Received: 31 January 2022 Revised: 23 May 2022 Accepted: 21 June 2022 DOI: 10.1111/andr.13221 ORIGINAL ARTICLE Common genetic variation in KATNAL1 non-coding regions is involved in the susceptibility to severe phenotypes of male infertility Miriam Cerván-Martín1,2 Lara Bossini-Castillo1,2 Andrea Guzmán-Jiménez1,2 Rocío Rivera-Egea3,4 Nicolás Garrido4,5 Saturnino Lujan5 Gema Romeu5 Samuel Santos-Ribeiro6,7 IVIRMA Group# Lisbon Clinical Group# José Antonio Castilla2,8,9 María del Carmen Gonzalvo2,8 Ana Clavero2,8 Vicente Maldonado10 Francisco Javier Vicente2,11 Miguel Burgos1 Rafael Jiménez1 Sara González-Muñoz1,2 Josvany Sánchez-Curbelo12 Olga López-Rodrigo12 Iris Pereira-Caetano13 Patricia Isabel Marques14,15 Filipa Carvalho14,16 Alberto Barros14,16 Lluís Bassas12 Susana Seixas14,15 João Gonçalves17,18 Sara Larriba19 Alexandra Manuel Lopes14,15 Rogelio Jesús Palomino-Morales2,20 Francisco David Carmona1,2 1 Departamento de Genética e Instituto de Biotecnología, Centro de Investigación Biomédica, Universidad de Granada, Granada, Spain 2 Instituto de Investigación Biosanitaria ibs. GRANADA, Granada, Spain 3 Andrology Laboratory and Sperm Bank, IVIRMA Valencia, Valencia, Spain 4 IVI Foundation, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Valencia, Spain 5 Servicio de Urología, Hospital Universitari i Politecnic La Fe e Instituto de Investigación Sanitaria La Fe (IIS La Fe), Valencia, Spain 6 IVI-RMA Lisbon, Lisbon, Portugal 7 Department of Obstetrics and Gynecology, Faculty of Medicine, University of Lisbon, Lisbon, Portugal 8 Unidad de Reproducción, UGC Obstetricia y Ginecología, HU Virgen de las Nieves, Granada, Spain 9 CEIFER-GAMETIA Biobank, Granada, Spain 10 UGC de Obstetricia y Ginecología, Complejo Hospitalario de Jaén, Jaén, Spain 11 UGC de Urología, HU Virgen de las Nieves, Granada, Spain 12 Laboratory of Seminology and Embryology, Andrology Service-Fundació Puigvert, Barcelona, Spain 13 Departamento de Genética Humana, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisbon, Portugal 14 Instituto de Investigação e Inovação em Saúde, Universidade do Porto (I3S), Porto, Portugal 15 Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal 16 Serviço de Genética, Departamento de Patologia, Faculdade de Medicina da Universidade do Porto, Porto, Portugal 17 Departamento de Genética Humana, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisbon, Portugal 18 ToxOmics—Centro de Toxicogenómica e Saúde Humana, Nova Medical School, Lisbon, Portugal 19 Human Molecular Genetics Group, Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Barcelona, Spain Miriam Cerván-Martín, Lara Bossini-Castillo and Andrea Guzmán-Jiménez contributed equally. # See Supporting Information for complete affiliation. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2022 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology. Andrology. 2022;10:1339–1350. wileyonlinelibrary.com/journal/andr 1339 20472927, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.13221 by Readcube (Labtiva Inc.), Wiley Online Library on [17/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1340 CERVÁN-MARTÍN ET AL . 20 Departamento de Bioquímica y Biología Molecular I, Universidad de Granada, Granada, Spain Correspondence F. David Carmona, Centro de Investigación Abstract Biomédica (CIBM), Universidad de Granada, Parque Tecnológico Ciencias de la Salud, Avda. Background: Previous studies in animal models evidenced that genetic mutations del Conocimiento S/N, 18016-Armilla, of KATNAL1, resulting in dysfunction of its encoded protein, lead to male infertility Granada, Spain. Email: dcarmona@ugr.es through disruption of microtubule remodelling and premature germ cell exfoliation. Rogelio J. Palomino-Morales, Centro de Subsequent studies in humans also suggested a possible role of KATNAL1 single- Investigación Biomédica (CIBM), Universidad de Granada, Parque Tecnológico Ciencias de la nucleotide polymorphisms in the development of male infertility as a consequence of Salud, Avda. del Conocimiento S/N, severe spermatogenic failure. 18016-Armilla, Granada, Spain. Email: rpm@ugr.es 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 Rogelio Jesus Palomino-Morales and Francisco cohort of infertile men because of severe spermatogenic failure. David Carmona share senior authorship. Materials and methods: A total of 715 infertile men because of severe spermato- Funding information genic failure, including 210 severe oligospermia and 505 non-obstructive azoospermia Spanish Ministry of Economy and Competitiveness through the Spanish National patients, as well as 1058 unaffected controls were genotyped for three KATNAL1 Plan for Scientific and Technical Research and single-nucleotide polymorphism taggers (rs2077011, rs7338931 and rs2149971). Innovation, Grant/Award Number: SAF2016-78722-R;PID2020-120157RB-I00; Case–control association analyses by logistic regression assuming different models Instituto de Salud Carlos III, Grant/Award and in silico functional characterisation of risk variants were conducted. Number: DTS18/00101; Generalitat de Catalunya, Grant/Award Number: Results: Genetic associations were observed between the three analysed taggers and 2017SGR191; Ramón y Cajal, Grant/Award different severe spermatogenic failure groups. However, in all cases, the haplotype Number: RYC-2014-16458; Juan de la Cierva Incorporación, Grant/Award Number: model (rs2077011*C | rs7338931*T | rs2149971*A) better explained the observed IJC2018-038026-I; Andalusian Government, associations than the three risk alleles independently. This haplotype was associated Grant/Award Number: PY20_00212;B-CTS-584-UGR20; FCT with non-obstructive azoospermia (adjusted p = 4.96E-02, odds ratio = 2.97), Sertoli- post-doctoral fellowship, Grant/Award cell only syndrome (adjusted p = 2.83E-02, odds ratio = 5.16) and testicular sperm Number: SFRH/BPD/120777/2016; FCT/MCTES, Grant/Award Numbers: extraction unsuccessful outcomes (adjusted p = 8.99E-04, odds ratio = 6.13). The in UID/BIM/00009/2016, UIDB/00009/2020; silico analyses indicated that the effect on severe spermatogenic failure predisposition ISCIII SNS/Dpt. Salut Generalitat de Catalunya, Grant/Award Number: CES09/020 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. KEYWORDS KATNAL1, male infertility, SNP, spermatogenesis, splicing 1 INTRODUCTION energy of nucleotide hydrolysis through the catalytic p60 subunit and the centrosome-targeting regulatory p80 subunit.4–6 Spermatogenesis is a multistep process that relies on both the phys- Microtubules are major components of the cytoskeleton, which ical and metabolic support provided by Sertoli cells.1,2 It has been provides structural stability in every cell type. Regarding the sper- estimated that over 2000 genes are involved in the regulation of matogenesis process, microtubules play an essential role in both the spermatogenesis, and it is plausible to consider that any disruption establishment of Sertoli cell/germ cell interactions and the maturation of their function may ultimately lead to a large variety of fertility of male gametes by supporting cell division and by taking part in sperm issues.3 One of the key members of such a regulatory network is the head remodelling and sperm tail formation.7 Interestingly, reports in human katanin p60 subunit A-like 1 (KATNAL1) gene, located in human mutant mice have shown that a loss-of-function mutation in Katnal1, chromosome 13, a member of the Katanin family, which belongs to the which is expressed in both Sertoli cells and the germ line, may lead AAA ATPase superfamily. The main function of the proteins encoded to male infertility through disruption of microtubule remodelling and by these genes is to split and disassemble microtubules using the premature germ cell exfoliation from the seminiferous epithelium.8 20472927, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.13221 by Readcube (Labtiva Inc.), Wiley Online Library on [17/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License CERVÁN-MARTÍN ET AL . 1341 Subsequent studies in bovine models also associated this abnormal gene study to evaluate the possible association of the genetic variation phenotype with the presence of a splice variant of the gene that pro- in KATNAL1 with idiopathic SpF risk in a large Iberian population duces a loss of the microtubule interacting and trafficking domain of European descent. The case–control cohort comprised a total resulting in KATNAL1 dysfunction.9 of 715 infertile men because of severe SpF (including 505 NOA Although the human KATNAL1 protein has a considerably high patients and 210 SO patients) and 1058 unaffected male controls sequence identity with its bovine and murine orthologues (99% and matched by age, ethnicity and geographical origin, as previously 93%, respectively), no association of KATNAL1 genetic variants with described.17,18 human male infertility was observed in a case–control study performed Our study complied with the ethical guidelines and was conducted by Fedick et al.10 However, some of the single-nucleotide polymor- in accordance with the Declaration of Helsinki. The study protocol and phisms (SNP) located in the 3′UTR of KATNAL1 were posited to predict the informed written consent, which was signed by all participants, was male infertility based on a borderline statistical significance of the approved by the Ethics Committee ‘CEIM/CEI Provincial de Granada’ association, and the authors eventually speculated that these may have (Andalusia, Spain) at the session held on January 26, 2021 (approval been because of limited statistical power, as only 105 non-obstructive number: 1/21). In addition, each participating centre received prior azoospermia (NOA) cases and 242 normozoospermic controls were ethical approval in compliance with the requirements of their local analysed.10 regulatory authorities. Interestingly, genetic variants of the KATNAL2 and KATNB1 genes, The control group was composed of 700 population-representative which encode two additional members of the katanin family that men (with a self-reported fatherhood) and 358 samples from interact with KATNAL1 to maintain the integrity of Sertoli cells and men with normal semen analyses (sperm number and motil- to allow the production of male germ cells,11 have been associated ity), all of them matching the geographical origin and ethnicity of with the development of male subfertility because of oligo-astheno- cases. teratozoospermia, a condition characterised by low sperm counts, SpF cases were recruited in different private fertility clinics as well teratozoospermia (abnormal sperm shape) and asthenozoospermia as in public health centres and hospitals from Spain and Portugal. (poor sperm movement).12 Patient recruitment relied on exhaustive medical examination and Two of the most extreme forms of male infertility caused by severe clinical tests by experts in this clinical field. Two high-speed centrifu- spermatogenic failure (SpF) are severe oligozoospermia (SO), charac- gation processes in two different semen samples were performed terised by very low sperm counts in the ejaculate (<5 million sperma- to establish the diagnosis, according to the guidelines of the World tozoa/ml), and NOA, defined by a complete absence of spermatozoa Health Organization.19 NOA was defined as a total absence of sperm in the ejaculate without any obstruction of the post-testicular genital cells in the ejaculate, while SO was diagnosed whenever a sperm con- tract.13 Although there are some known genetic causes of SpF, includ- centration below 5 million/ml was observed. In order to select patients ing Y chromosome microdeletions, karyotype abnormalities, deficits with SpF of idiopathic origin, each medical file was scrutinised in order in gonadotropin and/or sex steroid hormones, and high-penetrance to extract all the available information regarding eventual karyotype monogenic mutations, the aetiology of SpF in most patients remains analysis, Y chromosome microdeletion screening, physical examina- unknown.14 Increasing evidence clearly suggests that the idiopathic tion and endocrine analysis of luteinising hormone, follicle stimulating form of male infertility represents a complex trait, in which common hormone and testosterone. Only infertile men with a normal history variation of the human genome, such as the SNPs, may be involved in of testicular development, normal karyotype and the absence of its predisposition and development.15 Indeed, genome-wide associa- Yq AZF deletions were selected. A testicular biopsy was obtained tion studies have identified different SNPs associated with SpF risk, from 277 NOA patients (which represents 54.85% of the total NOA which are located in non-coding regions that regulate the expression group) to assess their specific histological phenotype and to perform of nearby genes.15 testicular sperm extraction (TESE) techniques for subsequent in vitro Taking all the above into account, we decided to investigate the fertilisation reproductive treatment as previously described.17,18 possible influence of genetic variation in the 5′ and 3′ regions of KAT- Thus, NOA patients were classified further into the following major NAL1 on the genetic susceptibility for SpF. To address this question, we subgroups according to the histological analysis: (1) Sertoli-cell only designed a genetic variant panel including three tagger SNPs that cov- syndrome group (SCO, if a total absence of germ cells was observed), ered most of the common variation in the region. Then, we analysed the (2) maturation arrest of germ cells group (MA, patients with > 90% genetic association of this locus with specific subtypes of severe SpF in of maturation arrest of the germ line either at the spermatogonia or a large cohort of Iberian men. primary spermatocyte stages) and (3) hypospermatogenesis group (HS, including patients with extremely low cellularity but with all cell types of the germ line present in few testicular locations). Additionally, 2 MATERIALS AND METHODS we also established two additional subgroups based on the TESE outcome of NOA patients, that is, TESEneg (if no viable sperm cell was 2.1 Study design and sample collection retrieved from the biopsy) and TESEpos (including NOA patients with a successful sperm retrieval), as detailed elsewhere.20 All the available Following the STrengthening the REporting of Genetic Association information regarding the main clinical features of our study cohort is Studies (STREGA) reporting guidelines,16 we designed a candidate shown in Table S1. 20472927, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.13221 by Readcube (Labtiva Inc.), Wiley Online Library on [17/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1342 CERVÁN-MARTÍN ET AL . Hilden, Germany), the Wizard Genomic DNA Purification Kit Protocol (Promega, Madison, WI, USA) or the MagNA Pure LC-DNA LV Isolation kit I (Roche, Basel, Switzerland), following the manufacturer’s recom- mendations. Genotyping was performed with TaqMan SNP genotyping technology (Applied Biosystems, Foster City, CA, USA) using spe- cific predesigned TaqMan probes (assay IDs: C___1409936_10, C__62793736_10 and C__15864138_10) and a 7900HT Fast Real-Time PCR System (Applied Biosystems), as previously described.18 2.4 Statistical analysis The statistical power of our study was estimated using the CaTS Power Calculator for Genetic Studies.23 The estimated power values of our study, accordingly with different expected ORs, are shown in Table S2. The software Plink v1.924 and R were used to perform all the statistical analyses. First, we evaluated the possible deviance from Hardy– F I G U R E 1 Genetic architecture of the KATNAL1 gene and the Weinberg equilibrium (HWE) of both the case and control cohorts at position of each analysed tagger. (A) Recombination rate across the a 5% significance level. To test for association, we conducted case– gene. (B) Linkage disequilibrium pattern of the region according to the control comparisons of the allele and genotype frequencies between all Dʹ statistic (Dʹ = 1, bright red; Dʹ < 1, shades of red) in the European (EUR) population of the 1000 Genomes Project. The promoter case groups (SpF, NOA, SO, MA, HS and TESEneg) and the control group location is represented with a red line assuming additive, dominant, recessive and two degrees of freedom (genotypic) models. In addition, cases showing a specific clinical phe- notype/TESE outcome were also compared against those not showing 2.2 Candidate gene and SNP selection it, in order to eliminate infertility as a possible confounding variable. p- Values, odds ratios (ORs) and 95% confidence intervals were calculated We performed a thorough search in the available literature to identify by means of logistic regression on the genotypes and using geograph- candidate genes potentially involved in male infertility issues. The ical origin (Spain or Portugal) as a covariate. Possible multiple testing KATNAL1 gene was selected because of its function in microtubule effects were controlled for by using the Benjamini and Hochberg split and disassembling, which is essential for proper cell division and step-up false discovery rate (FDR-BH) correction.25 p-Values <0.05 sperm remodelling and formation.7 Moreover, a potential implication after FDR-BH correction were considered statistically significant. of KATNAL1 in the development of male infertility both in humans and Haplotype-based logistic regression tests were also performed to in animal models has also been previously reported.8–10 Considering analyse putative combined effects of the KATNAL1 selected taggers the most likely complex aetiology of idiopathic SpF,15 our hypothesis (assuming any potential allele combination with a frequency higher was that deregulation of the expression levels of KATNAL1 may impact than 1% in the control population). In this case, multiple testing cor- the correct formation of microtubules, thus triggering male fertility rection was performed by permutation tests (10,000 permutations) problems. to estimate empirical p-values as implemented in Plink. To evalu- With that aim, we downloaded the genotype information of the ate whether the haplotype model would better explain the observed European cohort of the 1000 Genome Project Phase III (1KGPh3).21 associations than the model considering individual SNP effects, we Then, we followed a SNP tagging strategy as implemented in Haploview compared the goodness of fit of both models using Plink. In short, to V.4.222 to identify taggers (SNPs representative of haplotype blocks) assess whether a significant improvement occurred in fit when the hap- covering all the common genetic variation (r2 ≥ 0.8) within the main lotype effect was considered, we calculated the deviance (defined as regulatory regions of the gene (including the promoter and both the -2 × the log likelihood), and if statistically significant differences were 5′ and 3′UTR regions). Three KATNAL1 taggers were selected using observed, we assumed that the haplotype model was more informative this method: rs2077011, rs7338931 and rs2149971. Figure S1 shows in explaining the association. the linkage disequilibrium (LD) pattern between such taggers. Further- more, the haplotype architecture of KATNAL1 and the specific location of the analysed genetic variants are summarised in Figures 1, S2 and S3. 2.5 In silico characterisation of associated variants 2.3 Genotyping We decided to evaluate any functional implications of the observed Genomic DNA was extracted from peripheral white blood cells of all associations by using different bioinformatics tools and by exploring participants using the QIAamp DNA Blood Midi/Maxi kit (Qiagen, publicly available annotation data of the human genome. First, we 20472927, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.13221 by Readcube (Labtiva Inc.), Wiley Online Library on [17/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License CERVÁN-MARTÍN ET AL . 1343 identified all proxies of the analysed taggers (Dʹ > 0.8) in the European In order to further analyse the suggestive association between population of the 1KGPh3 using LDLink.26 Subsequently, a priori- rs7338931 and SO, we carried out another association test consider- tisation of proxies was also conducted, as previously described,17 ing the SO group as cases and the NOA group as controls, consequently in an attempt to understand the potential causal molecular and removing the confounding factor of having SpF. This comparison cellular mechanisms that could explain the observed associations yielded statistically significant differences in the allele/genotype using tools and resources such as the portals of GTEx,27 Single Cell frequencies of the tested groups under the additive, dominant and Expression Atlas,28 ENCODE,29 Haploreg v.4.1,30 SNPnexus31 and genotypic models (pADD = 2.76E-02, OR = 0.76; pDOM = 1.36E-02, RegulomeDB.32 The relevance of the functional scores used in the OR = 0.63; pGENO = 4.58E-02). Nonetheless, only the FDR-adjusted prioritisation analysis is described in Tables S3 and S4. p-value of the dominant model was significant (pDOM = 4.07E-02) (Table S5). No additional evidence of a possible association between the three 3 RESULTS analysed taggers and SO or NOA was observed in any of the different models tested (Tables 1 and S5). The genotyping success rate reached >99% for the three analysed SNPs, and no significant deviation from the HWE (p < 0.05) was observed either in cases or controls. Moreover, the minor allele fre- 3.3 Susceptibility to non-obstructive azoospermia quencies of both control groups were concordant with those described histological subphenotypes and unsuccessful for the Iberian subpopulation and the European super population testicular sperm extraction (EUR) of the 1KGPh321 (consequently, no significant difference in either the allele or genotype frequencies were observed between Our results suggested a subphenotype-specific genetic association them). between the KATNAL1 3′ variant rs2149971 and SCO when this subgroup was compared against the control group in the additive, dom- inant and genotypic tests (pADD = 1.76E-02, OR = 1.69; pDOM = 1.32E- 3.1 Genetic association analysis for 02, OR = 1.82; pGENO = 4.52E-02) (Table 1). The associations under spermatogenic failure overall the additive and dominant tests were also significant when adjusted by multiple testing (pADD-FDR = 4.98E-02; pDOM-FDR = 3.96E-02). The In the first step, we evaluated whether the taggers’ allele and genotype comparison between the SCO group against the non-SCO NOA group frequencies of the SpF group differed from those of the unaffected con- (i.e., including MA and HS) showed similar effect sizes toward risk for trol population. No statistically significant differences were detected rs2149971*A assuming additive (OR = 1.44) and dominant (OR = 1.50) when the additive or recessive effects of the minor alleles were con- models. However, such tests did not produce significant p-values (Table sidered (Table 1). However, significant p-values were observed under S5), likely because of the considerably lower statistical power of this the dominant and genotypic models for the rs2077011 KATNAL1 analysis in comparison with the SCO versus fertile control model. variant (pDOM = 1.55E-02, OR = 0.78; pGENO = 3.07E-02), with the first On the other hand, significant p-values were also obtained in the remaining significant following FDR correction (pDOM-FDR = 4.66E-02). comparison between the MA group and the non-MA group (i.e., that comprising SCO and HS) for rs7338931 under the dominant and geno- typic models (pDOM = 3.89E-02, OR = 0.48; pGENO = 4.53E-02), but 3.2 Susceptibility to male infertility phenotypes the statistical significance in both cases was lost after FDR correction defined by semen analysis (Table S5). Finally, the group including the NOA patients with a negative TESE Subsequently, we compared the NOA and SO groups against the outcome (TESEneg) was compared against the unaffected control pop- control cohort (Table 1). A trend towards an association between ulation. This comparison revealed a potential trend of an association rs2077011 and NOA was evident under the additive model between rs7338931 and TESEneg under the additive and genotypic (pADD = 5.90E-02, OR = 0.85). Such a suggestive association models (pADD = 5.58E-02, OR = 1.28; pGENO = 5.06E-02) and a sta- reached statistical significance when the dominant and genotypic tistically significant association when the recessive model for the models were assumed (pDOM = 8.74E-03, OR = 0.75; pGENO = 1.67E- minor allele was assumed (pREC = 1.47E-02, OR = 1.61), even after 02), even after multiple testing correction (pDOM-FDR = 2.62E-02; FDR correction (pREC-FDR = 4.40E-02) (Table 1). Similar results for pGENO-FDR = 5.00E-02). rs7338931 were obtained when the TESEneg group was tested against On the other hand, the minor allele (T) of the rs7338931 KATNAL1 the TESEpos group of NOA patients (pADD = 5.87E-02, OR = 1.45; variant showed a protective effect against SO development in both pREC = 4.50E-03, OR = 2.64; pGENO = 1.27E-02). In this case, the the dominant and genotypic models (pDOM = 2.47E-02, OR = 0.66; p-value of both the recessive and the genotypic models remained sig- pGENO = 4.91E-02). However, the p-values lost their statistical signifi- nificant when multiple testing was considered (pREC-FDR = 1.35E-02; cance when multiple testing correction was applied (Table 1). pGENO-FDR = 3.81E-02) (Table S5). 1344 TA B L E 1 Analysis of the genotype and allele frequencies of the KATNAL1 taggers comparing subgroups of clinical phenotypes of male infertility and unsuccessful TESE (TESEneg) against the unaffected control group Additive model Recessive model Dominant model Genotypic Change Genotypes SNP (1/2) Cohorts (11/12/22) MAF OR [CI 95%] p p (FDR) OR [CI 95%] p p (FDR) OR [CI 95%] p p (FDR) p p (FDR) rs2149971 A/G Controls 8/165/878 0.086 NA NA NA NA NA NA NA NA NA NA NA (n = 1051) SpF (n = 705) 7/122/576 0.097 1.12 0.3690 NS 1.36 0.5703 NS 1.12 0.4075 NS 0.6531 NS [0.88–1.43] [0.47–4.00] [0.86–1.46] SO (n = 206) 2/29/175 0.080 0.85 0.4592 NS 1.37 0.7234 NS 0.81 0.3800 NS 0.5883 NS [0.56–1.30] [0.24–7.98] [0.52–1.29] NOA (n = 499) 5/93/401 0.103 1.20 0.1727 NS 1.31 0.6481 NS 1.22 0.1754 NS 0.3921 NS [0.92–1.56] [0.41–4.16] [0.92–1.61] SCO (n = 101) 1/26/74 0.139 1.69 0.0176 0.0498 1.31 [0.16– 0.8010 NS 1.82 0.0132 0.0396 0.0452 0.1357 [1.10–2.61] 10.67] [1.13–2.91] MA (n = 52) 1/7/44 0.087 0.97 0.9368 NS 2.68 [0.31– 0.3691 NS 0.88 0.7582 NS 0.5888 NS [0.48–1.97] 23.00] [0.41–1.93] HS (n = 48) 0/10/38 0.104 1.18 0.6450 NS NA [ NA– NA] NA NS 1.26 0.5261 NS NA NS [0.59–2.34] [0.61–2.62] TESEneg 2/33/106 0.131 1.62 0.0130 0.0389 1.87 0.4307 NS 1.70 0.0130 0.0391 0.0440 0.0759 (n = 141) [1.11–2.37] [0.39–8.95] [1.12–2.57] rs7338931 T/C Controls 232/545/272 0.481 NA NA NA NA NA NA NA NA NA NA NA (n = 1049) SpF (n = 705) 167/348/190 0.484 1.03 0.7208 NS 1.13 0.3065 NS 0.95 0.6853 NS 0.4377 NS [0.89–1.19] [0.89–1.44] [0.76–1.20] SO (n = 206) 43/93/70 0.435 0.85 0.1857 NS 1.05 0.8181 NS 0.66 0.0247 0.0741 0.0491 0.1473 [0.67–1.08] [0.70–1.58] [0.46–0.95] NOA (n = 499) 124/255/120 0.504 1.09 0.2639 NS 1.18 0.2096 NS 1.08 0.5695 NS 0.4478 NS [0.93–1.28] [0.91–1.52] [0.84–1.39] SCO (n = 101) 27/54/20 0.535 1.26 0.1274 NS 1.32 0.2413 NS 1.40 0.1967 NS 0.3138 NS [0.94–1.70] [0.83–2.11] [0.84–2.33] MA (n = 52) 16/19/17 0.490 1.03 0.8755 NS 1.63 0.1220 NS 0.68 0.2183 NS 0.0626 NS [0.69–1.55] [0.88–3.01] [0.37–1.25] HS (n = 48) 14/25/9 0.552 1.36 0.1637 NS 1.52 0.2042 NS 1.45 0.3267 NS 0.3691 NS [0.88–2.10] [0.80–2.92] [0.69–3.07] TESEneg 45/64/33 0.542 1.28 0.0558 NS 1.61 0.0147 0.0440 1.16 0.4904 NS 0.0506 NS (n = 142) [0.99–1.64] [1.10–2.36] [0.76–1.75] (Continues) CERVÁN-MARTÍN ET AL . 20472927, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.13221 by Readcube (Labtiva Inc.), Wiley Online Library on [17/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License CERVÁN-MARTÍN ET AL . TA B L E 1 (Continued) Additive model Recessive model Dominant model Genotypic Change Genotypes SNP (1/2) Cohorts (11/12/22) MAF OR [CI 95%] p p (FDR) OR [CI 95%] p p (FDR) OR [CI 95%] p p (FDR) p p (FDR) rs2077011 T/C Controls 101/456/493 0.313 NA NA NA NA NA NA NA NA NA NA NA (n = 1050) SpF (n = 704) 67/267/370 0.285 0.87 0.0813 NS 1.04 0.8200 NS 0.78 0.0155 0.0466 0.0307 0.092 [0.75–1.02] [0.74–1.47] [0.64–0.95] SO (n = 205) 19/85/101 0.300 0.96 0.7597 NS 1.12 0.7096 NS 0.90 0.5446 NS 0.7044 NS [0.74–1.24] [0.63–1.98] [0.65–1.26] NOA (n = 499) 48/182/269 0.279 0.85 0.0590 NS 1.05 0.8018 NS 0.75 0.0087 0.0262 0.0167 0.0500 [0.72–1.01] [0.72–1.52] [0.60–0.93] SCO (n = 102) 10/43/49 0.309 0.98 0.8982 NS 1.06 0.8732 NS 0.95 0.7949 NS 0.9370 NS [0.72–1.34] [0.53–2.10] [0.63–1.43] MA (n = 52) 6/15/31 0.260 0.79 0.3124 NS 1.40 0.4605 NS 0.61 0.0875 NS 0.1026 NS [0.50–1.25] [0.57–3.40] [0.34–1.08] HS (n = 48) 3/22/23 0.292 0.92 0.7262 NS 0.71 0.5716 NS 0.96 0.8964 NS 0.8516 NS [0.58–1.47] [0.21–2.35] [0.53–1.73] TESEneg 16/52/75 0.294 0.91 0.4899 NS 1.16 0.6122 NS 0.80 0.2247 NS 0.3076 NS (n = 143) [0.69–1.19] [0.66–2.02] [0.57–1.14] Note: Significant p-values are highlighted in bold. Abbreviations: CI, confidence interval; FDR, false discovery rate; HS, hypospermatogenesis; MA, maturation arrest; MAF, minor allele frequency; NA, not applicable; NOA, non-obstructive azoospermia; NS, not significant; OR, odds ratio; SCO, Sertoli cell only; SNP, single-nucleotide polymorphism; SO, severe oligospermia; SpF, spermatogenic failure. 1345 20472927, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.13221 by Readcube (Labtiva Inc.), Wiley Online Library on [17/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 20472927, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.13221 by Readcube (Labtiva Inc.), Wiley Online Library on [17/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1346 CERVÁN-MARTÍN ET AL . TA B L E 2 Case–control analysis of the haplotype containing the combination of the risk alleles of the three KATNAL1 taggers (rs2077011*C | rs7338931*T | rs2149971*A) according to different clinical features of male infertility Haplotype frequency Clinical feature (cases/controls) p-Value pPERM a OR [CI 95%] SpF 0.019/0.011 0.0345 0.1970 2.33 [1.06–5.10] NOA 0.023/0.011 0.0082 0.0496 2.97 [1.33–6.66] SO 0.012/0.011 0.9330 1.0000 0.94 [0.22–4.00] HS 0.025/0.011 0.1670 0.6253 3.33 [0.60–18.34] MA 0.034/0.011 0.0244 0.1351 5.00 [1.23–20.32] SCO 0.031/0.011 0.0040 0.0283 5.16 [1.69–15.79] TESEneg 0.034/0.011 0.0002 0.0009 6.13 [2.34–16.07] Note: Significant p-values are highlighted in bold. Abbreviations: CI, confidence interval; HS, hypospermatogenesis; MA, maturation arrest; NOA, non-obstructive azoospermia; OR, odds ratio; SCO, Sertoli cell only; SO, severe oligospermia; SpF, spermatogenic failure; TESEneg, unsuccessful testicular sperm extraction. a Permutation test p-value for 10,000 permutations. The 3′ KATNAL1 tagger, rs2149971, was also significantly asso- 3.5 In silico characterisation ciated with TESE outcome when the TESEneg group was com- pared against the fertile control group under additive, dominant and According to the Human Protein Atlas database,33 the testis repre- genotypic models (pADD = 1.30E-02, OR = 1.62; pDOM = 1.30E-02, sents the organ with the highest expression of KATNAL1 (Figure S4). OR = 1.70; pGENO = 4.40E-02) (Table 1), with the two first maintain- First, in order to determine the specific cell types of the human testis ing statistical significance after FDR adjustment (pADD-FDR = 3.89E- in which this gene is expressed, we queried the Single Cell Expression 02; pDOM-FDR = 3.91E-02). Although the comparison between the Atlas portal,28 which showed that KATNAL1 transcripts were mostly TESEneg and TESEpos groups did not yield significant p-values, the present in spermatocytes and early spermatids at puberty34 (Figure ORs observed for such models (OR = 1.68 assuming an additive effect S4). and OR = 1.62 under a dominant effect of the minor allele) were con- Subsequently, considering that our genetic study was performed sistent with those obtained from the more powered TESEneg versus following a tagging strategy (meaning that the analysed SNPs were fertile control analysis (Tables 1 and S5). not selected based on their possible functional evidence but on their No additional associations were observed in the subtype analyses representativeness of haplotype blocks), we decided to identify all of (Tables 1 and S5). their proxies (Dʹ > 0.8) in the European population of the 1KGPh3.21 A prioritisation analysis of the taggers and proxies was then con- ducted to elucidate the putative causal variants of the observed 3.4 Haplotype analysis KATNAL1 associations with male infertility features. All identified prox- ies were located in non-coding regions, namely, in introns and the To investigate whether the allelic combinations of SNPs located in the 5′ upstream region of KATNAL1 (Figures S2 and S3 and Table S8). different loci resulted in an increased risk of disease susceptibility or According to the GTEx project,27 a large number of the proxies of a high probability of unsuccessful TESE, a haplotype analysis includ- the 5′ tagger rs2077011 are expression quantitative trait loci (eQTLs) ing all combinations of the three KATNAL1 taggers was performed. The of KATNAL1 in different tissues but not in the testis, in which an haplotype containing the risk alleles of the three SNPs (rs2077011*C eQTL effect of such proxies was observed for other genes such as | rs7338931*T | rs2149971*A) was significantly associated with SpF HMGB1, MEDAG and RP11-374F3.5 (Table S8). Both these genes and (p = 3.45E-02, OR = 2.33), NOA (p = 8.22E-03, OR = 2.97), MA KATNAL1 are targets of the same enhancer elements according to (p = 2.44E-02, OR = 5.00), SCO (p = 4.03E-03, OR = 5.16) and ENCODE.29 TESEneg (p = 2.22E-04, OR = 6.13) (Tables 2 and S6). The haplotype Regarding the rs2149971 tagger (which covers the 3′ region associations with NOA, SCO and TESEneg remained significant after of KATNAL1), three of its proxies, that is, rs202093, rs617899 and multiple testing correction (Table 2). In all cases, a statistically sig- rs846483 are testis-specific eQTLs for KATNAL1 (Table S8), suggest- nificant improvement in the goodness of fit was observed when the ing that genetic variation of the 3′ region may influence the gene haplotype model was compared against the independent SNP models expression levels. Indeed, 14 proxies of rs2149971 are annotated (Tables 2 and S7). as testis-specific splicing quantitative trait loci (sQTL) for KATNAL1 20472927, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.13221 by Readcube (Labtiva Inc.), Wiley Online Library on [17/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License CERVÁN-MARTÍN ET AL . 1347 F I G U R E 2 Isoform expression of KATNAL1. (A) Isoform representation in the different tissues included in the GTEx project. (B) Gene model and transcripts per million (TPM) reads in testis. Source: GTEx Analysis Release V8 (dbGaP Accession phs000424.v8.p2) (Table S9). In this regard, the data extracted from the GTEx and 4 DISCUSSION Ensembl portals indicated an alternative splicing of KATNAL1 mRNA, leading to five different mRNA isoforms: (1) a 7618 bp transcript We designed a candidate gene study to evaluate the putative impli- with 11 exons (ENST00000380615.7) that encodes a 490 amino cation of KATNAL1 polymorphic positions in human male infertility. acid protein, which represents the primary transcript in the major- Our results showed that common genetic variants of the non-coding ity of analysed tissues; (2) another isoform of 1634 bp with 11 regions of this gene confer the risk of developing extreme phenotypes exons (ENST00000380617.7) that encodes a similar 490 amino of SpF and may be informative of the TESE outcome. acid protein and that constitutes the most abundant KATNAL1 tran- As previously mentioned, several studies point to KATNAL1 as a cru- script in the testis of healthy subjects; (3) two shorter isoforms cial gene for the spermatogenic process.8,9 In the genetic association of 797 and 566 bp with four exons each (ENST00000441394.1 study performed by Fedick et al.,10 the KATNAL1 SNPs that showed the and ENST00000414289.5, respectively) encoding two small pep- most promising trends towards association were rs17074420 (uncor- tides of 150 and 153 amino acids; and (4) one retained intron of rected p = 0.004, OR = 2.55) and rs17074416 (uncorrected p = 0.025, only 363 bp containing two exons of the 3′ region of the gene OR = 1.39), both of which are located in the 3′ end of the gene (specifi- (ENST00000480854.1), which does not produce a functional protein. cally rs17074420 in the 3′UTR and rs17074416 at 3.5 kb downstream The highest expression levels of the latter non-coding isoform among of KATNAL1). Interestingly, these SNPs are in LD (Dʹ > 0.8) with all analysed tissues are detected in the testis (Figure 2). rs2149971, the tagger in the 3′ region of the gene that was analysed in Notably, the minor alleles of the abovementioned sQTLs, which our study (rs2149971-rs17074416 Dʹ = 0.85, rs2149971-rs17074420 correlate with the rs2149971*A risk allele for SCO and TESEneg (Table Dʹ = 1.00, in the EUR population of the 1KGPh3). S9), are associated with an overrepresentation of the last exon of the Moreover, the effect size observed for the minor allele of rs2149971 largest isoforms, which corresponds to the first exon of the small non- (A) is consistent with those reported by Fedick et al.10 for the linked coding isoform, in comparison with the protective alleles (Figure S5). A minor alleles of rs17074416 (G) and rs17074420 (T), with all asso- comprehensive analysis of the alternative exon expression patterns in ciated with an increased risk of developing severe male infertility KATNAL1 highlighted that there are only 18 SNPs with a sQTL effect on phenotypes under the additive model (Table S10). Fedick et al.10 did this gene. Seventeen out of these 18 sQTLs act in the testis and increase not conduct the analyses according to specific NOA subtypes, as per- the expression of the previously mentioned exon. Moreover, 14 of them formed now in our study. This could be a possible explanation for the are linked to the identified SpF risk variants. Therefore, we hypothesise lack of association that they observed between NOA and the KATNAL1 that this effect might be very specific to the testicular tissue, likely variants rs17074416 and rs17074420 after adjusting for multiple test- related to an increased abundance of the short non-coding isoform ing. Indeed, a non-significant p-value was obtained for rs2149971 (ENST00000480854.1) and controlled by the identified SpF risk when the NOA phenotype was considered in our study (despite observ- haplotype. ing an OR = 1.20). Our data clearly suggest that this KATNAL1 variant Other testis-specific functional annotations overlapping the prox- is specifically associated with SCO and TESEneg with effect sizes of ies of the KATNAL1 taggers, including transcription factor-binding OR = 1.69 and 1.62, respectively. It would be interesting to evaluate sites, DNAse hypersensitivity, and chromatin epigenetic marks, among such associations in the case–control cohort included in the study by others, are shown in Table S8. Fedick et al.10 20472927, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.13221 by Readcube (Labtiva Inc.), Wiley Online Library on [17/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 1348 CERVÁN-MARTÍN ET AL . In this sense, SCO and TESEneg represent the most extreme NOA a statistically significant eQTL effect of this block on KATNAL1 but features. The latter is composed mainly of SCO and MA patients, and for other nearby genes, such as HMGB1. The protein encoded by this therefore, it is not surprising that rs2149971 was associated with both gene belongs to the non-histone chromosomal high mobility group SCO and TESEneg. It is likely that the lack of association between this protein family, which plays a major role in the establishment of chro- KATNAL1 SNP and MA could be because of the considerably reduced matin interactions by promoting DNA architectural changes.35 HMGB1 power of this analysis, as the MA subgroup only included 52 patients. is implicated in many biological processes, including female fertility, in The fact that MA is a less homogeneous phenotype than SCO or HS (as which the follicular fluid levels of its encoded protein have been cor- it considers arrests at different differentiation steps) could also be a related with the outcome of in vitro fertilisation with intracytoplasmic confounding factor, masking a putative association. sperm injection.36 Moreover, its paralog HMGB2 has been associated Regarding the two remaining taggers analysed, rs7338931 and with male infertility because of spermatogenic anomalies in murine rs2077011, our study also showed evidence of a potential associ- models.37 ation with different SpF phenotypes, which suggests a most likely Interestingly, an alteration of the normal expression of KATNAL1 involvement of genetic variation of KATNAL1 in the deregulation of transcripts by specific genotypes of promoter SNPs has been associ- the spermatogenic process that may lead to male infertility. Hence, the ated with sperm deformities in Chinese Holstein bulls (9). Hence, it haplotype analysis revealed that a combined effect of allelic variants could be possible that the genetic effect on SpF of the KATNAL1 vari- was more informative for explaining the associations observed than ants located in the 5′ end of the promoter (tagged by rs2077011) was the model of independent SNP effects. This is consistent with the hap- independent from that of the 3′ haplotype block (tagged by rs2149971) lotype structure of KATNAL1, which shows extensive LD across most and may influence the expression pattern of either KATNAL1 or other of the gene except for a recombination hotspot within the promoter nearby upstream genes, such as HMGB1. (Figure 1). Two of the analysed taggers, rs2149971 and rs7338931, Overall, this study provides additional insight regarding the role of located downstream of this recombination hotspot, were associated KATNAL1 in the different differentiation stages that take place dur- with the most severe SpF expression, defined by TESEneg (particularly ing spermatogenesis, most likely by facilitating the interaction between rs2149971, the tagger at the 3′ end of the gene also associated with Sertoli cells and germ cells through the regulation of microtubular SCO). Hence, the risk variants rs2149971*A and rs7338931*T (as well dynamics.8,38 Indeed, some compounds that inhibit KATNAL1 function, as the linked alleles of their proxies) may have a key role in the devel- such as calotropin, have been proposed as a non-hormonal male- opment of the most extreme phenotypes of NOA. The SNPs located specific contraceptives,39 emphasising the high relevance of this gene upstream of the recombination hotspot of the promoter (tagged by in the field of male infertility. rs2077011) seem to contribute to such phenotypes to a lesser extent, In conclusion, our results point to a relevant role of the KATNAL1 emphasising our suspicion that the causal variants are mostly located gene in the development of SpF. The insight provided by this study within the 3′UTR, as proposed by Fedick et al.10 may help to develop more efficient diagnostic and prognostic tools that With regard to the functional implication of the possible causal could anticipate both the diagnosis and TESE outcome prior to con- SNPs tagged by the 3′ tagger rs2149971, it should be noted that sidering a testis biopsy, thus preventing NOA patients with extreme 14 of its proxies were annotated as sQTLs of KATNAL1 in the testis. phenotypes from undergoing unnecessary surgeries. The minor alleles of the SNPs comprising this haplotype block, which correlate with the risk rs2149971*A allele for SCO and TESEneg, ACKNOWLEDGEMENTS are associated with an increased expression of a small isoform We thank the National DNA Bank Carlos III (University of Salamanca, (ENST00000480854.1), composed only of the last two exons of the Spain) for supplying part of the control DNA samples from Spain gene, that does not produce a functional KATNAL1 protein. Accord- and all the participants for their essential collaboration. This arti- ing to GTEx project data,27 this short isoform is normally expressed cle is related to the Ph.D. doctoral thesis of Miriam Cerván-Martín. at low levels in healthy human testes. Therefore, the presence of the This work was supported by the Spanish Ministry of Economy and risk alleles of the SCO-associated 3′ haplotype block may unbalance Competitiveness through the Spanish National Plan for Scientific the KATNAL1 isoform ratio, likely by overrepresenting the truncated and Technical Research and Innovation (refs. SAF2016-78722-R and ENST00000480854.1 variant in the transcript pool of the cell and thus PID2020-120157RB-I00), the ‘Instituto de Salud Carlos III’ (Fondo reducing the relative counts of the functional full-length isoforms.27 It de Investigaciones Sanitarias)/Fondo Europeo de Desarrollo Regional would be interesting to evaluate the KATNAL1 isoform ratio in our NOA ‘Una manera de hacer Europa’ (FIS/FEDER) (ref. DTS18/00101 to cohort (or, at least, in any of the patients carrying the risk variants). Sara Larriba), the Generalitat de Catalunya (ref. 2017SGR191), the However, no testicular tissue was available for mRNA expression anal- ‘Ramón y Cajal’ program (ref. RYC-2014-16458) and the ‘Juan de yses during the development of this study, which represents an evident la Cierva Incorporación’ program (ref. IJC2018-038026-I), as well as limitation. the Andalusian Government through the R&D&i Projects Grants for On the other hand, different proxies of the 5′ tagger rs2077011, Universities and Public Research Entities (ref. PY20_00212), which located upstream of the recombination hotspot of the promoter include FEDER funds. Andrea Guzmán-Jiménez was a recipient of a (Figure 1), seem to modulate the expression levels of KATNAL1 in dif- grant from the Spanish Ministry of Education and Professional Train- ferent tissues.27 However, the GTEx data for the testis did not show ing (‘Becas de Colaboración en Departamentos Universitarios para 20472927, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/andr.13221 by Readcube (Labtiva Inc.), Wiley Online Library on [17/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License CERVÁN-MARTÍN ET AL . 1349 el curso académico 2020/2021’). Patricia I. Marques is supported by 6. McNally KP, Bazirgan OA, McNally FJ. Two domains of p80 katanin the FCT post-doctoral fellowship (SFRH/BPD/120777/2016), financed regulate microtubule severing and spindle pole targeting by p60 katanin. J Cell Sci. 2000;113(Pt 9):1623-1633. from the Portuguese State Budget of the Ministry for Science, Tech- 7. O’Donnell L, O’Bryan MK. Microtubules and spermatogenesis. Semin nology and High Education and from the European Social Fund, Cell Dev Biol. 2014;30:45-54. doi:10.1016/j.semcdb.2014.01.003 available through the ‘Programa Operacional do Capital Humano’. João 8. Smith LB, Milne L, Nelson N, et al. KATNAL1 regulation of sertoli cell Gonçalves was partially funded by FCT/MCTES through national funds microtubule dynamics is essential for spermiogenesis and male fertil- ity. PLoS Genet. 2012;8:e1002697. doi:10.1371/journal.pgen.1002697 attributed to the Centre for Toxicogenomics and Human Health— 9. Zhang X, Wang C, Zhang Y, et al. Association between an alternative ToxOmics (UID/BIM/00009/2016 and UIDB/00009/2020). Sara Lar- promoter polymorphism and sperm deformity rate is due to modu- riba is sponsored by the Researchers Consolidation Program (ISCIII lation of the expression of KATNAL1 transcripts in Chinese Holstein SNS/Dpt. Salut Generalitat de Catalunya) (CES09/020). bulls. Anim Genet. 2014;45:641-651. doi:10.1111/age.12182 10. Fedick AM, Eckert K, Thompson K, et al. Lack of association of KATNAL1 gene sequence variants and azoospermia in humans. CONFLICT OF INTEREST J Assist Reprod Genetics. 2014;31:1065-1071. doi:10.1007/s10815- The authors declare no competing financial interests. 014-0269-1 11. Dunleavy JEM, Okuda H, O’Connor AE, et al. Katanin-like 2 (KAT- NAL2) functions in multiple aspects of haploid male germ cell devel- AUTHOR CONTRIBUTIONS opment in the mouse. PLoS Genet. 2017;13:e1007078. doi:10.1371/ Miriam Cerván-Martín, Lara Bossini-Castillo and Andrea Guzmán- journal.pgen.1007078 Jiménez analysed and interpreted the data and drafted the manuscript. 12. Wei X, Liu W, Zhu X, et al. Biallelic mutations in KATNAL2 cause male infertility due to oligo-astheno-teratozoospermia. Clin Genet. Miguel Burgos, Rafael Jiménez and Sara González-Muñoz participated 2021;100:376-385. doi:10.1111/cge.14009 in the analysis and interpretation of the data and revised the differ- 13. Tournaye H, Krausz C, Oates RD. Novel concepts in the etiology of ent manuscript versions. Rocío Rivera-Egea, Nicolás Garrido, Saturnino male reproductive impairment. Lancet Diab Endocrinol. 2017;5:544- Lujan, Gema Romeu, Samuel Santos-Ribeiro, José A. Castilla, M. Car- 553. doi:10.1016/S2213-8587(16)30040-7 14. Krausz C, Riera-Escamilla A. Genetics of male infertility. Nat Rev Urol. men Gonzalvo, Ana Clavero, Vicente Maldonado, F. Javier Vicente, 2018;15:369-384. doi:10.1038/s41585-018-0003-3 Josvany Sánchez-Curbelo, Olga López-Rodrigo, Iris Pereira-Caetano, 15. Cervan-Martin M, Castilla JA, Palomino-Morales RJ, Carmona FD. Patricia I. Marques, Filipa Carvalho, Alberto Barros, Lluís Bassas, Genetic landscape of nonobstructive azoospermia and new perspec- Susana Seixas, João Gonçalves, Sara Larriba and Alexandra M. Lopes tives for the clinic. J Clin Med. 2020;9. doi:10.3390/jcm9020300 contributed to the acquisition of the data, DNA patient’s and con- 16. Little J, Higgins JP, Ioannidis JP, et al. Strengthening the reporting of genetic association studies (STREGA): an extension of the STROBE trol samples and critically revised the content of the final manuscript. statement. Hum Genet. 2009;125:131-151. doi:10.1007/s00439-008- Rogelio J. Palomino-Morales and F. David Carmona made substantial 0592-7 contributions to the conception and design of the present study and 17. Cervan-Martin M, Bossini-Castillo L, Rivera-Egea R, et al. Evaluation were involved in manuscript drafting. All authors approved the final of male fertility-associated loci in a European population of patients with severe spermatogenic impairment. 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