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Background: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are short (21 to 35 nucleotides in length) and noncoding and are found almost exclusively in germ cells, where they regulate aberrant expression of transposable elements and postmeiotic gene expression. Critical to the processing of piRNAs is the protein poly(A)-specific RNase-like domain containing 1 (PNLDC1), which trims their 3' ends and, when disrupted in mice, causes azoospermia and male infertility.Methods: We performed exome sequencing on DNA samples from 924 men who had received a diagnosis of nonobstructive azoospermia. Testicular-biopsy samples were analyzed by means of histologic and immunohistochemical tests, in situ hybridization, reverse-transcriptase-quantitative-polymerase-chain-reaction assay, and small-RNA sequencing.Results: Four unrelated men of Middle Eastern descent who had nonobstructive azoospermia were found to carry mutations in PNLDC1: the first patient had a biallelic stop-gain mutation, p.R452Ter (rs200629089; minor allele frequency, 0.00004); the second, a novel biallelic missense variant, p.P84S; the third, two compound heterozygous mutations consisting of p.M259T (rs141903829; minor allele frequency, 0.0007) and p.L35PfsTer3 (rs754159168; minor allele frequency, 0.00004); and the fourth, a novel biallelic canonical splice acceptor site variant, c.607-2A→T. Testicular histologic findings consistently showed error-prone meiosis and spermatogenic arrest with round spermatids of type Sa as the most advanced population of germ cells. Gene and protein expression of PNLDC1, as well as the piRNA-processing proteins PIWIL1, PIWIL4, MYBL1, and TDRKH, were greatly diminished in cells of the testes. Furthermore, the length distribution of piRNAs and the number of pachytene piRNAs was significantly altered in men carrying PNLDC1 mutations.Conclusions: Our results suggest a direct mechanistic effect of faulty piRNA processing on meiosis and spermatogenesis in men, ultimately leading to male infertility. (Funded by Innovation Fund Denmark and others.). [ABSTRACT FROM AUTHOR]

Background: The effects of vitamin D are exerted by interaction with the vitamin D receptor (VDR) and vitamin D binding protein (VDBP). Polymorphisms in VDR or VDBP genes may affect vitamin D levels, influencing the pathogenesis of asthma and atopy. The aim of this study was to investigate the possible association of VDR and VDBP gene single-nucleotide polymorphisms (SNP), 25-hydroxyvitamin D (25(OH)D), blood eosinophils and total IgE level in subjects with asthma in comparison with healthy individuals., Methods: This case-control study enrolled 63 subjects with asthma (45 allergic and 18 non-allergic) and 32 healthy subjects were involved in the study. Sensitization of subjects to inhaled allergens was determined by a skin prick test, lung function was evaluated by spirometry. Blood eosinophil count was determined by standard methods. Serum 25(OH)D and total IgE levels were evaluated by ELISA. Polymorphisms in the VDR and VDBP genes on the 12q13.11 and 4q13.3 chromosomal region were analyzed using TaqMan SNP Genotyping Assay probes., Results: In asthma patients with vitamin D deficiency (< 20 ng/ml) the allele G of rs11168293 of VDR was more common than in those having insufficiency (20-30 ng/ml) of vitamin D (63% and 31%, p < 0.05). Moreover, asthmatic subject with rs11168293 G allele has significant higher blood eosinophil count compared to asthmatic without the rs11168293 G allele (8.5 ± 12.3% vs. 5.1 ± 1.5%, p < 0.05). Significantly higher IgE level was found in subjects with allergic asthma with the allele A of rs7041 on VDBP gene than in those without this allele (540 ± 110 and 240 ± 80 IU/ml, p < 0.05)., Conclusions: The association of polymorphisms in VDBP and VDR gene, the rs11168293 G allele and the rs7041 A allele, with 25(OH)D, blood eosinophil and total IgE level in asthma, let us suggest that vitamin D, VDR and VDBP gene polymorphisms are important in pathogenesis of asthma despite its form in relation to atopy., (© 2023. The Author(s).)

In 2020, 878,348 newly reported cases and 444,347 deaths related to head and neck cancer were reported. These numbers suggest that there is still a need for molecular biomarkers for the diagnosis and prognosis of the disease. In this study, we aimed to analyze mitochondria-related mitochondrial transcription factor A ( TFAM) and DNA polymerase γ ( POLG) single-nucleotide polymorphisms (SNPs) in the head and neck cancer patient group and evaluate associations between SNPs, disease characteristics, and patient outcomes. Genotyping was performed using TaqMan probes with Real-Time polymerase chain reaction. We found associations between TFAM gene SNPs rs11006129 and rs3900887 and patient survival status. We found that patients with the TFAM rs11006129 CC genotype and non-carriers of the T allele had longer survival times than those with the CT genotype or T-allele carriers. Additionally, patients with the TFAM rs3900887 A allele tended to have shorter survival times than non-carriers of the A allele. Our findings suggest that variants in the TFAM gene may play an important role in head and neck cancer patient survival and could be considered and further evaluated as prognostic biomarkers. However, due to the limited sample size ( n = 115), further studies in larger and more diverse cohorts are needed to confirm these findings.

Background: Puberty marks the transition from childhood to adulthood and is initiated by activation of a pulsatile GnRH secretion from the hypothalamus. MKRN3 functions as a pre-pubertal break on the GnRH pulse generator and hypothalamic expression and circulating levels of MKRN3 decrease peri-pubertally. In rodents, microRNA miR-30b seems to directly target hypothalamic MKRN3 expression - and in boys, circulating levels of miR-30b-5p increase when puberty is pharmacologically induced. Similarly, miR-200b-3p and miR-155-5p have been suggested to inhibit expression of other proteins potentially involved in the regulation of GnRH secretion. Here we measure circulating levels of these three miRNAs as boys progress through puberty., Materials and Methods: Forty-six boys from the longitudinal part of the Copenhagen Puberty Study were included. All boys underwent successive clinical examinations including estimation of testis size by palpation. miR-30b-5p, miR-200b-3p, and miR-155-5p were measured in serum by RT-qPCR using a kit sensitive to the phosphorylation status of the miRNAs. Thirty-nine boys had miRNA levels measured in three consecutive samples (pre-, peri-, and post-pubertally) and seven boys had miR-30b-5p levels measured in ten consecutive samples during the pubertal transition., Results: When circulating levels of miR-30b-5p in pre- and peri-pubertal samples were compared with post-pubertal levels, we observed a significant increase of 2.3 and 2.2-fold (p-value<6.0×10 -4 ), respectively, and a larger fraction of miR-30b-5p appeared to be phosphorylated post-pubertally indicating an increase in its bioactivity. We also observed a negative correlation between circulating levels of miR-30b-5p and MKRN3. The inter-individual variation in circulating miR-30b levels was substantial and we could not define a clinical threshold for miR-30b-5p suggestive of imminent puberty. Also, miR-155-5p showed significantly increasing levels from the peri- to the post-pubertal stage (p=3.0×10 -3 ), whereas miR-200b-3p did not consistently increase., Conclusion: Both circulating levels of miR-30b-5p and its bioactivity increase during the pubertal transition in boys supporting its role in the activation of the HPG axis at the onset of physiologically normal puberty., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mørup, Stakaitis, Main, Golubickaite, Hagen, Juul and Almstrup.)

Non-obstructive azoospermia (NOA) is the most severe form of male infertility and typically incurable. Defining the genetic basis of NOA has proven challenging, and the most advanced classification of NOA subforms is not based on genetics, but simple description of testis histology. In this study, we exome-sequenced over 1000 clinically diagnosed NOA cases and identified a plausible recessive Mendelian cause in 20%. We find further support for 21 genes in a 2-stage burden test with 2072 cases and 11,587 fertile controls. The disrupted genes are primarily on the autosomes, enriched for undescribed human "knockouts", and, for the most part, have yet to be linked to a Mendelian trait. Integration with single-cell RNA sequencing data shows that azoospermia genes can be grouped into molecular subforms with synchronized expression patterns, and analogs of these subforms exist in mice. This analysis framework identifies groups of genes with known roles in spermatogenesis but also reveals unrecognized subforms, such as a set of genes expressed across mitotic divisions of differentiating spermatogonia. Our findings highlight NOA as an understudied Mendelian disorder and provide a conceptual structure for organizing the complex genetics of male infertility, which may provide a rational basis for disease classification., (© 2022. The Author(s).)

Most glioblastoma studies incorporate the layer of tumor molecular subtype based on the four-subtype classification system proposed in 2010. Nevertheless, there is no universally recognized and convenient tool for glioblastoma molecular subtyping, and each study applies a different set of markers and/or approaches that cause inconsistencies in data comparability and reproducibility between studies. Thus, this study aimed to create an applicable user-friendly tool for glioblastoma classification, with high accuracy, while using a significantly smaller number of variables. The study incorporated a TCGA microarray, sequencing datasets, and an independent cohort of 56 glioblastomas (LUHS cohort). The models were constructed by applying the Agilent G4502 dataset, and they were tested using the Affymetrix HG-U133a and Illumina Hiseq cohorts, as well as the LUHS cases. Two classification models were constructed by applying a logistic regression classification algorithm, based on the mRNA levels of twenty selected genes. The classifiers were translated to a RT-qPCR assay and validated in an independent cohort of 56 glioblastomas. The classification accuracy of the 20-gene and 5-gene classifiers varied between 90.7-91% and 85.9-87.7%, respectively. With this work, we propose a cost-efficient three-class (classical, mesenchymal, and proneural) tool for glioblastoma molecular classification based on the mRNA analysis of only 5-20 genes, and we provide the basic information for classification performance starting from the wet-lab stage. We hope that the proposed classification tool will enable data comparability between different research groups.

Infertility affects around 7% of the male population and can be due to severe spermatogenic failure (SPGF), resulting in no or very few sperm in the ejaculate. We initially identified a homozygous frameshift variant in FKBP6 in a man with extreme oligozoospermia. Subsequently, we screened a total of 2,699 men with SPGF and detected rare bi-allelic loss-of-function variants in FKBP6 in five additional persons. All six individuals had no or extremely few sperm in the ejaculate, which were not suitable for medically assisted reproduction. Evaluation of testicular tissue revealed an arrest at the stage of round spermatids. Lack of FKBP6 expression in the testis was confirmed by RT-qPCR and immunofluorescence staining. In mice, Fkbp6 is essential for spermatogenesis and has been described as being involved in piRNA biogenesis and formation of the synaptonemal complex (SC). We did not detect FKBP6 as part of the SC in normal human spermatocytes, but small RNA sequencing revealed that loss of FKBP6 severely impacted piRNA levels, supporting a role for FKBP6 in piRNA biogenesis in humans. In contrast to findings in piRNA-pathway mouse models, we did not detect an increase in LINE-1 expression in men with pathogenic FKBP6 variants. Based on our findings, FKBP6 reaches a "strong" level of evidence for being associated with male infertility according to the ClinGen criteria, making it directly applicable for clinical diagnostics. This will improve patient care by providing a causal diagnosis and will help to predict chances for successful surgical sperm retrieval., Competing Interests: Declaration of interests C.N.J. has an Investigator-led grant from Logixx Pharma Ltd, UK., (Crown Copyright © 2022. Published by Elsevier Inc. All rights reserved.)

Background: The occurrence of allergic conditions, for example allergic asthma, rhinitis, and atopic dermatitis, is rising worldwide. These allergic conditions are associated with poor life quality. Vitamin D is proposed to be linked with increased risk and severe forms of allergic diseases., Aims: This review article aimed to evaluate the vitamin D level role and polymorphisms of vitamin D receptor gene (VDR) in atopy., Methods & Materials: We analyzed publications that were focusing on levels of vitamin D and/or polymorphism analysis of vitamin D receptor gene in allergic asthma, rhinitis, and atopic dermatitis patients., Results: We noticed that levels of vitamin D are extensively studied in atopy by many research groups, however, polymorphisms of vitamin D receptor gene and their link with levels of vitamin D lack comprehensive data. There is evidence that vitamin D may be associated with anti-inflammatory effects in allergic diseases. Some of VDR polymorphisms also may play a role in pathogenesis of these diseases. However, the data from different studies are controversial., Discussion: The results of different studies are usually inconsistent, most probably due to populational bias or differences in methodology. Even though, more evidence shows a positive impact of vitamin D on the risk and outcomes of allergic diseases, especially atopic dermatitis, and asthma., Conclusions: There is controversial data about the level of vitamin D and its role in atopy; however, more evidence shows a positive impact on the risk and outcomes of allergic diseases., (© 2021 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Cervical cancer is the fourth most common type of cancer in women worldwide, with high incidence and mortality rates, particularly in developing countries. There are human papillomavirus vaccines and cytological screening programs available; however, there are no molecular markers that would aid the prognosis of the course of the disease or prediction of the outcomes of the patients. The aim of the present study was to investigate the associations between single nucleotide polymorphisms (SNPs) of the mitochondrial transcription factor A ( TFAM ) gene (rs11006132, rs11006129, rs1937, rs16912174, rs16912202 and rs3900887), and the clinical parameters and tumor phenotype of patients with cervical cancer. DNA isolated from patients with cervical cancer (n=172) was used for genotyping using Real-Time PCR using TaqMan probes. It was revealed that the TFAM rs3900887 TT and AT genotypes were associated with a lower risk of developing larger tumors. The results showed an association between the rs3900887 SNP and tumor phenotype, indicating TFAM rs3900887 as a potential biomarker for tumor size in cervical cancer., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Golubickaite et al.)

Purpose: Breast cancer is the most frequent female cancer, leading to relapse with distant metastasis of approximately one-third of patients. Cancer is usually considered a genetic disease involving mutations in nuclear DNA. However, genes, coding for mitochondrial proteins or regulatory molecules, are rarely under consideration. This study aimed to analyse 10 single nucleotide variants in POLG and TFAM genes and assess their association with tumour phenotype and disease outcome., Materials and Methods: A total of 234 breast cancer patients were included in this study. Variations were determined with Real-Time PCR using TaqMan ® probes., Results: We found that patients with POLG rs2307441 TT and CT genotypes had a lower probability for vascular invasion than those with CC genotype ( p =  0.001). Patients with POLG rs2072267 AG genotype were predisposed for progression compared with GG genotype ( p =  0.015). TFAM rs3900887 TT genotype was associated with a higher probability for positive oestrogen receptors ( p =  0.003) and lymphatic invasion ( p =  0.001) in comparison to AA genotype, patients with TT ( p =  0.000) were more likely to have positive lymph nodes., Conclusions: Our data suggest that variations in POLG and TFAM genes are important determinacies of tumour phenotype and disease outcome in breast cancer patients.

Circulating miRNAs secreted by testicular germ cell tumors (TGCT) show great potential as novel non-invasive biomarkers for diagnosis of TGCT. Seminal plasma (SP) represents a biofluid closer to the primary site. Here, we investigate whether small RNAs in SP can be used to diagnose men with TGCTs or the precursor lesions, germ cell neoplasia in situ (GCNIS). Small RNAs isolated from SP from men with TGCTs ( n = 18), GCNIS-only ( n = 5), and controls ( n = 25) were sequenced. SP from men with TGCT/GCNIS ( n = 37) and controls ( n = 22) were used for validation by RT-qPCR. In general, piRNAs were found at lower levels in SP from men with TGCTs. Ten small RNAs were found at significantly (q-value < 0.05) different levels in SP from men with TGCT/GCNIS than controls. Random forests classification identified sets of small RNAs that could detect either TGCT/GCNIS or GCNIS-only with an area under the curve of 0.98 and 1 in ROC analyses, respectively. RT-qPCR validated hsa-miR-6782-5p to be present at 2.3-fold lower levels ( p = 0.02) in the SP from men with TGCTs compared with controls. Small RNAs in SP show potential as novel biomarkers for diagnosing men with TGCT/GCNIS but validation in larger cohorts is needed.

Cervical cancer is one of the most common cancers in women worldwide. Human papillomaviruses are known to be the main, but not the only risk factor, of this cancer type. Despite all the knowledge on this cancer type, it is still a challenge to predict the course of the disease, and therefore, minimally invasive biomarkers are needed. This study aimed to analyze single-nucleotide variants in the POLG gene and assess the associations with tumor phenotype and patient outcome. A total of 172 cervical cancer patients were included in this study. Clinical and tumor data were gathered from medical records retrospectively. Single nucleotide variations were determined using TaqMan probes with Real-Time PCR. Significant associations between POLG rs3087374 and cervical cancer patients' tumor histological type, stage, and tumor size were determined. The CA genotype and A allele of rs3087374 increased the probability of adenocarcinoma histological tumor type, IIIA stage, and T3 tumor size compared to CC genotype and C allele, respectively. Furthermore, patients with AA genotype in rs2072267 had longer metastasis-free survival than those with the GG genotype. Our data suggest that mitochondrial polymerase gamma encoded by nuclear POLG gene is important for specific tumor phenotype formation and patient outcome in cervical cancer.