different endogamous populations of India
comprisingvariouscaste,tribeandreligiousgroups(Fig. 1).
Care was taken to avoid blood relatives up to the first cousin
during the sampling. Population groups studied belong to
five geographical regions of India viz., north India, north-
east India, east India, west India and south India cutting
acrossallthefourmajorlinguisticgroupsofthecountryviz.,
the Austro-Asiatic, Dravidian, Indo-European and Tibeto-
Burman. Informed written consent was taken from all the
subjects before collecting the samples.
Genotyping
Genomic DNA was isolated using salting out method [29].
MTHFR C677T SNP was genotyped by RFLP method and
for this PCR amplification was done following the standard
protocol [30]. The product was digested with HinfI
restriction enzyme. MTHFR A1298C SNP was typed by
allele specific PCR amplification [31]. In both cases, the
PCR products were genotyped by agarose gel electropho-
resis and genotypes were visualised under long wave UV
light after staining with ethidium bromide. A total of 1,061
DNA samples could be genotyped for MTHFR C677T and
997 for MTHFR A1298C.
Statistics
Genotype and allele frequencies were calculated by gene
counting method using POPGENE software [32]. Confor-
mity to Hardy–Weinberg equilibrium (HWE) in each
population group was performed by goodness of fit Chi-
square test. Haplotype frequencies were determined using
HAPLOPOP software [33]. Statistical significance was
checked at 5% level of probability.
Results
Genotypes
Table 1 shows that of the 23 studied population groups, 8
were found not following the HWE for MTHFR C677T
SNP. In case of A1298C SNP, as many as 14 population
groups deviated from the genetic equilibrium. Geographi-
cally, significant deviation from HWE was seen in north,
west and south India for C677T SNP, whereas in case of
A1298C SNP, it was observed in north, north-east and
south India.
MTHFR gene was found to be polymorphic at both
C677T and A1298C sites in almost all the population
groups studied, barring few groups in each case (Table 1).
In the total sample, MTHFR 677 CC, CT and TT genotypes
Fig. 1 Map of India showing the details of population groups studied
from different geographical regions
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were found to be 82.28, 15.27 and 2.45%, respectively. The
TT genotype was present in all north and south Indian
populations with the highest frequency being found in the
Rajput of north India (7.84%). On the other hand, it was
found absent in seven populations of north-east and east
India viz., the Kom, Meitei, Paite, Thadou, Kabui, Munda,
Oraon and Naikda of west India. In the present study,
polymorphism of A1298C was found in all but two popu-
lations viz., Aimol of north-east India and Nayakpod of
south India. In the total sample, frequencies of AA, AC and
CC genotypes were found to be 70.1, 18.5 and 11.4%,
respectively. The highest CC frequency was found in the
Brahmin of north India (22%).
The frequencies of 677T and 1298C alleles were 10.08
and 20.66%, respectively in the total sample. The T allele
of MTHFR C677T SNP was most frequent among the
Sindhi (23.75%), but it was absent in three population
groups viz., the Kom and Thadou of north-east India and
Munda of east India. Geographically, the allele was found
to be highest in north India with an average frequency of
16.70%, followed by north-east India (9.88%), west India
(7.83%), south India (7.57%) and east India (1.11%).
Linguistically, the Indo-European group showed the high-
est average T allele frequency (12.76%), followed by
Tibeto-Burman (9.88%), Dravidian (6.50%) and Austro-
Asiatic (nil). The C allele of MTHFR A1298C SNP was
found to be most frequent in the Oraon (46.43%). Geo-
graphically, the highest average C allele frequency was
found in east India (34.63%), followed by west India
(26.98%), south India (24.29%), north India (16.78%) and
north-east India (13.50%). Linguistically, the Dravidian
populations showed the highest average C allele frequency
Table 1 Genotype number and allele frequency (%) distribution of MTHFR C677T and A1298C SNPs in various Indian population groups
Population groupMTHFR 677MTHFR 1298
n
CCCT TTT (%)
P-value
n
AA AC CCC (%)
P-value
North India
Punjabi413731 6.100.009a
0.011a
38 3134 14.47 0.000a
0.000a
0.000a
0.000a
0.000a
Brahmin51408313.73503631125.00
Jat4730 14321.280.399
0.031a
47412410.64
Rajput513611418.63514416 12.75
Sindhi402315223.750.88538292721.05
North-east India
Aimol503217119.000.4985050000.00–
0.000a
Kom3838000.00–33 3012 7.58
Meitei463790 9.780.490 42 2714119.050.650
0.001a
Paite 4441303.410.849 3734215.41
Thadou4242000.00– 35 1519130.000.098
0.000a
0.000a
0.010a
Muslim 6243 181 16.130.60455446514.55
Bamon473412114.890.98046363718.48
Kabui412813015.850.24931254212.90
East India
Munda494900 0.00–46343922.830.000a
Oraon4543202.220.91542 1025746.430.232
West India
Patelia4034518.750.123
0.003a
4440226.820.000a
Damor5046315.00471328642.550.154
0.000a
Naikda4738909.570.496
0.001a
39304517.95
Kathodi5044428.00481723840.62 0.905
South India
Brahmin 5045327.000.000a
0.000a
0.040a
4927 81436.73 0.000a
Nayakpod3533114.293838000.00–
Thoti47387211.70
4825 176 30.210.235
Kolam4842517.29 0.077
0.000a
4323 14630.230.114
0.000a
Total1,061 873 1622610.08
997 699 18411420.66
aDeviated significantly from HWE (P B 0.05)
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(28.72%), followed by Austro-Asiatic (22.83%), Indo-
European (21.32%) and Tibeto-Burman (13.50%).
Haplotypes
Table 2 shows that all the four possible haplotypes of the
two SNPs were observed in this study. In the total sample,
the most preponderant haplotype was 677C-1298A with a
frequency of 0.693, followed by 677C-1298C (0.201),
677T-1298A (0.099) and 677T-1298C (0.008). The least
common mutant haplotype, 677T-1298C, was observed in
seven population groups viz., the Kathodi and Naikda from
west India, Meitei and Muslim from north-east India and
Brahmin, Thoti and Kolam from south India.
Discussion
MTHFR 677T allele frequency in the present study
(10.08%) was lower than the average frequency (14%)
reported by Indian Genome Variation Consortium [27].
The frequencies of both 677T and 1298C alleles had shown
large variation among the present population groups
ranging from complete absence to 23.75 and 46.43%,
respectively. The incidence of 677T allele was found to be
invariably higher among castes compared to tribes. The
incidence was also found to be comparatively higher
among the north Indian populations, followed by those of
north-east, west, south and east India. Linguistically, 677T
allele frequency was highest among the Indo-European,
followed by Tibeto-Burman, Dravidian and Austro-Asiatic.
The presence of TT genotype among all non-tribal popu-
lations except Meitei with Mongoloid background, and also
a high frequency of 677T allele among the caste popula-
tions particularly from north India which are linguistically
Indo-European compared to tribal populations, is indicative
of the gene flow into caste populations of north India from
Eurasian populations where the 677T allele frequency is
very high [34].
Globally, 677T allele is found to be highest in Europe
with a frequency ranging from 24.1 to 64.3%, followed by
North America (6–64.3%), East Asia (2–55%), South
America (2–48.7%), Asia (2.5–45%), Africa (0–35.5%),
Siberia (8–31.5%) and Oceania (2.9–28.6%) [35]. It is only
in African populations that the allele is often reported
absent. Considering the higher frequencies of 677T
observed in Europe and North America, it can be presumed
that the allele originated in Europe in the late stage of
human evolution. The migration, settlements and coloni-
sation might have helped in spreading this allele to other
regions. North America which has more of European
migrants has higher 677T allele frequency than South
America, mainly inhabited by indigenous populations.
Higher frequency of this allele among the Caucasoid
groups compared to Mongoloid and Negroid groups also
supports the above hypothesis. However, we cannot rule
out the possible multi centric origin of this mutant allele as
it is observed to be relatively high among some South East
Asian populations [36] and African populations [35].
Lighter skin colour and more exposure to ultraviolet rays is
a disadvantage for 677T allele carriers because of dermal
photolysis of folate [37] and thus a south to north cline of
decrease in the allele frequency is apparent in European
populations [38]. On the other hand, in the present study
and as reported elsewhere, a reverse trend was observed,
i.e., lower frequency of 677T among dark skin coloured
tribes of south and east India, and higher frequency of the
allele among the light skin coloured Castes of north and
north-east India. Moreover, since this allele is an important
clinical marker associated with many complex disorders,
the dietary habits might have had a significant influence in
the propagation of 677T in the world populations. In
Africa, malnutrition and infectious diseases impairing
Table 2 Haplotype frequencies of MTHFR gene in 23 population
groups of India
Population
group
677
C-1298 A
677
C-1298 C
677
T-1298 A
677
T-1298 C
Punjabi
Brahmina
0.7840.1490.068 0.000
0.6100.2500.1400.000
Jat0.6890.0940.2030.015
Rajput0.6860.1270.1860.000
Sindhi0.541 0.216 0.2430.000
Aimol0.8060.0000.1940.000
Kom0.9240.0760.0000.000
Meitei0.7490.1550.0600.035
Paite0.9050.0540.0410.000
Thadou0.7120.2880.0000.000
Muslim0.6970.1300.1570.015
Bamon0.6740.1850.1410.000
Kabui0.6940.1290.1770.000
Munda0.772 0.228 0.0000.000
Oraon0.5260.4490.0260.000
Patelia0.8330.0830.0830.000
Damor0.6960.0870.2170.000
Naikda0.7410.1440.0800.036
Kathodi
Brahminb
0.5420.373 0.0540.031
0.5820.3470.0510.020
Nayakpod0.9830.0000.0170.000
Thoti0.585 0.3000.0950.020
Kolam0.6590.2680.0610.012
Total0.6930.2010.0990.008
aNorth India
bSouth India
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intestinal absorption of folic acid are common [39]. Like-
wise, the economic status of many tribal groups in India is
low compared to the caste groups leading to a poor living
conditions and malnutrition in the former. As adequate
folic acid intakes may raise the MTHFR 677T frequency in
a population [40] the low frequency of the allele among
African and tribal groups in the present study may be
related to folate deficiency. In the Indian context, apart
from folate status, vitamin B12, another cofactor in meth-
ylation of homocysteine to mithionine needs attention as
most of the Indian populations depend more on vegetarian
diet which is low in vitamin B12.
MTHFR 1298C allele was found more preponderant
compared to 677T allele in the present study. In fact, unlike
677T, 1298C was polymorphic in all the population groups
exceptamongtheAimolandNayakpodtribes.Thiscouldbe
explained by the fact that 677T emerged later than 1298C
[41] and the lesser detrimental effect caused by 1298C
comparedto677T.Incontrasttothedistributionof677T,the
incidence of 1298C was found to be highest among the east
Indianpopulations,followedbywest,south,northandnorth-
east Indian populations. Linguistically, the allele was most
frequent among the Dravidian speaking populations, fol-
lowedbyAustro-Asiatic,Indo-EuropeanandTibeto-Burman
populations. The 1298C was more or less uniformly dis-
tributed among castes and tribes. Globally, the allele was
found with high frequency in East Asia ranging from 18 to
70%, followed by Asia (17–44.6%), Europe (24–40%),
Siberia (38%), Africa (13–32.3%), South America (0–15%)
and North America (14.7%) [42]. The present data showed a
higher frequency of 1298C (20.66%) than that of Americas
(0–15%).AstudyamongtheAhirandJatofHaryanainIndia
had reported the 677T allele frequency to be 3 and 6%,
respectively [43]. In another study, 1298C and 677T allele
frequencies were reported as 38.9 and 10.4%, respectively
[44].Thelatterallelefrequencyissimilartothepresentstudy
while that of the former allele frequency is about two times.
In recent years a large number of studies on MTHFR
gene, particularly on C677T SNP has been conducted
because of clinical importance of the gene. The enzymatic
function of the gene is reduced up to 70% by 677T
mutation which leads to hyperhomocysteinemia [8]. Sim-
ilarly, the MTHFR 1298C mutation also reduces the
enzyme activity but to a lesser extent [8]. However, this
detrimental effect of mutations can be compensated by the
supplementation of folic acid [2]. In other words, apart
from mating patterns, migratory histories and ethnic
backgrounds, environment, i.e., folic acid in the present
case plays an important role in the propagation of these two
mutant alleles in human populations.
The role of MTHFR in the patho-physiology of various
complex disorders is still debatable despite the fact that
hyperhomocysteinemia is associated with these disorders.
The homozygote 677TT genotype was reported to have a
four-fold higher frequency among neonates in comparison
to aborted foetuses [26]. In another study, both T allele and
TT genotype were reported to be significantly higher in the
age group 0–24 years compared to 25 years and above
[40]. A similar study showed that these allele and genotype
gradually increased in each quarter and were found to be
highest in subjects born in the last quarter of the twentieth
century [45]. These studies indicate a selective advantage
for T allele in human populations or a lesser disadvantage
of carrying it, which could possibly be because of the
improved nutritional status, especially with folic acid and
vitamin B12 supplementation in course of time.
The haplotype distribution showed the presence of less
common mutant haplotype, 677T-1298C among eight
population groups with a frequency ranging from 0.012 to
0.036 (Table 2). In the total sample, the haplotype fre-
quency was found to be 0.008. The frequency observed in
the present study population groups was comparable to that
of Africa (0.005) [46]. The distribution of the haplotype
showed a large global variation; it was reported to be nil in
Pakistan and Brazil, 0.013 in China, 0.07 in Europe, 0.026
in Mexico, 0.23 in USA and 0.015 in Denmark [46]. A
meta-analysis mainly on European and Western popula-
tions estimated the frequency of this rare haplotype to be
ranging from 0.0023 to 0.0034 [47]. In the present study
haplotype 677C-1298C was more frequent (0.201) than
haplotype 677T-1298A (0.099) which is also indicative of
more deleteriousness of 677T allele compared to that of the
1298C.
The occurrence of MTHFR 677T and 1298C was usu-
ally reported in trans [10, 48, 49] and occasionally in cis
[8, 26] configurations. The identification of cis MTHFR
mutation is significant because it allows more than two
mutant alleles to be present in the foetus which would
result in a selective disadvantage because of the expression
of severe phenotypes, a category that would include
both neural tube defects and spontaneous abortions [10].
This could explain the low frequency of 677TT-1298AC
and 677CT-1298CC genotype combinations (0.94% both
combined) and absence of 677TT-1298CC combination
in the present study. Similar findings have been reported
elsewhere and no sample was observed to have haplotypes
677TT-1298CC, 677TT-1298AC and 677CT-1298CC [50].
This also supports the earlier observations that the presence
of more than two mutant alleles may decrease the viability
of foetus and possibly may be selectively disadvantageous
in a population [10, 27].
In conclusion, the present study demonstrates that the
distributions of both MTHFR 677T and 1298C alleles are
greatly heterogeneous in the Indian population groups. The
caste-tribe difference was found acute in case of the former
allele than the latter allele. This study also highlights the
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serious consequences of having more than two mutant
alleles in an individual. Significant departures from HWE
in several population groups indicated that selective pres-
sures were acting on these alleles. Since both MTHFR
C677T and A1298C are important clinical markers influ-
enced by food habits, further studies are desirable taking
into account the dietary habits of populations for an in-
depth understanding of these SNPs in India. Nonetheless,
this study has provided baseline data for future studies
from multi-ethnic India, where each population group has
its own strict mating pattern, dietary habit and history of
origin. The present study provides baseline data on hap-
lotypes of C677T and A1298C SNPs in people of India and
also suggests that sub-lethal alleles of MTHFR gene may
attain balanced polymorphism in a population depending
on the surrounding cultural and environmental variables.
Therefore, unlike the present population genetics study, the
disease association studies need to be comprehended taking
all the bio-social determinants of the population into
consideration.
Acknowledgments
University Grant Commission (UGC) for Special Assistant Pro-
gramme (SAP) and also the Department of Biotechnology, Govt. of
India for the financial support for the collection of samples. We are
also thankful to Dr. Deepti Shukla, Dr. Harpreet Kaur, Dr. Yaiphaba
Meitei and Dr. Somibabu Meitei for helping in the collection of blood
samples.
We would like to extend our gratitude to the
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