Exome sequencing and functional analyses revealed CETN1 variants leads to impaired cell division and male fertility.

Human spermatogenesis requires an orchestrated expression of numerous genes in various germ cell subtypes. Therefore, the genetic landscape of male infertility is highly complex. Known genetic factors alone account for at least 15% of male infertility. However, ~40% of infertile men remain undiagnosed and are classified as idiopathic infertile men. We performed exome sequencing in 47 idiopathic infertile men (discovery cohort), followed by replication study (40 variants in 33 genes) in 844 infertile men and 709 controls using Sequenom MassARRAY® based genotyping. We report 17 variants in twelve genes that comprise both previously reported (DNAH8, DNAH17, FISP2 and SPEF2) and novel candidate genes (BRDT, CETN1, CATSPERD, GMCL1, SPATA6, TSSK4, TSKS and ZNF318) for male infertility. The latter have a strong biological nexus to human spermatogenesis and their respective mouse knockouts are concordant with human phenotypes. One candidate gene CETN1, identified in this study, was sequenced in another independent cohort of 840 infertile and 689 fertile men. Further, CETN1 variants were functionally characterized using biophysical and cell biology approaches. We demonstrate that CETN1 variant- p.Met72Thr leads to multipolar cells, fragmented nuclei during mitosis leading to cell death and show significantly perturbed ciliary disassembly dynamics. Whereas CETN1-5' UTR variant; rs367716858 leads to loss of a methylation site and increased reporter gene expression in vitro. We report a total of eight novel candidate genes identified by exome sequencing, which may have diagnostic relevance and can contribute to improved diagnostic workup and clinical management of male infertility.

TEX13B is essential for metabolic reprogramming during germ cell differentiation.

STUDY QUESTION: What is the functional significance of Tex13b in male germ cell development and differentiation? SUMMARY ANSWER: Tex13b regulates male germ cell differentiation by metabolic reprogramming during spermatogenesis. WHAT IS KNOWN ALREADY: Studies in mice and humans suggest that TEX13B is a transcription factor and is exclusively expressed in germ cells. STUDY DESIGN, SIZE, DURATION: We sequenced the coding regions of TEX13B in 628 infertile men and 427 ethnically matched fertile control men. Further, to identify the molecular function of Tex13b, we created a Tex13b knockout and conditional overexpression system in GC-1spg (hereafter, GC-1) cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Our recent exome sequencing study identified novel candidate genes for male infertility. TEX13B was found to be one of the potential candidates, hence we explored the role of TEX13B in male infertility within a large infertile case-control cohort. We performed functional analyses of Tex13b in a GC-1 cell line using CRISPR-Cas9. We differentially labelled the cell proteins by stable isotope labelling of amino acids in cell culture (SILAC) and performed mass spectrometry-based whole-cell proteomics to identify the differential protein regulation in knockout cells compared to wild-type cells. We found that Tex13b knockout leads to downregulation of the OXPHOS complexes and upregulation of glycolysis genes, which was further validated by western blotting. These results were further confirmed by respirometry analysis in Tex13b knockout cells. Further, we also performed a conditional overexpression of TEX13B in GC-1 cells and studied the expression of OXPHOS complex proteins by western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a rare variant, rs775429506 (p.Gly237Glu), exclusively in two non-obstructive-azoospermia (NOA) men, that may genetically predispose these men for infertility. Further, we demonstrated that Tex13b functions in the transcription regulation of OXPHOS complexes. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We examined the function of Tex13b in GC-1 in vitro by knocking out and conditional overexpression, for understanding the function of Tex13b in germ cells. Unfortunately, this could not be replicated in either an animal model or in patient-derived tissue due to the non-availability of an animal model or patient's testis biopsies. WIDER IMPLICATIONS OF THE FINDINGS: This study identified that Tex13b plays an important role in male germ cell development and differentiation. The findings of this study would be useful for screening infertile males with spermatogenic failure and counselling them before the implementation of assisted reproduction technique(s). STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by the Council of Scientific and Industrial Research (CSIR) under the network project (BSC0101 and MLP0113) and SERB, the Department of Science and Technology, Government of India (J C Bose Fellowship: JCB/2019/000027). The authors do not have any competing interest.

Mutations in the desert hedgehog (DHH) gene in the disorders of sexual differentiation and male infertility.

PURPOSE: To identify the contribution of mutations in the Desert Hedgehog (DHH) gene to the disorders of sexual differentiation (DSD) and male infertility. METHODS: The study included a total 430 subjects, including 47 gonadal dysgenesis cases, 6 patients with undescended testis and infertility characterized by azoospermia, 125 infertile male patients characterized by oligoasthenozoospermia, 24 patients with oligoasthenoteratozoospermia, and 200 ethnically matched normozoospermic fertile men who had fathered a child in the last two years. Sequencing of the complete coding region of the DHH gene was undertaken to find its contribution to the DSD and male infertility. RESULTS: We observed four novel mutations in the DHH gene in the cases with different reproductive anomalies. A synonymous substitution, c. 543C>T (p.His181His) was observed in 6.6% oligoasthenozoospermic infertile males and 1.5% normozoospermic fertile control samples (RR = 4.4077, 95%CI 1.19-16.29). Another synonymous substitution, c.990G>A (p.Ala330Ala) was observed in an infertile patient with unilateral undescended testis (case #12). Insertion of G at c.1156insG (p.Arg385fs) was observed in a case with bilateral undescended testis and azoospermia (case #23). In gonadal dysgenesis category, two mutations, insertion of G at c.1156insG (p.Arg385fs) and c.997A>G (p.Thr333Ala) substitution were observed in one case (case #34). These mutations were completely absent in control samples. CONCLUSION: Mutations in the DHH gene impact reproduction with mild mutations affecting fertility, and severe or multiple mutations resulting in gonadal dysgenesis.

Repeated implantation failure versus repeated implantation success: discrimination at a metabolomic level.

STUDY QUESTION: Is there any difference at the serum metabolic level between women with recurrent implantation failure (RIF) and women with recurrent implantation success (RIS) when undergoing in vitro fertilization (IVF)? SUMMARY ANSWER: Eight metabolites, including valine, adipic acid, l-lysine, creatine, ornithine, glycerol, d-glucose and urea, were found to be significantly up-regulated in women with RIF when compared with women with RIS. WHAT IS KNOWN ALREADY: Despite transfer of three high-grade embryos per cycle, RIF following three or more consecutive IVF attempts occurs in a group of infertile women. Conversely, there is a group of women who undergo successful implantation each cycle, yet have a poor obstetric history. STUDY DESIGN, SIZE, DURATION: This study was conducted over a period of 10 years (January 2004-October 2014). Groups of 28 women with RIF (age ≤40 years and BMI ≤28) and 24 women with RIS (age and BMI matched) were selected from couples with primary infertility reporting at the Institute of Reproductive Medicine, Kolkata, India. Women recruited in the RIF group had history of implantation failure in at least three consecutive IVF attempts, in which three embryos of high-grade quality were transferred in each cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples were collected from both the groups during the implantation window following overnight fasting for at least 10 h (7-10 days post ovulation). Samples were analyzed using a 700 MHz NMR spectrometer and acquired spectra were subjected to chemometric and statistical analysis. Serum levels of endothelial nitric oxide synthase (eNOS) were measured using an enzyme immunoassay technique. MAIN RESULTS AND THE ROLE OF CHANCE: Valine, adipic acid, l-lysine, creatine, ornithine, glycerol, d-glucose and urea were found to be significantly down-regulated in women with RIS when compared with those with RIF, with fold change values of 0.81, 0.82, 0.79, 0.80, 0.78, 0.68, 0.76 and 0.74, respectively. Further, serum eNOS was found to be significantly lower in women with RIF when compared with RIS (P < 0.05), indicating possible impairment in nitric oxide production. Metabolites, mostly related to energy metabolism, lipid metabolism and the arginine metabolic pathway were found to be considerably altered and are likely to be associated with the RIF phenomenon. However, the interplay between these molecules in RIF is complex and holds merit for further exploration. LIMITATIONS, REASONS FOR CAUTION: In-depth studies of the arginine metabolic pathway in endometrial tissues seem necessary to validate our findings. A limitation of the present study is that the metabolic level changes, eNOS and nitric oxide levels have not been investigated in the endometrial tissues of the two groups of women. It would be interesting to investigate whether there exists a direct link between metabolic dysregulation and genetic factors that affects implantation in RIF women. WIDER IMPLICATIONS OF THE FINDINGS: We speculate that tissue metabolomics can provide an improved understanding of the metabolic dysfunction associated with RIF. The identification of serum metabolic marker(s) in women with RIS may help with strategies of early therapeutic intervention, which may improve the chances of implantation significantly in women otherwise susceptible to IVF failure. STUDY FUNDING/COMPETING INTERESTS: One of the authors, S.R.C. acknowledges the Council of Scientific and Industrial Research (CSIR), Government of India [No: 9/81(1228)/14, EMR-I] for financial support.

CAG repeat variation in the mtDNA polymerase gamma is not associated with oligoasthenozoospermia.

Variations in the trinucleotide-CAG repeat number of the catalytic subunit of the mitochondrial DNA polymerase gamma (POLG) have been speculated to be associated with male infertility. The ten CAG repeats (10/10) were found to be the most common allele (88%), absence of which was found to be associated with male infertility. As no study on Indian population was conducted so far to support this view, we investigated the distribution of the POLG-CAG repeats in 509 oligoasthenozoospermic and 241 normozoospermic control Indian men from the same ethnic background. Our study suggested that the distribution of common allele (10/10) was almost similar in both infertile (75%) and normozoospermic (75.5%) men. Further, we had analysed the CAG repeat number in as many as 1306 Indian men belonging to different ethnic, geographical and linguistic backgrounds and found the common allele 10/10 at a frequency of 78.4%. Our study, therefore, suggests that the 10-CAG repeat is the most common allele present in Indian populations, but its absence and the occurrence of the other mutant homozygous (non 10/non 10) genotype should not be understood as being specific to infertility. It, thus, suggests that the POLG-CAG repeat variation is not associated with male infertility in Indian populations, and hence is not a useful marker for screening infertile men.

High frequencies of Non Allelic Homologous Recombination (NAHR) events at the AZF loci and male infertility risk in Indian men.

Deletions in the AZoospermia Factor (AZF) regions (spermatogenesis loci) on the human Y chromosome are reported as one of the most common causes of severe testiculopathy and spermatogenic defects leading to male infertility, yet not much data is available for Indian infertile men. Therefore, we screened for AZF region deletions in 973 infertile men consisting of 771 azoospermia, 105 oligozoospermia and 97 oligoteratozoospermia cases, along with 587 fertile normozoospermic men. The deletion screening was carried out using AZF-specific markers: STSs (Sequence Tagged Sites), SNVs (Single Nucleotide Variations), PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism) analysis of STS amplicons, DNA sequencing and Southern hybridization techniques. Our study revealed deletion events in a total of 29.4% of infertile Indian men. Of these, non-allelic homologous recombination (NAHR) events accounted for 25.8%, which included 3.5% AZFb deletions, 2.3% AZFbc deletions, 6.9% complete AZFc deletions, and 13.1% partial AZFc deletions. We observed 3.2% AZFa deletions and a rare long AZFabc region deletion in 0.5% azoospermic men. This study illustrates how the ethnicity, endogamy and long-time geographical isolation of Indian populations might have played a major role in the high frequencies of deletion events.

Novel NR5A1 Pathogenic Variants Cause Phenotypic Heterogeneity in 46,XY Disorders of Sex Development.

Steroidogenic factor 1 (NR5A1/SF1) is a key transcription factor that is known to regulate the development of adrenal glands and gonads and is also involved in steroidogenesis. Several pathogenic NR5A1 variants have been reported to cause 46,XY disorders of sex development (DSD), with varying clinical phenotypes ranging from hypospadias to complete gonadal dysgenesis. Most often, the primary cause of DSD is due to variants in gene(s) related to gonadal development or the steroidogenic pathway. In the present study, we have analyzed 64 cases of 46,XY DSD for pathogenic NR5A1 variants. We report a total of 3 pathogenic variants of which 2 were novel (p.Gly22Ser and p.Ser143Asn) and 1 was already known (p.Ser32Asn). Functional studies have revealed that the 2 mutations p.Gly22Ser and p.Ser32Asn could significantly affect DNA binding and transactivation abilities. Further, these mutant proteins showed nuclear localization with aggregate formation. The third mutation, p.Ser143Asn, showed unspeckled nuclear localization and normal DNA binding, but the ability of transcriptional activation was significantly reduced. In conclusion, we recommend screening for NR5A1 pathogenic variants in individuals with features of 46,XY DSD for better diagnosis and management.

Androgen insensitivity syndrome: do trinucleotide repeats in androgen receptor gene have any role?

AIM: To investigate the role of CAG and GGN repeats as genetic background affecting androgen insensitivity syndrome (AIS) phenotype. METHODS: We analyzed lengths of androgen receptor (AR)-CAG and GGN repeats in 69 AIS cases, along with 136 unrelated normal male individuals. The lengths of repeats were analyzed using polymerase chain reaction (PCR) amplification followed by allelic genotyping to determine allele length. RESULTS: Our study revealed significantly shorter mean lengths of CAG repeats in patients (mean 18.25 repeats, range 14-26 repeats) in comparison to the controls (mean 22.57 repeats, range 12-39 repeats) (two-tailed P < 0.0001). GGN repeats, however, did not differ significantly between patients (mean 21.48 repeats) and controls (mean 21.21 repeats) (two-tailed P = 0.474). Among patients' groups, the mean number of CAG repeats in partial androgen insensitivity cases (mean 15.83 repeats) was significantly less than in complete androgen insensitivity cases (mean 19.46 repeats) (two-tailed P < 0.0001). CONCLUSION: The findings suggest that shorter lengths of repeats in the AR gene might act as low penetrance genetic background in varying manifestation of androgen insensitivity.

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.

A novel human sex-determining gene linked to Xp11.21-11.23.

CONTEXT: The molecular basis for about 70-80% of 46,XY sex-reversed females remains unexplained, because they carry normal copies of the genes (SRY, SOX9, DAX1, DMRT, SF1, WT1) involved in sex determination pathway. OBJECTIVE: The objective of this study is to map the chromosomal locus responsible for an unexplained sex-reversed phenotype. DESIGN: The study implemented a genome-wide scan using families with multiple sex-reversed individuals. SETTING: The patients, along with the family members, were selected from different hospitals/reproductive centers. PARTICIPANTS: Sex-reversed individuals and their siblings and parents participated in the study. MAIN OUTCOME MEASURES: Identification of the chromosomal locus responsible for sex reversal in these families and sequence analysis of candidate genes were the main outcome measures. RESULTS: Parametric linkage analysis revealed a maximum two-point LOD score of 5.70 with marker DXS991 (Xp11.21) and 4.57 with marker DXS1039 (Xp11.23-Xp11.22), and a multipoint LOD score of 5.77 with marker DXS991 and 5.22 with marker DXS1039. The two markers (DXS991 and DXS1039) with highest LOD score span approximately 3.41 cM (75.79-79.2 cM) on the short arm of the X-chromosome. CONCLUSION: Our findings provide evidence for a major susceptibility locus for sex reversal/gonadal dysgenesis on the short arm of the X-chromosome (Xp11.21-11.23). Furthermore, molecular exploration of the expression of candidate genes in the embryonic gonad/gonadal ridge will help in the identification of the underlying gene for sex reversal.

Male infertility: no evidence of involvement of androgen receptor gene among Indian men.

Spermatogenesis is collaboratively controlled by testosterone and follicle stimulating hormone. Testosterone and its immediate metabolite dihydrotestosterone affect their roles through the androgen receptor (AR). Mutations in the AR gene have been shown to cause partial to complete androgen insensitivity or infertility in otherwise normal males. The dependence of germ cells upon Sertoli and Leydig cells for their differentiation into sperms and deletion studies of the AR gene in animal models indicate a direct or indirect role of the AR gene in spermatogenesis. Although a few studies worldwide have reported AR mutations in male infertility, no similar study has been conducted on Indian populations. Therefore, we undertook this study to look at the contribution of AR mutations in male infertility among Indian men. We have sequenced the complete coding region of the AR gene in a total of 399 infertile samples, comprising 277 azoospermic, 100 oligozoospermic, and 22 oligoteratozoospermic samples. A total of 100 healthy males with proven fertility and the same ethnicity as the experimental group served as controls. Sequence analysis revealed no mutation in any of these samples. Our study suggests that mutations in the AR gene are less likely to cause azoospermia and oligozoospermia; however, it was difficult to rule out its effect in oligoteratozoospermia, as the sample size was small.

No association of androgen receptor GGN repeat length polymorphism with infertility in Indian men.

Androgens, acting through the androgen receptor (AR), play a role in secondary sexual differentiation from the prenatal stage to adulthood, including spermatogenesis. The AR gene has 2 polymorphic trinucleotide repeats (CAG and GGN) in exon 1. The CAG repeat length polymorphism has been well studied in a variety of medical conditions, including male infertility. Many of these studies have shown an association of the expanded CAG repeats with male infertility, although this is not true for all populations. The GGN repeat, in contrast, has been less thoroughly studied. Thus far, only 4 reports worldwide have analyzed the GGN repeat, alone or in combination with the CAG repeat, in male infertility cases. No such study has been undertaken on infertile Indian men. Therefore, we have analyzed AR-GGN repeats in a total of 595 Indian males, including 277 azoospemric, 97 oligozoospermic, and 21 oligoteratozoospermic cases, along with 200 normozoospermic controls. The analysis revealed no difference in the mean number or the range of the repeat between cases (mean = 21.51 repeats, range 15-26 repeats) and controls (mean 21.58 repeats, range 15-26 repeats). Furthermore, no difference was observed when azoospermic (mean = 21.53 repeats, range 15-26 repeats), oligozoospermic (mean = 21.46 repeats, range 15-26 repeats), and oligoteratozoospermic cases (mean = 21.48, range 19-26 repeats) were compared individually with the controls.

CAG repeat expansion in the androgen receptor gene is not associated with male infertility in Indian populations.

CAG repeat expansion in exon 1 of the androgen receptor (AR) gene has been reported to be associated with male infertility in some but not all populations. Until now, studies have not been carried out to examine this among Indian populations. For the first time, we have analyzed the CAG repeat motif in the AR gene in 280 men with azoospermia and in 201 men with normal fertility. The mean number of CAG repeats in the AR gene of men with azoospermia was 21.7 +/- 0.18, with a high incidence of repeat number 22. Among fertile-control men, the mean number of CAG repeats was 22.4 +/- 0.19, with a predominance of repeat number 23. The highest number of CAG repeats (32) was found with low frequency in both fertile and azoospermic groups. Comparison of fertile men and those with azoospermia on the basis of CAG repeats revealed that the number of CAG repeats in both groups were similar, as revealed with a paired t test (t = 0.04; P =.967). Expansion of the CAG repeat in the AR gene is therefore not associated with male infertility in Indian populations. This suggests that what is true for one population may not be true for other populations.

Y chromosome deletions in azoospermic men in India.

Genetic factors cause about 10% of male infertility. Azoospermia factors (AZFa, AZFb, AZFc) are considered to be the most important for spermatogenesis. We therefore made an attempt to evaluate the genetic cause of azoospermia, Y chromosome deletion in particular, in Indian men. We have analyzed a total of 570 men, including 340 azoospermic men and 230 normal control subjects. DNA samples were initially screened with 30 sequence-tagged site (STS) markers representing AZF regions (AZFa, AZFb, AZFc). Samples, with deletion in the above regions were mapped by STS walking. Further, the deletions were confirmed by Southern hybridization using the probes from both euchromatic and heterochromatic regions. Of the total 340 azoospermic men analyzed, 29 individuals (8.5%) showed Y chromosome deletion, of which deletion in AZFc region was the most common (82.8%) followed by AZFb (55.2%) and AZFa (24.1%). Microdeletions were observed in AZFa, whereas macrodeletions were observed in AZFb and AZFc regions. Deletion of heterochromatic and azoospermic regions was detected in 20.7% of the azoospermic men. In 7 azoospermic men, deletion was found in more than 8.0 Mb spanning AZFb and AZFc regions. Sequence analysis at the break points on the Y chromosome revealed the presence of L1, ERV, and other retroviral repeat elements. We also identified a approximately 240-kb region consisting of 125 bp tandem repeats predominantly comprised of ERV elements in the AZFb region. Histological study of the testicular tissue of the azoospermic men, who showed Y chromosome deletion, revealed complete absence of germ cells and presence of only Sertoli cells.

L712V mutation in the androgen receptor gene causes complete androgen insensitivity syndrome due to severe loss of androgen function.

Inability to respond to the circulating androgens is named as androgen insensitivity syndrome (AIS). Mutations in the androgen receptor (AR) gene are the most common cause of AIS. A cause and effect relationship between some of these mutations and the AIS phenotype has been proven by in vitro studies. Several other mutations have been identified, but need to be functionally validated for pathogenicity. Screening of the AR mutations upon presumptive diagnosis of AIS is recommended. We analyzed a case of complete androgen insensitivity syndrome (CAIS) for mutations in the AR gene. Sequencing of the entire coding region revealed C>G mutation (CTT-GTT) at codon 712 (position according to the NCBI database) in exon 4 of the gene, resulting in replacement of leucine with valine in the ligand-binding domain of the AR protein. No incidence of this mutation was observed in 230 normal male individuals analyzed for comparison. In vitro androgen binding and transactivation assays using mutant clone showed approximately 71% loss of ligand binding and about 76% loss of transactivation function. We conclude that CAIS in this individual was due to L712V substitution in the androgen receptor protein.

G708E mutation in the androgen receptor results in complete loss of androgen function.

End-organ resistance to androgens, called androgen insensitivity syndrome (AIS), is a rare disorder. The most common cause of AIS is mutations(s) in the androgen receptor (AR) gene; however, a significant number of these mutations have not been functionally analyzed. In the present study, we analyzed a case of complete AIS for mutations in the AR gene. Sequencing of the entire coding region of the AR gene revealed a 2650G>A mutation (mRNA sequence reference) in exon 4 of the gene, resulting in replacement of glycine with glutamate at codon 708 in the ligand-binding domain of the AR protein. The mutation was absent in 200 normal male individuals analyzed to look at its occurrence in general population. In vitro androgen-binding and transactivation assays showed that the mutation resulted in approximately 65% loss of ligand binding and almost complete loss of transactivation function. Complete AIS in this individual was due to a G708E substitution in the AR protein.

Mitochondrial DNA variations associated with recurrent pregnancy loss among Indian women.

Several genetic factors have been found to be associated with recurrent pregnancy loss (RPL). However, not many attempts have been made to associate the mitochondrial DNA (mtDNA) variations with RPL. Therefore, we have analyzed the complete mtDNA of 100 women with RPL and 12 aborted fetal tissues. Our analysis revealed a total of 681 variations, most of which were in NADH Dehydrogenase (ND) genes that encode mitochondrial enzyme Complex I. Presence of T4216C variation (ND1 gene) in 9% of the RPL women and several pathogenic, and novel mutations suggest the role of mtDNA variations in RPL.

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.

Ala 586 Asp mutation in androgen receptor disrupts transactivation function without affecting androgen binding.

OBJECTIVE: To understand the pathogenesis of the androgen insensitivity syndrome. DESIGN: Familial case study. SETTING: Medical and Evolutionary Genetics Laboratory, Centre for Cellular and Molecular Biology, Hyderabad, India. PATIENT(S): Two affected sisters and other unaffected family members. INTERVENTION(S): The hormone levels were measured by RIA. Histology was done by standard protocols. DNA isolation and direct DNA sequencing was undertaken for mutation identification. Site-directed mutagenesis was used for incorporation of mutation in the androgen receptor clone. Functional assays were done using COS-1 cell cultures. MAIN OUTCOME MEASURE(S): Phenotype, hormone levels, DNA mutations, ligand binding, transactivation function of androgen-androgen receptor complex. RESULT(S): The patients exhibited a female phenotype despite the 46,XY chromosome complement. Both of the affected individuals had higher levels of T and LH. C1760A (coding DNA sequence reference) substitution (Ala 586 Asp) in the AR gene was observed in all of the affected individuals. The mutation did not result in a loss of ligand binding but instead in almost complete loss of transactivation function. CONCLUSION(S): The Ala 586 Asp mutation resulted in a complete loss of transactivation function of the androgen-androgen receptor complex but did not affect ligand binding. In vitro assays confirmed the pathogenic nature of this mutation.

APOB gene signal peptide deletion polymorphism is not associated with infertility in Indian men.

Apolipoprotein B (APOB) plays a key role in lipoprotein metabolism and plasma lipid transport. It has been shown that about two-thirds of male mice heterozygous for ApoB were infertile. Moreover, a 3-codon deletion polymorphism (rs11279109) in the signal peptide region of the APOB gene has been shown to be a risk factor for infertility in Slovenian men, but its association with infertility in Indian men has not been evaluated to date. Hence, in the present study, we have genotyped this polymorphism in 545 Indian men, including 294 infertile and 251 fertile men. Our results show that the distribution of this deletion polymorphism was consistent with the Hardy-Weinberg equilibrium in both infertile and fertile men. No statistically significant difference was observed in the distribution of the APOB signal peptide deletion polymorphism between infertile and fertile men (chi(2) = 0.156, P = .925 for genotypes; chi(2) = 0.015, P = .903 for alleles). Moreover, no significant difference was observed when infertile and fertile men were categorized on the basis of presence (D/D and D/W genotypes) or absence (W/W genotypes) of deletion (odds ratio, 0.955; 95% confidence interval, 0.644-01.418; P = .820). Our study concludes that the APOB gene deletion polymorphism is not a risk factor for the development of infertility in Indian men.