- Email: [email protected]
Polymorphisms in the TFAM and PGC1-α genes and their association with polycystic ovary syndrome among South Indian women
Accepted Manuscript Polymorphisms in the TFAM and PGC1-α genes and their association with polycystic ovary syndrome among South Indian women
Tumu Venkat Reddy, Suresh Govatati, Mamata Deenadayal, Sisinthy Shivaji, Manjula Bhanoori PII: DOI: Reference:
S0378-1119(17)30823-5 doi:10.1016/j.gene.2017.10.010 GENE 42229
To appear in:
Gene
Received date: Revised date: Accepted date:
12 July 2017 13 September 2017 6 October 2017
Please cite this article as: Tumu Venkat Reddy, Suresh Govatati, Mamata Deenadayal, Sisinthy Shivaji, Manjula Bhanoori , Polymorphisms in the TFAM and PGC1-α genes and their association with polycystic ovary syndrome among South Indian women. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2017.10.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: Polymorphisms in the TFAM and PGC1-α genes and their association with polycystic ovary syndrome among South Indian women Names of Authors: Tumu Venkat Reddya, Suresh Govatatia, Mamata Deenadayalb, Sisinthy
PT
Shivajic,d and Manjula Bhanooria,*
c
Infertility Institute and Research Centre (IIRC), Secundrabad, INDIA
NU
b
SC
Department of Biochemistry, Osmania University, Hyderabad, INDIA
Centre for Cellular and Molecular Biology (CCMB), Hyderabad, INDIA
d
MA
a
RI
Institutional Affiliations:
Presently at: Prof Brien Holden Eye Research Centre, L V Prasad Eye
PT E
D
Institute, Hyderabad, INDIA
CE
*Address for Correspondence:
AC
Dr. Manjula Bhanoori, Assistant Professor, Department of Biochemistry, Osmania University, Hyderabad – 500 007, INDIA E-mail: [email protected] Tel: 00-91-9989661469 Fax: 00-91-40-27097044
ACCEPTED MANUSCRIPT Full postal address and contact information of all authors 1. Mr. Tumu Venkat Reddy, C/O Dr. Manjula Bhanoori, Department of Biochemistry, Osmania University, Hyderabad – 500 007, INDIA E-mail: [email protected]
PT
2. Dr. Suresh Govatati, C/O Dr. Manjula Bhanoori, Department of Biochemistry,
RI
Osmania University, Hyderabad – 500 007, INDIA
SC
E-mail: [email protected] 3. Dr. Mamata Deenadayal,
MA
E-mail: [email protected]
NU
Infertility Institute and Research Centre (IIRC), Secundrabad, INDIA
4. Dr. Shivaji Sisinthy,
D
Centre for Cellular and Molecular Biology (CCMB), Hyderabad – 500 007, INDIA
PT E
E-mail: [email protected] 5. Dr. Manjula Bhanoori,
CE
Assistant Professor, Department of Biochemistry, Osmania University, Hyderabad – 500 007, INDIA
AC
E-mail: [email protected] Tel: 00-91-9989661469; Fax: 00-91-40-27097044
ACCEPTED MANUSCRIPT 1. Introduction Mitochondria are cytoplasmic organelles, whose prime function is to generate energy through oxidative phosphorylation, and execution of apoptosis (Attardi and Schatz, 1988; Lee HC et al., 2005; Wallace, 2008). Mitochondria possess their own genome (mitochondrial DNA, mtDNA), a 16,569 bp circular double-stranded molecule. The mitochondrial genome is
PT
maintained within nucleoprotein complexes known as nucleoids. Mitochondrial transcription
RI
factor A (TFAM) is the predominant architectural mtDNA packaging protein of the nucleoid,
SC
where it is involved in both transcription and replication of mtDNA. It is a nuclear encoded protein that promotes the expression of the mitochondrial encoded 13 proteins, that are
NU
essential for oxidative phosphorylation (OXPHOS), 2 rRNAs and 22 tRNAs, which are essential for protein translation on mitochondrial ribosomes (Ekstrand et al., 2004). TFAM is
MA
a high mobility group (HMG) -box protein, which binds to mtDNA promoters, and lead to bending and opening of DNA for the initiation of transcription. The proper replication and
PT E
al., 1997; Scarpulla et al., 2008).
D
transcription of the mtDNA is essential to maintain the mitochondrial function (Montoya et
TFAM activates transcription of each mtDNA strand by binding to an element of
CE
approximately 30 nucleotides present in both the light-strand and the heavy-strand promoters
AC
as a monomer (Parisi and Clayton, 1991). It also interacts with mitochondrial transcription factor B (TFB1M and TFB2M) proteins. A common +35 G/C single nucleotide polymorphism (SNP), rs1937, located in the first exon of TFAM, is a missense mutation leading to an amino acid substitution from serine to threonine at 12th position (Ser12Thr) (Reyes et al., 2002). This variant may have an effect on the function of TFAM and as a result it influences the mitochondrial transcription and thus the disease susceptibility. Till date, no known functional significance has been observed for this polymorphism. Association of alterations in the TFAM gene has been reported in several human diseases such as
ACCEPTED MANUSCRIPT Alzheimer's and Parkinson in which mitochondrial dysfunction plays an important role (Alvarez et al., 2008; Belin et al., 2007). PCOS is associated with decreased antioxidant concentrations, and is thus considered an oxidative state (Palacio et al., 2006). The decrease in mitochondrial O2 consumption and GSH levels along with increased ROS production
PT
explains the mitochondrial dysfunction in PCOS patients (Victor et al., 2011). Peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGC-1α) is a
RI
member of a family of nuclear coregulatory proteins that are involved in the modulation of
SC
gene transcription (Lin et al., 2005). Rather than directly binding to DNA, these coregulators form large multiprotein complexes with nuclear receptors (NRs) and transcription factors
NU
(TFs) to regulate transcriptional activities (O’Malley and Kumar, 2009). PGC-1α binds to and
MA
coactivates the transcriptional function of NRF-1 on the promoter of TFAM, a direct regulator of mitochondrial DNA replication/transcription (Lin et al., 2005). Three commonly
D
studied polymorphisms of PGC-1α gene are namely the rs8192678 (G/A) polymorphism,
PT E
rs13131226 (intron 2) (T/C) polymorphism and the rs2970856 (intron 5) (T/C) polymorphism. The most common coding single nucleotide polymorphism (SNP), rs8192678 (G/A), is located within the eighth exon and results in an amino acid change from Glycine to
CE
Serine (Gly482Ser). This polymorphism is associated with the risk of developing diabetes in
AC
several populations (Andrulionyte et al., 2005; Barroso et al., 2006). However, the potential differences in the PGC-1α variants were not evaluated in PCOS patients of Indian origin. The present study was undertaken to investigate the frequency of the aforementioned polymorphisms in TFAM and PGC-1α genes as well as their relationship with clinical and hormonal characteristics in South Indian women with PCOS. In addition, to better understand genetic contributions to the pathophysiology of PCOS, the mtDNA copy number with TFAM and PGC-1α variants was analyzed in PCOS patients and controls.
ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1 Study subjects A total of one hundred and eighteen South Indian women of reproductive age with PCOS were recruited at the infertility institute and research center (IIRC), Secunderabad, India. The diagnosis of PCOS was based on the Rotterdam ESHRE/ASRM-Sponsored PCOS
PT
Consensus Workshop Group (Rotterdam, 2004). PCOS was diagnosed when the phenotypes
RI
of the patients satisfied two of the following three criteria: 1) oligomenorrhea or amenorrhea,
SC
2) clinical or biochemical hyperandrogenism, and 3) ultrasonographic polycystic ovarian morphology. Polycystic ovaries were identified by transvaginal ultrasonography following
NU
the Rotterdam criteria, which defines PCOS as the presence of 12 or more small (2 to 9 mm) follicles in each ovary. Women with cardiovascular disease, diabetes, other endocrine
MA
disorders such as congenital adrenal hyperplasia, hyperprolactinemia, cushing’s syndrome or androgen-secreting tumors, were excluded from this study. They had no smoking and no
D
caffeine consumption habits. In addition, laboratory tests associated with PCOS revealing,
PT E
oligoovulation (cycles longer than 35 days or less than 26 days), elevated free testosterone levels (serum testosterone concentration >2.5 nmol/l or plasma testosterone >40 pmol/l),
AC
as cases.
CE
hirsutism (total Ferriman-Gallwey score ≥7), and an elevated LH/FSH ratio were considered
One hundred and ten women of reproductive age with regular menses, normal glucose tolerance, no hirsutism, normal androgen levels and no family history of diabetes, were recruited as control subjects. BMI of all subjects was calculated as: weight in kilograms (kg) divided by the square of height in metres (m2). The demographic and biochemical characteristics of South Indian PCOS women and controls were summarized in table 1. Written informed consent was obtained from all the participants for the collection of samples
ACCEPTED MANUSCRIPT and subsequent analysis. The study was approved by the review board of the Osmania University, Hyderabad. Peripheral blood samples (5 ml) were collected from all the subjects in sterile EDTA coated vacutainers for DNA isolation and plasma was removed followed by storage at −20 °C
PT
until further analysis was performed.
RI
2.2 DNA extraction
Genomic DNA was extracted from 1 ml of EDTA anti-coagulated whole blood by
NU
2.3 Genotyping of TFAM and PGC-1α
SC
salting out procedure method as described earlier (Tumu et al., 2013).
MA
The genotyping of TFAM and PGC-1α was carried out in a randomized, blinded fashion. The genotyping of TFAM and PGC-1α SNPs were analyzed by polymerase chain reaction (PCR) followed by RFLP analysis. The predesigned primers used for genotyping of
PT E
D
TFAM and PGC-1α (Palacín et al., 2011; Zhu et al. 2009) and the details of PCR-RFLP genotyping experiments are shown in Table 2.
CE
2.4 Quantification of mitochondrial DNA copy number by real time PCR The mtDNA copy number was measured using a real time quantitative polymerase
AC
chain reaction (qRT-PCR) using an Applied Biosystems 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). For the analysis of the nuclear DNA, forward primer 5'-GCCAATCTCAGTCCCTTCCC-3'
and
the
reverse
primer
5'-
TCGGTGAGGATCTTCATGAGGTA-3' (complementary to the sequences of the GAPDH gene) were used to amplify 177-bp product. For analysis of the mtDNA, forward primer 5'GGGCTACTACAACCCTTCGCT-3'
and
the
reverse
primer
5'-
GAGGCCTAGGTTGAGGTTGAC-3' (complementary to the sequences of the NADH dehydrogenase subunit 1 (ND1) gene) were used to amplify 153-bp product. The DNA (10
ACCEPTED MANUSCRIPT ng) was mixed with 10 ul SYBR Green I Master Mix (TaKaRa, USA) that contained 10 pmol of forward and reverse primer in a final volume of 20 ul. The qRT-PCR conditions consisted of initiation at 50°C for 2 min, 95°C for 10 sec followed by 40 cycles of denaturation at 95°C for 5 sec, annealing at 59°C for 30 sec, and extension at 72°C for 1 min. The value of the threshold cycle number (Ct) of the GAPDH gene and the ND1 gene were determined for each
PT
individual quantitative PCR run. Each measurement was performed at least two times and
RI
normalized in each experiment against control DNA sample. Ct value differences used to quantify mtDNA copy number relative to the GAPDH gene were calculated as follows:
SC
Relative copy number (Rc) = 2∆Ct, where ∆Ct is the CtGAPDH- CtND1. Reasonably good
NU
reproducibility was observed both within and between runs. The intraassay coefficients of variation of Ct values were around 2.9 % and 3.6% for ND1 and GAPDH gene, respectively.
MA
The interassay coefficients of variation of Ct values were around 4.1% and 5.4% for ND1 and GAPDH gene, respectively. To reduce the variations, all parameters throughout the study
2.5 Statistical analysis
PT E
D
were measured by the same person.
All statistical analyses were performed using the SPSS 17.0 software (SPSS
CE
Company, Chicago, IL, USA). The genotype distribution among cases and controls was
AC
performed using the Fisher’s exact test. The chi-square (χ2), odds ratio and 95% confidence interval (CI) values were calculated using the online Vassar Stats Calculator (http://www.faculty.vassar.edu/lowry/VassarStats.html). Values were expressed as mean ± standard deviation unless otherwise indicated. Student’s t-test was used to determine the differences between two subgroups. When, more than two subgroups were present, one-way ANOVA was used to calculate the differences of mtDNA copy number, followed by the least significant difference test. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D’) for pair-wise linkage disequilibrium (LD) were assessed by
ACCEPTED MANUSCRIPT Haploview Software (Barrett et al., 2005). For all the statistical tests, values of p<0.05 was considered to indicate a statistically significant difference. 3. Results 3.1 Genotyping of exon 1 (+35G/C) TFAM polymorphism
PT
We investigated the variations in the exon 1 of TFAM and found one previously reported polymorphism in the coding (rs1937) sequence. The +35G/C SNP genotype
RI
distribution and allele frequencies among the cases and controls are summarized in table 3.
SC
There is no statistically significant differences in the genotype (OR-1.7871; 95%CI -0.9631 to 3.316; P = 0.0639) and allele (OR-0.606; 95%CI-0.3409 to 1.0774; P= 0.0857) frequencies
NU
between PCOS patients and controls (Table 3). Together, these data indicated that TFAM
MA
variation did not contribute to the risk for PCOS in South Indian population. 3.2 Comparison of clinical data and mtDNA copy number between individuals with
PT E
D
different TFAM genotypes in the PCOS group
The results of a further stratified analysis of the TFAM +35G/C genotypes in PCOS subjects are shown in table 4. There were no significant differences in any of the following
CE
parameters among women with PCOS with different TFAM +35G/C genotypes: Age, BMI,
AC
FSH, LH and LH:FSH Ratio. We have also studied the mtDNA content in relation with the aforementioned variant at the TFAM gene, and found no statistically significant differences among the genotypes in PCOS group (P = 0. 426; Table 4). 3.3 Genotyping of PGC1-α polymorphisms In the present study we genotyped three polymorphisms of PGC1-α gene (rs8192678, rs13131226 and rs2970856) by PCR-RFLP analysis. rs8192678 (G/A) polymorphism
ACCEPTED MANUSCRIPT The genotypic distribution of the PGC-1α rs8192678 (G/A) polymorphism was in agreement with Hardy-Weinberg equilibrium for both PCOS patients and control groups. Statistically significant variation in the distribution of the GG, GA, and AA genotypes of the PGC-1α rs8192678 (G/A) polymorphism was observed in PCOS patients compared to control group (OR-2.488; 95%CI-1.0673 to 5.7998; P = 0.04743, Table 5). There was significant
PT
reduction of the wild type genotype (GG) frequency and elevation of the mutant genotype
RI
(AA) frequency in patients as compared to controls (Table 5). The allele frequency also showed a similar trend (OR-1.6091; 95%CI-1.0955 to 2.3634; P = 0.015) indicating that the
SC
‘A’ allele might confer risk to PCOS.
NU
rs13131226 (T/C) polymorphism
MA
The genotypes (P = 0.059) and alleles frequencies (P = 0.109) of the rs13131226 (T/C) polymorphism were not statistically significant between PCOS cases and controls
PT E
both in cases and controls.
D
(Table 5). The genotype and allele distribution showed high prevalence of wild type allele (T)
CE
rs2970856 (T/C) polymorphism
AC
The genotypes (P = 0.7464) and alleles frequencies (P = 0.27473) of the rs2970856 (T/C) polymorphism were not statistically significant between PCOS cases and controls (Table 5). The genotype and allele distribution showed high prevalence of wild type allele (T) both in cases and controls. 3.4 Comparison of clinical data and mtDNA copy number between individuals with different PGC1-α genotypes in the PCOS group The results of a further stratified analysis of the PGC1-α rs8192678 (G/A) genotypes in PCOS subjects are shown in table 6. There were no significant differences in any of the
ACCEPTED MANUSCRIPT following parameters among women with PCOS with different PGC1-α rs8192678 (G/A) genotypes: Age, BMI, FSH, LH and LH:FSH Ratio. However, patients with the AA genotype demonstrated significantly lower levels of MCN compared with patients with GA and GG genotypes (P = 0.032).
PT
The results of a further stratified analysis of the PGC1-α rs13131226 (T/C) genotypes in PCOS subjects are shown in table 6. There were no significant differences in any of the
SC
genotypes: Age, BMI, FSH, LH, LH:FSH Ratio and MCN.
RI
following parameters among women with PCOS with different PGC1-α rs13131226 T/C
NU
The results of a further stratified analysis of the PGC1-α rs2970856 (T/C) genotypes in PCOS subjects are shown in Table 6. There were no significant differences in any of the
MA
following parameters among women with PCOS with different PGC1-α rs2970856 (T/C) genotypes: Age, BMI, FSH, LH:FSH Ratio and MCN, except for LH (P = 0.030). Patients
3.5 Haplotype analysis
PT E
and CC genotypes.
D
with the CT genotype demonstrated significantly higher levels of LH when compared to TT
CE
To analyze the additive effect of PGC-1α SNPs on risk of developing PCOS, the
AC
haplotype frequencies for multiple loci were calculated (Table 7; Figure 1). Linkage disequilibrium (LD) coefficients (D') were calculated to express the strength of linkage between studied SNPs of PGC-1 α gene among cases and controls (Figure 1). Our data indicates the +1564G/15056881T/23825992T as the most common PGC-1α haplotype in South Indian women. The relative risk of each haplotype was calculated by using this as reference (Table 7). Bonferroni correction was used to adjust the significance level of a statistical test to protect against Type I errors. Since we have 8 haplotypes, the Bonferroni correction should be 0.05/8 = 0.00625. Therefore, a P-value less than 0.00625 was
ACCEPTED MANUSCRIPT considered significant. Our results showed different pattern of LD between patients and controls. However, we did not observe significant LD between any of the three loci analysed. 4. Discussion Previous studies have shown that PCOS is a type of complex, heterogeneous diseases
PT
(Teede et al., 2010), and despite its high prevalence the etiology of the disease is still largely unknown. PCOS is associated with decreased antioxidant concentrations, and it is considered
RI
an oxidative state (Palacio et al., 2006). The decrease in mitochondrial O2 consumption and
SC
reduced Glutathione (GSH) levels along with increased reactive oxygen species (ROS) production explains the mitochondrial dysfunction in PCOS patients (Victor et al., 2011).
NU
PGC-1 α has known to be a master regulator of mitochondrial biogenesis. PGC-1α stimulates
MA
PPAR γ and nuclear respiratory factors (NRF-1 and NRF-2) dependent transcriptions; in addition, PGC-1 α also binds to and coactivates the transcriptional function of NRF-1 on the
D
promoter of mitochondrial transcription factor A (TFAM), a direct regulator of mitochondrial
PT E
DNA replication/transcription (Lin et al., 2005). Analysis of mutations in the TFAM and PGC-1 α genes may provide insights on the role of mitochondrial biogenesis in PCOS.
CE
Biogenesis of the mitochondrial oxidative phosphorylation enzyme complex requires the concordant expression of mtDNA and nDNA genes, which both encode mitochondrial
AC
proteins as well as their regulatory factors. A recent study has shown that a reduction in the expression of nuclear genes can be mostly attributed to a reduction of the mitochondrial function in the insulin-resistant women with PCOS (Skov et al., 2007). One of these main control factors is TFAM, which plays a main role in regulating mtDNA transcription and replication (Virbasius and Scarpulla, 1994; Montoya et al., 1997; Wang et al., 1999). An association of alterations in the TFAM gene has been reported in several human diseases such as Alzheimer's and Parkinson in which mitochondrial dysfunction plays a main role (Alvarez
ACCEPTED MANUSCRIPT et al., 2008 and Belin et al., 2007), but in PCOS it remains elusive. So, we studied the association of +35 G/C (rs1937) single-nucleotide polymorphism in the first exon of the TFAM gene with PCOS. We did not find statistically significant differences in the genotype frequencies for the TFAM +35G/C polymorphism between women with PCOS and controls. In addition, we studied the effect of this polymorphism on PCOS phenotypes, we analyzed
PT
biochemical variables according to this polymorphism, but we did not find a significant
RI
association between clinical parameters of the PCOS and each genotype.
SC
We further aimed to determine whether the peripheral blood mtDNA content observed in our PCOS patients would show some relation to gene variant of main
NU
mitochondrial copy number regulator, TFAM and found that no significant differences in the
MA
leukocyte mtDNA copy number among the genotypes in PCOS patients. However, mtDNA content is related to several other factors such as regulation of nuclear encoded genes, the mechanisms regarding regulation of mtDNA content are not entirely clear. Then, we studied
PT E
D
the SNP’s of PGC-1α gene, which is the main regulator of mitochondrial biogenesis. The PGC-1α gene exists in several variants which differ in a single nucleotide from
CE
the major allele. Among these SNPs, particularly the rs8192678 G/A polymorphism has been most extensively studied in different populations. However, the molecular as well as
AC
functional background of this SNP is largely unknown. A recent study provided an indication that the ‘A’ gene variant impairs the transcription of TFAM, which plays a vital role in mtDNA replication and transcription (Choi et al., 2006). Ling et al., (2004) have demonstrated that the rs8192678 (G/A) polymorphism was associated with reduced PGC-1α mRNA expression, suggesting that rs8192678 (G/A) may be an important functional SNP. Several studies have been performed to elucidate the role of the PGC-1α gene in PCOS. A study in the skeletal muscle of PCOS patients showed lower levels of expression in the PGC-
ACCEPTED MANUSCRIPT 1 gene when compared with controls, which confirms an impaired mitochondrial oxidative metabolism in PCOS (Skov et al., 2007). Then, we studied the rs8192678 (G/A) SNP in PGC-1α gene, which changes Glycine to serine in codon 482 in the functional domain; this variant is associated with either type 2
PT
diabetes pathogenesis or insulin resistance in several populations (Vimaleswaran et al., 2005). In the present study, for the first time we report a significant association between PGC-1α
RI
rs8192678 (G/A) polymorphism and PCOS risk in South Indian women. The AA genotype
SC
frequency was significantly higher in PCOS patients (P = 0.03). In addition, the ‘A’ allele frequency was also significantly higher in PCOS patients than controls. Our results indicate
NU
that PGC-1 α as a candidate gene for PCOS. However, two previous studies have been
MA
investigated the possible association between PCOS and the PGC-1α rs8192678 (G/A) polymorphism. Wang et al. (2006) reported that the PGC-1α rs8192678 (G/A) polymorphism
D
was not associated with PCOS in Chinese population. Another study in Korean women also
PT E
found no significant differences in the distribution of the PGC-1α rs8192678 (G/A) polymorphism with PCOS (Chae at al., 2010). The discrepant results in different populations imply ethnic difference, demographic location and/or sample size of PGC-1α polymorphisms
CE
distribution and suggest that the PGC-1 α gene may be a susceptibility factor for PCOS in
AC
South Indian population.
In our study, in addition to PGC-1α rs8192678 (G/A) polymorphism, we have also studied rs13131226 (intron 2) (T/C) polymorphism and the rs2970856 (intron 5) (T/C) polymorphism in South Indian women. We observed that no significant distribution of genotypes or allele of these polymorphisms between patients with PCOS and controls. Further, our findings have the potential for future clinical utility in the identification of individuals at a higher risk of developing PCOS. Further studies with more subjects and
ACCEPTED MANUSCRIPT genotyping of the entire PGC-1α gene are necessary for the better understanding of the correlation between PGC-1α gene SNPs and PCOS. PGC-1 could stimulate mitochondrial proliferation, Wu et al, (1999) examined the mtDNA content in the muscle cells and showed that increased approximately 2-fold by PGC-
PT
1α (Wu et al., 1999). A study showed that reduced levels of PGC-1α could play a role in the down regulation of OXPHOS genes in PCOS (Skov et al., 2007). Reduced levels of PGC-1α
RI
may lead to decrease in mtDNA content. We further aimed to determine whether the studied
SC
PGC-1α polymorphisms have some relation to clinical characteristics and peripheral blood mtDNA content in our PCOS patients. We analysed the peripheral blood leukocyte mtDNA
NU
content in relation with the aforementioned variants at the PGC-1α gene. Present study
MA
showed that ‘Ser’ mutation leads to decrease in the levels of mtDNA content in PCOS subjects. We further analysed the biochemical variables that affect PCOS patients in each genotype of studied polymorphisms and found significant association between LH levels and
PT E
D
‘CC’ genotype of rs2970856 T/C polymorphism. However, the functional significance of this polymorphism is unknown.
CE
In conclusion, our study indicates that ‘Ser’ allele may be involved in the pathophysiology of PCOS. Our study also indicates mtDNA copy number is not associated
AC
with TFAM +35G/C SNP in PCOS patients. To the best of our knowledge, this is the first study exploring the association of PGC-1α rs8192678 G/A polymorphism, TFAM +35G/C polymorphism and mtDNA copy number with PCOS risk in Indian population. Further, our findings have the potential for future clinical utility in the identification of individuals at a higher risk of developing PCOS.
Conflicts of interest:
ACCEPTED MANUSCRIPT The authors declare no conflict of interest. Author Contribution TV Reddy: execution of experiments, analysis and interpretation of data, statistical analysis and drafting of manuscript.
PT
S. Govatati: acquisition of data. M. Deenadayal: acquisition of data.
RI
S. Shivaji: analysis and interpretation of data, drafting of manuscript.
SC
M. Bhanoori: conception and design of study, analysis and interpretation of data, statistical
NU
analysis, drafting of manuscript Funding
MA
This study was supported in part by grants from the OU-DST PURSE Programme-II07/2017, Department of Science and Technology (DST), India to Dr. Manjula Bhanoori.
D
Acknowledgments
PT E
We are most grateful to all of the patients who participated in the present study. Tumu Venkat Reddy would like to thank University Grants Commission (UGC), India for providing
AC
CE
Basic Science Research (BSR-SRF) fellowship.
References 1.
Alvarez, V., Corao, A.I., Sánchez-Ferrero, E., De Mena, L., Alonso-Montes, C., Huerta, C., Blázquez, M., Ribacoba, R., Guisasola, L.M., Salvador, C., García-Castro, M., 2008. Mitochondrial transcription factor A (TFAM) gene variation in Parkinson's disease. Neurosci. Lett. 432, 79-82.
2.
Andrulionyte, L., Zacharova, J., Chiasson, J.L., Laakso, M., Stop-Niddm Study Group, 2004. Common polymorphisms of the PPAR-γ2 (Pro12Ala) and PGC-1α (Gly482Ser)
ACCEPTED MANUSCRIPT genes are associated with the conversion from impaired glucose tolerance to type 2 diabetes in the STOP-NIDDM trial. Diabetologia. 47, 2176-2184. 3.
Attardi, G., Schatz, G., 1998. Biogenesis of mitochondria. Annu. Rev. Cell Bio. 1, 289331.
4.
Barrett, J.C., Fry, B., Maller, J.D.M.J., Daly, M.J., 2005. Haploview: analysis and
Barroso, I., Luan, J., Sandhu, M.S., Franks, P.W., Crowley, V., Schafer, A.J., O’rahilly,
RI
5.
PT
visualization of LD and haplotype maps. Bioinformatics, 21, 263-265.
SC
S., Wareham, N.J., 2006. Meta-analysis of the Gly482Ser variant in PPARGC1A in type 2 diabetes and related phenotypes. Diabetologia. 49, 501-505. Belin, A.C., Björk, B.F., Westerlund, M., Galter, D., Sydow, O., Lind, C., Pernold, K.,
NU
6.
Rosvall, L., Håkansson, A., Winblad, B., Nissbrandt, H., 2007. Association study of
MA
two genetic variants in mitochondrial transcription factor A (TFAM) in Alzheimer's and Parkinson's disease. Neurosci. Lett. 420, 257-262. Chae, S.J., Kim, J.J., Choi, Y.M., Kim, J.M., Cho, Y.M., Moon, S.Y., 2010. Peroxisome
D
7.
PT E
proliferator-activated receptor-γ and its coactivator-1α gene polymorphisms in korean women with polycystic ovary syndrome. Gynecol Obstet Invest. 70, 1-7. Choi, Y.S., Hong, J.M., Lim, S., Ko, K.S., Pak, Y.K., 2006. Impaired coactivator
CE
8.
activity of the Gly 482 variant of peroxisome proliferator-activated receptor γ
AC
coactivator-1α (PGC-1α) on mitochondrial transcription factor A (Tfam) promoter. Biochem. Biophys. Res. Commun. 344, 708-712. 9.
Ekstrand, M.I., Falkenberg, M., Rantanen, A., Park, C.B., Gaspari, M., Hultenby, K., Rustin, P., Gustafsson, C.M., Larsson, N.G., 2004. Mitochondrial transcription factor A regulates mtDNA copy number in mammals. Hum. Mol. Gen. 13, 935-944.
ACCEPTED MANUSCRIPT 10.
Lee, H.C., Wei, Y.H., 2005. Mitochondrial biogenesis and mitochondrial DNA maintenance of mammalian cells under oxidative stress. Int. J. Biochem. Cell Biol. 37, 822-834.
11.
Lin, J., Handschin, C., Spiegelman, B.M., 2005. Metabolic control through the PGC-1 family of transcription coactivators. Cell Metab. 1, 361-370. Ling, C., Poulsen, P., Carlsson, E., Ridderstråle, M., Almgren, P., Wojtaszewski, J.,
PT
12.
RI
Beck-Nielsen, H., Groop, L., Vaag, A., 2004. Multiple environmental and genetic
SC
factors influence skeletal muscle PGC-1α and PGC-1β gene expression in twins. The Journal of clinical investigation, 114, 1518-1526.
Montoya, J., Perez-Martos, A., Garstka, H.L., Wiesner, R.J., 1997. Regulation of
NU
13.
mitochondrial transcription by mitochondrial transcription factor A. Mol. Cell.
14.
MA
Biochem. 174, 227-230.
O'Malley, B.W., Kumar, R., 2009 Nuclear receptor coregulators in cancer biology.
Palacín, M., Alvarez, V., Martín, M., Díaz, M., Corao, A.I., Alonso, B., Díaz-Molina,
PT E
15.
D
Cancer Res.69, 8217-22.
B., Lozano, I., Avanzas, P., Morís, C., Reguero, J.R., 2011. Mitochondrial DNA and
CE
TFAM gene variation in early-onset myocardial infarction: evidence for an association to haplogroup H. Mitochondrion. 11, 176-181. Palacio, J.R., Iborra, A.,
AC
16.
lcova Gallova,
., Badia, R., Martinez, P., 2006. The
presence of antibodies to oxidative modified proteins in serum from polycystic ovary syndrome patients. Clin. Exp. Immunol. 144, 217-222. 17.
Parisi, M., Clayton, D.A., 1991. Similarity of human mitochondrial transcription factor 1 to high mobility group proteins. Science. 252, 965.
ACCEPTED MANUSCRIPT 18.
Reyes, A., Mezzina, M., Gadaleta, G., 2002. Human mitochondrial transcription factor A (mtTFA): gene structure and characterization of related pseudogenes. Gene. 291, 223-232.
19.
Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. 2004. Revised 2003 consensus on diagnostic criteria and long-term health risks related to
Scarpulla, R.C., 2008 Transcriptional paradigms in mammalian mitochondrial
21.
SC
biogenesis and function. Physiol. Rev. 88, 611-38.
RI
20.
PT
polycystic ovary syndrome. Fertil. Steril. 81, 19–25.
Skov, V., Glintborg, D., Knudsen, S., Jensen, T., Kruse, T.A., Tan, Q., Brusgaard, K.,
NU
Beck-Nielsen, H., Højlund, K., 2007. Reduced expression of nuclear-encoded genes involved in mitochondrial oxidative metabolism in skeletal muscle of insulin-resistant
22.
MA
women with polycystic ovary syndrome. Diabetes, 56, 2349-2355. Teede, H., Deeks, A., Moran, L., 2010. Polycystic ovary syndrome: a complex
D
condition with psychological, reproductive and metabolic manifestations that impacts
23.
PT E
on health across the lifespan. BMC Med. 8, 41. Tumu, V.R., Govatati, S., Guruvaiah, P., Deenadayal, M., Shivaji, S., Bhanoori, M.,
CE
2013. An interleukin-6 gene promoter polymorphism is associated with polycystic ovary syndrome in South Indian women. J. Assist. Reprod. Genet. 301541-1546. Victor, V.M., Rocha, M., Bañuls, C., Alvarez, A., de Pablo, C., Sanchez-Serrano, M.,
AC
24.
Gomez, M., Hernandez-Mijares, A., 2011. Induction of oxidative stress and human leukocyte/endothelial cell interactions in polycystic ovary syndrome patients with insulin resistance. J. Clin. Endocrinol. Metab. 96, 3115-3122. 25.
Vimaleswaran, K.S., Radha, V., Ghosh, S., Majumder, P.P., Deepa, R., Babu, H.N.S., Rao, M.R.S., Mohan, V., 2005. Peroxisome proliferator‐ activated receptor‐ γ
ACCEPTED MANUSCRIPT co‐ activator‐ 1α (PGC‐ 1α) gene polymorphisms and their relationship to Type 2 diabetes in Asian Indians. Diabetic Med. 22, 1516-1521. 26.
Virbasius, J.V., Scarpulla, R.C., 1994. Activation of the human mitochondrial transcription factor A gene by nuclear respiratory factors: a potential regulatory link between nuclear and mitochondrial gene expression in organelle biogenesis. Proc. Natl.
PT
Acad. Sci. U.S.A. 91, 1309-1313.
Wallace, D.C., 2008. Mitochondria as chi. Genetics. 179, 727-735.
28.
Wang, Y., Wu, X., Cao, Y., Yi, L., Fan, H., Chen, J., 2006. Polymorphisms of the
RI
27.
SC
peroxisome proliferator–activated receptor-γ and its coactivator-1α genes in Chinese
29.
NU
women with polycystic ovary syndrome. Fertil Steril. 85, 1536-1540. Wang, J., Wilhelmsson, H., Graff, C., Li, H., Oldfors, A., Rustin, P., Brüning, J.C.,
atrioventricular
MA
Kahn, C.R., Clayton, D.A., Barsh, G.S., Thorén, P., 1999. Dilated cardiomyopathy and conduction blocks induced by heart-specific inactivation of
Wu, Z., Puigserver, P., Andersson, U., Zhang, C., Adelmant, G., Mootha, V., Troy, A.,
PT E
30.
D
mitochondrial DNA gene expression. Nature Genet. 21, 133-137.
Cinti, S., Lowell, B., Scarpulla, R.C., Spiegelman, B.M., 1999. Mechanisms controlling
CE
mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell. 98, 115-124.
Zhu, S., Liu, Y., Wang, X., Wu, X., Zhu, X., Li, J., Ma, J., Gu, H.F., Liu, Y., 2009.
AC
31.
Evaluation of the association between the PPARGC1A genetic polymorphisms and type 2 diabetes in Han Chinese population. Diabetes Res Clin Pract. 86, 168-172.
ACCEPTED MANUSCRIPT Figure legends Figure: 1 LD analysis of cases and controls are shown separately (A and B). Haploview plots are presented along with the SNPs studied. The pair wise linkage disequilibrium values (D' = 0– 100) of all SNPs are given in each diamond. A value of 100 represents maximum possible
AC
CE
PT E
D
MA
NU
SC
RI
PT
linkage disequilibrium. (A) LD analysis of cases and (B) LD analysis of controls.
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
Fig. 1
ACCEPTED MANUSCRIPT Table 1
Demographic and clinical characteristics of PCOS and control group
Controls
118
110
Age (years)
28.31±5.78
BMI (kg/m2)
22.85±5.85
FSH (mIU/ml)
LH (mIU/ml)
RI
Variable
SC
Number
0.0748
22.87±3.33
0.9750
5.67±1.6
6.18±3.38
0.1425
7.72±2.22
5.14±1.49
˂0.0001
0.98±0.69
˂0.0001
PT E
D
MA
NU
29.71±6.03
1.44±0.55
CE
LH:FSH ratio
p-value a
PT
PCOS
AC
Data are given as mean ± S.D.
p a values obtained by comparison of variables between controls and PCOS by Student’s t test
ACCEPTED MANUSCRIPT Table 2. PCR conditions and product size for the studied polymorphisms S.
SNP
Gene
Primer sequences 5′ to 3′
Locati
Mode
PCR
PCR
Referen
N
on
of
condition
produ
ce
o
(Base
analys
ct
change
is
(bp)
) TFA M
Exon1
RFLP
5′CCCCGCCCCCATCTACCG A3′
Denaturati on96°C(5min ) 35cycle:94°C 50 sec, 59°C - 45 sec 72°C-50 sec Extension72°C-10 min
GG: 200b p+ 126b p
Denaturati on96°C(5min ) 35cycle:94°C 50 sec, 60°C - 45 sec 72°C-50 sec Extension72°C-10 min
GG: 366b p+ 245b p
PT
Dde I 1 ( rs1937)
5′GACGTCCTGGGCCCTGCT G3′
MA
NU
SC
RI
+35 G/C
PGC -1α
Exon8
RFLP
5′CAAGTCCTCAGTCCTCAC 3′
D
Msp I (rs819267 8)
PT E
2
5′GGGGTCTTTGAGAAAAT AAGG3′
AC
CE
+482 G/A
3
Alu I (rs131312 26)
PGC -1α
Intron2 T/C
RFLP
5′TGACCAGCCATATCCCAA GT3′ 5′CAGAGACCAGTTTCCAC AGT 3′
Denaturati on96°C(5min ) 35cycle:94°C 50
(Palacín et al., 2011)
GC: 326b p+ 200b p+ 126b p CC: 326b p
(Zhu et al., 2009)
GA: 611b p+ 366b p+ 245b p AA: 611b p
(Zhu et CC: 120b p+ 169b p
al., 2009)
ACCEPTED MANUSCRIPT sec, 59°C - 45 sec 72°C-50 sec Extension72°C-10 min
CT: 289b p+ 120b p+ 169b p
PGC -1α
Intron5
RFLP
5′TGAAAAAGACACATTCC TGA3′
5′GGATAATTCATACAACTT CC3′
PT E
D
MA
NU
SC
T/C
CE
Denaturati on96°C(5min ) 35cycle:94°C 50 sec, 59°C - 45 sec 72°C-50 sec Extension72°C-10 min
RI
Alu I (rs297085 6)
AC
4
PT
TT: 289b p
CC: 389b p+ 44bp TC: 433b p+ 389b p+ 44bp TT: 433b p
(Zhu et al., 2009)
ACCEPTED MANUSCRIPT Table 3. Genotype and allele frequencies of TFAM +35G/C polymorphism in PCOS patients and controls Genotypes / Controls (%) Cases (%)
χ2 P-value
Odds ratio
95%CI
Alleles Genotypes 83 (70.33)
GC
21 (19.10)
35 (29.66)
CC
0
0
G
199 (90.45)
201 (85.16)
C
21 (9.55)
35(14.83)
1.0 (Reference) 0.0639a
0.9631 to 3.316
1.0 (Reference)
NU
0.0857b
Fisher’s exact test (3 × 2 table at 2 df ),
b
Fisher’s exact test (2 × 2 table at 1 df ),
MA
a
PT E
D
CI, confidence interval
CE
1.7871
SC
Alleles
AC
PT
89 (80.90)
RI
GG
0.606
0.3409 to 1.0774
ACCEPTED MANUSCRIPT
Table 4. Genotypes frequencies of studied polymorphism (TFAM) along with clinical characteristics and MCN in PCOS subjects
Variable
PT
PCOS (n=118) GC (n=35)
RI
GG (n=83) 28.43±5.69
Weight
55.62±14.52
BMI (kg/m-2)
22.38±5.87
FSH(mIU/mL)
LH(mIU/mL)
0.7333
0.2322
23.67±3.98
0.2370
5.56±1.63
5.93±1.51
0.2523
7.8±1.02
7.54±1.98
0.3493
1.50±0.61
1.32±0.36
0.1063
1.39±0.43
1.32±0.48
0.426
D
MA
NU
59.11±14.17
PT E
MCN
CE
LH:FSH Ratio
28.03±6.09
SC
Age
p a value
AC
Data are given as mean ± S.D. p a values obtained by comparison of variables between controls and PCOS by Student’s t test
ACCEPTED MANUSCRIPT Table 5. Genotype and allele distribution of PGC1-α polymorphisms in PCOS patients and controls of South Indian women Genotypes/Alleles
Controls
χ2 P-
Cases (%)
(%)
Odds ratio
95 % CI
values
Hw-0.6547 52 (47.27)
38 (32.21)
GA
47 (42.72)
60 (50.84)
AA
11 (10.00)
20 (16.95)
G
151 (68.64)
136 (57.63)
A
69 (31.36)
100 (42.37)
MA
D
rs13131226
0.015b
1.00
1.7469
0.9915 to 3.0777
2.488
1.0673 to 5.7998
1.00
1.00
1.6091
1.0955 to 2.3634
Hw-0.7131
PT E
Genotypes
0.047a
NU
Alleles
1.00
RI
GG
SC
Genotypes
PT
rs8192678
54 (49.09)
50 (42.37)
1.00
1.00
TC
47 (42.72)
55 (46.61)
1.2638
0.7311 to 2.1846
1.56
0.6137 to 3.9654
1.00
1.00
1.2462
0.8395 to 1.85
1.00
1.00
Alleles T C
9 (8.18)
13 (11.01)
155 (71.45)
155 (65.68)
65 (29.54)
81 (34.32)
AC
CC
CE
TT
0.541a
0.274
b
rs2970856 Hw-0.8539
Genotypes TT
67 (60.90)
66 (55.93)
ACCEPTED MANUSCRIPT TC
37 (33.63)
45 (38.13)
CC
6 (5.45)
7 (5.93)
T
171 (29.54)
177 (75.00)
C
49 (22.27)
59 (25.00)
0.7464a
1.2346
0.7109 to 2.1442
1.1843
0.3779 to 3.7112
1.00
1.00
1.1633
0.7543 to 1.7941
Alleles
b
PT
0.27473
AC
CE
PT E
D
MA
NU
SC
RI
Hw-Hardy-Weinberg equilibrium CI Confidence Interval, a Fisher’s exact test (3×2 table at 2 df) P<0.05 b Fisher’s exact test (2×2 table at 1 df) P<0.05
ACCEPTED MANUSCRIPT Table 6. PGC-1 α genotypes of studied Polymorphisms by clinical variables along with MCN in PCOS subjects p
p rs13131226 (T/C)
rs2970856
val
val p val ues
rs8192678 (G/A) ues
(T/C) ues
a
29.19
27.86
28.25
0.5
28.22
28.75
26.84
Age
±7.19
±5.55
±3.14
59
±6.03
±5.67
BMI
22.68
22.40
24.68
0.3
23.08
22.63
(kg/m-2)
±6.60
±5.84
±4.19
17
±5.25
FSH(mI
5.46±
5.80±
5.78±
0.5
5.47±
U/mL)
1.44
1.77
1.35
69
1.50
LH(mIU
7.38±
7.93±
/mL)
2.32
2.23
LH:FSH
1.38±
1.51±
Ratio
0.37
PT
CC
TT
0.5
a
TC
CC
27.75
28.61
32.17
0.1
RI
TC
±5.49
66
±6.23
±4.57
±8.13
85
NU
AA
22.93
0.9
23.27
22.07
24.00
0.5
±8.02
21
±3.98
±7.92
±5.40
00
5.73±
6.23±
0.2
5.46±
6.04±
5.29±
0.1
1.79
1.01
99
1.60
1.52
1.98
43
±5.90
MA
GA
MCN
PT E
D
GG
SC
TT
CE
a
0.4
7.22±
7.98±
8.61±
0.0
7.24±
8.36±
8.14±
0.0
2.02
81
2.24
2.27
1.56
71
1.88
2.55
2.03
30
1.32±
0.4
1.37±
1.53±
1.39±
0.3
1.43±
1.41±
1.79±
0.2
0.71
0.36
96
0.42
0.71
0.14
27
0.61
0.35
1.00
36
1.47±
1.33±
1.26±
0.0
1.32±
1.37±
1.42±
0.2
1.37±
1.36±
1.37±
0.9
0.46
0.47
0.32
32
0.45
1.47
0.28
04
0.47
0.42
0.50
91
AC
7.89±
Data are given as mean ± S.D. p a values obtained by comparison of variables between genotypes in PCOS by one-way ANNOVA test followed by least significance test.
ACCEPTED MANUSCRIPT
Table 7. Haplotype frequencies of PGC-1α polymorphisms in PCOS patients and controls Haplotypes
Haplotype frequency P-valuea
15056881
23825992
G/A
T/C
T/C
G
T
G
95% CI
T
94 (39.83)
102 (46.36)
Reference
C
T
20 (8.47)
23 (9.74)
0.86249
1.05
0.5469 to 2.0537
G
T
C
12 (5.08)
15 (6.82)
1.15
0.5129 to 2.5875
G
C
C
10 (4.23)
11 (5.00)
0.97477
1.01
0.4117 to 2.496
A
T
T
35 (14.83)
25 (11.36)
0.1596
0.66
0.3668 to 1.1814
A
C
T
30 (12.71)
21 (9.55)
0.16685
0.64
0.3456 to 1.2041
A
T
C
10 (4.23)
10 (4.54)
0.86249
0.92
0.3672 to 2.3131
A
C
C
25 (10.59)
0.0442
0.48
0.2318 to 0.9908
0.73121
SC
NU
MA
D
AC
CE
PT E
Fisher’s exact (2x2 table at I df); P < 0.05
PT
Controls (%)
CI: confidence interval a
OR
PCOS (%)
RI
+1564
13 (5.91)
ACCEPTED MANUSCRIPT Abbreviations: CI, confidence interval; D', disequilibrium coefficient; G, glycine; GG, glycine/glycine; GS, glycine/serine; FSH, follicle-stimulating hormone; IIRC, infertility institute and research centre; LD, linkage disequilibrium; LH, luteinizing hormone; MCN, mtDNA copy number; NRF,
nuclear
respiratory
factor;
NRs,
nuclear
receptors;
OXPHOS,
oxidative
PT
phosphorylation; PCOS, polycystic ovarian syndrome; PCR-RFLP, polymerase chain
RI
reaction-restriction fragment length polymorphism; PGC-1α, peroxisome proliferator
SC
activated receptor gamma coactivator-1 alpha; ROS, reactive oxygen species; SNPs, single nucleotide polymorphisms; SS, serine/serine; TFAM, mitochondrial transcription factor A;
AC
CE
PT E
D
MA
NU
TFs, transcription factors.
ACCEPTED MANUSCRIPT Highlights
The study showed significant differences between the distribution of the genotypes and alleles of the PGC-1α rs8192678 (G/A) polymorphism in PCOS patients and control groups
We observed patients with the AA genotype showed significantly lower levels of
PT
MCN compared with patients with GA and GG genotypes of rs8192678 (G/A)
Our study indicates ‘Ser’ allele of rs8192678 SNP could be involved in the
SC
RI
polymorphism.
pathophysiology of PCOS.
NU
The study showed significantly higher levels of LH in patients with the PGC1-α
CE
PT E
D
MA
rs2970856 CT genotype when compared to other genotypes.
AC
Tumu Venkat Reddy, Suresh Govatati, Mamata Deenadayal, Sisinthy Shivaji, Manjula Bhanoori PII: DOI: Reference:
S0378-1119(17)30823-5 doi:10.1016/j.gene.2017.10.010 GENE 42229
To appear in:
Gene
Received date: Revised date: Accepted date:
12 July 2017 13 September 2017 6 October 2017
Please cite this article as: Tumu Venkat Reddy, Suresh Govatati, Mamata Deenadayal, Sisinthy Shivaji, Manjula Bhanoori , Polymorphisms in the TFAM and PGC1-α genes and their association with polycystic ovary syndrome among South Indian women. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2017.10.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: Polymorphisms in the TFAM and PGC1-α genes and their association with polycystic ovary syndrome among South Indian women Names of Authors: Tumu Venkat Reddya, Suresh Govatatia, Mamata Deenadayalb, Sisinthy
PT
Shivajic,d and Manjula Bhanooria,*
c
Infertility Institute and Research Centre (IIRC), Secundrabad, INDIA
NU
b
SC
Department of Biochemistry, Osmania University, Hyderabad, INDIA
Centre for Cellular and Molecular Biology (CCMB), Hyderabad, INDIA
d
MA
a
RI
Institutional Affiliations:
Presently at: Prof Brien Holden Eye Research Centre, L V Prasad Eye
PT E
D
Institute, Hyderabad, INDIA
CE
*Address for Correspondence:
AC
Dr. Manjula Bhanoori, Assistant Professor, Department of Biochemistry, Osmania University, Hyderabad – 500 007, INDIA E-mail: [email protected] Tel: 00-91-9989661469 Fax: 00-91-40-27097044
ACCEPTED MANUSCRIPT Full postal address and contact information of all authors 1. Mr. Tumu Venkat Reddy, C/O Dr. Manjula Bhanoori, Department of Biochemistry, Osmania University, Hyderabad – 500 007, INDIA E-mail: [email protected]
PT
2. Dr. Suresh Govatati, C/O Dr. Manjula Bhanoori, Department of Biochemistry,
RI
Osmania University, Hyderabad – 500 007, INDIA
SC
E-mail: [email protected] 3. Dr. Mamata Deenadayal,
MA
E-mail: [email protected]
NU
Infertility Institute and Research Centre (IIRC), Secundrabad, INDIA
4. Dr. Shivaji Sisinthy,
D
Centre for Cellular and Molecular Biology (CCMB), Hyderabad – 500 007, INDIA
PT E
E-mail: [email protected] 5. Dr. Manjula Bhanoori,
CE
Assistant Professor, Department of Biochemistry, Osmania University, Hyderabad – 500 007, INDIA
AC
E-mail: [email protected] Tel: 00-91-9989661469; Fax: 00-91-40-27097044
ACCEPTED MANUSCRIPT 1. Introduction Mitochondria are cytoplasmic organelles, whose prime function is to generate energy through oxidative phosphorylation, and execution of apoptosis (Attardi and Schatz, 1988; Lee HC et al., 2005; Wallace, 2008). Mitochondria possess their own genome (mitochondrial DNA, mtDNA), a 16,569 bp circular double-stranded molecule. The mitochondrial genome is
PT
maintained within nucleoprotein complexes known as nucleoids. Mitochondrial transcription
RI
factor A (TFAM) is the predominant architectural mtDNA packaging protein of the nucleoid,
SC
where it is involved in both transcription and replication of mtDNA. It is a nuclear encoded protein that promotes the expression of the mitochondrial encoded 13 proteins, that are
NU
essential for oxidative phosphorylation (OXPHOS), 2 rRNAs and 22 tRNAs, which are essential for protein translation on mitochondrial ribosomes (Ekstrand et al., 2004). TFAM is
MA
a high mobility group (HMG) -box protein, which binds to mtDNA promoters, and lead to bending and opening of DNA for the initiation of transcription. The proper replication and
PT E
al., 1997; Scarpulla et al., 2008).
D
transcription of the mtDNA is essential to maintain the mitochondrial function (Montoya et
TFAM activates transcription of each mtDNA strand by binding to an element of
CE
approximately 30 nucleotides present in both the light-strand and the heavy-strand promoters
AC
as a monomer (Parisi and Clayton, 1991). It also interacts with mitochondrial transcription factor B (TFB1M and TFB2M) proteins. A common +35 G/C single nucleotide polymorphism (SNP), rs1937, located in the first exon of TFAM, is a missense mutation leading to an amino acid substitution from serine to threonine at 12th position (Ser12Thr) (Reyes et al., 2002). This variant may have an effect on the function of TFAM and as a result it influences the mitochondrial transcription and thus the disease susceptibility. Till date, no known functional significance has been observed for this polymorphism. Association of alterations in the TFAM gene has been reported in several human diseases such as
ACCEPTED MANUSCRIPT Alzheimer's and Parkinson in which mitochondrial dysfunction plays an important role (Alvarez et al., 2008; Belin et al., 2007). PCOS is associated with decreased antioxidant concentrations, and is thus considered an oxidative state (Palacio et al., 2006). The decrease in mitochondrial O2 consumption and GSH levels along with increased ROS production
PT
explains the mitochondrial dysfunction in PCOS patients (Victor et al., 2011). Peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGC-1α) is a
RI
member of a family of nuclear coregulatory proteins that are involved in the modulation of
SC
gene transcription (Lin et al., 2005). Rather than directly binding to DNA, these coregulators form large multiprotein complexes with nuclear receptors (NRs) and transcription factors
NU
(TFs) to regulate transcriptional activities (O’Malley and Kumar, 2009). PGC-1α binds to and
MA
coactivates the transcriptional function of NRF-1 on the promoter of TFAM, a direct regulator of mitochondrial DNA replication/transcription (Lin et al., 2005). Three commonly
D
studied polymorphisms of PGC-1α gene are namely the rs8192678 (G/A) polymorphism,
PT E
rs13131226 (intron 2) (T/C) polymorphism and the rs2970856 (intron 5) (T/C) polymorphism. The most common coding single nucleotide polymorphism (SNP), rs8192678 (G/A), is located within the eighth exon and results in an amino acid change from Glycine to
CE
Serine (Gly482Ser). This polymorphism is associated with the risk of developing diabetes in
AC
several populations (Andrulionyte et al., 2005; Barroso et al., 2006). However, the potential differences in the PGC-1α variants were not evaluated in PCOS patients of Indian origin. The present study was undertaken to investigate the frequency of the aforementioned polymorphisms in TFAM and PGC-1α genes as well as their relationship with clinical and hormonal characteristics in South Indian women with PCOS. In addition, to better understand genetic contributions to the pathophysiology of PCOS, the mtDNA copy number with TFAM and PGC-1α variants was analyzed in PCOS patients and controls.
ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1 Study subjects A total of one hundred and eighteen South Indian women of reproductive age with PCOS were recruited at the infertility institute and research center (IIRC), Secunderabad, India. The diagnosis of PCOS was based on the Rotterdam ESHRE/ASRM-Sponsored PCOS
PT
Consensus Workshop Group (Rotterdam, 2004). PCOS was diagnosed when the phenotypes
RI
of the patients satisfied two of the following three criteria: 1) oligomenorrhea or amenorrhea,
SC
2) clinical or biochemical hyperandrogenism, and 3) ultrasonographic polycystic ovarian morphology. Polycystic ovaries were identified by transvaginal ultrasonography following
NU
the Rotterdam criteria, which defines PCOS as the presence of 12 or more small (2 to 9 mm) follicles in each ovary. Women with cardiovascular disease, diabetes, other endocrine
MA
disorders such as congenital adrenal hyperplasia, hyperprolactinemia, cushing’s syndrome or androgen-secreting tumors, were excluded from this study. They had no smoking and no
D
caffeine consumption habits. In addition, laboratory tests associated with PCOS revealing,
PT E
oligoovulation (cycles longer than 35 days or less than 26 days), elevated free testosterone levels (serum testosterone concentration >2.5 nmol/l or plasma testosterone >40 pmol/l),
AC
as cases.
CE
hirsutism (total Ferriman-Gallwey score ≥7), and an elevated LH/FSH ratio were considered
One hundred and ten women of reproductive age with regular menses, normal glucose tolerance, no hirsutism, normal androgen levels and no family history of diabetes, were recruited as control subjects. BMI of all subjects was calculated as: weight in kilograms (kg) divided by the square of height in metres (m2). The demographic and biochemical characteristics of South Indian PCOS women and controls were summarized in table 1. Written informed consent was obtained from all the participants for the collection of samples
ACCEPTED MANUSCRIPT and subsequent analysis. The study was approved by the review board of the Osmania University, Hyderabad. Peripheral blood samples (5 ml) were collected from all the subjects in sterile EDTA coated vacutainers for DNA isolation and plasma was removed followed by storage at −20 °C
PT
until further analysis was performed.
RI
2.2 DNA extraction
Genomic DNA was extracted from 1 ml of EDTA anti-coagulated whole blood by
NU
2.3 Genotyping of TFAM and PGC-1α
SC
salting out procedure method as described earlier (Tumu et al., 2013).
MA
The genotyping of TFAM and PGC-1α was carried out in a randomized, blinded fashion. The genotyping of TFAM and PGC-1α SNPs were analyzed by polymerase chain reaction (PCR) followed by RFLP analysis. The predesigned primers used for genotyping of
PT E
D
TFAM and PGC-1α (Palacín et al., 2011; Zhu et al. 2009) and the details of PCR-RFLP genotyping experiments are shown in Table 2.
CE
2.4 Quantification of mitochondrial DNA copy number by real time PCR The mtDNA copy number was measured using a real time quantitative polymerase
AC
chain reaction (qRT-PCR) using an Applied Biosystems 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). For the analysis of the nuclear DNA, forward primer 5'-GCCAATCTCAGTCCCTTCCC-3'
and
the
reverse
primer
5'-
TCGGTGAGGATCTTCATGAGGTA-3' (complementary to the sequences of the GAPDH gene) were used to amplify 177-bp product. For analysis of the mtDNA, forward primer 5'GGGCTACTACAACCCTTCGCT-3'
and
the
reverse
primer
5'-
GAGGCCTAGGTTGAGGTTGAC-3' (complementary to the sequences of the NADH dehydrogenase subunit 1 (ND1) gene) were used to amplify 153-bp product. The DNA (10
ACCEPTED MANUSCRIPT ng) was mixed with 10 ul SYBR Green I Master Mix (TaKaRa, USA) that contained 10 pmol of forward and reverse primer in a final volume of 20 ul. The qRT-PCR conditions consisted of initiation at 50°C for 2 min, 95°C for 10 sec followed by 40 cycles of denaturation at 95°C for 5 sec, annealing at 59°C for 30 sec, and extension at 72°C for 1 min. The value of the threshold cycle number (Ct) of the GAPDH gene and the ND1 gene were determined for each
PT
individual quantitative PCR run. Each measurement was performed at least two times and
RI
normalized in each experiment against control DNA sample. Ct value differences used to quantify mtDNA copy number relative to the GAPDH gene were calculated as follows:
SC
Relative copy number (Rc) = 2∆Ct, where ∆Ct is the CtGAPDH- CtND1. Reasonably good
NU
reproducibility was observed both within and between runs. The intraassay coefficients of variation of Ct values were around 2.9 % and 3.6% for ND1 and GAPDH gene, respectively.
MA
The interassay coefficients of variation of Ct values were around 4.1% and 5.4% for ND1 and GAPDH gene, respectively. To reduce the variations, all parameters throughout the study
2.5 Statistical analysis
PT E
D
were measured by the same person.
All statistical analyses were performed using the SPSS 17.0 software (SPSS
CE
Company, Chicago, IL, USA). The genotype distribution among cases and controls was
AC
performed using the Fisher’s exact test. The chi-square (χ2), odds ratio and 95% confidence interval (CI) values were calculated using the online Vassar Stats Calculator (http://www.faculty.vassar.edu/lowry/VassarStats.html). Values were expressed as mean ± standard deviation unless otherwise indicated. Student’s t-test was used to determine the differences between two subgroups. When, more than two subgroups were present, one-way ANOVA was used to calculate the differences of mtDNA copy number, followed by the least significant difference test. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D’) for pair-wise linkage disequilibrium (LD) were assessed by
ACCEPTED MANUSCRIPT Haploview Software (Barrett et al., 2005). For all the statistical tests, values of p<0.05 was considered to indicate a statistically significant difference. 3. Results 3.1 Genotyping of exon 1 (+35G/C) TFAM polymorphism
PT
We investigated the variations in the exon 1 of TFAM and found one previously reported polymorphism in the coding (rs1937) sequence. The +35G/C SNP genotype
RI
distribution and allele frequencies among the cases and controls are summarized in table 3.
SC
There is no statistically significant differences in the genotype (OR-1.7871; 95%CI -0.9631 to 3.316; P = 0.0639) and allele (OR-0.606; 95%CI-0.3409 to 1.0774; P= 0.0857) frequencies
NU
between PCOS patients and controls (Table 3). Together, these data indicated that TFAM
MA
variation did not contribute to the risk for PCOS in South Indian population. 3.2 Comparison of clinical data and mtDNA copy number between individuals with
PT E
D
different TFAM genotypes in the PCOS group
The results of a further stratified analysis of the TFAM +35G/C genotypes in PCOS subjects are shown in table 4. There were no significant differences in any of the following
CE
parameters among women with PCOS with different TFAM +35G/C genotypes: Age, BMI,
AC
FSH, LH and LH:FSH Ratio. We have also studied the mtDNA content in relation with the aforementioned variant at the TFAM gene, and found no statistically significant differences among the genotypes in PCOS group (P = 0. 426; Table 4). 3.3 Genotyping of PGC1-α polymorphisms In the present study we genotyped three polymorphisms of PGC1-α gene (rs8192678, rs13131226 and rs2970856) by PCR-RFLP analysis. rs8192678 (G/A) polymorphism
ACCEPTED MANUSCRIPT The genotypic distribution of the PGC-1α rs8192678 (G/A) polymorphism was in agreement with Hardy-Weinberg equilibrium for both PCOS patients and control groups. Statistically significant variation in the distribution of the GG, GA, and AA genotypes of the PGC-1α rs8192678 (G/A) polymorphism was observed in PCOS patients compared to control group (OR-2.488; 95%CI-1.0673 to 5.7998; P = 0.04743, Table 5). There was significant
PT
reduction of the wild type genotype (GG) frequency and elevation of the mutant genotype
RI
(AA) frequency in patients as compared to controls (Table 5). The allele frequency also showed a similar trend (OR-1.6091; 95%CI-1.0955 to 2.3634; P = 0.015) indicating that the
SC
‘A’ allele might confer risk to PCOS.
NU
rs13131226 (T/C) polymorphism
MA
The genotypes (P = 0.059) and alleles frequencies (P = 0.109) of the rs13131226 (T/C) polymorphism were not statistically significant between PCOS cases and controls
PT E
both in cases and controls.
D
(Table 5). The genotype and allele distribution showed high prevalence of wild type allele (T)
CE
rs2970856 (T/C) polymorphism
AC
The genotypes (P = 0.7464) and alleles frequencies (P = 0.27473) of the rs2970856 (T/C) polymorphism were not statistically significant between PCOS cases and controls (Table 5). The genotype and allele distribution showed high prevalence of wild type allele (T) both in cases and controls. 3.4 Comparison of clinical data and mtDNA copy number between individuals with different PGC1-α genotypes in the PCOS group The results of a further stratified analysis of the PGC1-α rs8192678 (G/A) genotypes in PCOS subjects are shown in table 6. There were no significant differences in any of the
ACCEPTED MANUSCRIPT following parameters among women with PCOS with different PGC1-α rs8192678 (G/A) genotypes: Age, BMI, FSH, LH and LH:FSH Ratio. However, patients with the AA genotype demonstrated significantly lower levels of MCN compared with patients with GA and GG genotypes (P = 0.032).
PT
The results of a further stratified analysis of the PGC1-α rs13131226 (T/C) genotypes in PCOS subjects are shown in table 6. There were no significant differences in any of the
SC
genotypes: Age, BMI, FSH, LH, LH:FSH Ratio and MCN.
RI
following parameters among women with PCOS with different PGC1-α rs13131226 T/C
NU
The results of a further stratified analysis of the PGC1-α rs2970856 (T/C) genotypes in PCOS subjects are shown in Table 6. There were no significant differences in any of the
MA
following parameters among women with PCOS with different PGC1-α rs2970856 (T/C) genotypes: Age, BMI, FSH, LH:FSH Ratio and MCN, except for LH (P = 0.030). Patients
3.5 Haplotype analysis
PT E
and CC genotypes.
D
with the CT genotype demonstrated significantly higher levels of LH when compared to TT
CE
To analyze the additive effect of PGC-1α SNPs on risk of developing PCOS, the
AC
haplotype frequencies for multiple loci were calculated (Table 7; Figure 1). Linkage disequilibrium (LD) coefficients (D') were calculated to express the strength of linkage between studied SNPs of PGC-1 α gene among cases and controls (Figure 1). Our data indicates the +1564G/15056881T/23825992T as the most common PGC-1α haplotype in South Indian women. The relative risk of each haplotype was calculated by using this as reference (Table 7). Bonferroni correction was used to adjust the significance level of a statistical test to protect against Type I errors. Since we have 8 haplotypes, the Bonferroni correction should be 0.05/8 = 0.00625. Therefore, a P-value less than 0.00625 was
ACCEPTED MANUSCRIPT considered significant. Our results showed different pattern of LD between patients and controls. However, we did not observe significant LD between any of the three loci analysed. 4. Discussion Previous studies have shown that PCOS is a type of complex, heterogeneous diseases
PT
(Teede et al., 2010), and despite its high prevalence the etiology of the disease is still largely unknown. PCOS is associated with decreased antioxidant concentrations, and it is considered
RI
an oxidative state (Palacio et al., 2006). The decrease in mitochondrial O2 consumption and
SC
reduced Glutathione (GSH) levels along with increased reactive oxygen species (ROS) production explains the mitochondrial dysfunction in PCOS patients (Victor et al., 2011).
NU
PGC-1 α has known to be a master regulator of mitochondrial biogenesis. PGC-1α stimulates
MA
PPAR γ and nuclear respiratory factors (NRF-1 and NRF-2) dependent transcriptions; in addition, PGC-1 α also binds to and coactivates the transcriptional function of NRF-1 on the
D
promoter of mitochondrial transcription factor A (TFAM), a direct regulator of mitochondrial
PT E
DNA replication/transcription (Lin et al., 2005). Analysis of mutations in the TFAM and PGC-1 α genes may provide insights on the role of mitochondrial biogenesis in PCOS.
CE
Biogenesis of the mitochondrial oxidative phosphorylation enzyme complex requires the concordant expression of mtDNA and nDNA genes, which both encode mitochondrial
AC
proteins as well as their regulatory factors. A recent study has shown that a reduction in the expression of nuclear genes can be mostly attributed to a reduction of the mitochondrial function in the insulin-resistant women with PCOS (Skov et al., 2007). One of these main control factors is TFAM, which plays a main role in regulating mtDNA transcription and replication (Virbasius and Scarpulla, 1994; Montoya et al., 1997; Wang et al., 1999). An association of alterations in the TFAM gene has been reported in several human diseases such as Alzheimer's and Parkinson in which mitochondrial dysfunction plays a main role (Alvarez
ACCEPTED MANUSCRIPT et al., 2008 and Belin et al., 2007), but in PCOS it remains elusive. So, we studied the association of +35 G/C (rs1937) single-nucleotide polymorphism in the first exon of the TFAM gene with PCOS. We did not find statistically significant differences in the genotype frequencies for the TFAM +35G/C polymorphism between women with PCOS and controls. In addition, we studied the effect of this polymorphism on PCOS phenotypes, we analyzed
PT
biochemical variables according to this polymorphism, but we did not find a significant
RI
association between clinical parameters of the PCOS and each genotype.
SC
We further aimed to determine whether the peripheral blood mtDNA content observed in our PCOS patients would show some relation to gene variant of main
NU
mitochondrial copy number regulator, TFAM and found that no significant differences in the
MA
leukocyte mtDNA copy number among the genotypes in PCOS patients. However, mtDNA content is related to several other factors such as regulation of nuclear encoded genes, the mechanisms regarding regulation of mtDNA content are not entirely clear. Then, we studied
PT E
D
the SNP’s of PGC-1α gene, which is the main regulator of mitochondrial biogenesis. The PGC-1α gene exists in several variants which differ in a single nucleotide from
CE
the major allele. Among these SNPs, particularly the rs8192678 G/A polymorphism has been most extensively studied in different populations. However, the molecular as well as
AC
functional background of this SNP is largely unknown. A recent study provided an indication that the ‘A’ gene variant impairs the transcription of TFAM, which plays a vital role in mtDNA replication and transcription (Choi et al., 2006). Ling et al., (2004) have demonstrated that the rs8192678 (G/A) polymorphism was associated with reduced PGC-1α mRNA expression, suggesting that rs8192678 (G/A) may be an important functional SNP. Several studies have been performed to elucidate the role of the PGC-1α gene in PCOS. A study in the skeletal muscle of PCOS patients showed lower levels of expression in the PGC-
ACCEPTED MANUSCRIPT 1 gene when compared with controls, which confirms an impaired mitochondrial oxidative metabolism in PCOS (Skov et al., 2007). Then, we studied the rs8192678 (G/A) SNP in PGC-1α gene, which changes Glycine to serine in codon 482 in the functional domain; this variant is associated with either type 2
PT
diabetes pathogenesis or insulin resistance in several populations (Vimaleswaran et al., 2005). In the present study, for the first time we report a significant association between PGC-1α
RI
rs8192678 (G/A) polymorphism and PCOS risk in South Indian women. The AA genotype
SC
frequency was significantly higher in PCOS patients (P = 0.03). In addition, the ‘A’ allele frequency was also significantly higher in PCOS patients than controls. Our results indicate
NU
that PGC-1 α as a candidate gene for PCOS. However, two previous studies have been
MA
investigated the possible association between PCOS and the PGC-1α rs8192678 (G/A) polymorphism. Wang et al. (2006) reported that the PGC-1α rs8192678 (G/A) polymorphism
D
was not associated with PCOS in Chinese population. Another study in Korean women also
PT E
found no significant differences in the distribution of the PGC-1α rs8192678 (G/A) polymorphism with PCOS (Chae at al., 2010). The discrepant results in different populations imply ethnic difference, demographic location and/or sample size of PGC-1α polymorphisms
CE
distribution and suggest that the PGC-1 α gene may be a susceptibility factor for PCOS in
AC
South Indian population.
In our study, in addition to PGC-1α rs8192678 (G/A) polymorphism, we have also studied rs13131226 (intron 2) (T/C) polymorphism and the rs2970856 (intron 5) (T/C) polymorphism in South Indian women. We observed that no significant distribution of genotypes or allele of these polymorphisms between patients with PCOS and controls. Further, our findings have the potential for future clinical utility in the identification of individuals at a higher risk of developing PCOS. Further studies with more subjects and
ACCEPTED MANUSCRIPT genotyping of the entire PGC-1α gene are necessary for the better understanding of the correlation between PGC-1α gene SNPs and PCOS. PGC-1 could stimulate mitochondrial proliferation, Wu et al, (1999) examined the mtDNA content in the muscle cells and showed that increased approximately 2-fold by PGC-
PT
1α (Wu et al., 1999). A study showed that reduced levels of PGC-1α could play a role in the down regulation of OXPHOS genes in PCOS (Skov et al., 2007). Reduced levels of PGC-1α
RI
may lead to decrease in mtDNA content. We further aimed to determine whether the studied
SC
PGC-1α polymorphisms have some relation to clinical characteristics and peripheral blood mtDNA content in our PCOS patients. We analysed the peripheral blood leukocyte mtDNA
NU
content in relation with the aforementioned variants at the PGC-1α gene. Present study
MA
showed that ‘Ser’ mutation leads to decrease in the levels of mtDNA content in PCOS subjects. We further analysed the biochemical variables that affect PCOS patients in each genotype of studied polymorphisms and found significant association between LH levels and
PT E
D
‘CC’ genotype of rs2970856 T/C polymorphism. However, the functional significance of this polymorphism is unknown.
CE
In conclusion, our study indicates that ‘Ser’ allele may be involved in the pathophysiology of PCOS. Our study also indicates mtDNA copy number is not associated
AC
with TFAM +35G/C SNP in PCOS patients. To the best of our knowledge, this is the first study exploring the association of PGC-1α rs8192678 G/A polymorphism, TFAM +35G/C polymorphism and mtDNA copy number with PCOS risk in Indian population. Further, our findings have the potential for future clinical utility in the identification of individuals at a higher risk of developing PCOS.
Conflicts of interest:
ACCEPTED MANUSCRIPT The authors declare no conflict of interest. Author Contribution TV Reddy: execution of experiments, analysis and interpretation of data, statistical analysis and drafting of manuscript.
PT
S. Govatati: acquisition of data. M. Deenadayal: acquisition of data.
RI
S. Shivaji: analysis and interpretation of data, drafting of manuscript.
SC
M. Bhanoori: conception and design of study, analysis and interpretation of data, statistical
NU
analysis, drafting of manuscript Funding
MA
This study was supported in part by grants from the OU-DST PURSE Programme-II07/2017, Department of Science and Technology (DST), India to Dr. Manjula Bhanoori.
D
Acknowledgments
PT E
We are most grateful to all of the patients who participated in the present study. Tumu Venkat Reddy would like to thank University Grants Commission (UGC), India for providing
AC
CE
Basic Science Research (BSR-SRF) fellowship.
References 1.
Alvarez, V., Corao, A.I., Sánchez-Ferrero, E., De Mena, L., Alonso-Montes, C., Huerta, C., Blázquez, M., Ribacoba, R., Guisasola, L.M., Salvador, C., García-Castro, M., 2008. Mitochondrial transcription factor A (TFAM) gene variation in Parkinson's disease. Neurosci. Lett. 432, 79-82.
2.
Andrulionyte, L., Zacharova, J., Chiasson, J.L., Laakso, M., Stop-Niddm Study Group, 2004. Common polymorphisms of the PPAR-γ2 (Pro12Ala) and PGC-1α (Gly482Ser)
ACCEPTED MANUSCRIPT genes are associated with the conversion from impaired glucose tolerance to type 2 diabetes in the STOP-NIDDM trial. Diabetologia. 47, 2176-2184. 3.
Attardi, G., Schatz, G., 1998. Biogenesis of mitochondria. Annu. Rev. Cell Bio. 1, 289331.
4.
Barrett, J.C., Fry, B., Maller, J.D.M.J., Daly, M.J., 2005. Haploview: analysis and
Barroso, I., Luan, J., Sandhu, M.S., Franks, P.W., Crowley, V., Schafer, A.J., O’rahilly,
RI
5.
PT
visualization of LD and haplotype maps. Bioinformatics, 21, 263-265.
SC
S., Wareham, N.J., 2006. Meta-analysis of the Gly482Ser variant in PPARGC1A in type 2 diabetes and related phenotypes. Diabetologia. 49, 501-505. Belin, A.C., Björk, B.F., Westerlund, M., Galter, D., Sydow, O., Lind, C., Pernold, K.,
NU
6.
Rosvall, L., Håkansson, A., Winblad, B., Nissbrandt, H., 2007. Association study of
MA
two genetic variants in mitochondrial transcription factor A (TFAM) in Alzheimer's and Parkinson's disease. Neurosci. Lett. 420, 257-262. Chae, S.J., Kim, J.J., Choi, Y.M., Kim, J.M., Cho, Y.M., Moon, S.Y., 2010. Peroxisome
D
7.
PT E
proliferator-activated receptor-γ and its coactivator-1α gene polymorphisms in korean women with polycystic ovary syndrome. Gynecol Obstet Invest. 70, 1-7. Choi, Y.S., Hong, J.M., Lim, S., Ko, K.S., Pak, Y.K., 2006. Impaired coactivator
CE
8.
activity of the Gly 482 variant of peroxisome proliferator-activated receptor γ
AC
coactivator-1α (PGC-1α) on mitochondrial transcription factor A (Tfam) promoter. Biochem. Biophys. Res. Commun. 344, 708-712. 9.
Ekstrand, M.I., Falkenberg, M., Rantanen, A., Park, C.B., Gaspari, M., Hultenby, K., Rustin, P., Gustafsson, C.M., Larsson, N.G., 2004. Mitochondrial transcription factor A regulates mtDNA copy number in mammals. Hum. Mol. Gen. 13, 935-944.
ACCEPTED MANUSCRIPT 10.
Lee, H.C., Wei, Y.H., 2005. Mitochondrial biogenesis and mitochondrial DNA maintenance of mammalian cells under oxidative stress. Int. J. Biochem. Cell Biol. 37, 822-834.
11.
Lin, J., Handschin, C., Spiegelman, B.M., 2005. Metabolic control through the PGC-1 family of transcription coactivators. Cell Metab. 1, 361-370. Ling, C., Poulsen, P., Carlsson, E., Ridderstråle, M., Almgren, P., Wojtaszewski, J.,
PT
12.
RI
Beck-Nielsen, H., Groop, L., Vaag, A., 2004. Multiple environmental and genetic
SC
factors influence skeletal muscle PGC-1α and PGC-1β gene expression in twins. The Journal of clinical investigation, 114, 1518-1526.
Montoya, J., Perez-Martos, A., Garstka, H.L., Wiesner, R.J., 1997. Regulation of
NU
13.
mitochondrial transcription by mitochondrial transcription factor A. Mol. Cell.
14.
MA
Biochem. 174, 227-230.
O'Malley, B.W., Kumar, R., 2009 Nuclear receptor coregulators in cancer biology.
Palacín, M., Alvarez, V., Martín, M., Díaz, M., Corao, A.I., Alonso, B., Díaz-Molina,
PT E
15.
D
Cancer Res.69, 8217-22.
B., Lozano, I., Avanzas, P., Morís, C., Reguero, J.R., 2011. Mitochondrial DNA and
CE
TFAM gene variation in early-onset myocardial infarction: evidence for an association to haplogroup H. Mitochondrion. 11, 176-181. Palacio, J.R., Iborra, A.,
AC
16.
lcova Gallova,
., Badia, R., Martinez, P., 2006. The
presence of antibodies to oxidative modified proteins in serum from polycystic ovary syndrome patients. Clin. Exp. Immunol. 144, 217-222. 17.
Parisi, M., Clayton, D.A., 1991. Similarity of human mitochondrial transcription factor 1 to high mobility group proteins. Science. 252, 965.
ACCEPTED MANUSCRIPT 18.
Reyes, A., Mezzina, M., Gadaleta, G., 2002. Human mitochondrial transcription factor A (mtTFA): gene structure and characterization of related pseudogenes. Gene. 291, 223-232.
19.
Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. 2004. Revised 2003 consensus on diagnostic criteria and long-term health risks related to
Scarpulla, R.C., 2008 Transcriptional paradigms in mammalian mitochondrial
21.
SC
biogenesis and function. Physiol. Rev. 88, 611-38.
RI
20.
PT
polycystic ovary syndrome. Fertil. Steril. 81, 19–25.
Skov, V., Glintborg, D., Knudsen, S., Jensen, T., Kruse, T.A., Tan, Q., Brusgaard, K.,
NU
Beck-Nielsen, H., Højlund, K., 2007. Reduced expression of nuclear-encoded genes involved in mitochondrial oxidative metabolism in skeletal muscle of insulin-resistant
22.
MA
women with polycystic ovary syndrome. Diabetes, 56, 2349-2355. Teede, H., Deeks, A., Moran, L., 2010. Polycystic ovary syndrome: a complex
D
condition with psychological, reproductive and metabolic manifestations that impacts
23.
PT E
on health across the lifespan. BMC Med. 8, 41. Tumu, V.R., Govatati, S., Guruvaiah, P., Deenadayal, M., Shivaji, S., Bhanoori, M.,
CE
2013. An interleukin-6 gene promoter polymorphism is associated with polycystic ovary syndrome in South Indian women. J. Assist. Reprod. Genet. 301541-1546. Victor, V.M., Rocha, M., Bañuls, C., Alvarez, A., de Pablo, C., Sanchez-Serrano, M.,
AC
24.
Gomez, M., Hernandez-Mijares, A., 2011. Induction of oxidative stress and human leukocyte/endothelial cell interactions in polycystic ovary syndrome patients with insulin resistance. J. Clin. Endocrinol. Metab. 96, 3115-3122. 25.
Vimaleswaran, K.S., Radha, V., Ghosh, S., Majumder, P.P., Deepa, R., Babu, H.N.S., Rao, M.R.S., Mohan, V., 2005. Peroxisome proliferator‐ activated receptor‐ γ
ACCEPTED MANUSCRIPT co‐ activator‐ 1α (PGC‐ 1α) gene polymorphisms and their relationship to Type 2 diabetes in Asian Indians. Diabetic Med. 22, 1516-1521. 26.
Virbasius, J.V., Scarpulla, R.C., 1994. Activation of the human mitochondrial transcription factor A gene by nuclear respiratory factors: a potential regulatory link between nuclear and mitochondrial gene expression in organelle biogenesis. Proc. Natl.
PT
Acad. Sci. U.S.A. 91, 1309-1313.
Wallace, D.C., 2008. Mitochondria as chi. Genetics. 179, 727-735.
28.
Wang, Y., Wu, X., Cao, Y., Yi, L., Fan, H., Chen, J., 2006. Polymorphisms of the
RI
27.
SC
peroxisome proliferator–activated receptor-γ and its coactivator-1α genes in Chinese
29.
NU
women with polycystic ovary syndrome. Fertil Steril. 85, 1536-1540. Wang, J., Wilhelmsson, H., Graff, C., Li, H., Oldfors, A., Rustin, P., Brüning, J.C.,
atrioventricular
MA
Kahn, C.R., Clayton, D.A., Barsh, G.S., Thorén, P., 1999. Dilated cardiomyopathy and conduction blocks induced by heart-specific inactivation of
Wu, Z., Puigserver, P., Andersson, U., Zhang, C., Adelmant, G., Mootha, V., Troy, A.,
PT E
30.
D
mitochondrial DNA gene expression. Nature Genet. 21, 133-137.
Cinti, S., Lowell, B., Scarpulla, R.C., Spiegelman, B.M., 1999. Mechanisms controlling
CE
mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell. 98, 115-124.
Zhu, S., Liu, Y., Wang, X., Wu, X., Zhu, X., Li, J., Ma, J., Gu, H.F., Liu, Y., 2009.
AC
31.
Evaluation of the association between the PPARGC1A genetic polymorphisms and type 2 diabetes in Han Chinese population. Diabetes Res Clin Pract. 86, 168-172.
ACCEPTED MANUSCRIPT Figure legends Figure: 1 LD analysis of cases and controls are shown separately (A and B). Haploview plots are presented along with the SNPs studied. The pair wise linkage disequilibrium values (D' = 0– 100) of all SNPs are given in each diamond. A value of 100 represents maximum possible
AC
CE
PT E
D
MA
NU
SC
RI
PT
linkage disequilibrium. (A) LD analysis of cases and (B) LD analysis of controls.
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
CE
PT E
D
MA
Fig. 1
ACCEPTED MANUSCRIPT Table 1
Demographic and clinical characteristics of PCOS and control group
Controls
118
110
Age (years)
28.31±5.78
BMI (kg/m2)
22.85±5.85
FSH (mIU/ml)
LH (mIU/ml)
RI
Variable
SC
Number
0.0748
22.87±3.33
0.9750
5.67±1.6
6.18±3.38
0.1425
7.72±2.22
5.14±1.49
˂0.0001
0.98±0.69
˂0.0001
PT E
D
MA
NU
29.71±6.03
1.44±0.55
CE
LH:FSH ratio
p-value a
PT
PCOS
AC
Data are given as mean ± S.D.
p a values obtained by comparison of variables between controls and PCOS by Student’s t test
ACCEPTED MANUSCRIPT Table 2. PCR conditions and product size for the studied polymorphisms S.
SNP
Gene
Primer sequences 5′ to 3′
Locati
Mode
PCR
PCR
Referen
N
on
of
condition
produ
ce
o
(Base
analys
ct
change
is
(bp)
) TFA M
Exon1
RFLP
5′CCCCGCCCCCATCTACCG A3′
Denaturati on96°C(5min ) 35cycle:94°C 50 sec, 59°C - 45 sec 72°C-50 sec Extension72°C-10 min
GG: 200b p+ 126b p
Denaturati on96°C(5min ) 35cycle:94°C 50 sec, 60°C - 45 sec 72°C-50 sec Extension72°C-10 min
GG: 366b p+ 245b p
PT
Dde I 1 ( rs1937)
5′GACGTCCTGGGCCCTGCT G3′
MA
NU
SC
RI
+35 G/C
PGC -1α
Exon8
RFLP
5′CAAGTCCTCAGTCCTCAC 3′
D
Msp I (rs819267 8)
PT E
2
5′GGGGTCTTTGAGAAAAT AAGG3′
AC
CE
+482 G/A
3
Alu I (rs131312 26)
PGC -1α
Intron2 T/C
RFLP
5′TGACCAGCCATATCCCAA GT3′ 5′CAGAGACCAGTTTCCAC AGT 3′
Denaturati on96°C(5min ) 35cycle:94°C 50
(Palacín et al., 2011)
GC: 326b p+ 200b p+ 126b p CC: 326b p
(Zhu et al., 2009)
GA: 611b p+ 366b p+ 245b p AA: 611b p
(Zhu et CC: 120b p+ 169b p
al., 2009)
ACCEPTED MANUSCRIPT sec, 59°C - 45 sec 72°C-50 sec Extension72°C-10 min
CT: 289b p+ 120b p+ 169b p
PGC -1α
Intron5
RFLP
5′TGAAAAAGACACATTCC TGA3′
5′GGATAATTCATACAACTT CC3′
PT E
D
MA
NU
SC
T/C
CE
Denaturati on96°C(5min ) 35cycle:94°C 50 sec, 59°C - 45 sec 72°C-50 sec Extension72°C-10 min
RI
Alu I (rs297085 6)
AC
4
PT
TT: 289b p
CC: 389b p+ 44bp TC: 433b p+ 389b p+ 44bp TT: 433b p
(Zhu et al., 2009)
ACCEPTED MANUSCRIPT Table 3. Genotype and allele frequencies of TFAM +35G/C polymorphism in PCOS patients and controls Genotypes / Controls (%) Cases (%)
χ2 P-value
Odds ratio
95%CI
Alleles Genotypes 83 (70.33)
GC
21 (19.10)
35 (29.66)
CC
0
0
G
199 (90.45)
201 (85.16)
C
21 (9.55)
35(14.83)
1.0 (Reference) 0.0639a
0.9631 to 3.316
1.0 (Reference)
NU
0.0857b
Fisher’s exact test (3 × 2 table at 2 df ),
b
Fisher’s exact test (2 × 2 table at 1 df ),
MA
a
PT E
D
CI, confidence interval
CE
1.7871
SC
Alleles
AC
PT
89 (80.90)
RI
GG
0.606
0.3409 to 1.0774
ACCEPTED MANUSCRIPT
Table 4. Genotypes frequencies of studied polymorphism (TFAM) along with clinical characteristics and MCN in PCOS subjects
Variable
PT
PCOS (n=118) GC (n=35)
RI
GG (n=83) 28.43±5.69
Weight
55.62±14.52
BMI (kg/m-2)
22.38±5.87
FSH(mIU/mL)
LH(mIU/mL)
0.7333
0.2322
23.67±3.98
0.2370
5.56±1.63
5.93±1.51
0.2523
7.8±1.02
7.54±1.98
0.3493
1.50±0.61
1.32±0.36
0.1063
1.39±0.43
1.32±0.48
0.426
D
MA
NU
59.11±14.17
PT E
MCN
CE
LH:FSH Ratio
28.03±6.09
SC
Age
p a value
AC
Data are given as mean ± S.D. p a values obtained by comparison of variables between controls and PCOS by Student’s t test
ACCEPTED MANUSCRIPT Table 5. Genotype and allele distribution of PGC1-α polymorphisms in PCOS patients and controls of South Indian women Genotypes/Alleles
Controls
χ2 P-
Cases (%)
(%)
Odds ratio
95 % CI
values
Hw-0.6547 52 (47.27)
38 (32.21)
GA
47 (42.72)
60 (50.84)
AA
11 (10.00)
20 (16.95)
G
151 (68.64)
136 (57.63)
A
69 (31.36)
100 (42.37)
MA
D
rs13131226
0.015b
1.00
1.7469
0.9915 to 3.0777
2.488
1.0673 to 5.7998
1.00
1.00
1.6091
1.0955 to 2.3634
Hw-0.7131
PT E
Genotypes
0.047a
NU
Alleles
1.00
RI
GG
SC
Genotypes
PT
rs8192678
54 (49.09)
50 (42.37)
1.00
1.00
TC
47 (42.72)
55 (46.61)
1.2638
0.7311 to 2.1846
1.56
0.6137 to 3.9654
1.00
1.00
1.2462
0.8395 to 1.85
1.00
1.00
Alleles T C
9 (8.18)
13 (11.01)
155 (71.45)
155 (65.68)
65 (29.54)
81 (34.32)
AC
CC
CE
TT
0.541a
0.274
b
rs2970856 Hw-0.8539
Genotypes TT
67 (60.90)
66 (55.93)
ACCEPTED MANUSCRIPT TC
37 (33.63)
45 (38.13)
CC
6 (5.45)
7 (5.93)
T
171 (29.54)
177 (75.00)
C
49 (22.27)
59 (25.00)
0.7464a
1.2346
0.7109 to 2.1442
1.1843
0.3779 to 3.7112
1.00
1.00
1.1633
0.7543 to 1.7941
Alleles
b
PT
0.27473
AC
CE
PT E
D
MA
NU
SC
RI
Hw-Hardy-Weinberg equilibrium CI Confidence Interval, a Fisher’s exact test (3×2 table at 2 df) P<0.05 b Fisher’s exact test (2×2 table at 1 df) P<0.05
ACCEPTED MANUSCRIPT Table 6. PGC-1 α genotypes of studied Polymorphisms by clinical variables along with MCN in PCOS subjects p
p rs13131226 (T/C)
rs2970856
val
val p val ues
rs8192678 (G/A) ues
(T/C) ues
a
29.19
27.86
28.25
0.5
28.22
28.75
26.84
Age
±7.19
±5.55
±3.14
59
±6.03
±5.67
BMI
22.68
22.40
24.68
0.3
23.08
22.63
(kg/m-2)
±6.60
±5.84
±4.19
17
±5.25
FSH(mI
5.46±
5.80±
5.78±
0.5
5.47±
U/mL)
1.44
1.77
1.35
69
1.50
LH(mIU
7.38±
7.93±
/mL)
2.32
2.23
LH:FSH
1.38±
1.51±
Ratio
0.37
PT
CC
TT
0.5
a
TC
CC
27.75
28.61
32.17
0.1
RI
TC
±5.49
66
±6.23
±4.57
±8.13
85
NU
AA
22.93
0.9
23.27
22.07
24.00
0.5
±8.02
21
±3.98
±7.92
±5.40
00
5.73±
6.23±
0.2
5.46±
6.04±
5.29±
0.1
1.79
1.01
99
1.60
1.52
1.98
43
±5.90
MA
GA
MCN
PT E
D
GG
SC
TT
CE
a
0.4
7.22±
7.98±
8.61±
0.0
7.24±
8.36±
8.14±
0.0
2.02
81
2.24
2.27
1.56
71
1.88
2.55
2.03
30
1.32±
0.4
1.37±
1.53±
1.39±
0.3
1.43±
1.41±
1.79±
0.2
0.71
0.36
96
0.42
0.71
0.14
27
0.61
0.35
1.00
36
1.47±
1.33±
1.26±
0.0
1.32±
1.37±
1.42±
0.2
1.37±
1.36±
1.37±
0.9
0.46
0.47
0.32
32
0.45
1.47
0.28
04
0.47
0.42
0.50
91
AC
7.89±
Data are given as mean ± S.D. p a values obtained by comparison of variables between genotypes in PCOS by one-way ANNOVA test followed by least significance test.
ACCEPTED MANUSCRIPT
Table 7. Haplotype frequencies of PGC-1α polymorphisms in PCOS patients and controls Haplotypes
Haplotype frequency P-valuea
15056881
23825992
G/A
T/C
T/C
G
T
G
95% CI
T
94 (39.83)
102 (46.36)
Reference
C
T
20 (8.47)
23 (9.74)
0.86249
1.05
0.5469 to 2.0537
G
T
C
12 (5.08)
15 (6.82)
1.15
0.5129 to 2.5875
G
C
C
10 (4.23)
11 (5.00)
0.97477
1.01
0.4117 to 2.496
A
T
T
35 (14.83)
25 (11.36)
0.1596
0.66
0.3668 to 1.1814
A
C
T
30 (12.71)
21 (9.55)
0.16685
0.64
0.3456 to 1.2041
A
T
C
10 (4.23)
10 (4.54)
0.86249
0.92
0.3672 to 2.3131
A
C
C
25 (10.59)
0.0442
0.48
0.2318 to 0.9908
0.73121
SC
NU
MA
D
AC
CE
PT E
Fisher’s exact (2x2 table at I df); P < 0.05
PT
Controls (%)
CI: confidence interval a
OR
PCOS (%)
RI
+1564
13 (5.91)
ACCEPTED MANUSCRIPT Abbreviations: CI, confidence interval; D', disequilibrium coefficient; G, glycine; GG, glycine/glycine; GS, glycine/serine; FSH, follicle-stimulating hormone; IIRC, infertility institute and research centre; LD, linkage disequilibrium; LH, luteinizing hormone; MCN, mtDNA copy number; NRF,
nuclear
respiratory
factor;
NRs,
nuclear
receptors;
OXPHOS,
oxidative
PT
phosphorylation; PCOS, polycystic ovarian syndrome; PCR-RFLP, polymerase chain
RI
reaction-restriction fragment length polymorphism; PGC-1α, peroxisome proliferator
SC
activated receptor gamma coactivator-1 alpha; ROS, reactive oxygen species; SNPs, single nucleotide polymorphisms; SS, serine/serine; TFAM, mitochondrial transcription factor A;
AC
CE
PT E
D
MA
NU
TFs, transcription factors.
ACCEPTED MANUSCRIPT Highlights
The study showed significant differences between the distribution of the genotypes and alleles of the PGC-1α rs8192678 (G/A) polymorphism in PCOS patients and control groups
We observed patients with the AA genotype showed significantly lower levels of
PT
MCN compared with patients with GA and GG genotypes of rs8192678 (G/A)
Our study indicates ‘Ser’ allele of rs8192678 SNP could be involved in the
SC
RI
polymorphism.
pathophysiology of PCOS.
NU
The study showed significantly higher levels of LH in patients with the PGC1-α
CE
PT E
D
MA
rs2970856 CT genotype when compared to other genotypes.
AC