Bi-allelic Mutations in M1AP Are a Frequent Cause of Meiotic Arrest and Severely Impaired Spermatogenesis Leading to Male Infertility
2020; Elsevier BV; Volume: 107; Issue: 2 Linguagem: Inglês
10.1016/j.ajhg.2020.06.010
ISSN1537-6605
AutoresMargot J. Wyrwoll, Şehime Gülsün Temel, Liina Nagirnaja, Manon S. Oud, Alexandra M. Lopes, Godfried W. van der Heijden, James S. Heald, Nadja Rotte, Joachim Wistuba, Marius Wöste, Susanne Ledig, Henrike Krenz, Roos M. Smits, Filipa Carvalho, João Gonçalves, Daniela Fietz, Burcu Türkgenç, Mahmut Çerkez Ergören, Murat Çetinkaya, Murad Başar, Semra Kahraman, Kevin McEleny, Miguel J. Xavier, Helen Turner, Adrian Pilatz, Albrecht Röpke, Martin Dugas, Sabine Kliesch, Nina Neuhaus, Kenneth I. Aston, Donald F. Conrad, Joris A. Veltman, Corinna Friedrich, Frank Tüttelmann,
Tópico(s)Genomics and Rare Diseases
ResumoMale infertility affects ∼7% of men, but its causes remain poorly understood. The most severe form is non-obstructive azoospermia (NOA), which is, in part, caused by an arrest at meiosis. So far, only a few validated disease-associated genes have been reported. To address this gap, we performed whole-exome sequencing in 58 men with unexplained meiotic arrest and identified the same homozygous frameshift variant c.676dup (p.Trp226LeufsTer4) in M1AP, encoding meiosis 1 associated protein, in three unrelated men. This variant most likely results in a truncated protein as shown in vitro by heterologous expression of mutant M1AP. Next, we screened four large cohorts of infertile men and identified three additional individuals carrying homozygous c.676dup and three carrying combinations of this and other likely causal variants in M1AP. Moreover, a homozygous missense variant, c.1166C>T (p.Pro389Leu), segregated with infertility in five men from a consanguineous Turkish family. The common phenotype between all affected men was NOA, but occasionally spermatids and rarely a few spermatozoa in the semen were observed. A similar phenotype has been described for mice with disruption of M1ap. Collectively, these findings demonstrate that mutations in M1AP are a relatively frequent cause of autosomal recessive severe spermatogenic failure and male infertility with strong clinical validity. Male infertility affects ∼7% of men, but its causes remain poorly understood. The most severe form is non-obstructive azoospermia (NOA), which is, in part, caused by an arrest at meiosis. So far, only a few validated disease-associated genes have been reported. To address this gap, we performed whole-exome sequencing in 58 men with unexplained meiotic arrest and identified the same homozygous frameshift variant c.676dup (p.Trp226LeufsTer4) in M1AP, encoding meiosis 1 associated protein, in three unrelated men. This variant most likely results in a truncated protein as shown in vitro by heterologous expression of mutant M1AP. Next, we screened four large cohorts of infertile men and identified three additional individuals carrying homozygous c.676dup and three carrying combinations of this and other likely causal variants in M1AP. Moreover, a homozygous missense variant, c.1166C>T (p.Pro389Leu), segregated with infertility in five men from a consanguineous Turkish family. The common phenotype between all affected men was NOA, but occasionally spermatids and rarely a few spermatozoa in the semen were observed. A similar phenotype has been described for mice with disruption of M1ap. Collectively, these findings demonstrate that mutations in M1AP are a relatively frequent cause of autosomal recessive severe spermatogenic failure and male infertility with strong clinical validity. Around 7% of all men in Western societies experience infertility,1Juul S. Karmaus W. Olsen J. Regional differences in waiting time to pregnancy: pregnancy-based surveys from Denmark, France, Germany, Italy and Sweden. The European Infertility and Subfecundity Study Group.Hum. Reprod. 1999; 14: 1250-1254Crossref PubMed Scopus (147) Google Scholar which is primarily diagnosed by semen analysis comprising sperm concentration and count as the most relevant parameters. More than 10% of all infertile men exhibit azoospermia2Tüttelmann F. Ruckert C. Röpke A. Disorders of spermatogenesis: Perspectives for novel genetic diagnostics after 20 years of unchanged routine.Med. Genetik. 2018; 30: 12-20Google Scholar—the absence of spermatozoa in the ejaculate. Azoospermia constitutes the most challenging and clinically severe form of male infertility and is further classified into obstructive azoospermia (OA) with normal spermatogenesis and non-obstructive azoospermia (NOA) due to impaired spermatogenesis. In some men, a few spermatozoa can be identified after centrifugation of the semen, which is denoted as cryptozoospermia. From the biological point of view, NOA and cryptozoospermia are closely related, just as they are in their clinical implications, i.e., virtually no chance of natural conception.3World Health OrganizationWHO laboratory manual for the examination and processing of human semen. World Health Organization, 2010Google Scholar The variable spermatogenic impairment in these men correlates to a diverse spectrum of testicular histological phenotypes. This spectrum includes Sertoli cell-only (SCO), over maturation arrest, and hypospermatogenesis, and all of these can be complete, focal, or mixed. Maturation arrest most frequently presents as meiotic arrest in which spermatocytes are the most advanced germ cell types in the testes. If germ cell arrest is complete, no mature spermatozoa develop; testicular biopsy and sperm extraction (TESE) will not be successful and assisted reproductive technology (ART) will not be possible. In a large fraction of severely disturbed spermatogenesis, a genetic origin is assumed,4Lee J.Y. Dada R. Sabanegh E. Carpi A. Agarwal A. Role of genetics in azoospermia.Urology. 2011; 77: 598-601Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar and affected men are routinely screened for chromosomal aberrations and Y chromosome azoospermia factor (AZF) microdeletions. Yet these diagnostic tests only establish a causal diagnosis in 15%–20% of azoospermia-affected individuals.2Tüttelmann F. Ruckert C. Röpke A. Disorders of spermatogenesis: Perspectives for novel genetic diagnostics after 20 years of unchanged routine.Med. Genetik. 2018; 30: 12-20Google Scholar Recently, monogenic alterations associated with germ cell arrest in human males have been described.5Yatsenko A.N. Georgiadis A.P. Röpke A. Berman A.J. Jaffe T. Olszewska M. Westernströer B. Sanfilippo J. Kurpisz M. Rajkovic A. et al.X-linked TEX11 mutations, meiotic arrest, and azoospermia in infertile men.N. Engl. J. Med. 2015; 372: 2097-2107Crossref PubMed Scopus (148) Google Scholar,6Gershoni M. Hauser R. Yogev L. Lehavi O. Azem F. Yavetz H. Pietrokovski S. Kleiman S.E. A familial study of azoospermic men identifies three novel causative mutations in three new human azoospermia genes.Genet. Med. 2017; 19: 998-1006Crossref PubMed Scopus (55) Google Scholar However, according to a standardized clinical validity assessment, the X chromosome gene TEX11 (MIM: 300311) currently remains the only one in which variants are associated with male infertility with strong evidence.7Oud M.S. Volozonoka L. Smits R.M. Visser L.E. Ramos L. Veltman J.A. A systematic review and standardized clinical validity assessment of male infertility genes.bioRxiv. 2018; https://doi.org/10.1101/425553Crossref Scopus (0) Google Scholar Given the large number of genes in which variants are known to cause meiotic arrest in mice, the vast majority of mutations causing this phenotype in humans are yet to be identified. To this end, we first screened the exomes of well-characterized men with complete bilateral meiotic arrest and identified bi-allelic loss-of-function (LoF) variants in the gene encoding meiosis 1 associated protein (M1AP) in three unrelated men. In mice, M1ap is primarily expressed in male germ cells throughout spermatogenesis, and its knockout leads to infertility due to meiotic arrest and severe oligozoospermia.8Arango N.A. Li L. Dabir D. Nicolau F. Pieretti-Vanmarcke R. Koehler C. McCarrey J.R. Lu N. Donahoe P.K. Meiosis I arrest abnormalities lead to severe oligozoospermia in meiosis 1 arresting protein (M1ap)-deficient mice.Biol. Reprod. 2013; 88: 76Crossref PubMed Scopus (7) Google Scholar,9Arango N.A. Huang T.T. Fujino A. Pieretti-Vanmarcke R. Donahoe P.K. Expression analysis and evolutionary conservation of the mouse germ cell-specific D6Mm5e gene.Dev. Dyn. 2006; 235: 2613-2619Crossref PubMed Scopus (8) Google Scholar Our subsequent analyses in four independent cohorts and a consanguineous Turkish family, as well as in vitro analyses of a recurring M1AP frameshift variant, corroborated that disruption of M1AP is associated with a variable spectrum of severely impaired spermatogenesis, mostly at meiosis and resulting in azoospermia, but also compatible with sparse postmeiotic germ cell development and retrieval of sperm in some instances. We originally selected 64 azoospermic but otherwise healthy males who attended the Centre of Reproductive Medicine and Andrology (CeRA), University Hospital Münster (n = 51) or the Clinic for Urology, Pediatric Urology and Andrology, Gießen (n = 13) for couple infertility. All men were diagnosed with complete bilateral germ cell arrest at the spermatocyte stage after the evaluation of at least 100 seminiferous tubules in tissue sections of both testes accompanied by a negative TESE outcome, i.e., no sperm could be recovered. This is a subset of all individuals included in our large-scale Male Reproductive Genomics (MERGE) study, which currently comprises 735 men with lacking or severe quantitatively impaired spermatogenesis and 53 individuals with normal spermatogenesis (OA and controls) (Figure S1). Specifically, we performed whole-exome sequencing (WES; for details, see Supplemental Methods) in 569 men with NOA, 116 with cryptozoospermia, and 50 with severe oligozoospermia (sperm concentration < 5 M/mL). Chromosomal aberrations and AZF deletions were excluded in this and all other cohorts and subjects (detailed below). All participants gave written informed consent and the study protocol was approved by the respective ethics committees and institutional review boards (details in Supplemental Data). We identified likely causal variants in the three genes TEX11, STAG3, and SYCP2 in six of the men with complete meiotic arrest.5Yatsenko A.N. Georgiadis A.P. Röpke A. Berman A.J. Jaffe T. Olszewska M. Westernströer B. Sanfilippo J. Kurpisz M. Rajkovic A. et al.X-linked TEX11 mutations, meiotic arrest, and azoospermia in infertile men.N. Engl. J. Med. 2015; 372: 2097-2107Crossref PubMed Scopus (148) Google Scholar,10van der Bijl N. Röpke A. Biswas U. Wöste M. Jessberger R. Kliesch S. Friedrich C. Tüttelmann F. Mutations in the stromal antigen 3 (STAG3) gene cause male infertility due to meiotic arrest.Hum. Reprod. 2019; 34: 2112-2119PubMed Google Scholar,11Schilit S.L.P. Menon S. Friedrich C. Kammin T. Wilch E. Hanscom C. Jiang S. Kliesch S. Talkowski M.E. Tüttelmann F. et al.SYCP2 Translocation-Mediated Dysregulation and Frameshift Variants Cause Human Male Infertility.Am. J. Hum. Genet. 2020; 106: 41-57Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar The WES data of the remaining 58 men were filtered for rare (minor allele frequency [MAF] < 0.01 according to the Genome Aggregation Database12Karczewski K.J. Francioli L.C. Tiao G. Cummings B.B. Alföldi J. Wang Q. Collins R.L. Laricchia K.M. Ganna A. Birnbaum D.P. et al.The mutational constraint spectrum quantified from variation in 141,456 humans.bioRxiv. 2020; https://doi.org/10.1101/531210Crossref Scopus (0) Google Scholar [gnomAD]) bi-allelic LoF variants. Affected genes were prioritized with regard to the level of expression in the testes and previous evidence for an association with infertility in either human or model species (Figure S1). The highest-ranked gene was M1AP because three unrelated men (M330, M864, and M1792, Table 1) carried the same homozygous LoF variant (c.676dup, MAF = 0.0021, no homozygotes in gnomAD12Karczewski K.J. Francioli L.C. Tiao G. Cummings B.B. Alföldi J. Wang Q. Collins R.L. Laricchia K.M. Ganna A. Birnbaum D.P. et al.The mutational constraint spectrum quantified from variation in 141,456 humans.bioRxiv. 2020; https://doi.org/10.1101/531210Crossref Scopus (0) Google Scholar), the M1AP mRNA displayed the highest expression in the testis (according to both the Genotype-Tissue Expression [GTEx] project13Lonsdale J. Thomas J. Salvatore M. Phillips R. Lo E. Shad S. Hasz R. Walters G. Garcia F. Young N. et al.GTEx ConsortiumThe Genotype-Tissue Expression (GTEx) project.Nat. Genet. 2013; 45: 580-585Crossref PubMed Scopus (3155) Google Scholar and the Human Protein Atlas [HPA]), and it was shown to play a crucial role in spermatogenesis in mice.8Arango N.A. Li L. Dabir D. Nicolau F. Pieretti-Vanmarcke R. Koehler C. McCarrey J.R. Lu N. Donahoe P.K. Meiosis I arrest abnormalities lead to severe oligozoospermia in meiosis 1 arresting protein (M1ap)-deficient mice.Biol. Reprod. 2013; 88: 76Crossref PubMed Scopus (7) Google Scholar,9Arango N.A. Huang T.T. Fujino A. Pieretti-Vanmarcke R. Donahoe P.K. Expression analysis and evolutionary conservation of the mouse germ cell-specific D6Mm5e gene.Dev. Dyn. 2006; 235: 2613-2619Crossref PubMed Scopus (8) Google Scholar The c.676dup variant in M1AP was confirmed by Sanger sequencing (GenBank: NM_138804.4, the longest isoform with highest testis expression; for primer sequences see Table S1) in all affected men from the MERGE cohort. Testis biopsies of all three individuals were collected for TESE and research use. These were fixed in Bouin's solution and embedded in paraffin, and subsequently, sections were stained with periodic acid-Schiff (PAS) as previously described.14Nieschlag E. Behre H.M. Nieschlag S. Andrology: Male reproductive health and dysfunction.Third Edition. Springer-Verlag Berlin Heidelberg, 2010Crossref Scopus (3) Google Scholar These were re-analyzed to confirm complete meiotic arrest (Figure 1). Because DNA from the three individuals' parents was not available, and to exclude a hemizygous deletion on the other allele, quantitative PCR (qPCR) of M1AP's exon 5 was performed on gDNA (primers and conditions in Table S1 and the Supplemental Methods). This excluded an intragenic deletion in all individuals (Table S2). No regions of homozygosity (ROHs) involving M1AP were detected for any of the affected men, rendering consanguinity of their parents unlikely. We also did not notice evidence for consanguinity between the men (analysis by H3M2 and vcftools algorithms,15Magi A. Tattini L. Palombo F. Benelli M. Gialluisi A. Giusti B. Abbate R. Seri M. Gensini G.F. Romeo G. Pippucci T. H3M2: detection of runs of homozygosity from whole-exome sequencing data.Bioinformatics. 2014; 30: 2852-2859Crossref PubMed Scopus (54) Google Scholar data not shown).Table 1Genetic and Clinical Data of Infertile Men with M1AP VariantsIndividualAge, OriginM1AP VariantFertility ParametersTesticular Phenotype, TESE OutcomeM33038 years, Germanyc.[676dup];[676dup], p.[Trp226LeufsTer4];[Trp226LeufsTer4]FSH, 9; LH, 5.3; T, 14.6; TV, 17/23; azoospermiameiotic arrest (0/0% tubules with ES, 0/0% RS, 91/99% SC, 6/1% SG, 3/0% SCO, 0/0% TS), TESE negativeM86441 years, Germanyc.[676dup];[676dup], p.[Trp226LeufsTer4];[Trp226LeufsTer4]FSH, 4.7; LH, 1.5; T, 9.6; TV, 19/26; azoospermiameiotic arrest (0/0% tubules with ES, 0/0% RS, 71/91% SC, 10/4% SG, 17/1% SCO, 2/4% TS), TESE negativeM179236 years, Germanyc.[676dup];[676dup], p.[Trp226LeufsTer4];[Trp226LeufsTer4]FSH, 7.8; LH, 5.1; T, 10.1; TV, 15/15; azoospermiameiotic arrest (0/0% tubules with ES, 0/0% RS, 96/97% SC, 0/2% SG, 3/1% SCO, 1/0% TS), TESE negativeM194343 years, Croatiac.[676dup];[797G>A], p.[Trp226LeufsTer4];[Arg266Gln]FSH, 5.5; LH, 3.2; T, 43.6; TV, 28/20; cryptozoospermia or azoospermiaaSemen contained none or below 10 spermatozoa/sample on repeated analyses.N/AM206226 years, Polandc.[676dup];[676dup], p.[Trp226LeufsTer4];[Trp226LeufsTer4]FSH, 3.5; LH, 3.6; T, 18.6; TV, 26/23; cryptozoospermia or azoospermiaaSemen contained none or below 10 spermatozoa/sample on repeated analyses.N/AY12634 years, Portugalc.[676dup];[949G>A], p.[Trp226LeufsTer4];[Gly317Arg]FSH, 6.7; LH, 3.2; T, N/A; TV, N/A; azoospermiamaturation arrest at round spermatid stage, (quantification N/A), TESE negativeP8644 years, Portugalc.[148T>C];[1289T>C], p.[Ser50Pro];[Leu430Pro]FSH, N/A; LH, N/A; T, N/A; TV, N/A; azoospermiadispersed Sertoli cells, some tubules contained only spermatogonia, (quantification N/A), TESE negativeRU0169141 years, the Netherlandsc.[676dup];[676dup], p.[Trp226LeufsTer4];[Trp226LeufsTer4]FSH, 5; LH, 2.0; T, 11.3; TV, N/A; azoospermiapredominant meiotic arrest with occasional spermatids, (unilateral TESE: 4% tubules with ES, 5% RS, 88% SC, 2% SG, 0% SCO, 0% TS), TESE positiveMI-0006-P33 years, UKc.[676dup];[676dup], p.[Trp226LeufsTer4];[Trp226LeufsTer4]FSH, 10.4; LH, N/A; T, 15.4; TV, 20/20; azoospermiapredominant meiotic arrest with occasional postmeiotic germ cells (quantification N/A), TESE negativeT102428 years, Turkeyc.[1166C>T];[1166C>T], p.[Pro389Leu];[Pro389Leu]FSH, 8.3; LH, 4.4; T, 8.8; TV, 15/15; azoospermiamaturation arrest at round spermatid stage, (quantification N/A), TESE negativeF1: II-116Tu C. Wang Y. Nie H. Meng L. Wang W. Li Y. Li D. Zhang H. Lu G. Lin G. et al.An M1AP homozygous splice-site mutation associated with severe oligozoospermia in a consanguineous family.Clin. Genet. 2020; 97: 741-746Crossref PubMed Scopus (2) Google Scholar (Tu et al.)34 years, Chinac.[1435−1G>A];[1435−1G>A], p.?FSH, 3.65; LH, 3.17; T, 18.24; TV, 12/12; severe oligozoospermiaN/AAbbreviations are as follows: FSH, follicle-stimulating hormone (IU/L); LH, luteinizing hormone (IU/L); T, testosterone (nmol/L); TV, testicular volume right/left (mL); ES, elongating spermatids; RS, round spermatids; SC, spermatocytes; SG, spermatogonia; SCO, Sertoli cell-only; TS, tubular shadows; N/A, not available. Reference values: FSH 1–7 IU/L, LH 2–10 IU/L, T > 12 nmol/L, TV > 15 mL per testis.a Semen contained none or below 10 spermatozoa/sample on repeated analyses. Open table in a new tab Abbreviations are as follows: FSH, follicle-stimulating hormone (IU/L); LH, luteinizing hormone (IU/L); T, testosterone (nmol/L); TV, testicular volume right/left (mL); ES, elongating spermatids; RS, round spermatids; SC, spermatocytes; SG, spermatogonia; SCO, Sertoli cell-only; TS, tubular shadows; N/A, not available. Reference values: FSH 1–7 IU/L, LH 2–10 IU/L, T > 12 nmol/L, TV > 15 mL per testis. By screening the complete MERGE cohort, we identified an additional man (M2062) carrying the same homozygous LoF variant, c.676dup (Figure S2), and another man (M1943) carrying c.676dup in combination with a second rare missense variant, c.797G>A (p.Arg266Gln). This missense variant is consistently predicted as pathogenic by all in silico prediction programs (PolyPhen-2, SIFT, MutationTaster, and HOPE17Venselaar H. Te Beek T.A.H. Kuipers R.K.P. Hekkelman M.L. Vriend G. Protein structure analysis of mutations causing inheritable diseases. An e-Science approach with life scientist friendly interfaces.BMC Bioinformatics. 2010; 11: 548Crossref PubMed Scopus (468) Google Scholar). Both men had varying azoospermia and cryptozoospermia in repeated semen analyses and did not undergo a testicular biopsy. In contrast, no individuals with two rare M1AP variants predicted as pathogenic via in silico programs were observed in the remaining MERGE cohort or in the individuals with spermatogenic impairment and other testicular phenotypes, such as SCO (n = 213), or normal spermatogenesis (OA or controls, n = 53). Next, through collaborations established within the International Male Infertility Genomics Consortium (IMIGC), three cohorts of infertile men in whom WES was performed in independent studies (details are provided in the Supplemental Methods) were screened for bi-allelic variants in M1AP: 930 men with unexplained NOA from the Genetics of Male Infertility Initiative (GEMINI) study, 283 men with unexplained azoospermia (n = 214) or oligozoospermia (n = 69) who presented at Radboud University Medical Center (Radboudumc, Nijmegen), and 48 men with unexplained azoospermia (n = 36) or oligozoospermia (n = 12) recruited at The Newcastle upon Tyne Hospitals NHS Foundation Trust (Newcastle, UK). From these 1,261 individuals in total, we identified four additional infertile men with likely bi-allelic variants in M1AP. Clinical data of all individuals carrying M1AP variants is shown in Table 1. The variants, gnomAD frequencies, in silico predictions, and classification according to the guidelines of the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP)18Richards S. Aziz N. Bale S. Bick D. Das S. Gastier-Foster J. Grody W.W. Hegde M. Lyon E. Spector E. et al.ACMG Laboratory Quality Assurance CommitteeStandards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet. Med. 2015; 17: 405-424Crossref PubMed Scopus (9296) Google Scholar are given in Table 2 (for detailed variant description and interpretation, see Supplemental Note). On the basis of RNA analyses from public sources (GTEx and HPA), M1AP might also be expressed in the bone marrow and other tissues, such as the pituitary. However, no blood-system-related or hormonal abnormalities were noted in any of the affected men reported herein.Table 2Assessment of M1AP VariantscDNA ChangeProtein ChangeIn Silico Prediction for Missense Variants (PolyPhen-2/SIFT/MutationTaster)MAFaOverall MAF is presented. This is, for example, slightly higher for the recurring variant c.676dup in non-Finnish Europeans with an MAF of 0.0038. By contrast, this variant has not been reported in East and South Asian populations. (gnomAD)MAF (Local Controls)ConservationClassification According to ACMG-AMP Guidelines18Richards S. Aziz N. Bale S. Bick D. Das S. Gastier-Foster J. Grody W.W. Hegde M. Lyon E. Spector E. et al.ACMG Laboratory Quality Assurance CommitteeStandards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet. Med. 2015; 17: 405-424Crossref PubMed Scopus (9296) Google Scholarc.676dupp.Trp226LeufsTer4N/A0.0021aOverall MAF is presented. This is, for example, slightly higher for the recurring variant c.676dup in non-Finnish Europeans with an MAF of 0.0038. By contrast, this variant has not been reported in East and South Asian populations.0.0088N/Apathogenicc.1435−1G>A16Tu C. Wang Y. Nie H. Meng L. Wang W. Li Y. Li D. Zhang H. Lu G. Lin G. et al.An M1AP homozygous splice-site mutation associated with severe oligozoospermia in a consanguineous family.Clin. Genet. 2020; 97: 741-746Crossref PubMed Scopus (2) Google Scholarp.?N/A0NDN/Apathogenicc.148T>Cp.Ser50ProT/P/D0NDplatypusuncertain significancec.797G>Ap.Arg266GlnD/D/D0.0002NDzebrafishuncertain significancec.949G>Ap.Gly317ArgD/D/D0.00007NDplatypusuncertain significancec.1166C>Tp.Pro389LeuD/D/D0.00001NDtetraodonuncertain significancec.1289T>Cp.Leu430ProD/D/D0.000008NDplatypusuncertain significanceAbbreviations are as follows: D, damaging, deleterious, or disease-causing; B, benign; T, tolerated; P, polymorphism; MAF, minor allele frequency; N/A, not applicable; ND, not determined.a Overall MAF is presented. This is, for example, slightly higher for the recurring variant c.676dup in non-Finnish Europeans with an MAF of 0.0038. By contrast, this variant has not been reported in East and South Asian populations. Open table in a new tab Abbreviations are as follows: D, damaging, deleterious, or disease-causing; B, benign; T, tolerated; P, polymorphism; MAF, minor allele frequency; N/A, not applicable; ND, not determined. Two individuals of Portuguese origin (Y126 and P86) analyzed within the GEMINI study each carried two different variants in M1AP (Figure S2). Individual Y126 carried the missense variant c. 949G>A (p.Gly317Arg) and the recurrent frameshift variant c.676dup and had germ cell arrest at the round spermatid stage. Individual P86 had the two missense variants c.148T>C (p.Ser50Pro) and c.1289T>C (p.Leu430Pro), suggesting compound heterozygosity. Testicular histology showed severely disturbed spermatogenesis. Individuals RU01691 from the Netherlands and MI-0006-P from the UK were also homozygous for the frameshift variant c.676dup. RU01691's parents were both heterozygous carriers. His testicular biopsy showed bilateral severe hypospermatogenesis with predominantly meiotic arrest; sporadically, spermatids were present, and material was cryopreserved for intracytoplasmic sperm injection (ICSI) but hitherto not used. Individual MI-0006-P had azoospermia and predominant meiotic arrest with rare postmeiotic germ cells (Figure S2). In parallel, WES was performed in two infertile, azoospermic brothers from a consanguineous Turkish family as well as in their fertile brother. The index T1024 (V.2; Figure 2) presented at Istanbul Memorial Hospital, Assisted Reproductive Technologies and Reproductive Genetics Centre and Bursa Uludag University Faculty of Medicine Hospital, Turkey because of couple infertility. Testicular histology demonstrated maturation arrest at round spermatid stage, and no sperm could be recovered by TESE. The WES data were analyzed focusing on rare homozygous variants shared between both infertile brothers but not found in the fertile brother. The two affected men carried rare homozygous missense variants in the autosomal genes AMPD2, CELSR2, CEP164, and M1AP as well as rare hemizygous variants in the X chromosome genes ATG4A and ENOX2. Of these genes, only M1AP has been described in the context of infertility. Both infertile men carried the homozygous missense variant c.1166C>T (p.Pro389Leu) (MAF = 0.00001), which was also found in a heterozygous state in the fertile brother, in M1AP. No homozygous individuals with this variant have been described in any public databases, whereas it was found in a homozygous state in three additional infertile males from this family: two third cousins once removed from the maternal side and one second cousin once removed from the paternal side. We did identify both a fertile man and a fertile woman (IV.13 and V.6, respectively, in Figure 2A) as heterozygous carriers of the same variant (example result of Sanger sequencing for subject V.6 shown in Figure 2B). In a complementary approach, an updated version of the population sampling probability (PSAP) pipeline19Wilfert A.B. Chao K.R. Kaushal M. Jain S. Zöllner S. Adams D.R. Conrad D.F. Genome-wide significance testing of variation from single case exomes.Nat. Genet. 2016; 48: 1455-1461Crossref PubMed Scopus (21) Google Scholar was used to prioritize potentially causative variants. PSAP models the significance of observing a single person's genotype in comparison to genotype frequencies in unaffected populations. This enabled us to rank all variants per individual by following the prioritization criteria MAF ≤ 0.01, CADD ≥ 20, and PopScore ≤ 0.005.20Kasak L. Punab M. Nagirnaja L. Grigorova M. Minajeva A. Lopes A.M. Punab A.M. Aston K.I. Carvalho F. Laasik E. et al.GEMINI ConsortiumBi-allelic Recessive Loss-of-Function Variants in FANCM Cause Non-obstructive Azoospermia.Am. J. Hum. Genet. 2018; 103: 200-212Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar The bi-allelic M1AP LoF variants were ranked in the first position for M1792 and in the third position for M330, M864, and M2062 in the discovery cohort (Table S3). The missense variant c.797G>A and the duplication c.676dup of M1943 were ranked in the ninth position under a compound heterozygous recessive disease model. The five men identified in the follow-up analyses exhibited highly ranked M1AP variants as well: position seven in RU01691, position four in Y126, position seven in P86, position two in MI-0006-P, and position three in T1024. The rare but recurring M1AP variant c.676dup warranted further analyses. It is located in exon 5 of 11 and causes a frameshift and premature stop codon (p.Trp226LeufsTer4) as confirmed by testicular cDNA sequencing of exon 5 of individual M864 (Figure S3). This results in a truncated protein as shown by heterologous expression of mutated M1AP in HEK293T and subsequent immunoblot analysis displaying a protein band of almost 23 kD (Supplemental Methods and Figure 3), indicating a protein lacking 57% of its normal length. This is in line with the analysis of individual M864's testicular RNA that resulted in an equal band compared to control testis RNA, excluding elimination of the mRNA through nonsense-mediated decay (Figure S3). Still, because of the non-native, ectopic expression of the protein in HEK293T cells, it remains possible that no product is translated by the mutant mRNA in vivo. The relevance of the homozygous frameshift variant c.676dup in M1AP is further supported by the exceptionally low PSAP-PopScore (9.7 × 10−7) and the high prioritization (Table S3). Moreover, the expected mode of inheritance for M1AP is autosomal recessive according to a general prediction,21Cassa C.A. Weghorn D. Balick D.J. Jordan D.M. Nusinow D. Samocha K.E. O'Donnell-Luria A. MacArthur D.G. Daly M.J. Beier D.R. Sunyaev S.R. Estimating the selective effects of heterozygous protein-truncating variants from human exome data.Nat. Genet. 2017; 49: 806-810Crossref PubMed Scopus (42) Google Scholar fitting our observations of bi-allelic variants in the affected men. According to the ACMG-AMP guidelines,18Richards S. Aziz N. Bale S. Bick D. Das S. Gastier-Foster J. Grody W.W. Hegde M. Lyon E. Spector E. et al.ACMG Laboratory Quality Assurance CommitteeStandards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet. Med. 2015; 17: 405-424Crossref PubMed Scopus (9296) Google Scholar this variant is categorized as pathogenic (Table S4). The fact that the same frameshift variant, c.676dup, was also found in individuals from Croatia, Poland, the Netherlands, UK, and Portugal, suggests that it is relatively prevalent in European populations most likely originating from a founder mutation
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