Exome sequencing identified mutations in the WNT1 and COL1A2 genes in osteogenesis imperfecta cases.

BACKGROUND: Osteogenesis imperfecta (OI) is a heritable connective tissue disorder characterized by bone deformities, fractures and reduced bone mass. OI can be inherited as a dominant, recessive, or X-linked disorder. The mutational spectrum has shown that autosomal dominant mutations in the type I collagen-encoding genes are responsible for OI in 85% of the cases. Apart from collagen genes, mutations in more than 20 other genes, such as CRTAP, CREB3L1, MBTPS2, P4HB, SEC24D, SPARC, FKBP10, LEPRE1, PLOD2, PPIB, SERPINF1, SERPINH1, SP7, WNT1, BMP1, TMEM38B, and IFITM5 have been reported in OI. METHODS AND RESULTS: To understand the genetic cause of OI in four cases, we conducted whole exome sequencing, followed by Sanger sequencing. In case #1, we identified a novel c.506delG homozygous mutation in the WNT1 gene, resulting in a frameshift and early truncation of the protein at the 197th amino acid. In cases #2, 3 and 4, we identified a heterozygous c.838G > A mutation in the COL1A2 gene, resulting in a p.Gly280Ser substitution. The clinvar frequency of this mutation is 0.000008 (GnomAD-exomes). This mutation has been identified by other studies as well and appears to be a mutational hot spot. These pathogenic mutations were found to be absent in 96 control samples analyzed for these sites. The presence of these mutations in the cases, their absence in controls, their absence or very low frequency in general population, and their evaluation using various in silico prediction tools suggested their pathogenic nature. CONCLUSIONS: Mutations in the WNT1 and COL1A2 genes explain these cases of osteogenesis imperfecta.

Small RNAs, spermatogenesis, and male infertility: a decade of retrospect.

Small non-coding RNAs (sncRNAs), being the top regulators of gene expression, have been thoroughly studied in various biological systems, including the testis. Research over the last decade has generated significant evidence in support of the crucial roles of sncRNAs in male reproduction, particularly in the maintenance of primordial germ cells, meiosis, spermiogenesis, sperm fertility, and early post-fertilization development. The most commonly studied small RNAs in spermatogenesis are microRNAs (miRNAs), PIWI-interacting RNA (piRNA), small interfering RNA (siRNA), and transfer RNA-derived small RNAs (ts-RNAs). Small non-coding RNAs are crucial in regulating the dynamic, spatial, and temporal gene expression profiles in developing germ cells. A number of small RNAs, particularly miRNAs and tsRNAs, are loaded on spermatozoa during their epididymal maturation. With regard to their roles in fertility, miRNAs have been studied most often, followed by piRNAs and tsRNAs. Dysregulation of more than 100 miRNAs has been shown to correlate with infertility. piRNA and tsRNA dysregulations in infertility have been studied in only 3-5 studies. Sperm-borne small RNAs hold great potential to act as biomarkers of sperm quality and fertility. In this article, we review the role of small RNAs in spermatogenesis, their association with infertility, and their potential as biomarkers of sperm quality and fertility.

COVID-19 vaccination does not affect male sexual functions.

BACKGROUND: COVID-19 infection has been linked with erectile dysfunction, which has also raised apprehensions about the impact of COVID-19 vaccination on male sexual functions. The purpose of this study was to investigate the impact of COVID-19 vaccination on male sexual functions, such as erectile function, orgasmic function, sexual desire, intercourse satisfaction, and overall satisfaction. METHODS: We used International Index of Erectile Function (IIEF) questionnaire for data collection. Mixed methods were adopted for this study, which consisted of Google online form distribution and the distribution of hard copies of the form to those who were not internet friendly. All data were entered in a spreadsheet and scores were assigned to each response according to the standard scores given in the IIEF questionnaire. Fifteen questions, one corresponding to each question in the IIEF questionnaire, were included to assess the impact of COVID-19 vaccination on each sexual function. RESULTS: In the first part of analysis, we calculated sexual function scores and men reporting low sexual function scores (~ 15%) were excluded, providing us with 465 individuals for further analysis. Regarding the impact of COVID-19 vaccination on male sexual functions, 71% individuals reported no impact, 3% reported a decline, 2.7% reported an improvement, and 23.3% could not assess the impact. We also performed analysis on the basis of age-groups of the participants and the duration after vaccination, finding that there was no impact irrespective of the age of subjects or the length of period after vaccination. CONCLUSIONS: COVID-19 vaccination does not affect male sexual functions, including erectile function, orgasmic function, sexual desire, intercourse satisfaction, and overall sexual satisfaction.

Synthesis of green emissive terbium(III) complexes for displays: Optical, electrochemical and photoluminescent analysis.

A series of heteroleptic terbium(III) complexes with fluorinated 2-thenoyltrifluoroacetone (TTFA) and other heteroaromatic units have been synthesized. The developed heteroleptic complexes were inspected via elemental study, cyclic voltammetry, thermal analysis and spectroscopic investigations. Optical band-gap data proposed the conducting property of prepared complexes. The photoluminescence emission profiles illustrated peaks based on terbium(III) cation (Tb3+ ) positioned at ~617, 586, 546 and 491 nm, imputed to 5 D4 to 7 FJ (J = 3,4,5,6) transitions separately. Most intense peak at 546 nm corresponding to 5 D4 → 7 F5 transition is accountable for the green emissive character of developed complexes. The luminous character of complexes reveals the sensitization of Tb3+ by ligands. Color parameters further corroborates the green emanation of Tb3+ complexes. The photometric characteristics of complexes recommended their usages in designing display devices.

Challenges and technological interventions in rice-wheat system for resilient food-water-energy-environment nexus in North-western Indo-Gangetic Plains: A review.

Rice (Oryza sativa L.)-wheat (Triticum aestivum L.) cropping system in north-western Indo-Gangetic Plains performed a crucial role in the national food security. However, the widespread and intensive cultivation of this system has led to serious problems such as declining groundwater table (~1 meter year-1) with sharp increase in number of districts under over-exploitation category, residue burning, higher greenhouse gases emission and herbicide resistance in weeds, causing stagnant crop productivity and lesser profitability. In this review article, an attempt has been made to discuss the major issues pertaining to intensive rice-wheat cultivation amidst climate vagaries and futuristic approach to address these challenges. Different tillage- and crop-specific recommendations such as adoption of direct seeded rice, diversification with lesser resource guzzling crops such as maize (Zea mays L.) at least on the periodic manner especially in light-medium soils, inclusion of summer legumes and alternative tillage systems (permanent beds and zero tillage with residue retention) have been suggested to address these issues. However, crop performance under these techniques has been found to be location, soil and cultivar specific. The absence of aerobic tailored genotypes and weeds have been identified as the major constraints in adoption of direct seeded rice. The integrated strategies of conservation tillage, crop breeding program and resource conserving region- and soil-specific agronomic measures with crop diversification would be helpful in tackling the sustainability issues. It requires future efforts on developing crop genotypes suited to conservation tillage, effective weed control strategies and trainings and demonstrations to farmers to switch from conventional rice-wheat system to alternative cropping systems.

Radon and thoron exhalation rate in the soil of Western Haryana, India.

This study reports the exhalation rates of radon and thoron from surface soil collected from 60 rural sites of district Hisar, Haryana, India. The exhalation rates of Rn222 (radon) and Rn220 (thoron) were measured by portable SMART RnDuo (AQTEK SYSTEMS) using a mass accumulation chamber which was equipped with a scintillation material-coated cell. Dose rates due to natural gamma radiations ranged from 0.526 to 1.139 mSv y-1. The Rn222 mass exhalation rate in soil samples varied from 0.14 to 94.65 mBq kg-1 h-1. Thoron surface exhalation rates ranged from 46.42 to 619.88 Bq m-2 h-1. This study gives an idea about the differences in Rn222 and Rn220 exhalation at different locations which may be due to variations in geological features of the locations and characteristics of the topsoil. The findings show that usage of study area soil as building material is safe.

Proposing a clinical algorithm for better diagnosis of hypophosphatasia in resource-limiting situations.

Early diagnosis of hypophosphatasia (HPP) is challenging. Here, we propose to broaden the diagnostic criteria of HPP by reviewing published data on BMD and fractures in HPP patients. Non-osteoporotic fractures and higher than normal lumbar BMD were recurrent in HPP patients and could be included as diagnostic criteria. HPP is a genetic disorder caused by autosomal recessive or dominant loss-of-function mutations in the ALPL gene that encodes for tissue-nonspecific alkaline phosphatase (TNSALP). Expressive genetic heterogeneity and varying severity of TNSALP deficiency lead to a wide-ranging presentation of skeletal diseases at different ages that coupled with HPP's rarity and limitation of biochemical and mutational studies present serious hurdles to early diagnosis and management of HPP. To widen the scope of HPP diagnosis, we assessed the possibility of areal bone mineral density (BMD) as an additional clinical feature of this disease. PubMed, Web of Science, and ScienceDirect were searched with the following keywords: ("Hypophosphatasia OR HPP") AND ("Bone Mineral Density OR BMD") AND "Human". Studies and case reports of subjects with age ≥ 18 years and having BMD data were included. We pooled data from 25 publications comprising 356 subjects (90 males, 266 females). Only four studies had a control group. Biochemical hallmarks, pyridoxal 5'-phosphate (PLP) and phosphoethanolamine (PEA), were reported in fifteen and six studies, respectively. Twenty studies reported genetic data, nineteen studies reported non-vertebral fractures, all studies reported lumbar spine (LS) BMD, and nineteen reported non-vertebral BMD. Higher than normal and normal BMD at LS were reported in three and two studies, respectively. There was marked heterogeneity in BMD at the non-vertebral sites. Higher than normal or normal LS BMD in an adult with minimal or insufficient fractures, pseudofractures, non-healing fractures, fragility fractures, and stress fractures may be included in the diagnostic protocol of HPP. However, genetic testing is recommended for a definitive diagnosis.

Qualitative and quantitative assessment of sperm miRNAs identifies hsa-miR-9-3p, hsa-miR-30b-5p and hsa-miR-122-5p as potential biomarkers of male infertility and sperm quality.

BACKGROUND: In contrast with the preceding stages of the germ cells, spermatozoa are unusually rich in small non-coding RNAs in comparison to the coding RNAs. These small RNAs may have had an essential role in the process of spermatogenesis or may have critical roles in the post-fertilization development. Sporadic efforts have identified a few differentially expressed miRNAs in infertile individuals, which do not replicate in other studies. METHODS: In order to identify miRNAs signatures of infertility or poor sperm quality, we compared miRNA differential expression data across nine datasets, followed by their analysis by real-time PCR in a case-control study. This was followed by the validation of potential biomarkers in yet another set of cases and controls. For this, total RNA was isolated from 161 sperm samples. miRNA expression levels in infertile cases and fertile controls were measured using TaqMan real-time PCR. Meta-analyses of two miRNAs (hsa-miR-9-3p and hsa-miR-122-5p) were performed using Comprehensive Meta-Analysis Software (version 2). All statistical analyses were performed with the help of GraphPad Prism Software (version 8). RESULTS: Literature search identified seven miRNAs (hsa-let-7a-5p, hsa-miR-9-3p, hsa-miR-22-5p, has-miR-30b-5p, hsa-miR-103-3p, hsa-miR-122-5p and hsa-miR-335-5p) showing consistent dysregulation in infertility across a minimum of four studies. In the discovery phase, six miRNAs showed strong association with infertility with four (hsa-miR-9-3p, hsa-miR-30b-5p, hsa-miR-103-3p and hsa-miR-122-5p) showing consistent differential regulation across all sub-groups. Receiver operating characteristic (ROC) curve analysis showed that the area under curve of > 0.75 was achieved by three (hsa-mir-9-3p, hsa-miR-30b-5p and hsa-miR-122-5p) miRNAs. In the validation phase, these three miRNAs showed consistent association with infertility (hsa-mir-9-3p, hsa-miR-30b-5p, and hsa-miR-122-5p). Meta-analysis on hsa-miR-122-5p showed its significant quantitative association with infertility [Hedge's g = -2.428, p = 0.001 (Random effects)]. CONCLUSIONS: Three miRNAs (hsa-miR-9-3p, hsa-miR-30b-5p and hsa-miR-122-5p) have strong linkage with infertility and a high potential as sperm quality biomarkers.

Genistein lowers fertility with pronounced effect in males: Meta-analyses on pre-clinical studies.

Genistein, an isoflavonoid, is found in a plethora of plant-based foods, and has been approved for use in various therapies. A couple of studies in adult men observed a negative correlation between genistein exposure and reproductive parameters. To assess the effects of genistein exposure on reproduction and fertility in males and females, we performed quantitative meta-analyses by pooling data from published studies on animals that assessed various reproductive parameters. Pooled analysis showed significant decreases in sperm count in males exposed to genistein during adulthood (Hedges's g = -2.51, p = 0.013) and in utero (Hedges's g = -0.861, p = 0.016) compared with controls. In males exposed to genistein in utero, serum testosterone levels decreased (Hedges's g = -6.301, p = 0.000) and luteinizing hormone (LH) (Hedges's g = 7.127, p = 0.000) and FSH (Hedges's g = 6.19, p = 0.000) levels increased in comparison with controls. In females, the number of corpora lutea (Hedges's g = -2.103, p = 0.019) and the litter size (Hedges's g = -1.773, p-value = 0.000) decreased; however, female reproductive hormones remained unaffected. These meta-analyses show that genistein has detrimental effects on male reproductive system and on the progression and sustenance of pregnancy, with more pronounced adverse impact in males, particularly when exposed in utero.

Polymorphisms in the HSF2, LRRC6, MEIG1 and PTIP genes correlate with sperm motility in idiopathic infertility.

The aim of this study was to investigate the association of 24 functionally important single nucleotide polymorphisms (SNPs) with male infertility. In this cross-sectional study, we genotyped 24 functionally important single nucleotide polymorphisms in 24 infertility candidate genes in 500 oligo-/astheno-/oligoastheno-/normo-zoospermic infertile men with idiopathic infertility. Sequenom iPlex gold assay was used for genotyping. Sperm count and motility were compared between prevalent genotypes at each test locus. We did not observe any significant difference in the average sperm count between the alternate genotypes for the loci in the KLK3, LRRC6, MEIG1, HSF2, ESR2 and PTIP genes. However, we observed a significant difference in sperm motility between the alternate genotypes for the loci in the LRRC6, MEIG1, HSF2 and PTIP genes. Polymorphisms in the LRRC6 (rs200321595), MEIG1 (rs150031795), HSF2 (rs143986686) and PTIP (rs61752013) genes show association with sperm motility.

HCG therapy in azoospermic men with lower or borderline testosterone levels and the prognostic value of Y-deletion analysis in its outcome.

The purpose of this study was to investigate the efficacy of hCG therapy in hypogonadotropic hypogonadic (HH) azoospermic males along with dissecting the prognostic value of Y-deletion analysis in these patients. Fifty-eight azoospermic infertile males with diminished testosterone levels (≤400 ng/dl) and hypogonadism symptoms were subjected to human chorionic gonadotropin (hCG) therapy, and Y-deletion analysis was undertaken. Post-treatment, 43% (25/58) patients showed improvement in sperm count with 8.6% (5/58) turning severe oligozoospermic, 24.14% (14/58) patients turning oligozoospermic and 10.54% (6/58) turning normozoospermic. Among responders, the mean sperm concentration was 8.47 ± 13.16 million/ml, sperm count was 17.05 ± 26.17 million, sperm motility was 52.59% ± 25.09% and sperm progressive motility was 26.91% ± 20.51%. Seventeen out of 25 (68%) responders and 11/33 (33%) nonresponders showed an improvement in libido post-therapy. A Y-deletion was observed in 8% (2/25) responders and in 39.39% (13 out of 33) nonresponders. The Y-deletions were more often found in nonresponders in comparison with the responders (Fisher's exact probability test, p = .007, one tailed). We conclude that hCG therapy in hypogonadotropic azoospermic males is effective in improving andrological parameters and sperm production and that Y-chromosome deletion analysis has prognostic significance in predicting the success of hCG therapy.

Coding and regulatory transcriptome comparisons between fertile and infertile spermatozoa identify RNA signatures of male infertility.

The aim of the present study was to identify RNA-based signatures of male infertility by sperm transcriptome analysis. In this study, deep sequencing analyses of coding (mRNA) and regulatory (miRNA) transcriptomes were performed by pooling 15 oligo/oligoasthenozoospermic infertile sperm and 9 normozoospermic fertile sperm samples. Furthermore, interesting candidates were selected for validation by real-time PCR. The comparison of miRNAs between cases and controls identified 94 differentially expressed miRNAs, of which at least 38 have known functions in spermatogenesis. In transcriptome (mRNA) data, a total of 60,505 transcripts were obtained. The comparison of coding RNAs between cases and controls revealed 11,688 differentially expressed genes. miRNA-mRNA paired analysis revealed that 94 differentially expressed miRNAs could potentially target 13,573 genes, of which 6419 transcripts were actually differentially expressed in our data. Out of these, 3303 transcripts showed inverse correlation with their corresponding regulatory miRNAs. Moreover, we found that most of the genes of miRNA-mRNA pairs were involved in male germ cell differentiation, apoptosis, meiosis, spermiogenesis and male infertility. In conclusion, we found that a number of sperm transcripts (miRNAs and mRNAs) have a very high potential of serving as infertility/sperm quality markers.

SPATA16 promoter hypermethylation and downregulation in male infertility.

Spermatogenesis associated 16 (SPATA16) gene plays an important role in acrosome formation. In this study, we analysed SPATA16 promoter methylation in 29 oligozoospermic infertile and 16 normozoospermic fertile sperm samples and in testicular biopsy from 16 non-obstructive azoospermic and 2 obstructive azoospermic individuals. Next, we analysed SPATA16 level in sperm from 8 oligozoospermic infertile, 6 normozoospermic fertile, 9 IVF failed normozoospermic and 10 IVF successful normozoospermic samples by immunoblotting. This was followed by the analysis of SPATA16 expression in testicular biopsy from azoospermic individuals (n = 8) in comparison to normozoospermic individuals (n = 2). Oligozoospermic infertile sperm samples showed significantly higher methylation in the SPATA16 promoter region. Similarly, testicular biopsy from azoospermic men also showed significantly higher level of DNA methylation. Sub-group analysis of infertile sperm and testicular biopsy samples showed a direct correlation between DNA methylation and the degree of spermatogenic impairment. Oligozoospermic infertile samples and IVF failed samples showed reduced SPATA16 expression in comparison to normozoospermic fertile and IVF successful samples, respectively. Human biopsy analysis showed a significant decrease in SPATA16 expression in hypospermatogenesis, maturation arrest and Sertoli cell only syndrome. In conclusion, hypermethylation in SPATA16 promoter shows a highly significant correlation with infertility, which is consistent with its down-regulation in infertility.

Red emissive ternary europium complexes: synthesis, optical, and luminescence characteristics.

Solid ternary europium complexes consisting of fluorinated ß-diketone (thenoyltrifluoroacetone, TTFA) and heteroaromatic bidentate auxiliary ligands were synthesized. The luminescence features of the complexes were estimated using various spectral measurements and clearly proved that the Eu3+ ion is efficiently sensitized by ligands by an antenna effect. Photoluminescence excitation spectra have shown that Eu(III) complexes are excited effectively in the ultraviolet (UV) region and the corresponding emission spectra consist of characteristic peaks attributed to the 5 D0 →7 FJ transitions of the europium ion with the strongest emission peak at 611 nm (5 D0 →7 F2 ). From photoluminescence (PL) data, decay time, Judd-Ofelt parameters, transition rates, and quantum efficiency of the complexes were also determined. The Commission Internationale de l'éclairage (CIE) colour coordinates indicated the bright red emission of ternary europium complexes. Correlated colour temperature values indicated the utilization of these complexes in display devices. Judd-Ofelt and photophysical parameters were also estimated theoretically using LUMPAC software. Various frontier molecular orbitals and their respective energy were determined. These red emissive europium complexes could be utilized for fabricating solid-state lighting systems.

Mononuclear luminous ß-diketonate Ln(III) complexes with heteroaromatic auxiliary ligands: synthesis and luminescence characteristics.

A series of lanthanide (samarium and terbium) ß-diketonates with heteroaromatic auxiliary ligands was synthesized. The prepared complexes were characterized through electrochemical, thermal, and spectroscopic analyses. Infrared analysis revealed the binding of the respective metal ion to oxygen and nitrogen atoms of diketone and ancillary ligands. Thermogravimetry/differential thermogravimetry profiles provided thermal information and specified the high thermal stability of the prepared complexes. The complexes exhibited the sharp and structured Ln-based emission in the visible region upon irradiation in the ultraviolet range. Photophysical analysis demonstrated the green and orange-red emission due to the respective characteristic transitions of Tb3+ and Sm3+ ions. Photophysical properties affirmed the luminous behaviour of the synthesized complexes. These luminous lanthanide complexes could be used as emitting materials in the design of organic light-emitting diodes.

Defining the role of a caffeic acid 3-O-methyltransferase from Azadirachta indica fruits in the biosynthesis of ferulic acid through heterologous over-expression in Ocimum species and Withania somnifera.

MAIN CONCLUSION: The recombinant caffeic acid 3-O-methyltransferase gene has been cloned and characterized from Neem. The gene is involved in ferulic acid biosynthesis, a key intermediate component of lignin biosynthesis. Azadirachta indica (Neem) is a highly reputed traditional medicinal plant and is phytochemically well-known for its limonoids. Besides limonoids, phenolics are also distinctively present, which add more medicinal attributes to Neem. Caffeic acid is one of such phenolic compound and it can be converted enzymatically into another bioactive phytomolecule, ferulic acid. This conversion requires transfer of a methyl group from a donor to caffeic acid under the catalytic action of an appropriate methyltransferase. In this study, caffeic acid 3-O-methyltransferase gene from Neem (NCOMT) fruits has been isolated and heterologously expressed in E. coli. The recombinant NCOMT enzyme was purified, which exhibited efficient catalytic conversion of caffeic acid into ferulic acid, a highly potential pharmaceutical compound. The purified recombinant enzyme was physico-kinetically characterized for its catalysis. The analysis of tissue-wide expression of NCOMT gene revealed interesting pattern of transcript abundance reflecting its role in the development of fruit tissues. Further, NCOMT was heterologously overexpressed in Withania somnifera and Ocimum species, to analyze its role in ferulic acid biosynthesis in planta. Thus, the study provides insight for the endogenous role of NCOMT in ferulic acid biosynthesis en route to lignin, an important structural component. To the best of our knowledge, NCOMT pertains to be the first enzyme of the secondary metabolism that has been purified and kinetically characterized from Neem. This study may also have important prospects of applications as the observation on heterologous expression of NCOMT showed its involvement in the maintenance of the in vivo pool of ferulic acid in the plants. Thus, the study involving NCOMT opens up new dimensions of metabolic engineering approaches for the biosynthesis of potential therapeutically important phytomolecules in heterologous systems.

Human Relevance of Preclinical Studies on the Skeletal Impact of Inflammatory Bowel Disease: A Systematic Review and Meta-Analysis.

Inflammatory bowel disease (IBD) is a relapsing chronic idiopathic inflammatory condition. The increased risks of fractures in the spine and decreased BMD at all weight-bearing skeletal sites have been reported in IBD patients. The understanding of the mechanisms of IBD-induced bone loss is far from complete. Appropriate animal models are a prerequisite for studying IBD-induced bone loss, which prompted us to undertake quantitative meta-analyses by pooling data from the available IBD models that assessed various bone parameters. Sufficient data for meta-analysis are obtained from chemically- but not genetically induced models. Among the chemically induced models, only the effects of dextran sulfate sodium (DSS) and 2,4,6-trinitrobenzene sulfonic acid (TNBS) on bone parameters have been reported. Meta-analysis showed that both DSS (Hedge's g = 2.124, p = 0.001) and TNBS (Hedge's g = 6.292, p = 0.000) increased inflammatory disease severity. In pooled analysis, bone volumes in femur (Hedge's g = - 3.42, p = 0.000) and tibia (Hedge's g = - 2.49, p = 0.000) showed significant losses upon DSS administration. Similarly, bone formation rate was significantly reduced upon IBD induction (Hedge's g = - 3.495, p = 0.006). Besides, cortical thickness was reduced and trabecular microstructure deteriorated by IBD induction. Insufficient data precluded us from determining the effect of IBD on bone strength and calciotropic hormones, as well as the impact of proinflammatory cytokines on bone turnover. This meta-analysis showed that IBD induction in rodents causes significant bone loss. Impaired osteoblast function appears to be the cause of this impact.

Exome sequencing identified compound heterozygous mutations in the SRD5A2 gene in a case of 46,XY ambiguous genitalia.

The disorders of sexual development (DSD) represent an array of phenotypes with ambiguous genitalia. The present case had microphallus with fused and bifid scrotum and was initially assigned androgen insensitivity syndrome; however, sequencing of the complete coding region of the androgen receptor gene failed to identify a causative mutation. We undertook whole exome sequencing for identification of the pathogenic mutation. The most promising pathogenic variants were genotyped using Sanger sequencing to confirm the genotypes. We found compound heterozygous mutations, c.169G>T and c.586G>A in the SRD5A2 gene in this case, resulting in a nonsense (p.Glu57Ter) and a nonsynonymous substitution (p.Gly196Ser), respectively. While the nonsense mutation would result in a truncated protein, p.Gly196Ser substitution has been previously reported to be pathogenic. The mutations were confirmed by Sanger sequencing. Sequencing of 96 normal male individuals did not show the above mutations, suggesting their pathogenic nature. In conclusion, we identified compound heterozygous pathogenic mutations, c.169G>T (p.Glu57Ter) and c.586G>A (p.Gly196Ser), in the SRD5A2 gene in a case of ambiguous genitalia. p.Glu57Ter is a novel mutation, which in compound heterozygote combination with Gly196Ser causes 5a reductase deficiency.

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.

Inflammation driven tumor-like signaling in prostatic epithelial cells by sexually transmitted Trichomonas vaginalis.

OBJECTIVES: To identify the sequence of inflammation-driven signaling cascades and other molecular events that might cause tumor-like transformation of prostatic cells. METHODS: Cytokine array analysis, Reactome and STRING analysis, immunoblotting, and immunocytochemistry were used to investigate the molecular mechanisms governing inflammation-driven adverse changes in human prostatic cells caused by the sexually transmitted infection, Trichomonas vaginalis, resulting in prostatitis, benign prostatic hyperplasia and prostate cancer. RESULTS: Array analysis showed upregulation of 23 cytokines within 24 h of infection of human prostatic epithelial RWPE-1 cells with the parasite, in vitro. Reactome and STRING analysis of array data identified interleukin-6, interleukin-8, nuclear factor kappa B, signal transducer and activator of transcription 3 and cyclooxygenase 2 as chief instigators of prostatic anomaly, which were found to be significantly upregulated by immunofluorescence and western blotting analyses. STRING further connected these instigators with macrophage migration inhibitory factor, PIM-1 and prostate-specific antigen; which was confirmed by their marked stimulation in infected prostatic cells by immunoblotting and immunocytochemistry. Upregulated proliferation markers, such as Ki67, proliferating cell nuclear antigen and B-cell lymphoma 2, suggested tumor-like signaling in infected RWPE-1 cells, which was further supported by downregulation of E-cadherin, upregulation of vimentin and activation of focal adhesion kinase. Prostate tumor DU145 cells were more sensitive to parasite invasion, and showed rapid upregulation with nuclear translocation of sensitive parameters, such as nuclear factor kappa B, signal transducer and activator of transcription 3, and macrophage migration inhibitory factor. The migration of DU145 cells augmented when incubated in spent media from parasite-infected RWPE-1 cells. CONCLUSION: The initiation of inflammation driven tumor-like cell signaling in parasite-infected human prostatic epithelial cells is apparent, with the prostate tumor (DU145) cells being more sensitive to T. vaginalis than normal (RWPE-1) prostatic cells.