Rogelio Palomino-Morales

STUDY QUESTION: What is the functional impact of the rs508485 genetic polymorphism, located in the 3′-untranslated region (UTR) region of the PIWIL4 gene, on non-obstructive azoospermia (NOA)? SUMMARY ANSWER: The rs508485 genetic variant contributes to the pathogenesis of extreme patterns of NOA by modulating PIWIL4 expression through microRNA (miRNA) interactions. WHAT IS KNOWN ALREADY: Male infertility represents a significant global health challenge with profound societal and economic consequences. One of the most severe forms of male infertility is NOA, which is characterized by severe spermatogenic failure (SPGF) of idiopathic origin in most cases. Cumulating knowledge increasingly suggests that this idiopathic form of NOA may represent a multifactorial condition involving complex interactions between genetic and environmental factors. The PIWI protein subfamily, particularly PIWIL4, plays a pivotal role in spermatogenesis by processing PIWI-interacting RNAs, which silence retrotransposons to protect genomic integrity. Genetic variations in this gene have been found to be associated with susceptibility to NOA. STUDY DESIGN, SIZE, DURATION: A case–control study was conducted in a European cohort including 1516 infertile men with SPGF and 2451 fertile controls. Logistic regression and functional assays were employed to investigate the functional role of the rs508485 polymorphism in PIWIL4. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were genotyped for the rs508485 polymorphism. Associations between the polymorphism and NOA phenotypes, including Sertoli cell-only (SCO) syndrome and testicular sperm extraction (TESE) outcomes, were assessed. In silico tools predicted miRNA binding effects, which were subsequently validated using luciferase reporter assays. MAIN RESULTS AND THE ROLE OF CHANCE: The T allele of rs508485 was significantly associated with the SCO phenotype (P = 2.69E-03, OR = 1.34) and unfavourable TESE outcomes (P = 1.09E-03, OR = 1.54). In silico analyses predicted that the rs508485 variant might alter binding sites in the 3′-UTR region of PIWIL4 for different miRNAs, such as hsa-miR-215-3p and hsa-miR-136-3p. Functional validation using luciferase assays confirmed that these miRNAs differentially bind to the T and C alleles of this polymorphism, influencing PIWIL4 regulation. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The study is limited to a single genetic polymorphism and functional assays were performed in vitro. Additional studies are required to validate these findings across diverse populations and explore additional genetic interactions. WIDER IMPLICATIONS OF THE FINDINGS: These findings highlight the critical role of miRNA regulation in extreme forms of male infertility by influencing the expression of essential spermatogenesis genes, such as PIWIL4. Our study sheds light on the genetic mechanisms underlying spermatogenesis and suggests potential therapeutic targets for NOA. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Spanish Ministry of Economy and Competitiveness through the Spanish National Plan for Scientific and Technical Research and Innovation (grant: PID2020-120157RB-I00 funded by MICIU/AEI/10.13039/501100011033; and grant: PID2023-152215OB-I00 funded by MICIU/AEI/10.13039/501100011033 and ERDF, EU). M.C.-M is beneficiary of a Juan de la Cierva 2022 postdoctoral fellowship (reference number JDC2022-048561-I) funded by MCIN/AEI/10.13039/501100011033 and European Union NextGenerationEU/PRTR. S.L. received support from “Generalitat de Catalunya” (grant 2021SGR052). S.L. is sponsored by the “Researchers Consolidation Program” from the SNS-Dpt. Salut Generalitat de Catalunya (Exp. CES09/020). S.G.-M. was funded by grant ref. FPU23/02674. S.S. was supported by funds from the Portuguese Foundation for Science and Technology (FCT; 10.54499/DL57/2016/CP1363/CT0019). The authors declare no competing interests.

STUDY QUESTION: Do the genetic determinants of idiopathic severe spermatogenic failure (SPGF) differ between generations? SUMMARY ANSWER: Our data support that the genetic component of idiopathic SPGF is impacted by dynamic changes in environmental exposures over decades. WHAT IS KNOWN ALREADY: The idiopathic form of SPGF has a multifactorial etiology wherein an interaction between genetic, epigenetic, and environmental factors leads to the disease onset and progression. At the genetic level, genome-wide association studies (GWASs) allow the analysis of millions of genetic variants across the genome in a hypothesis-free manner, as a valuable tool for identifying susceptibility risk loci. However, little is known about the specific role of non-genetic factors and their influence on the genetic determinants in this type of conditions. STUDY DESIGN, SIZE, DURATION: Case-control genetic association analyses were performed including a total of 912 SPGF cases and 1360 unaffected controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: All participants had European ancestry (Iberian and German). SPGF cases were diagnosed during the last decade either with idiopathic non-obstructive azoospermia (n = 547) or with idiopathic non-obstructive oligozoospermia (n = 365). Case-control genetic association analyses were performed by logistic regression models considering the generation as a covariate and by in silico functional characterization of the susceptibility genomic regions. MAIN RESULTS AND THE ROLE OF CHANCE: This analysis revealed 13 novel genetic association signals with SPGF, with eight of them being independent. The observed associations were mostly explained by the interaction between each lead variant and the age-group. Additionally, we established links between these loci and diverse non-genetic factors, such as toxic or dietary habits, respiratory disorders, and autoimmune diseases, which might potentially influence the genetic architecture of idiopathic SPGF. LARGE SCALE DATA: GWAS data are available from the authors upon reasonable request. LIMITATIONS, REASONS FOR CAUTION: Additional independent studies involving large cohorts in ethnically diverse populations are warranted to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS: Overall, this study proposes an innovative strategy to achieve a more precise understanding of conditions such as SPGF by considering the interactions between a variable exposome through different generations and genetic predisposition to complex diseases. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the “Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020)” (ref. PY20_00212, P20_00583), the Spanish Ministry of Economy and Competitiveness through the Spanish National Plan for Scientific and Technical Research and Innovation (ref. PID2020-120157RB-I00 funded by MCIN/ AEI/10.13039/501100011033), and the ‘Proyectos I+D+i del Programa Operativo FEDER 2020’ (ref. B-CTS-584-UGR20). ToxOmics-Centre for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology, is also partially supported by the Portuguese Foundation for Science and Technology (Projects: UIDB/00009/2020; UIDP/00009/2020). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

We conducted a genome-wide association study in a large population of infertile men due to unexplained spermatogenic failure (SPGF). More than seven million genetic variants were analysed in 1,274 SPGF cases and 1,951 unaffected controls from two independent European cohorts. Two genomic regions were associated with the most severe histological pattern of SPGF, defined by Sertoli cell-only (SCO) phenotype, namely the MHC class II gene HLA-DRB1 (rs1136759, P = 1.32E-08, OR = 1.80) and an upstream locus of VRK1 (rs115054029, P = 4.24E-08, OR = 3.14), which encodes a protein kinase involved in the regulation of spermatogenesis. The SCO-associated rs1136759 allele (G) determines a serine in the position 13 of the HLA-DRβ1 molecule located in the antigen-binding pocket. Overall, our data support the notion of unexplained SPGF as a complex trait influenced by common variation in the genome, with the SCO phenotype likely representing an immune-mediated condition.

Background: Previous studies in animal models evidenced that genetic mutations of KATNAL1, resulting in dysfunction of its encoded protein, lead to male infertility through disruption of microtubule remodelling and premature germ cell exfoliation. Subsequent studies in humans also suggested a possible role of KATNAL1 single‐nucleotide polymorphisms in the development of male infertility as a consequence of severe spermatogenic failure.Objectives: The main objective of the present study is to evaluate the effect of the common genetic variation of KATNAL1 in a large and phenotypically well‐characterised cohort of infertile men because of severe spermatogenic failure.Materials and methods: A total of 715 infertile men because of severe spermatogenic failure, including 210 severe oligospermia and 505 non‐obstructive azoospermia patients, as well as 1058 unaffected controls were genotyped for three KATNAL1 single‐nucleotide polymorphism taggers (rs2077011, rs7338931 and rs2149971). Case–control association analyses by logistic regression assuming different models and in silico functional characterisation of risk variants were conducted.Results: Genetic associations were observed between the three analysed taggers and different severe spermatogenic failure groups. However, in all cases, the haplotype model (rs2077011*C | rs7338931*T | rs2149971*A) better explained the observed associations than the three risk alleles independently. This haplotype was associated with non‐obstructive azoospermia (adjusted p = 4.96E‐02, odds ratio = 2.97), Sertoli‐cell only syndrome (adjusted p = 2.83E‐02, odds ratio = 5.16) and testicular sperm extraction unsuccessful outcomes (adjusted p = 8.99E‐04, odds ratio = 6.13). The in silico analyses indicated that the effect on severe spermatogenic failure predisposition could be because of an alteration of the KATNAL1 splicing pattern.Conclusions: Specific allelic combinations of KATNAL1 genetic polymorphisms may confer a risk of developing severe male infertility phenotypes by favouring the overrepresentation of a short non‐functional transcript isoform in the testis.

What is the contribution of the common variation of KATNAL1 to the development of severe spermatogenic failure (SPGF) in a phenotypically well-characterized cohort? An allelic combination of KATNAL1 single-nucleotide polymorphisms (SNPs) increases the risk to develop SPGF, likely by altering the expression and splicing pattern of the gene. Spermatogenesis is a process that requires an exhaustive control of gene expression, and subtle alterations of its molecular regulatory network can lead to male infertility. The aetiology of most SPGF cases remains unknown, and increasing evidence clearly suggests that the idiopathic form of SPGF represents a complex trait, in which genetic susceptibility is conferred by the accumulation of risk alleles in genetically predisposed men. In this regard, previous studies reported that rare genetic mutations and polymorphisms in the KATNAL1 locus lead to male infertility through the disruption of microtubule remodelling and premature germ cell exfoliation. We designed a case-control genetic association study including three SNPs (rs2077011, rs7338931, and rs2149971) in the 3' and 5' regulatory regions of KATNAL1, which tagged the common genetic variability in the region. The allele frequencies in the study population, composed of 715 infertile men diagnosed with idiopathic SPGF, were compared to those observed in a control group comprising 1058 fertile men from Spain and Portugal. The SPGF group comprised 210 men with severe oligospermia (SO) and 505 with non-obstructive azoospermia (NOA). The latter were phenotypically characterised according to the histological examination of testis biopsies and its outcome (Sertoli cell-only syndrome, SCO; maturation arrest, MA; hypospermatogenesis, HS; and testicular sperm extraction, TESE). After genotyping, case-control association analyses by logistic regression were conducted. In silico functional characterization of risk variants was also carried out using public multiomic databases and bioinformatic tools. Significant genetic associations with different SPGF patterns and/or TESE outcome were observed even after correction for multiple testing when independent SNP models were tested. However, in all cases, the haplotype model including the three risk alleles (rs2077011*C | rs7338931*T | rs2149971*A) was the most informative. This haplotype was specifically over-represented in the SPGF group (P = 3.45E-02, OR = 2.33), which includes all infertile men, and in the NOA group (P = 8.22E-03, OR = 2.97). In addition, subtype-specific associations were observed with the most severe subgroups, namely MA (P = 2.44E-02, OR = 5.00), SCO (P = 4.03E-03, OR = 5.16), and unsuccessful TESE outcome (P = 2.22E-04, OR = 6.13), which indicates the relevant role of KATNAL1 in spermatogenesis development. We prioritized the most likely causal variant/s based on in silico analyses addressing the possible functional implication of the tagged variants. We observed that an alteration of the KATNAL1 splicing pattern, by favouring the overrepresentation of a short non-functional transcript isoform in the testis, might be the cause behind the observed genetic association. The analysis of the testis transcriptome at the single cell level showed that KATNAL1 transcripts were mostly presented in spermatocytes and early spermatids at puberty, which correlates with its effect of premature exfoliation and loss of the germ cells. Although a previous low-powered study reported suggestive associations between KATNAL1 and SPGF, additional genetic association studies in independent populations should be conducted to confirm our findings. Moreover, the statistical power for the overall analysis was appropriate, but the subphenotype analyses were performed with reduced power due to smaller study groups. Our results suggest a relevant role of the common genetic variation of KATNAL1 in the susceptibility to develop the most severe histological phenotypes of NOA (i.e., SCO and MA). Studies like the one presented here may definitively help to develop future non-invasive molecular markers of TESE success. Not Applicable

We aimed to analyze the role of the common genetic variants located in the PIN1 locus, a relevant prolyl isomerase required to control the proliferation of spermatogonial stem cells and the integrity of the blood–testis barrier, in the genetic risk of developing male infertility due to a severe spermatogenic failure (SPGF). Genotyping was performed using TaqMan genotyping assays for three PIN1 taggers (rs2287839, rs2233678 and rs62105751). The study cohort included 715 males diagnosed with SPGF and classified as suffering from non-obstructive azoospermia (NOA, n = 505) or severe oligospermia (SO, n = 210), and 1058 controls from the Iberian Peninsula. The allelic frequency differences between cases and controls were analyzed by the means of logistic regression models. A subtype specific genetic association with the subset of NOA patients classified as suffering from the Sertoli cell-only (SCO) syndrome was observed with the minor alleles showing strong risk effects for this subset (OR_addrs2287839 = 1.85 (1.17–2.93), OR_addrs2233678 = 1.62 (1.11–2.36), OR_addrs62105751 = 1.43 (1.06–1.93)). The causal variants were predicted to affect the binding of key transcription factors and to produce an altered PIN1 gene expression and isoform balance. In conclusion, common non-coding single-nucleotide polymorphisms located in PIN1 increase the genetic risk to develop SCO.

Allosterism is a common phenomenon in protein biochemistry that allows rapid regulation of protein stability; dynamics and function. However, the mechanisms by which allosterism occurs (by mutations or post-translational modifications (PTMs)) may be complex, particularly due to long-range propagation of the perturbation across protein structures. In this work, we have investigated allosteric communication in the multifunctional, cancer-related and antioxidant protein NQO1 by mutating several fully buried leucine residues (L7, L10 and L30) to smaller residues (V, A and G) at sites in the N-terminal domain. In almost all cases, mutated residues were not close to the FAD or the active site. Mutations L→G strongly compromised conformational stability and solubility, and L30A and L30V also notably decreased solubility. The mutation L10A, closer to the FAD binding site, severely decreased FAD binding affinity (≈20 fold vs. WT) through long-range and context-dependent effects. Using a combination of experimental and computational analyses, we show that most of the effects are found in the apo state of the protein, in contrast to other common polymorphisms and PTMs previously characterized in NQO1. The integrated study presented here is a first step towards a detailed structural–functional mapping of the mutational landscape of NQO1, a multifunctional and redox signaling protein of high biomedical relevance.

HIF-1α is a master regulator of oxygen homeostasis involved in different stages of cancer development. Thus, HIF-1α inhibition represents an interesting target for anti-cancer therapy. It was recently shown that the HIF-1α interaction with NQO1 inhibits proteasomal degradation of the former, thus suggesting that targeting the stability and/or function of NQO1 could lead to the destabilization of HIF-1α as a therapeutic approach. Since the molecular interactions of NQO1 with HIF-1α are beginning to be unraveled, in this review we discuss: (1) Structure–function relationships of HIF-1α; (2) our current knowledge on the intracellular functions and stability of NQO1; (3) the pharmacological modulation of NQO1 by small ligands regarding function and stability; (4) the potential effects of genetic variability of NQO1 in HIF-1α levels and function; (5) the molecular determinants of NQO1 as a chaperone of many different proteins including cancer-associated factors such as HIF-1α, p53 and p73α. This knowledge is then further discussed in the context of potentially targeting the intracellular stability of HIF-1α by acting on its chaperone, NQO1. This could result in novel anti-cancer therapies, always considering that the substantial genetic variability in NQO1 would likely result in different phenotypic responses among individuals.