Rajender Singh

Longer CAG repeat length in the androgen receptor gene is associated with premature ovarian failure

BACKGROUND Premature ovarian failure (POF) is a disorder characterized by lack of ovulation and elevated serum gonadotrophin levels before the age of 40 years. The cause of POF in most cases is unknown. As mice lacking the Androgen receptor (Ar) gene reportedly have a POF-like phenotype, we hypothesize that, variations in the AR gene maybe one of the causative factors for POF in humans. Thus the objective of the study is to evaluate the number of CAG repeats in exon 1 of the AR gene in non-familial, non-syndromic cases of POF. METHODS A clinic-based case-control study. Seventy-eight patients with non-familial, non-syndromic POF, and 90 controls were recruited to investigate the CAG repeat numbers in exon 1 of the AR gene by PCR and Gene Scan analysis. RESULTS The mean CAG repeat length in exon 1 of the AR gene of women with POF was 23.6 +/- 3.8, which was significantly higher than controls (20.08 +/- 3.45) (P < 0.001). The biallelic mean CAG repeat ranged from 11 to 32 in the control women, compared to 16 to 30 in the POF patients. The 22 CAG repeat allele followed by the 24 CAG repeat allele was found to be at highest frequency (15.38 and 12.8%) in POF cases, although the 19 CAG repeat allele was observed at highest frequency (12.2%) in controls. CONCLUSIONS The observation suggests that the CAG repeat length is increased in women with POF as compared with controls, and may be pathogenic for POF, at least in a subset of Indian women.

Is there an inter-relationship between prostate specific antigen, kallikrein-2 and androgen receptor gene polymorphisms with risk of prostate cancer in north Indian population?

Androgen receptor (AR) and kallikrein (KLK-2) regulates the PSA (prostate specific antigen) transcription and activation, respectively. We investigated the individual and combined risk of KLK-2, PSA and AR gene polymorphism in histologically confirmed CaP patients and healthy controls from north India. DNA was extracted from peripheral blood leucocytes pellet of 277 subjects. AR repeats analysis was done by PCR-Genscan method. PSA and KLK-2 were genotyped by PCR-RFLP method. Kruskal-Wallis test and logistic regression was applied for mean comparison and risk determination. A significant association for CaP risk was observed with short AR-CAG repeats (OR=3.36, p<0.001) and CC genotype of KLK-2 (OR=2.78, p=0.031), however, no association was found with PSA and AR-GGN repeat polymorphism. PSA/GG genotype was significantly associated with higher Gleason score (> or =7) of tumor (OR=6.23, p<0.01). No association was observed with other confounding variables such as PSA and age with any of these polymorphisms. Thus, we hypothesize that these polymorphisms may influence the etiology of CaP and may have the probability to become appropriate marker either independently or in combination. The combined information on serum PSA level, PSA (G/A), KLK-2 (C/T) genotypes and AR (CAG; GGN repeat) may assist in the deterrence of unnecessary biopsies.

IL-4 Haplotype-590T,-34T and Intron-3 VNTR R2 Is Associated with Reduced Malaria Risk among Ancestral Indian Tribal Populations

Background Interleukin 4 (IL-4) is an anti-inflammatory cytokine, which regulates balance between TH1 and TH2 immune response, immunoglobulin class switching and humoral immunity. Polymorphisms in this gene have been reported to affect the risk of infectious and autoimmune diseases. Methods We have analyzed three regulatory IL-4 polymorphisms; -590C>T, -34C>T and 70 bp intron-3 VNTR, in 4216 individuals; including: (1) 430 ethnically matched case-control groups (173 severe malaria, 101 mild malaria and 156 asymptomatic); (2) 3452 individuals from 76 linguistically and geographically distinct endogamous populations of India, and (3) 334 individuals with different ancestry from outside India (84 Brazilian, 104 Syrian, and 146 Vietnamese). Results The -590T, -34T and intron-3 VNTR R2 alleles were found to be associated with reduced malaria risk (P<0.001 for -590C>T and -34C>T, and P = 0.003 for VNTR). These three alleles were in strong LD (r2>0.75) and the TTR2 (-590T, -34T and intron-3 VNTR R2) haplotype appeared to be a susceptibility factor for malaria (P = 0.009, OR = 0.552, 95% CI = 0.356 –0.854). Allele and genotype frequencies differ significantly between caste, nomadic, tribe and ancestral tribal populations (ATP). The distribution of protective haplotype TTR2 was found to be significant (χ2 3 = 182.95, p-value <0.001), which is highest in ATP (40.5%); intermediate in tribes (33%); and lowest in caste (17.8%) and nomadic (21.6%). Conclusions Our study suggests that the IL-4 polymorphisms regulate host susceptibility to malaria and disease progression. TTR2 haplotype, which gives protection against malaria, is high among ATPs. Since they inhabited in isolation and mainly practice hunter-gatherer lifestyles and exposed to various parasites, IL-4 TTR2 haplotype might be under positive selection.

A novel Arg615Ser mutation of androgen receptor DNA-binding domain in three 46,XY sisters with complete androgen insensitivity syndrome and bilateral inguinal hernia

OBJECTIVE To present clinical, genetic, biochemical, and molecular findings in a family with three sisters with complete androgen insensitivity syndrome (CAIS) and bilateral inguinal hernia. DESIGN Case report. SETTING Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Hyderabad, India; Centre for Cellular and Molecular Biology, Hyderabad, India. PATIENT(S) A family with four sisters of female phenotype, three of whom have CAIS and bilateral inguinal hernia. Their parents also were screened. INTERVENTION(S) Chromosomal analyses by harvesting lymphocytes from peripheral blood sample; hormonal analysis from serum; gene sequencing. MAIN OUTCOME MEASURE(S) Karyotype, testosterone, follicle-stimulating hormone, and luteinizing hormone evaluation, sex-determining region Y (SRY) and androgen receptor (AR) gene sequencing. RESULT(S) In three sisters of karyotype 46,XY, we found a novel mutation of the single nucleotide transition C to A at position 2205 (GenBank accession number GU784857), resulting in amino acid interchange arginine to serine at codon 615 in exon 3 [R615S] (GenBank Protein_id ADD26779.1). The mother (46,XX) is a heterozygous carrier, and the fourth sister (46,XX) and father (46,XY) are normal. CONCLUSION(S) We report a novel missense mutation in exon 3 of the androgen receptor gene. The nature of the mutation presented is in the highly conserved DNA-binding domain of the AR gene, implicating it in the phenotypes observed with severe undervirilization in three sisters.

A case of oligoasthenoteratozoospermia with AZFc deletion and persistent oxidative stress

Y-chromosomal microdeletions are associated with severe oligozoospermia or azoospermia. AZFc microdeletions have been always associated with severe oligozoospermia or azoospermia with a rare occurrence in individuals with other infertility phenotypes. We report here a rare case of an infertile man carrying AZFc deletion, whose semen picture is oligoasthenoteratozoospermia complexed with seminal oxidative stress. Anti-oxidant therapy could make no change in either oxidative stress biomarker levels of semen, seminal parameters or serum hormone levels. Therefore, oligoasthenoteratozoospermia in the present case correlates with AZFc deletion, and high content of abnormal sperm eventually might be responsible for persistently elevated reactive oxygen species levels. Understanding the function of genes in AZFc region could help decipher the exact cause of the phenotype in such cases.

Prevalence of oral cancer and pre-cancerous lesions and the association with numerous risk factors in North India: A hospital based study

Background: Oral cancer is one of the most common life-threatening diseases all over the world. Developing countries face several challenges to identify and remove potential risk factors. Chewing tobacco/pan masala is considered to be the most potent risk factor for oral precancerous lesions and oral cancer. Objectives: To investigate the relative occurrence of different oral pre-cancerous lesions and oral cancer in North India and to identify the associated risk factors. Materials and Methods: A hospital-based study was conducted and 471 subjects were recruited in the study. The subjects comprised patients with squamous cell carcinoma (n = 85), oral submucous fibrosis (n = 240), leukoplakia (n = 32), lichen planus (n = 15), and controls (n = 99). Statistical analysis of the data was done using Chi-square and regression analysis. Results: A strong correlation was observed between the presence of the chewing habit in all the oral precancerous lesions and oral cancer. Duration of the habit and intensity of habit ware also strongly correlated with the risk of oral precancerous lesions and oral cancer. Other factors such as alcohol and smoking were found to be less important in concern with oral cancer and precancerous lesions.

RHOG-DOCK1-RAC1 Signaling Axis Is Perturbed in DHEA-Induced Polycystic Ovary in Rat Model.

The function of RHOG, a RAC1 activator, was explored in the ovary during ovarian follicular development and pathological conditions. With the help of immunoblotting and immunolocalization, we determined the expression and localization of RHOG in normal (estrous cycle) and polycystic ovaries using Sprague Dawley (SD) rat model. Employing polymerase chain reaction and flow cytometry, we analyzed the transcript and expression levels of downstream molecules of RHOG, DOCK1, and RAC1 in the polycystic ovarian syndrome (PCOS) ovary along with normal antral follicular theca and granulosa cells after dehydroepiandrosterone (DHEA) supplementation. The effect of RHOG knockdown on DOCK1, VAV, and RAC1 expression was evaluated in the human ovarian cells (SKOV3), theca cells, and granulosa cells from SD rats with the help of flow cytometry. Oocyte at secondary follicles along with stromal cells showed optimal expression of RHOG. Immunoblotting of RHOG revealed its maximum expression at diestrus and proestrus, which was downregulated at estrus stage. Mild immunostaining of RHOG was also present in the theca and granulosa cells of the secondary and antral follicles. Polycystic ovary exhibited weak immunostaining for RHOG and that was corroborated by immunoblotting-based investigations. RHOG effectors DOCK1 and ELMO1 were found reduced in the ovary in PCOS condition/DHEA. RHOG silencing reduced the expression of DOCK1 and RAC1 in the theca and granulosa cells from SD rat antral follicles and that was mirrored in the human ovarian cells. Collectively, RHOG can mediate signaling through downstream effectors DOCK1 and RAC1 during ovarian follicular development (theca and granulosa cells and oocyte), but DHEA downregulated them in the PCOS ovary.The function of RHOG, a RAC1 activator, was explored in the ovary during ovarian follicular development and pathological conditions. With the help of immunoblotting and immunolocalization, we determined the expression and localization of RHOG in normal (estrous cycle) and polycystic ovaries using Sprague Dawley (SD) rat model. Employing polymerase chain reaction and flow cytometry, we analyzed the transcript and expression levels of downstream molecules of RHOG, DOCKI, and RACI in the polycystic ovarian syndrome (PCOS) ovary along with normal antral follicular theca and granulosa cells after dehydroepiandrosterone (DHEA) supplementation. The effect of RHOG knockdown on DOCKI, VAV, and RACI expression was evaluated in the human ovarian cells (SKOV3), theca cells, and granulosa cells from SD rats with the help of flow cytometry. Oocyte at secondary follicles along with stromal cells showed optimal expression of RHOG. Immunoblotting of RHOG revealed its maximum expression at diestrus and proestrus, which was downregulated at estrus stage. Mild immunostaining of RHOG was also present in the theca and granulosa cells of the secondary and antral follicles. Polycystic ovary exhibited weak immunostaining for RHOG and that was corroborated by immunoblotting-based investigations. RHOG effectors DOCK1 and ELMO1 were found reduced in the ovary in PCOS condition/DHEA. RHOG silencing reduced the expression of DOCK1 and RAC1 in the theca and granulosa cells from SD rat antral follicles and that was mirrored in the human ovarian cells. Collectively, RHOG can mediate signaling through downstream effectors DOCK1 and RAC1 during ovarian follicular development (theca and granulosa cells and oocyte), but DHEA downregulated them in the PCOS ovary.

C601S mutation in the androgen receptor results in partial loss of androgen function

The present study was undertaken on a case of partial androgen insensitivity syndrome to look at the etiology of the disorder. The patient exhibited a female phenotype despite 46,XY chromosome complement. Direct DNA sequencing of coding region of the androgen receptor gene in this case revealed a 2329G>C substitution (cDNA sequence reference) in exon 3 of the gene. The substitution resulted in replacement of Cys with Ser at codon 601 of the ligand-binding domain of the protein. Analyses on 200 control samples revealed absence of this substitution(s). In vitro assays were done using COS-1 cells. The mutation resulted in partial (∼40%) loss of ligand-binding and significant (∼70%) loss of downstream transactivation function. The mutation was absent in the controls. The findings are particularly interesting since another substitution at the same codon (TGC-TTC) has been reported in association with complete androgen insensitivity syndrome.

Sex Chromosomal Genes in Male Infertility

Y chromosome harbors the male-specific region (MSY) that regulates male sex determination and spermatogenesis. Y microdeletions are the most common cause of male infertility. These deletions are found in 15–20% of patients with idiopathic azoospermia and 7–10% of patients with severe oligozoospermia. Apart from microdeletions, partial deletions in the AZFc region result in loss of multiple copies of Y genes and increase the risk of infertility. A few studies have suggested that routine screening of these deletions could help in understanding the etiology, offering counseling and managing infertility by natural or assisted methods. X being a homologue chromosome of Y has drawn attention regarding the presence of spermatogenic genes. A number of theories and speculations have been put forward that are now supported by the identification of a number of testis-specific or testis-predominant genes present on the X chromosome. This chapter provides an overview of the Y deletions and X chromosome genes that affect spermatogenesis or male fertility.

Lifestyle, Fertility, and Infertility Management

With technological advancements, we have evolved into a species that is surrounded by a number of potential hazards that may endanger our own survival. One of the important aspects of the effect of modern lifestyle is increased incidence of infertility. Renewal being the most important requirement for a species, fertility loss can have radical consequences. Since fertility does not need to be earned, it is taken to be immune to changes in lifestyle and surroundings. While a number of poor lifestyle practices are adopted, we fall prey to other hazards inadvertently. We recognized the effect of lifestyle on fertility a little late, but fortunately it is neither too late nor too difficult to confront the lifestyle factors that may take a heavy toll on fertility. In this chapter, I have provided the most comprehensive review of a number of lifestyle factors that matter to fertility and simple ways to overcome their potential perils. Following a great lifestyle ardently may be a difficult task, but can have great therapeutic rewards.