Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive
Biomedicine, ACECR, Tehran, Iran
Department of Andrology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
*Corresponding Address:
P.O. BOX: 14115-154
Department of Genetics
Faculty of Biological Sciences
Tarbiat Modares
University
Tehran
Iran
Email:behmanesh@modares.ac.ir
Any use, distribution, reproduction or abstract of this publication in any medium, with the exception of commercial purposes, is permitted provided the original work is properly cited. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Mollaahmadi Fahimeh,
Moini Ashraf,
Salman Yazdi Reza,
Behmanesh Mehrdad.
The rs1127354 Polymorphism in ITPA Is Associated with
Susceptibility to Infertility.
Cell J.
2018;
20(1): 73-77.
Abstract
Objective
Infertility is a common human disorder which is defined as the failure to conceive for a period of 12 months
without contraception. Many studies have shown that the outcome of fertility could be affected by DNA damage. We
attempted to examine the association of two SNPs (rs1127354 and rs7270101) in ITPA, a gene encoding a key factor
in the repair system, with susceptibility to infertility.
Materials and Methods
This was a case-control study of individuals with established infertility. Blood samples were
obtained from 164 infertile patients and 180 ethnically matched fertile controls. Total genomic DNA were extracted
from whole blood using the standard salting out method, and stored at -20˚C. Genotyping were based on mismatch
polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in which PCR products were
digested with the XmnI restriction enzyme and run on a 12% polyacrylamide gel.
Results
All genotype frequencies in the control group were in Hardy-Weinberg equilibrium. A significant association
(in allelic, recessive and dominant genotypic models) was observed between infertile patients and healthy controls
based on rs1127354 (P=0.0001), however, no significant association was detected for rs7270101. Also, gender
stratification and analysis of different genotype models did not lead to a significant association for this single-
nucleotide polymorphis (SNP).
Conclusion
ITPA is likely to be a genetic determinant for decreased fertility. To the best of our knowledge, this is the
first report demonstrating this association, however, given the small sample size and other limitations, genotyping of
this SNP is recommended to be carried out in different populations with more samples.
Failure to conceive after 12 months of unprotected
regular intercourse is commonly defined as infertility (1).
A variety of factors may be involved in this process of
which genetic factors are perhaps among the best known
(2, 3). Also, a number of studies on humans and animal
models have suggested that inherited factors may be
involved in infertility since the ancestors were said to
have been similarly affected (4).
Despite the technological advancement in diagnostic
methods, the genetic factors of most infertility cases
are not known. Many genetic studies have proposed
that different genes might be responsible for male and
female infertility (5, 6). Genetic abnormalities, including
chromosomal aberrations and single gene mutations
are observed in about 15% of male and 10% of female
infertile subjects (7).
Oxidative stress (OS) is one of the main factors that
may influence fertility due to its role in the modulation
of gamete quality and interaction (6, 8, 9). Oocytes,
spermatozoa and embryos, and their environments are
influenced by free radicals such as reactive oxygen
species (ROS) (6).
Moreover, OS may cause mutations in the DNA
molecule. For example, excessive generation of OS may
lead to DNA damage in spermatozoa (10). OS and other
sources of DNA damage such as reactive nitrogen species
(RNS) can affect cellular nucleotides, however, they can
be repaired by DNA repair mechanisms.
Inosine triphosphatase encoded by ITPA is one of
the genes that serves as a key sanitizing enzyme of
cellular nucleotide pool. The enzyme ITPA catalyzes
the hydrolysis of rough purine nucleotides of inosine
triphosphate (d/ITP) and xanthine triphosphate (d/XTP) to
their monophosphate forms, preventing the accumulation
of deaminated nucleotides in DNA and RNA (11, 12).
Different studies have shown the association of ITPA
deficiency with systemic lupus erythematosus, anemia,
adverse reactions to thiopurine compounds, coronary
artery disease and other diseases (13-15). Since DNA
damage is likely to affect fertility, it is postulated here that
ITPA deficiency may also be associated with infertility.
Sumi et al. (16) showed that patients homozygous for
a 94C>A (Pro32Thr, rs1127354) variant display low or
absent enzyme activity.
Based on crystal structure studies, this variant disturbs
the affinity for nucleotides and therefore reduces the
catalytic activity of ITPA (17). Interestingly, this SNP has
a high frequency in the Asian population (19%) compared
with others (1-7%). We therefore selected this single-
nucleotide polymorphis (SNP) to examine its possible
association with infertility in the Iranian population.
Based on previous studies, other polymorphisms such
as rs7270101 were identified in this gene which affect
ITPAactivity, by causing alternative splicing and reducing
the expression of ITPA. The frequency of these SNPs is
different in various populations and their association with
some diseases have already been shown (18-20). This
paper hypothesized that the ITPA gene deficiency based
on rs7270101 and rs1127354 may be associated with
infertility in Iranian patients.
Materials and Methods
This study was a case-control study of individuals
with established infertility. Based on clinical diagnosis,
164 infertile patients (118 females and 41 males) were
selected who were referred to the Royan Institute
(Infertility Clinic & Reproductive Biomedicine
Center, Tehran, Iran) from July 2013 to October 2014.
Moreover, 180 ethnically matched fertile controls (132
females and 48 males) were randomly selected from
Tehran, Iran. Total genomic DNA was extracted from
500 µl of whole blood using the standard salting out
method and stored at -20°C.
Quality and quantity of extracted DNA was
evaluated by visualization on 1% agarose gel and
spectrophotometry, respectively. The age and sex
ratio of cases and controls are presented in Table 1.
This study adhered to the Declaration of Helsinki and
was approved by Tarbiat Modares University Ethics
Committee. Informed written consents were obtained
from all participating individuals prior to the sampling.
For genotyping of two target SNPs we used from
mismatch polymerase chain reaction-restriction fragment
length polymorphism (PCR-RFLP) strategy. To use this
method specific primers were designed by Oligo analyzer
software (version 7). For genotyping of:
rs7270101-F:
AAATTGACCGTATGTCTCTGGAATGTTT
and for
rs1127345-F:
CAGGTCGTTCAGATTCTAGGAGAAAAGT used as
the specific forward primers and a common reverse primer
of
R: CAAGAAGAGCAAGTGTGGGACAAG used for
PCR amplification used as the primers for PCR amplification.
The mismatched nucleotides in the forwards
primers are presented as underlined. PCR was performed
on 50 ng total DNA in a final volume of 20
µl using 10 µl of PCR Master Mix (Solis BioDyne,
Estonia) and 4 pM of each primer. The PCR cycling
conditions were an initial denaturation at 95°C for 10
minutes, followed by 35 cycles of 95°C for 20 seconds,
60°C for 45 seconds, and 72°C for 45 seconds.
After the amplification, the PCR products were digested
with the XmnI restriction enzyme (New England
BioLabs) according to the manufacturer’s instructions
and were run on a 12% polyacrylamide gel (Figes.1,,
2,). To verify the designed genotyping procedures the
DNA sequences of some randomly selected samples
for each genotype, was determined by an ABI automated
DNA sequencer (Macrogen, Korea).
- A mismatch PCR-RFLP technique used for genotyping of rs7270101
in ITPA gene. The presence of C allele in SNP position can be recognized
by XmnI as a restriction endonuclease enzyme. The length of produced
amplicon was 204 bp and in digestion process produces 175 bp and 25
bp fragments. The genotype of each sample is shown on top of the gel.
ladder is shown by M.
PCR-RFLP; Polymerase chain reaction-restriction fragment length
polymorphism and SNP; Single-nucleotide polymorphis.
Statistical analysis
Genotype frequencies were tested for deviation from
Hardy-Weinberg equilibrium (HWE). Moreover, allele
and genotype (total, dominant and recessive models)
frequencies were compared between the case and
control groups by Chi-Square test. Odd’s ratio (OR)
and its 95% confidence interval (CI) were obtained
to estimate the contribution of the risk factors.
Additionally, a Bonferroni-correction test was carried
out to determine the statistical significance level. A
P<0.025 was considered significant. All statistical
analyses were conducted using the statistical package
for the social sciences (SPSS) Version 20 (SPSS Inc.,
Chicago, IL) and GraphPad Prism 5.
- A mismatch PCR-RFLP technique used for genotyping of rs1127354.
The presence of C allele in SNP position can be recognized by XmnI as a
restriction endonuclease enzyme. Digestion process produces 230 bp and
26 bp fragments. The genotype of each sample is shown on top of the gel.
Ladder is shown by M letter.
PCR-RFLP; Polymerase chain reaction-restriction fragment length
polymorphism and SNP; Single-nucleotide polymorphis.
Results
Genotype frequencies of both SNPs were in HWE in
the control group (P>0.38), however, the genotype
distribution of rs1127354 deviated from HWE in
patients due to an excess of heterozygotes (P<0.05).
Also, a significant difference was found in rs1127354
genotype frequencies between infertile patients and
healthy fertile controls (P=0.0001, OR: 2.56, 95%
CI=1.86-3.53). Also, based on gender stratification,
a significance association was found between this
SNP and susceptibility to infertility in male and
female groups (P=0.02, OR: 1.8, 95% CI=0.97-
3.349 and P=0.0001, OR: 0.343, 95% CI=0.236-0.49,
respectively) (Table 2,).
Different genetic models (dominant=CC+AC/AA
and recessive=AA+AC/CC) also showed a significant
difference between infertile patients and fertile
controls (Table 3,).
Contrary to rs1127354, no significant association was
discovered between rs7270101 and risk of infertility
(P=0.57, OR: 1.73, 95% CI=0.86-2.43). Moreover, the
analysis of genotypes in the dominant (AA+AC/CC)
(P=0.86, OR: 0.9, 95% CI=0.31-2.6) and recessive
(AC+CC/AA) (P=0.57, OR: 1.29, 95% CI=0.61-2.79)
models showed no significant association between
rs7270101 and infertility. This lack of association was
also present at the allelic level (P=0.65, OR: 1.14, 95%
CI=0.62-2.1), and after gender stratification in males
(P=0.36, OR: 3.6, 95% CI=0.4-33.9) and females
(P=0.57, OR: 1.5, 95% CI=0.49-4.8) (Table 4,).
Association analysis of rs7270101 under different models
Genotype or allele
Infertile number (%)
Healthy number (%)
Analyze model
P value
OR (95% CI)
AA
151 (92.1)
162 (90)
Genotype
0.57
1.73 (0.862.43)
AC
6 (3.7)
11 (6.1)
Allele A/C
0.65
1.14 (0.62-2.1)
CC
7 (4.3)
7 (3.9)
Dominant (AA+AC/CC)
0.86
0.9 (0.31-2.6)
A
308 (93.9)
355 (93.06)
Recessive (AC+CC/AA)
0.57
1.29 (0.61-2.79)
C
20 (6.1)
25 (6.94)
Female/Female
0.5
1.5 (0.49 -4.8)
Male/Male
0.36
3.6 (0.43.9)
OR; Odd’s ratio and CI; Confidence interval.
Discussion
This is the first report which demonstrates this
association and therefore should be replicated in other
populations. This study was designed based on evidence
that OS may cause DNA and nucleotide pool damages.
It has been shown that deaminated triphosphate purine
nucleotides of d/ITP and XTP can be repaired in an ITPA-
dependent manner. It is well known that OS may affect
some key properties of sperm and ovum (21), however, no
previous study has examined the role of ITPA in infertility.
We found a significant association between rs1127354
in ITPA and infertility under different analysis models.
Although the important role of ITPA in the genome repair
and sanitization of nucleotide pool has been confirmed
by different studies, the association of this functional
SNP with infertility may shed further light into the
molecular mechanism of infertility. Behmanesh et al. (22)
demonstrated that Itpa knockout mice (Itpa −/−) die about
two weeks after birth with features of growth retardation
and cardiac myofiber disarray. In addition, homozygous
patients for the 94C>A (Pro32Thr, rs1127354) variant
display low or absent enzyme activity (16).
Interestingly, this polymorphism is more common
among Asian populations (11-19%) than other ethnic
groups such as Africans and Caucasians (1-7%) (13). All
these observations suggest that ITPA dysfunction may
affect the outcome of fertility, which must be considered
for further analyses in future molecular studies. The
effects of this SNP on ITPA activity has been investigated
in mercaptopurine metabolism (23), and ribavirin-induced
anemia and outcome of therapy in HCV patients (24).
Thompson et al. (20) reported that ITPA polymorphisms
reduce the amount of hemoglobin during treatment
with pegylated interferon. The association SNPs and
the expression level of ITPA has also been assessed in
different pathological situations (25, 26).
Low sample size was the main limitation of this study
which must be considered in future studies. Interestingly,
we found that the case group in this study was not in Hardy-
Weinberg equilibrium due to an excess of heterozygotes.
This may arise due to a strong association between an allele
and disease state, undetected population stratification,
genetic mistyping or inadequate sample size. However,
given that we observed no deviation in the matched
control group, it is most likely due to disease state. Recent
studies are concentrated on finding the molecular basis of
human disorders and in this way they investigate the role
of different molecular aspects of gene regulation. Based on
experimental data, certain SNPs in the genome may affect
the expression level of genes and therefore are important
in their regulation. Non-coding SNPs may increase the
susceptibility of disease development by affecting the
expression of nearby genes (27).
The intron 2 SNP rs7270101 is located downstream of
the 5’-splice donor site and upstream of the splice acceptor
polypyrimidine tract. Anumber of putative consensus branch-
site sequences are present in this small 92 bp intron (16) with
rs7270101 changing an adenosine nucleotide in one of these
sequences, thus possibly resulting in altered expression of
ITPA. While previous studies examined the role of the DNA
repair system in gametogenesis (22), this paper analyzed
the association of rs7270101 SNP in the ITPA gene with the
susceptibility to infertility in the Iranian population.
We observed no association at all levels, however,
since no previous study is available on this association,
no comparisons were possible. Moreover, all cases and
controls were not in HWE for rs7270101, even though
controls were randomly selected from ethnically matched
people. Undetected population stratification, genotyping
errors or inadequate sample size are the main factors for
Hardy-Weinberg disequilibrium. In order to check the
accuracy of the obtained results, a number of genotyped
samples were randomly selected for sequencing and ALL
genotypes were confirmed by this method. One possible
source of this disequilibrium may be due to the presence
of a degree of selective pressure on this SNP, which has
been previously observed for immunologically-related
SNPs (28, 29). Due to the importance of this functional
SNP and the main role of ITPA in the DNA repair system,
it is recommended that this association is assessed in other
populations with larger sample sizes.
Conclusion
We demonstrate that rs1127354 is associated with
infertility under different genetic models and also after
gender stratification. Our data is still preliminary and
additional studies may help define the actual role of
ITPA in infertility. Nevertheless, we did not observe this
association for the other SNP, rs7270101 with infertility.
Acknowledgements
The authors gratefully acknowledge the contribution
of the Royan institute and thank the patients and healthy
controls for their blood donations. The Iran National
Science Foundation and the Department of Research
Affairs of Tarbiat Modares University provided the
funding for this project. The authors declare that they
have no conflict of interest.
Author’s Contributions
Author’s Contributions
F.M.; Participated in study design, data collection
and evaluation and drafting. A.M.; Participated in study
design and sample collection. R.S.Y.; Performed sample
collection. M.B.; Participated in study design, data
collection and evaluation and responsible for overall
supervision. All authors read and approved the final manuscript.
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