Nishi Gupta

Strong Association of 677 C>T Substitution in the MTHFR Gene with Male Infertility - A Study on an Indian Population and a Meta-Analysis

Background Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme of folate and methionine metabolism, making it crucial for DNA synthesis and methylation. The objective of this study was to analyze MTHFR gene 677C>T polymorphism in infertile male individuals from North India, followed by a meta-analysis on our data and published studies. Methodology/Principal Findings We undertook genotyping on a total of 837 individuals including well characterized infertile (N = 522) and confirmed fertile (N = 315) individuals. The SNP was typed by direct DNA sequencing. Chi square test was done for statistical analysis. Published studies were searched using appropriate keywords. Source of data collection for meta-analysis included ‘Pubmed’, ‘Ovid’ and ‘Google Scholar’. Those studies analyzing 677C>T polymorphism in male infertility and presenting all relevant data were included in meta-analysis. The genotype data for infertile subjects and fertile controls was extracted from each study. Chi square test was done to obtain odds ratio (OR) and p-value. Meta-analysis was performed using Comprehensive Meta-analysis software (Version 2). The frequency of mutant (T) allele (p = 0.0025) and genotypes (CT+TT) (p = 0.0187) was significantly higher in infertile individuals in comparison to fertile controls in our case-control study. The overall summary estimate (OR) for allele and genotype meta-analysis were 1.304 (p = 0.000), 1.310 (p = 0.000), respectively, establishing significant association of 677C>T polymorphism with male infertility. Conclusions/Significance 677C>T substitution associated strongly with male infertility in Indian population. Allele and genotype meta-analysis also supported its strong correlation with male infertility, thus establishing it as a risk factor.

Differential Genes Expression between Fertile and Infertile Spermatozoa Revealed by Transcriptome Analysis

Background It was believed earlier that spermatozoa have no traces of RNA because of loss of most of the cytoplasm. Recent studies have revealed the presence of about 3000 different kinds of mRNAs in ejaculated spermatozoa. However, the correlation of transcriptome profile with infertility remains obscure. Methods Total RNA from sperm (after exclusion of somatic cells) of 60 men consisting of individuals with known fertility (n=20), idiopathic infertility (normozoospermic patients, n=20), and asthenozoospermia (n=20) was isolated. After RNA quality check on Bioanalyzer, AffymetrixGeneChip Human Gene 1.0 ST Array was used for expression profiling, which consisted of >30,000 coding transcripts and >11,000 long intergenic non-coding transcripts. Results Comparison between all three groups revealed that two thousand and eighty one transcripts were differentially expressed. Analysis of these transcripts showed that some transcripts [ribosomal proteins (RPS25, RPS11, RPS13, RPL30, RPL34, RPL27, RPS5), HINT1, HSP90AB1, SRSF9, EIF4G2, ILF2] were up-regulated in the normozoospermic group, but down-regulated in the asthenozoospermic group in comparison to the control group. Some transcripts were specific to the normozoospermic group (up-regulated: CAPNS1, FAM153C, ARF1, CFL1, RPL19, USP22; down-regulated: ZNF90, SMNDC1, c14orf126, HNRNPK), while some were specific to the asthenozoospermic group (up-regulated: RPL24, HNRNPM, RPL4, PRPF8, HTN3, RPL11, RPL28, RPS16, SLC25A3, C2orf24, RHOA, GDI2, NONO, PARK7; down-regulated: HNRNPC, SMARCAD1, RPS24, RPS24, RPS27A, KIFAP3). A number of differentially expressed transcripts in spermatozoa were related to reproduction (n = 58) and development (n= 210). Some of these transcripts were related to heat shock proteins (DNAJB4, DNAJB14), testis specific genes (TCP11, TESK1, TSPYL1, ADAD1), and Y-chromosome genes (DAZ1, TSPYL1). Conclusion A complex RNA population in spermatozoa consisted of coding and non-coding RNAs. A number of transcripts that participate in a host of cellular processes, including reproduction and development were differentially expressed between fertile and infertile individuals. Differences between comparison groups suggest that sperm RNA has strong potential of acting as markers for fertility evaluation.

Significant Impact of the MTHFR Polymorphisms and Haplotypes on Male Infertility Risk

Background Methylenetetrahydrofolate reductase (MTHFR) converts 5,10-methylene tetrahydrofolate to 5-methyl tetrahydrofolate and affects the activity of cellular cycles participating in nucleotide synthesis, DNA repair, genome stability, maintenance of methyl pool, and gene regulation. Genetically compromised MTHFR activity has been suggested to affect male fertility. The objective of the present study was to find the impact on infertility risk of c.203G>A, c.1298A>C, and c.1793G>A polymorphisms in the MTHFR gene. Methods PCR-RFLP and DNA sequencing were used to genotype the common SNPs in the MTHFR gene in 630 infertile and 250 fertile males. Chi-square test was applied for statistical comparison of genotype data. Linkage disequilibrium between the SNPs and the frequency of common haplotypes were assessed using Haploview software. Biochemical levels of total homocysteine (tHcy) and folic acid were measured. Meta-analysis on c.1298A>C polymorphism was performed using data from ten studies, comprising 2734 cases and 2737 controls. Results c.203G>A and c.1298A>C were found to be unrelated to infertility risk. c.1793G>A was protective against infertility (P = 0.0008). c.677C>T and c.1793G>A were in significant LD (D’ = 0.9). Folic acid and tHcy level did not correlate with male infertility. Pooled estimate on c.1298A>C data from all published studies including our data showed no association of this polymorphism with male infertility (Odds ratio = 1.035, P = 0.56), azoospermia (Odds ratio = 0.97, P = 0.74), or oligoasthenoteratozoospermia (Odds ratio = 0.92, p = 0.29). Eight haplotypes with more than 1% frequency were detected, of which CCGA was protective against infertility (p = 0.02), but the significance of the latter was not seen after applying Bonferroni correction. Conclusion Among MTHFR polymorphisms, c.203G>A and c.1298A>C do not affect infertility risk and c.1793G>A is protective against infertility. Haplotype analysis suggested that risk factors on the MTHFR locus do not extend too long on the DNA string.

Gr/gr deletions on Y-chromosome correlate with male infertility: an original study, meta-analyses, and trial sequential analyses.

We analyzed the AZFc region of the Y-chromosome for complete (b2/b4) and distinct partial deletions (gr/gr, b1/b3, b2/b3) in 822 infertile and 225 proven fertile men. We observed complete AZFc deletions in 0.97% and partial deletions in 6.20% of the cases. Among partial deletions, the frequency of gr/gr deletions was the highest (5.84%). The comparison of partial deletion data between cases and controls suggested a significant association of the gr/gr deletions with infertility (P = 0.0004); however, the other partial deletions did not correlate with infertility. In cohort analysis, men with gr/gr deletions had a relatively poor sperm count (54.20 ± 57.45 million/ml) in comparison to those without deletions (72.49 ± 60.06), though the difference was not statistically significant (p = 0.071). Meta-analysis also suggested that gr/gr deletions are significantly associated with male infertility risk (OR = 1.821, 95% CI = 1.39–2.37, p = 0.000). We also performed trial sequential analyses that strengthened the evidence for an overall significant association of gr/gr deletions with the risk of male infertility. Another meta-analysis suggested a significant association of the gr/gr deletions with low sperm count. In conclusion, the gr/gr deletions show a strong correlation with male infertility risk and low sperm count, particularly in the Caucasian populations.

MTHFR 677C>T Polymorphism and the Risk of Breast Cancer: Evidence from an Original Study and Pooled Data for 28031 Cases and 31880 Controls

Background Methylenetetrahydrofolate reductase (MTHFR) acts at an important metabolic point in the regulation of cellular methylation reaction. It assists in the conversion of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. The latter aids in remethylation of homocysteine to de novo methionine that is required for DNA synthesis. The objective of this study was to examine the effect of MTHFR 677 C>T polymorphism on the risk of breast cancer in the Indian sub-continent. Methods and Results We genotyped 677 C>T locus in 1096 individuals that were classified into cases (N=588) and controls (N=508). Genotype data were analyzed using chi-square test. No significant difference was observed in the distribution of genotypes between cases and controls in north Indian (P = 0.932), south Indian (P = 0.865), and pooled data (P = 0.680). To develop a consensus regarding the impact of 677C>T polymorphism on breast cancer risk, we also conducted a meta-analysis on 28031 cases and 31880 controls that were pooled from sixty one studies. The overall summary estimate upon meta-analysis suggested no significant correlation between the 677C>T substitution and breast cancer in the dominant model (Fixed effect model: OR = 0.97, P=0.072, Random effects model: OR = 0.96, P = 0.084) or the recessive model (Fixed effect model: OR = 1.05, P = 0.089; Random effects model: OR= 1.08, P= 0.067). Conclusion 677 C>T substitution does not affect breast cancer risk in the Indo-European and Dravidian populations of India. Analysis on pooled data further ruled out association between the 677 C>T polymorphism and breast cancer. Therefore, 677 C>T substitution does not appear to influence the risk of breast cancer.

The TNP1 haplotype – GCG is associated with azoospermia

Transition nuclear proteins (TNP1 and TNP2) are the major nuclear proteins that replace somatic histones during spermatogenesis. TNPs are required for the maintenance of normal spermatogenesis. Moreover, spermatogenesis was found to be compromised in both Tnp1 and Tnp2 null mice. As no study is available on the role of these genes in Indian infertile men, we have sequenced the entire TNP1 and TNP2 genes in 320 infertile men and 280 control fertile men drawn from two states in India. We identified 18 variants, including 8 previously known and 10 novel. Of the 10 novel variants, 3 were found only in azoospermic men, of which 2 (g.-688A>T in TNP1 and g.1030G>A in TNP2) were predicted to affect the transcription factor binding sites and therefore can cause deregulation of gene expression. Haplotype association analysis showed a significant omnibus association (omnibus χ(2) = 7.87, p = 0.0195) for the single nucleotide polymorphisms (SNPs) in the TNP1 gene with azoospermia. The frequency of the haplotype GCG (H3) was increased in azoospermic men (53.1%) compared with fertile men (43%; χ(2) = 7.964, p = 0.005). However, similar analysis of the TNP2 gene did not show any association with infertility. Furthermore, expression analysis of the TNP1 gene in obstructive azoospermic men showed that haplotypes of the TNP1 gene do not affect its expression level. Our results suggest that the individual SNPs of the TNP1 and TNP2 genes are not associated with infertility; however, the haplotype GCG of the TNP1 gene is a risk factor for azoospermia.

CYP1B1 gene polymorphisms correlate with an increased risk of urinary bladder cancer in India

PURPOSE The urinary bladder is the target of several toxic compounds, which makes the bladder more prone to cancer. Cytochrome P450 1B1 enzyme is present in tumor tissues and metabolizes the polyaromatic carcinogens and activates several procarcinogens that cause DNA damage. We examined the functional single-nucleotide polymorphisms in the CYP1B1 gene to study their association with the urinary bladder cancer. METHODS We recruited 234 cases of pure urothelial and 258 bladder cancer-free control samples from the individuals visiting the clinic for various investigations. We genotyped 4 CYP1B1 single-nucleotide polymorphisms using the polymerase chain reaction-restriction fragment length polymorphism analysis. The genotype data were analyzed by the Chi-square test. Haplotypes were constructed to evaluate the joint effect of the 4 polymorphisms. RESULTS Overall, 3 polymorphisms-rs10012, rs150799650, and rs1056827 (odds ratio [OR] = 2.34, CI: 1.59-3.45, P<0.0001; OR = 2.59, CI: 1.78-3.77, P<0.0001; OR = 2.32, CI: 1.57-3.44, P<0.0001, respectively) were found to correlate with urinary bladder cancer. Haplotype analysis suggested GTTC, GTTG, and ATGC to be the risk factors for bladder cancer. CONCLUSIONS Overall, 3 polymorphisms, rs10012, rs1056827, and rs150799650 in the CYP1B1 gene correlate with urinary bladder cancer significantly in the Indo-European population of Uttar Pradesh, India. Further investigations in other populations are advised.

Mutations in the prostate specific antigen ( PSA / KLK3 ) correlate with male infertility

Prostate specific antigen (PSA/KLK3) is known to be the chief executor of the fragmentation of semenogelins, dissolution of semen coagulum, thereby releasing sperm for active motility. Recent research has found that semenogelins also play significant roles in sperm fertility by affecting hyaluronidase activity, capacitation and motility, thereby making PSA important for sperm fertility beyond simple semen liquefaction. PSA level in semen has been shown to correlate with sperm motility, suggesting that PSA level/activity can affect fertility. However, no study investigating the genetic variations in the KLK3/PSA gene in male fertility has been undertaken. We analyzed the complete coding region of the KLK3 gene in ethnically matched 875 infertile and 290 fertile men to find if genetic variations in KLK3 correlate with infertility. Interestingly, this study identified 28 substitutions, of which 8 were novel (not available in public databases). Statistical comparison of the genotype frequencies showed that five SNPs, rs266881 (OR = 2.92, P < 0.0001), rs174776 (OR = 1.91, P < 0.0001), rs266875 (OR = 1.44, P = 0.016), rs35192866 (OR = 4.48, P = 0.025) and rs1810020 (OR = 2.08, P = 0.034) correlated with an increased risk of infertility. On the other hand, c.206 + 235 T > C, was more freuqent in the control group, showing protective association. Our findings suggest that polymorphisms in the KLK3 gene correlate with infertility risk.

RETRACTED ARTICLE: Transcriptome profile alterations in asthenozoospermic and idiopathic infertile spermatozoa uncovered by microarray

This article has been retracted at the request of the Editor-in- Chief. The original submission was made without the approval of the previously listed co-author Dr. Rajender Singh. In addition, the article is being retracted because the corresponding author is not the owner of the data and has no right to publication.