Open Journal of Genetics, 2013, 3, 59-66 OJGen
http://dx.doi.org/10.4236/ojgen.2013.32A3009 Published Online August 2013 (http://www.scirp.org/journal/ojgen/)
The I550V polymorphism in the renal human
sodium/dicarboxylate cotransporter 1 (hNaDC-1) gene is
associated with the risk for urolithiasis in adults from
Southeastern, Mexico
Martha Medina-Escobedo1*, Diana Franco-Bocanegra1, Salha Villanueva-Jorge1,
Lizbeth González-Herrera2
1Departamento de Investigación en Enfermedades Renales, Hospital General O’Horán, Secretaria de Salud de Yucatán, Mérida,
México
2Laboratorio de Genética, Centro de Investigaciones Regionales, Universidad Autónoma de Yucatán, Mérida, México
Received 10 July 2013; revised 31 July 2013; accepted 5 August 2013
Copyright © 2013 Martha Medina-Escobedo et al. This is an open access article distributed under the Creative Commons Attribu-
tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
ABSTRACT
Urolithiasis (UL) is an endemic disease in Southeast-
ern, Mexico. In order to evaluate the association of
I550V polymorphism in the hNaDC-1 gene with risk
for hypocitraturia and/or for UL; 139 adults with UL
and 132 adults without UL, were included under a
case-control association study. Citrate levels in 24-h
urine were quantified (citraturia). The polymorphism
I550V-hNaDC-1 was determined by PCR-RFLP. Sta-
tistical analysis was performed using the STATA10.2
software. Comparison of genotype a nd allele frequen-
cies between subjects with and without UL showed
significant differences for genotype bb (OR = 2.34, CI:
1.19 - 4.59, p = 0.01) and for allele b (OR = 1.62, CI:
1.15 - 2.28, p = 0.005), suggesting an association with
the risk for UL. Comparison of genotype and allele
frequencies between subjects with hypocitraturia and
subjects with normocitraturia, did not show any sig-
nificant difference (p > 0.05), suggesting that this
polymorphism is not associated with the risk of hypo-
citraturia. Interestingly, the risk for UL was in-
creased due to an additive effect of hypocitraturia
with the genotype bb (OR = 6.6, CI: 2.38 - 18.28, p =
0.0002) or with the allele b (OR: 4.2, CI = 2.52 - 6.97,
p < 0.0001) in the studied population.
Keywords: Hypocitraturia; hNaDC-1; Urolithiasis;
Mexico; Yucatan
1. INTRODUCTION
The population of Yucatan, Mexico, is characterized by a
high prevalence of urolithiasis (UL) [1]. Hypocitraturia,
which is considered as a urine citrate excretion below
320 mg in 24 hours [2], represents the most common me-
tabolic abnormality in adults with UL [3]. Hypocitraturia
is known to be a major risk factor for the development of
lithiasis [4-7], because of citrate acts as a calcium che-
lating agent, which reduces the formation of oxalate and
calcium phosphate, thus inhibiting the generation of uri-
nary calculus [8].
In nephrons, urine citrate is reabsorbed from the glo-
merular filtrate in the apical membrane of kidney proxi-
mal tubule cells by means of the transmembrane protein
known as human sodium/dicarboxylate cotransporter 1
(NaDC-1), through a sodium-dependent mechanism [9].
In these cells, NaDC-1 is responsible for the reabsorption
of other di- and tri-carboxylic acids most of which are
citric acid cycle intermediates, such as fumarate, succi-
nate and α-ketoglutarate, which are compounds involved
in numerous important physiological processes in the
body [10].
NaDC-1 is a protein with 593 amino acids, which con-
tains 11 transmembrane domains. The gene hNaDC-1
belongs to the family of genes encoding the SLC13 sol-
ute carriers [11] and; it is located on locus 17p13.2. The
coding portion of the hNaDC-1 gene contains 1953 base
pairs in 12 exons [9]. Certain single nucleotide poly-
morphisms (SNPs) in the hNaDC-1 gene, have been de-
scribed to produce differences in the rate of gene expres-
*Corresponding author.

M. Medina-Escobedo et al. / Open Journal of Genetics 3 (2013) 59-66 60
sion as well as, significant variation in the affinity to so-
dium and to various substrates in NaDC-1 protein; such
conditions would alter the rate of tubular reabsorption of
these compounds [12].
One of these SNPs, I550V, consists of a substitution of
adenine by guanine at position 1716, in exon 12, which
produces a change in amino acid 550 of NaDC-1 protein
from isoleucine to valine. This SNP has been associated
with the risk of hypocitraturia in recurrent renal stone
formers [13]. However, those results have not yet been
reproduced in any other population. Also, such study
found no direct association between the I550V SNP and
UL [13], nor the influence of the SNP on either the rate
of gene expression or the NaD C-1 transport activity have
been proven through in vitro studies [12]. Based on the
above and in order to provide evidence to help determine
the role performed by this SNP in the genetic risk of UL,
this study focuses on evaluating the association of I550V
SNP in hNaDC-1 gene with the genetic risk for develop
hypocitraturia and/or urolithiasis in a population of adult
subjects from Southeastern Mexico, where UL is highly
prevalent and considered endemic.
2. MATERIALS AND METHODS
2.1. Population
We performed a case-control association study. The sam-
ple consisted of 271 subjects, 95 men and 176 women,
above 17 years old. The case group included 139 subjects
with urolithiasis and 132 subjects without urolithiasis
were used as controls. UL subjects were 51 (36.7%) men
and 88 (63.3%) women, with mean age of 34 ± 10.5
years; whereas control subjects were 44 (33.3%) men
and 88 (66.7%) women, with mean age of 36.2 ± 10.6
years. The presence or absence of UL in each subject was
confirmed by ultrasound or X-ray. Subjects with urinary
tract obstruction diseases, diabetes mellitus, hypertension,
asthma, bronchitis, kidney failure, metabolic acidosis and
those with prolonged medical therapy for any disease;
were excluded. Likewise, the control group did not in-
clude subjects with a history of chronic disease or of diu-
retic drug intake. It should be noted that during the study
the subjects continued their usual diet without modifica-
tions. All selected subjects were born and had lived for at
least three generations in Yucatan, Mexico. We also used
anthropologic and demographic parameters such as lan-
guage, birth place, surnames, genealogy, and history of
lifestyle, among others, to match ethnically control with
case subjects belonging to the same ethnic group of Mes-
tizos, defined as individuals born in the country having a
Spanish-derived last name, and with a family history of
Mexican ancestors back at least to the third generation.
In addition, we previously determined the absence of
substructure or population stratification within the popu-
lation of Yucatan by using 16 autosomal STR markers
[14], which may represent a confounder.
Informed consent was obtained from cases and con-
trols according to the recommendations of the Helsinki
Declaration. This study was approved by the Bioethics
Committee of Hospital General Agustin O’Horan de Me-
rida, Yucatan, México.
2.2. Determination of Urine Citrate Levels
(Citraturia)
To determine citrate excretion, a 24-h urine sample was
collected. The concentration of citrate in urine was deter-
mined using a commercial citrate determination kit (Boe-
hringer-Mannheim Cat # 10139076035) and citraturia
was expressed in terms of mg/day measured in 24-hour
urine samples. Additionally, urea and creatinine serum
levels were measured to verify proper kidney function
[15,16].
2.3. Genotyping
Leukocyte DNA extraction with anticoagulant (EDTA)
from peripheral blood was carried out by a conventional
phenol-chloroform method [17]. The polymerase chain
reaction technique (PCR) was used to amplify a 520 bp
fragment of exon 12 at the hNaDC1 gene, wherein the
I550V SNP is located. Each 25 μl PCR reaction con-
tained 18.3 μl of sterile H2O, 2.5 μl of 5X Green reaction
buffer (PromegaCat#M5005), 1.5 mM MgCl2, 0.4 μM
dNTPs, 0.3 μM of each primer; and 0.65u of Taq poly-
merase enzyme (Promega). The sequences of the primers
used were 5’-ACGGGAGGACTTCCCAGAGA-3’ for
the forward primer and 5’-CAGGCGCACACATATC-
GCA-3’ for the reverse primer, previously described by
Okamoto et al. 2007 [13]. The prepared samples were
placed in an Applied Biosystems Veriti thermocycler
with the following PCR conditions: initial denaturation
for 1 min at 94˚C, 30 cycles of denaturation for 1 min at
94˚C, annealing for 1 min at 59˚C and extension for 1
min at 72˚C and finally 10 min final extension at 72˚C,
followed by cooling for 5 min at 4˚C. The amplified
DNA was incubated at 50˚C overnight in the presence of
the restriction endonuclease Bcl I (Bacillus caldolyticus I)
[13]. Each restriction reaction contained 10 μl of ampli-
fied DNA, 7.62 μl of sterile H2O, 2 μl of buffer C, 0.2
BSA and 1.8u of Bcl I restriction enzyme Promega Cat #
R4654). The Bcl I enzyme cuts only those fragments
which have adenine (B allele) instead of guanine (b al-
lele). When cut, the 520 bp fragment produces two frag-
ments, one with 354 bp and one with 166 bp. After in-
cubation, the samples were again subject to polyacryla-
mide gel electrophoresis, from which genotypes were
identified, according to Okamoto et al. [13].

M. Medina-Escobedo et al. / Open Journal of Genetics 3 (2013) 59-66 61
2.4. Statistical Analysis
Mean levels of citrate excretion between cases and con-
trols, as well as among genotypes were compared with
Student t test. A value of p < 0.05 was considered sig-
nificant. Hardy-Weinberg equilibrium deviations were
determined in the studied population by means of Chi
square test. Genotype and allele frequencies of I550V
SNP were also evaluated and compared between cases
and controls in order to determine significant associa-
tions. To determine whether the studied genotypes or al-
leles conferred a significant increase to the risk of hypo-
citraturia and/or UL, we calculated the odds ratio (OR)
with a confidence interval (CI) of 95%, using the Woolf
approximation. Significance was considered when p <
0.05. All statistical tests were performed using the
STATA version 10.2 software.
3. RESULTS
Genotype and allele frequencies of the I550V polymer-
phism in hNaDC gene in 139 subjects with urolithiasis
and 132 subjects without urolithiasis are shown in Table
1. Genotype frequencies for the I550V polymorphism in
hNaDC-1 gene were distributed according to Hardy-
Weinberg equilibrium expectations (p > 0.05). The three
I550V-hNaDC-1 SNP genotypes were observed in the
studied population, being the heterozygous genotype Bb
the most frequent in control subjects, whereas the ho-
mozygous bb genotype was the most frequent in subjects
with UL. The frequency of the allele b in the control
population was 47.73%. Comparison of genotype and
allele frequencies between subjects with and without UL,
showed significant differences for the homozygous
genotype bb (p = 0.017); and for the allele b (p = 0.005);
suggesting that the I550V polymorphism in hNaDC gene
is associated with the genetic risk for UL in the studied
population.
Mean levels of citrate excretion obtained in subjects
with UL were 309 ± 223 mg/24h, which were signifi-
cantly lower than mean levels of citrate excretion in con-
trol subjects of 408 ± 187 mg/24h (p = 0.0001). This
finding remarked the hipocitraturic condition en cases of
UL. Moreover, hypocitraturia (<320 mg/24h) was sig-
nificantly more frequent in cases of UL (61.9%, N = 86),
than in control subjects (38.1%, N = 53), suggesting that
hypocitraturia might be an associated risk factor for UL
in the studied population (OR = 2.5, CI: 1.49 - 4.20, p <
0.001).
According to the levels of citrate excretion, studied
subjects were stratified in hypocitraturia and normocit-
raturia in order to evaluate its association with the I550V
polymorphism in hNaDC-1 gene, independently of the
UL. We identified an overall of 138 subjects with hypo-
citraturia and 132 subjects with normocitraturia in our
population. Genotype and allele frequencies of the I550V
polymorphism in hNaDC-1 gene in subjects with hypo-
citraturia and subjects with normocitraturia, as well as
the comparison of frequencies on an association analysis
are listed in Table 2. Genotype frequencies for the I550V
polymorphism in hNaDC-1 gene was distributed accord-
ing to Hardy-Weinberg equilibrium expectations (p >
0.05). The heterozygous genotype Bb was the most fre-
quent in both subjects with hypocitraturia (50.72%) and
normocitraturia (40.60%). The comparison of genotype
and allele frequencies of I550V polymorphism in hNaDC-
1 gene between subjects with hypocitraturia and subjects
with a normal level of citrate excretion (normocitraturia)
did not show significant difference for any genotype or
allele (p > 0.05), suggesting that the I550V polymer-
phism in hNaDC-1 gene is not associated with the risk
for low levels of citrate excretion or with the risk for hy-
pocitraturia within the studied population.
Mean levels of citrate excretion according to genotype
for I550V-NaDC-1 polymorphism, are shown in Table 3.
Mean levels of citrate excretion for each genotype were
compared within genotypes, showing no significant dif-
Table 1. Genotype and allele frequencies of the I550V polymorphism in hNaDC-1 gene in subjects with urolithiasis (UL) and
subjects without lithiasis, Hardy-Weinberg expectations; and association analysis (OR, IC, p), N (%).
Cases vs. control
Genotypes and Alleles of the
SNP I550V in hNaDC-1 gene Subjects with urolithiasisSubjects without urolithiasis
OR 95% CI p
N = 139 N = 132
2N = 278 2N = 264
BB 29 (20.86) 34 (25.76) Reference
Bb 54 (38.85) 70 (53.03) 0.90 0.49 - 1.66 0.757
bb 56 (40.29) 28 (21.21) 2.34 1.19 - 4.59 0.017**
Allele B 112 (40.29) 138 (52.27) Reference
Allele b 166 (59.71) 126 (47.73) 1.62 1.15 - 2.28 0.005**
Hardy-Weinberg (p) (0.291) (0.905)
**significant.

M. Medina-Escobedo et al. / Open Journal of Genetics 3 (2013) 59-66 62
Table 2. Genotype and allele frequencies of the I550V polymorphism in hNaDC-1 gene in subjects with hypocitraturia and subjects
with normocitraturia, Hardy-Weinberg expectations; and association analysis (OR, IC, p), N (%).
Cases vs. control
Genotypes and Alleles of the
SNP I550V in hNaDC-1 gene Subjects with hipocitraturiaSubjects with normocitraturia
OR 95% CI p
N = 138 N = 133
2N = 276 2N = 266
BB 28 (20.29) 35 (26.32) Reference
Bb 70 (50.72) 54 (40.60) 1.62 0.87 - 2.98 0.125
bb 40 (28.99) 44 (33.08) 1.13 0.58 - 2.18 0.740
Allele B 126 (45.65) 124 (46.62) Reference
Allele b 150 (54.35) 142 (53.38) 1.03 0.74 - 1.45 0.444
Hardy-Weinberg (p) (0.998) (0.328)
Ta b l e 3 . Comparison of mean citrate excretion (mg/24hr) according genotypes for the SNP I550V-hNaDC-1 in the studied overall
population.
Significance (p) versus:
Genotype Mean of citrate excretion
(mg/24hrs.) Standard deviation
BB Bb bb
BB N = 63 374.71 216.22 --- 0.296 0.912
Bb N = 124 340.09 207.80 0.296 ---- 0.307
bb N = 84 370.77 214.65 0.912 0.307 ----
ferences (p > 0.05), which leads towards the point that
citrate excretion itself is not associated with any allele or
genotype for the I550V polymorphism in gene.
Case and control subjects were also compared inde-
pendently between two groups according hypocitraturia
or normocitraturia; and within these groups into the ge-
notype and allele frequencies for the I550V polymer-
phism in the hNaDC-1 gene (intra-group analysis). Sig-
nificant differences were found in the group with normo-
citraturia for both the bb genotype (OR = 2.52, CI: 1.00 -
6.31, p = 0.038), and b allele (OR = 1.82, CI: 1.10 - 3.00,
p = 0.023), whereas subjects with hypocitraturia did not
show any significant difference for any genotype or al-
lele (Table 4). This finding supports that the I550V poly-
morphism in the hNaDC-1 gene might be associated with
UL itself, rather than hypocitraturia.
Given that the bb genotype and hypocitraturia were in-
dependently found to be risk factors associated with the
presence of UL; the additive effect of these factors was
assessed, in order to determine if the risk is increased
when both factors occur concurrently. The genotype fre-
quencies of the SNP I550V-hNaDC-1 of subjects with
UL and hypocitraturia were compared with frequencies
for the reference conditions of control subjects with nor-
mocitraturia and BB or Bb genotype (Table 5). Signifi-
cant associations were obtained for these comparisons (p
< 0.05); except for subjects with UL, hypocitraturia and
genotype Bb (p = 0.06). The highest risk was associated
with subjects with UL, hypocitraturia and bb genotype,
OR = 6.6, CI: 2.38 - 18.28, p = 0.0002, when compare
with homozygous BB; and OR = 8.18, CI: 3.18 - 21.03, p
= 0.000; when compare with heterozygous Bb. These
findings suggest that the presence of bb genotype added
to the presence of hypocitraturia, noticeably increases the
risk for UL.
Comparison of mean urine citrate levels (mg/24h.) stra-
tified according to genotypes for the SNP I550V-hNaDC-
1; showed significant differences between subjects with
UL and control subjects for the heterozygous genotype
Bb (0.002) and for the homozygous genotype bb (0.004)
(Table 6); suggesting that these genotypes might be im-
plicated in the lower urine citrate excretion in presence
of urolithiasis.
4. DISCUSSION
The results of this study confirmed that hypocitraturia
was present with a significantly higher frequency in sub-
jects with UL (61.9%) than in controls (38.1%), repre-
senting an identifiable risk factor for UL in the study
population (p < 0.05). This observation is consistent with
results of previous studies which evaluated the same as-
sociation in other populations [4-7] and; with the results
obtained in a previous study conducted in the Yucatan
population, in which hypocitraturia was found in 63.6%
of patients with UL [3]. Given that we did not find any
association between I550V-hNaDC-1 genotype and hy-
pocitraturia itself, the genetic contribution of this SNP
towards hypocitraturia was not demonstrated in this
Mexican population; so the possible genetic basis should

M. Medina-Escobedo et al. / Open Journal of Genetics 3 (2013) 59-66 63
Table 4. Genotype and allele frequencies of the I550V polymorphism in hNaDC-1 gene in subjects with urolithiasis and subjects
without urolithiasis, according to hipocitraria or normocitraturia under an intragroup analysis; (OR, IC, p), N (%).
Cases vs. control
Genotypes and Alleles of the
SNP I550V in hNaDC-1 gene Subjects with urolithiasisSubjects without urolithiasis
OR 95% CI p
Normocitraturia N = 53 N = 80
BB 12 (22.64) 23 (28.75) Reference
Bb 16 (30.19) 38 (47.50) 0.80 0.34 - 2.00 0.648
bb 25 (47.17) 19 (23.75) 2.52 1.00 - 6.31 0.038**
Allele B 40 (37.74) 84 (52.50) Reference
Allele b 66 (62.26) 76 (47.50) 1.82 1.10 - 3.00 0.023**
Hipocitraturia N = 86 N = 52
BB 17 (19.77) 11 (21.15) Reference
Bb 38 (44.19) 32 (61.54) 0.76 0.31 - 1.87 0.654
bb 31 (36.04) 9 (17.31) 2.22 0.77 - 6.44 0.178
Allele B 72 (41.86) 54 (51.92) Reference
Allele b 100 (58.14) 50 (48.08) 1.50 0.91 - 2.44 0.107
**significant.
Table 5. Genotype frequencies of the I550V polymorphism in hNaDC-1 gene in subjects with urolithiasis and hipocitraturia versus
the reference condition of subjects without urolithiasis, normocitraturia and BB or Bb (across group analysis; OR, IC, p), N (%).
Cases vs. control
Genotypes and Alleles of the SNP
I550V in hNaDC-1 gene Subjects with urolithiasisSubjects without urolithiasis
OR 95% CI p
Normocitraturia and homozygous
BB 2 (22.64) 23 (28.75) Reference
versus
Hipocitraturia and
BB 17 (19.77) 11 (21.15) 2.96 1.05 - 8.30 0.045**
Bb 38 (44.19) 32 (61.54) 2.27 0.98 - 5.28 0.063
bb 31 (36.04) 9 (17.31) 6.06 2.38 - 18.28 0.0002**
Normocitraturia and Heterozygous
Bb 16 (30.19) 38 (47.50) Reference
versus
Hipocitraturia and
BB 17 (19.77) 11 (21.15) 3.67 1.40 - 9.56 0.009**
Bb 38 (44.19) 32 (61.54) 2.82 1.33 - 5.96 0.006**
bb 31 (36.04) 9 (17.31) 8.18 3.18 - 21.03 0.000**
**significant.
be in relation to different implicated genes or different
SNPs. However, we were able to demonstrate that the
polymorphism I550V-hNaDC-1 might contribute to
the genetic susceptibility to develop urolithiasis; show-
ing a higher risk for subjects with hypocitraturia and the
bb genotype. Implication of the SNP I550V-hNaDC-1 in
the mechanism of stone formation might be due to the
isoleucine-to-valine alteration at position 550 in hNaDC-
1 is located in the last trans membrane domain and the
C-terminal half, which contains amino acid residues that
determine substrate recognition and cation affinity; there-
fore, altering the function for transferring citrate. It is
also possible that the rate of transcription or stability, of
hNaDC-1 is modified by the SNP [13].
It is worth to note that the intestinal absorption of cit-
rate, is the main source of plasma citrate [18]. The ab-
sorbed citrate is utilized mainly in the liver, and also ful-
fills the function of promoting the sodium and water ab-
sorption in the jejunum [19]. In the intestine, the absorp-
tion of citrate is carried out by the same transporter,
NaDC-1 as in the renal proximal tubules. The presence
of this protein in the intestine was documented by

M. Medina-Escobedo et al. / Open Journal of Genetics 3 (2013) 59-66 64
Table 6. Comparison of mean citrate excretion (mg/24h.) ac-
cording to genotypes for the SNP I550V-hNaDC-1, between
cases and control.
Genotype
Subjects with
Urolithiasis
N = 139
Subjects without
Urolithiasis
N = 132
Significance p
BB 339.89 ± 216.22
N = 29
395.75 ± 164.11
N = 34 0.259
Bb 277.54 ± 222.19
N = 54
391.63 ± 182.82
N = 70 0.002**
bb 324.87 ± 202.11
N = 56
470.62 ± 212.53
N = 28 0.004**
**significant.
Northern blot analysis, in which, mRNA of NaDC-1 was
detected in intestinal tissue [9]. The fact that the same
protein regulates citrate absorption in both the intestine
as in the renal proximal tubules is interesting in the sense
that one SNP affecting the recognition of the NaDC-1
substrate would result in a poor citrate absorption at the
intestine level, which could be reflected with the onset of
hypocitraturia [20]. Poor intestinal citrate absorption
should not be dismissed as a possible cause for the high
frequency of hypocitraturia in the Yucatan population.
Compared with other UL associated SNPs, I550V SNP
has been scarcely studied. The only study regarding this
SNP, analyzed in a human population before the present
work was carried on, was in Japan [13]. The results ob-
tained in the present study markedly contrast those re-
ported in the Japan study [13], in which, when comparing
the genotype and allele frequencies of this SNP between
subjects with hypocitraturia and subjects with normocit-
raturia, significant differences were revealed for both
genotype (p = 0.0007), and allele (p = 0.00005), exhibit-
ing a higher frequency of the BB genotype and B allele
in subjects with hypocitraturia, pointing to these factors
as associated to the presence of hypocitraturia. Those
data contrast with findings in our study in which no sig-
nificant differences neither in genotype nor in allele fre-
quencies between subjects with hypocitraturia and nor-
mocitraturia, were shown.
Okamoto et al., 2007 [13] explain the results suggest-
ing that the b allele of the I550V SNP causes a change in
the structure of the transporter, which would result in a
decrease of its affinity to the substrate. With this, the rate
of reabsorption of the filtered citrate would be reduced
and, therefore, citrate excretion increased, whereby the b
allele would be conferring a protection factor against hy-
pocitraturia, and hence against UL. Nevertheless, these
results have not been reproduced in any other population
in the world, and the reported association between geno-
type I550V and citraturia has not been confirmed through
in vitro studies. A site-directed mutagenesis technique
study in COS-7 cells has reported no significant differ-
ences in the rate of gene expression or in the transport
activity of NaDC-1 among the different genotypes of
I550V SNP [12].
Lack of association between I550V SNP and citraturia
obtained in this study, might be explained because of the
NaDC-1 is capable of carrying other di- and tri-carbox-
ylic acids, by having greater affinity with these than with
citrate, as are fumarate and succinate as well as methyl-,
dimethyl succinate, which can be present in the luminal
liquid [21]. It has been reported from in vitro studies that
fumarate and succinate significantly inhibit citrate uptake
by NaDC-1 in brush border membrane vesicles through a
competitive inhibition mechanism [19]. Likewise, it is
known that sulfate may inhibit citrate transport; this in-
hibition, unlike that of succinate and fumarate is non-
competitive, since sulfate hinders the binding of other
substrates to the transporter, but without being carried in
to the tubular cell interior [22]. It is possible that the
transporter saturation by these luminal components causes
an inhibition of citrate transport, which may explain the
observed lack of association among I550V genotype and
citraturia in this study. Studies of the urinary profile, tak-
ing into account the determination of the concentration
of succinate and fumarate in plasma and urine, are needed
to shed some light on this point.
Another explanation is that other SNPs, either by them-
selves, or through an interaction with I550V SNP; might
have effects on the amount of transporters existing in the
membrane, which could influence substrate concentra-
tion at which the transporter reaches saturation.
In the Mexican population analyzed for this study, sig-
nificant differences were found with respect to I550V-
hNaDC-1 genotype and allele frequencies between cases
and controls, suggesting an association between the b
allele and the presence of UL, opposite to what was ob-
tained in the Japanese population studied by Okamoto et
al., 2007, [13] who did not evidence such association.
Given that in the present study, we did not find any asso-
ciation of the bb genotype with citraturia, whereas a pos-
sitive association was found with the presence of UL, the
outcome suggests the existence of another mechanism,
unrelated to urinary citrate excretion, whereby the trans-
porter is involved in the lithogenic process.
As mentioned above, in addition to I550V, other poly-
morphisms that have implications on the activity and
functioning of hNaDC-1 have been identified [12]. It is
possible that some alleles of other polymorphisms are
associated with the b allele in I550V SNP through link-
age disequilibrium [23] and; that this would explain the
association found between b allele, bb genotype and UL.
The association among these polymorphisms and the pre-
sence of UL has not been evaluated whatsoever.
The complete lack of association between genotype
and citraturia was confirmed once again by dividing the

M. Medina-Escobedo et al. / Open Journal of Genetics 3 (2013) 59-66 65
study population into subjects with hypocitraturia and
normocitraturia and comparing the genotype and allele
frequencies between cases and controls, within each
group. In this case, as mentioned before, frequency dif-
ferences were obtained, pointing at the bb genotype and
the b allele as risk factors for UL, only in the group of
subjects with normocitraturia. The above confirms that
the citraturia is not associated with genotype in this po-
pulation, since the association between genotype and UL
was present in subjects with normal levels of urinary
citrate.
The fact that the same association was not found in the
group of subjects with hypocitraturia may be due to cit-
raturia itself representing a heavier risk factor than the
genotype (which is consistent with the higher value of
OR obtained for hypocitraturia than that for genotype),
which implies that subjects with hypocitraturia develop
UL more often, even in the absence of the risk genotype.
Another important aspect to consider, especially when
dealing with multicausal origin conditions is the fact that
gene expression is a mechanism capable of changing ac-
cording to outer environmental stimuli, performing what
is known as gene-environment interactions. UL is known
to be a multifactorial pathology resulting from the inter-
action between environmental influences and genetic as
well as hormonal factors [24,25]. The differences be-
tween the results obtained in the Japanese population [13]
and; those reported in this paper can also owe to the
many variations that exist in the environmental condi-
tions to which the subjects in both populations are ex-
posed, same which, through repression or induction me-
chanisms could produce differences in hNaDC-1 gene ex-
pression, conversely, the combination of a certain geno-
type with diverse environmental variables could produce
a difference in the effect of this genotype on various
phenotypic features
With regard to the increased risk for UL due to the in-
teraction of both risk factors observed in this study: hy-
pocitraturia and the bb genotype or b allele, might be
explained because UL is a disorder with a multifactorial
etiology, the presence of only one of the risk factors is
not sufficient to develop the disease, however, these risk
factors show additive effect [26], in this study we showed
that the presence of two risk factors significantly in-
creases the risk of acquiring the disease compared with
the presence of each of these factors independently.
In order to find the answer to complete the new ques-
tions arisen from this study, the dynamics of di- and tri-
carboxylic acid transport must be analyzed further; as
well as the determination of plasma citrate; and other
intermediates of the citric acid cycle in urine as part of
the UL patient’s metabolic profile.
In conclusion, our study demonstrated that the poly-
morphism I550V in the hNaDC-1 gene is associated with
the risk for urolithiasis. Moreover, the risk for UL might
increase due to an additive effect of hypocitraturia with
the genotype bb or with the allele b in the population of
Southeastern, Mexico.
5. ACKNOWLEDGEMENTS
We thank Fundación Mexicana para la Salud Capítulo Peninsular A.C.
and CONACYT Sectoral Funds, Registration No.2008-1-86740 for the
necessary funding to carry out the present work.
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