New Genetic Point Mutations in Male Infertility

C H A P T E R

3 New Genetic Point Mutations in Male Infertility Xavier Vendrell Sistemas Genómicos Ltd, Valencia, Spain

INTRODUCTION Human reproductive incompetence is either expressed as infertility or subfertility and is an imbricated trait with several etiological causes and heterogeneous phenotypic presentation. Nowadays, in developed countries, close to 15% of couples have problems to reproduce, which represents a serious global disability that has been recognized by the World Human Organization in the WHO/World Bank World report on disability [1]. In fact, the diagnosis and treatment of the failure of the reproductive system is a milestone in private and public health care programs. There are several “nonbiological” causes of a decreased birth rate in modern societies such as the delay in the active search for the first conception, new social models, environmental pollution, and chemical exposition. The analysis of these factors is out of the objective of this chapter. However, it is important to note that it could mask the biological basis of the disorder. Traditionally, it is assumed that the infertility causes are shared between males and females and, in particular cases, it is extremely difficult to determine the exact contribution to the reproductive failure from each partner. In this scenario, it is typically assumed that the so-called male factor is responsible for close to half of the clinical causes of this phenotype. The etiology of male infertility has been thoroughly studied in the past decades. International specialist groups assume several causes: congenital or acquired urogenital abnormalities, malignancies, urogenital tract infections, increased scrotal temperature (e.g., because of varicocele), endocrine disturbances, immunological factors and genetic abnormalities (see a deep review in Ref. [2]). In 30%–40% of cases, any cause associated to male infertility is found after a complete diagnostic work-up. This idiopathic situation is assumed to be caused by several factors and a lot of research is directed to explain part of this inconclusive diagnosis. Under the definition of “idiopathic,” there are a few proposed causes such as: endocrine disruption because of environmental pollution, reactive oxygen species, or genetic and epigenetic variations. It is highly relevant to elucidate the genetic basis of the idiopathic male factor infertility.

Reproductomics https://doi.org/10.1016/B978-0-12-812571-7.00004-6

47

© 2018 Elsevier Inc. All rights reserved.

48

3.  New Genetic Point Mutations in Male Infertility

Nowadays, the advances of assisted reproductive techniques allow men with suboptimal reproductive prognosis to conceive a viable zygote. In these circumstances, the lack of knowledge about the inheritance of this trait could have an impact on future generations, through passing pathological traits to offspring. Therefore, an accurate genetic diagnosis is crucial in the frame of reproductive genetic counseling concerning the effectiveness and safety of treatments. From the genetic point of view, the human spermatogenesis depends on the synchronized action of thousands of genes whose transcripts are expressed mainly in the germ tissues. The role of a few known genetic regions’ variations on sperm’s function is clear (e.g., AZF regions on Y chromosome, sex chromosomal aneuploidies, or chromosomal rearrangements). On the other hand, there are an increasing number of studies that suggest the implication of new genes. These studies will be focused upon in the present chapter. In the recent years, the efforts of researchers have been to identify candidate genes involved in spermatogenesis. Global genome-based approaches have yielded close to 3000 genes related with spermatogenesis or germ cell function [3,4]. Recently, new technological approaches are available and offer novel ways to conduct the experiments. Comparative genome hybridization (CGH)based microarrays, single-nucleotide polymorphisms (SNPs)-based microarrays, exome sequencing or whole-genome sequencing are used to interrogate the genomic constitution of an organism, yielding different levels of resolutions (reviewed in Refs. [5,6]). The massive data analysis is revealing new knowledge and the findings show a sophisticated network of complex molecular pathways. However, the causative effects of new genes on reproductive failure remain to be proven. Furthermore, to identify the genes related to spermatogenesis is not the only challenge. The discovery of thousands of small noncoding RNA acting on the mRNA stability as well as translation and protein modification offers new data to understand this highly regulated process. In this sense, the importance of epigenetics (reviewed in Ref. [7]) and the role of micro-RNA in the regulation of spermatogenesis have been highlighted recently [8,9], and both of these issues will be the aim of other chapters. The objective of this chapter is to review the current knowledge concerning the genetic role on male reproductive failure, emphasizing new discoveries in relation to new point variants in the genome sequence of specific nuclear genes. In some cases, there is a clear correlation between new specific variants and infertile and subfertile traits. On the contrary, there are cases where the discovery of new changes in the genome and its relationship with male factor are purely speculative.

GENES AND MALE FACTOR INFERTILITY The fertility function in men is a highly coordinated process where many pathways are involved and are genetically regulated. These include the development of the urogenital system; differentiation of the spermatogonial stem cells; formation of the spermatid acrosome and flagellum; acquisition of motility in the ejaculated spermatozoa; searching mechanism to oocyte-corona-cumulus complex arrival; chemical, enzymatic, and mechanical equipment to penetrate the membranes when joining with the female machinery and finally completing the fertilization and activation of genomes. In parallel, the endocrine regulation of gonadal function and the physiological mechanism of erection or ejaculation is genetically directed.



Genes and Male Factor Infertility

49

From the genetic point of view, several genetic causes, with different degrees of certainty, center around the analysis of infertile males. The chromosomal abnormalities explain close to 5% of infertility in males (see review [10]) and increase to 15% in cases of azoospermy [11]. Altered somatic karyotypes, including aneuploidy (e.g., Klinefelter syndrome) or chromosomal translocations (reciprocal and robertsonian translocations), are clearly related with infertility or subfertility in male populations (reviewed in Refs. [10,12]). In addition, the aneuploidy of sperm chromosomes has been deeply related with subfertility (reviewed in Ref. [13]). Furthermore, Copy Number Variations (CNVs) have been described in association with fertility impairment. CNVs are complete gains or losses of sequences distributed along the genome, conferring variability. In male infertility, the only CNV clearly associated with spermatogenic failure is AZF microdeletions on the Y chromosome [6,14–16]. However, new candidate CNVs have been revised recently in autosomes and the X chromosome with strong correlation with spermatogenic failure [16,17]. It is difficult to study the reproductive function as a single process in order to find new causative genome variations. From the clinical research point of view, the study of the etiopathogenesis of male infertility obligates authors to separate the investigations in detached biological pathways (deeply reviewed in Ref. [18]). The analysis of genes related with distinct routes causing different forms of infertility has been focused upon by researchers in the past years [3]. Several strategies have been proposed using different tools (reviewed in Ref. [5]), which include Sanger sequencing for candidate genes, genome-whole association (GWA)-microarray’s based analysis, whole exome or genome sequencing based on next generation sequencing (NGS) or NGS’s resequencing studies. Conclusively, a plethora of genes expressed in the male germ line, and other tissues nondirectly related with testicular function, have been reported. Following Matzuk and Lamb’s proposal [18], we can differentiate three big groups of phenotypes in abnormal male reproductive function. These are: (i) male sexual differentiation disorders and gonadal development impairment, (ii) spermatogenesis alterations causing defects on spermatic functions, and (iii) systemic disorders affecting fertility. The last category is out of the scope of this chapter. There are a large number of systemic disorders (single-gene, congenital or nongenetic disorders) that present infertility or subfertility as a secondary trait and are included in a syndromic presentation. Some classical examples include autoimmune diseases, progressive neurological disorders (e.g., specific types of Charcot-Marie-Tooth disease or Duchenne’s muscular dystrophy), systemic cilia-proteins affectation (Kartagener syndrome), hematological disorders (e.g., sickle-cell anemia or thalassemias), urogenital abnormalities (e.g., Alport syndrome), anatomical malformations, infections, and toxic exposure [19]. In these cases, the diagnostic work-up is based on the main symptoms of the disorder and not for the disrupted fertility. The clinical diagnosis as well as the patient’s management and treatment options have special clinical protocols, with genetic counseling being highly recommended in all cases. Fig. 3.1 represents the distribution of genes (per chromosome) that has been related with these phenotypes, in order to have an overall view. Following the view of Matzuk and Lamb [18], genes have been separated into two big groups depending on the main traits associated to the gene function. In addition, genes have been associated with phenotypic presentation, with a total of 16 possible phenotypes being reported (see legend). The list of genes and phenotypes has been updated with data from recent publications. Several authors have described the implication of these genes with different levels of evidence. For instance, case-control

50 3.  New Genetic Point Mutations in Male Infertility

FIG. 3.1  See the legend in facing page.

Genes and Male Factor Infertility

Genes implicated in male sexual differentiation disorders and gonadal development impairment. < Genes implicated in spermatogenesis alterations and sperm function impairment. Superindexes: aambigous

genitalia, bgonadal dysgenesis, chyopospadias, cancer, gvas deferens malformation, hsex reversal and other disorders of sex iabnormal steroid biosynthesis, determination, jazoospermia, koligozoospermia, masthenozoospermia, nteratozoospermia, linflamatory efect, ocell maintenance, phypogonadism hypogonadotrop. dmicropenis, ecryptorchidism, ftestis

51

FIG.  3.1, CONT’D  Distribution of genes implicated in male infertility. The genes have been located in their chromosome cytobands. The figure represents ideograms of human chromosomes showing a G-banded pattern at resolution of 850 bands per haploid set (from NCBI’s Genome Decoration Page, https://www.ncbi.nlm.nih.gov/genome/tools/gdp). The genes are super indexed with letters that represent the reported associated phenotype (see legend). Two groups have been differentiated: genes related with sex male differentiation and gonadal development impairment (in blue) and genes related with spermatogenesis alteration and sperm function deficiency (in red). Information concerning phenotypes has been updated with data from [6,10,12,16–18,20–29].

52

3.  New Genetic Point Mutations in Male Infertility

studies with candidate genes have found more or less consistency in function of population size, with single-case studies observing an uncertain inheritance pattern. Even GWAs studies have reported genes directly related with complete testicular failure expressed as azoospermia. Furthermore, SNPs-based studies have suggested genetic susceptibility to male factor. In particular cases, the relationship between the genetic variant and the phenotype has been reported as a direct cause-effect relationship, which is responsible for the phenotype. However, in most of the cases, a collaborative action with other genes has been suggested (oligogenic model). Likewise, in particular cases, the authors propose the interaction of the gene products with other biomolecules, such as micro-RNA or epigenetic regulators, concluding that etiopathology remains to be elucidated. On the other hand, in many cases, functional studies are pending and the biological effect of the protein-coding genes is not fully understood. Finally, a very interesting issue that has been described in many studies is the crucial role of the ethnic background in order to explain incomplete penetrance and phenotype variability. The following subsections intend to update the knowledge related to genetic causes of male factor infertility, emphasizing new point variants recently described. The description of new discoveries has been separated by previously described phenotypes, i.e., male sexual differentiation disorders and spermatogenesis alterations.

Male Sexual Differentiation Disorders and Gonadal Development Impairment First of all, it is important to emphasize that eutherian mammals have a special early development, characterized by similar steps in both sexes until the fetal stage. At the initial fetal stages, males and females have two distinct pathways. The common primordial organization is driven by specific programs that, in males, conclude with the development of the testis (deeply reviewed in Ref. [30]). The primary sex determination in human males is genetically regulated by a large number of genes acting synergistically and antagonistically (see review [20]). The understanding of the genetic network responsible for the development of the testis has been the focus of developmental researchers for decades. Svingen and Koopman [30] reported on three special characteristics in the differentiation of male germinal tissue, which confer high complexity. These include: (1) the cell lineages implicated are bipotential, meaning the differentiation (into ovaries or testis) depends on the signals received; (2) the differentiation of the Sertoli cells regulates the differentiation of the rest of the cell types implicated in the formation of the testis, and (3) the influence of somatic factors on male germ cells. Eventually, all this orchestration fails, resulting in what we know as Disorders of sex Determination (DSD). The DSD has been defined as “congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical” [31]. It is estimated than around 50% of these patients have causative mutations in genes involved in differentiation. However, the molecular diagnosis is still only achieved in around 20% of cases, excluding cases of steroidogenic block, which is detected biochemically [31]. From the genetic point of view, point mutations and small deletions have been described in a few crucial genes in the early development (reviewed in Refs. [21,22]). In mammals, the Y chromosome plays a pivotal role in male sex determination and is essential for normal sperm production (reviewed in Refs. [23,32,33]). It is well known that alterations in the Y-chromosome genes affect male fertility. One of the clearly implicated genes in sexual differentiation in males is the Y-linked testis-determining gene SRY. The SRY’s protein is the



Genes and Male Factor Infertility

53

t­estis-determining factor, which initiates male sex determination by directing the development of supporting cell precursors (pre-Sertoli cells). Close to 15% of gonadal dysgenesis cases result from a mutation involving SRY. In many cases, the reported variants in SRY are de novo. However, there are cases with pathogenic variants inherited from a fertile father, which suggests incomplete penetrance or the concomitant action of other variants [34]. Another related Y-chromosome gene is the TSPY gene. This gene is involved in spermatic differentiation and proliferation, tumor suppression as well as cell cycle regulation, and it has been described in relation to sudden infant death with dysgenesis [23]. Other variants related with sexual developmental disorders have been reported in autosomal genes regulated by the Y-chromosome’s genes. Particularly, DSD has been reported to be associated to many genes, such as SOX9, GATA4, FOG2, NR5A1, WT, DHH, CBX2, ATRX, MAP3K1, and FGF9 (reviewed in Ref. [22]). The synergistic action of the NR5A gene and the SRY gene coactivating the action of the SOX9 gene is highly relevant. The key role of the SOX9 gene on sex differentiation has been highlighted in classical haploinsufficiency studies [35]. The product of the SOX9K gene acts during chondrocyte differentiation and, with steroidogenic factor 1, regulates the transcription of the anti-Muellerian hormone (AMH) gene. Deficiencies of this product lead to sex reversal. In this sense, it is important to highlight cases of testicular dysgenesis linked to heterozygous missense mutations in the NR5A1 gene. In these cases, men have normal development of external genitalia [36]. The NR5A1 gene product is a transcriptional activator. It appears to be essential for sexual differentiation and formation of the primary steroidogenic tissues as well as also regulating the AMH/Muellerian balance. Specifically, the point mutation p.R103Q in the NR5A1 gene has been associated to impaired activation of steroidogenic genes. Additionally, the specific point change p.R92W is the only monogenic variant described that has recurrently been associated with nonsyndromic errors in primary sex determination [37]. This point variant shows incomplete penetrance and its physiopathological function is unclear. However, a reduced ability to interact with β-catenin to synergistically activate the reporter gene has been reported [37,38]. These findings stress the importance of this variant on upregulation of the SOX9 gene expression (clearly explained in Ref. [20]). Finally, the new p.L230R point variant in the NR5A1 gene has been described to be related to nonclassical DSD’s phenotypes [39]. This fact emphasizes the importance of overlapping phenotypes. Authors have proposed the need to clearly distinguish between strict gonadal dysgenesis and androgen biosynthesis defects, thus allowing for a new classification of DSD based on new molecular findings. Point variants have been also identified in the cofactors GATA4 and FOG2 genes, which cooperatively interact with the NR5A1 protein for testis determination and differentiation [22]. The GATA4 gene participates in the regulation of other genes involved in embryogenesis and in myocardial differentiation and function, and it is also necessary for normal testicular development. In turn, the FOG2 gene’s protein interacts with GATA-family products regulating hematopoiesis and cardiogenesis in mammals. It has also been demonstrated that this protein can both activate and downregulate the expression of GATA-target genes. In particular, the heterozygous variant p.G221R in the GATA4 gene disrupts the synergistic activation of the AMH promoter, thus losing the ability to bind to the FOG2 protein [40]. However, the single-nucleotide variant p.S402R in the FOG2 gene exhibits a truncated protein, which lacks the ability to bind to the GATA4 protein. Other two point mutations in the FOG2 gene (e.g., the homozygous point variant p.M544I and de novo heterozygous variant p.R260Q) have

54

3.  New Genetic Point Mutations in Male Infertility

revealed its importance in this pathway. The combination of both of these variants eliminates FOG2’s interaction with the GATA4 protein [41]. Furthermore, another gene that plays a key role in the differentiation of male gonads is the DHH gene and mutations in this gene have been related with dysgenetic testis. DHH encodes a signaling molecule that plays an important role in regulating morphogenesis. The point homozygous mutation p.R124Q has been related with impairment of Sertolli cell-Leydig cell interaction in early testis formation [42]. Additionally, other cases of gonadal dysgenesis have been related with the variant p.G287 V in homozygosis in the HHAT gene [43], which is directly associated with the proteasome mediated degradation pathway. Furthermore, the key role of genes implicated with MAPKs (mitogen-activated protein kinases) in the initial stages of sex determination in mammals is well known. The MAPKs are key proteins that are evolutionarily conserved and associated with the signaling transduction pathway. Bashamboo and McElreavey [22] reviewed the role of six candidate pathogenic variants in the MAP3K1 gene: p.G616R, p.L189P, p.L189R, c.634-8 T>A, p.P153L, and c.21802A>G. The MAP3K1 gene codes to a serine/threonine kinase, which is part of some signal transduction cascades. Mutations are related with cases of sex reversal and gonadal dysgenesis. However, a direct relationship with a phenotype has not yet been established. Finally, other genes like DMRT1, SOXs, RSPO1, WNT4, and FOXL2 have been correlated with initial stages of sex differentiation. However, the information related with point mutations is diffuse and fragmented, and even though it has been deeply studied [21] the physiopathologic information related to specific variants must be confirmed. Unfortunately, classical genetic techniques for diagnosis or linkage studies in specific families of genes are tedious approaches; however, they are needed in order to conclusively identify novel pathogenic variants in this complicated process. In addition, another serious limitation consists of the lack of clear models for research. For example, the murine model to study the mechanism of sex determination and development in men is insufficient and, in some aspects, not fully appropriate, as the differences with the reproductive mouse systems are too large. In this context, the “omics” approaches are encouraging. Particularly, NextGeneration Sequencing (NGS) approaches are guiding the research to the identification of new genes or loci associated with these phenotypes, as previously stated. NGS strategies include a targeted approach on candidate or causative genes, sequencing of all the coding regions (exome) or whole-genome sequencing. These are very powerful approaches, both in diagnosis and clinical research. The application of exome sequencing in severe forms of DSD has been reported in close to 20% of patients where there was a misdiagnosis or atypical clinical presentation [30]. In addition, NGS applications have diagnosed close to 40% of patients with a wide phenotype spectrum. New genes such as TBC1D1, TBX18, HSD17B4, MBOAT, MTOR, and DVL1 have been postulated to have associations with syndromic forms of DSD. Nowadays, the challenge remains in determining the causative effect of the new variants. Researchers are centering their efforts on functional assays in order to determine the biological consequences of the new variants on the gene product. Other conditions associated with incomplete male virilization and compromised fertility are defects in the development of the genital tract. In this case, we are talking about hormonal, gonadal, or reproductive organ defects. It has been previously described alterations in genes responsible for the biosynthesis of steroid hormones, specifically the AR gene of androgen receptor, and in the signal pathway of AMH: the AMH, BMPR1A, and SMAD genes



Genes and Male Factor Infertility

55

­(transcription factor) (see review [18]). Furthermore, it is well established that mutations in the genes coding for enzymes and proteins supporting the early biosynthesis pathways cause classic androgen biosynthesis defects [44]. Mutations have been described in the StAR, CYP11A1, HSD3B2, CYP17A1, HSD17B3, and SRD5A2 genes as well as the POR and CYB5 genes, which are cofactors for the enzymatic reaction regulated by the CYP17 protein. Finally, from the genetic point of view, it is especially relevant to mention congenital abnormalities caused by abnormal genitourinary tract development. These malformations cause serious fertility impairment and the genetic etiology has been proven in some cases. In particular, these cases include cryptorchidism, hypospadias, and congenital absence (uni/bilateral) of the vas deferens (CBAVD). Extremely rare mutations have been described in the INSL3 and RXFP2 genes, which are implicated in the signal transduction pathways in tissues, which command the abdominal phase of testis descent [18]. However, a large body of evidence shows an association between CBAVD and the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It is well known that there is a clear correlation between the phenotype and the combination of mutations F508del (c.1521_1523delCTT) and p.R117H (c.350G > A) with the polymorphism 5T, localized at the splice acceptor site of exon 8. In addition, the penetrance of this conformation might be determined by the length of the adjacent TG repeats (TG)12_13 [45].

Spermatogenesis Alterations and Sperm Function Impairment Reduced sperm production is one of the more usual clinical traits detected during the clinical male examination of infertile couples. The affectation grade varies from low sperm counting (oligozoospermia) to complete absence of spermatozoa in the ejaculated seminal fluid (azoospermia). From the spermatogenesis alteration point of view, azoospermia is the most severe form of male infertility and has been studied for a long time. Approximately, 1% of men are azoospermic [46]. Azoospermia and low sperm counts might have a multifactorial etiology; but typically, it has been associated with a Sertolli-cell only phenotype, meiotic or maturation arrest, hypospermatogenesis (low cellularity) or normal spermatogenesis but an obstruction of the efferent vias. The biological pathways involved in these pathologies are highly regulated genetically. For example, defects in genes related with endocrine function, cellular proliferation and differentiation, DNA repair, apoptosis, recombination, chromatin remodeling, motility, cell-to-cell interactions, and mitosis and meiosis regulation could all possibly be implicated [47]. Mitchell and coworkers [17] differentiated between pretesticular, testicular, and posttesticular defects in order to organize the study of the genetic etiology of this imbricated network. Under this definition, pretesticular damage can be caused principally by endocrine abnormalities (low levels of sex steroids or gonadotropins). The cause might be congenital (e.g., single-gene defects), acquired (e.g., disorders of the hypothalamicpituitary axis), or iatrogenic (e.g., environmental factors). Secondly, posttesticular causes could be caused by ejaculatory disturbances or obstructions, which impede the transport of spermatozoa. The genetic variants related with these disturbances have been addressed in previous sections. Thirdly, the causes of inefficient spermatogenesis at the testicular level could be separated into three broad groups: (i) Y-chromosome deletions, (ii) germ cell aplasia (including Sertolli cell-only syndrome), or (iii) spermatogenesis arrest. Other causes of drastic reduction in sperm counts at the testicular level include traumas, neoplasia, infections, or inflammatory diseases. These processes have been deeply studied in humans and animal

56

3.  New Genetic Point Mutations in Male Infertility

­ odels. However, clear evidence does not really exist at the genetic level. Nowadays, abnorm mal karyotype and Y-linked CNVs remains as the only recurrent genetic cause of nonobstructive azoospermia (NOA). The correlation between chromosomal translocations, aneuploidy of sex chromosomes (e.g., 47,XXY), chromosome Y microdeletions affecting the AZF region, and hypoespermatogenesis is being intensively studied (see reviews [6,10,15,18]). Historically, the Y chromosome has been the center for the study of male infertility. Beyond the study of the AZF region and its CNVs (deeply reviewed in Ref. [48]), a few variants have been correlated with infertility phenotypes in more than 20 genes (reviewed in Ref. [23]). Recently, two reviews [24,49] collected the discoveries of new massive technologies in this field. In particular, it is important to highlight the role of variants in genes outside the AZF regions (e.g., the ZFY, SRY, RPS4Y1, and TG1FLY genes). However, the clinical association of the new variants has not been clearly established. Furthermore, Krausz and Casamonti [24] reviewed the role of the human protein-coding Y genes TSPY, VCY, XKRY, CDY, HSFY, RBMY1, PRY, BPY2, and DAZ as well as their autosomal or X-linked homologues in the infertility phenotype. The contribution of NGS technologies and the massive data analysis has revealed the variability of genotype-phenotype correlation concerning infertility phenotypes and the importance of noncoding transcripts from male-specific regions of Y chromosome (MYS) DNA. This fact suggests the possible regulatory function of these genic products [49]. Due to large population stratification and geographic variability of these genome regions, the establishment of a clear correlation between variants and particular phenotypes is challenging. Alternatively, the new approaches in genomics have revealed novel mutations related with infertile patients and severe reduced counting in autosomal genes. In particular, Miyamoto and colleagues [25] and most recently Mitchell and colleagues [17] have reviewed the role of new point mutations in the autosomal genes SYCP3, SYCE1, KLHL10, AURKC, and SPATA6. have described the high impact of the genes coding for synaptonemal complex proteins such as the SYCP3 and SYCE1 genes on human fertility [26]. It has been suggested that variants c.643delA, IVS7-16_19del, c.657 T > C, c.548 T > C, and 666A > G in the SYCP3 gene, and c.613C > T, c.197-2A > G in the SYCE1 gene, correlate with meiotic arrest. These genes codify to a DNA-binding protein in the synaptonemal complex involved in the key steps of germ cell meiosis. Variants in these genes cause meiotic arrest, sperm aneuploidy, or synaptic failure [26]. In regards to the KLHL10 gene, its product is a substrate-specific adapter of a CUL3based E3 ubiquitin-protein ligase complex, which mediates the ubiquitination and subsequent proteasomal degradation of target proteins during spermatogenesis. Single-nucleotide missense variants p.A313T and p.Q216P were detected in heterozygosis in severe oligozoospermic males. However, a new analysis performed in control men and revision of data in the Exome Aggregation Consortium database (ExAC; url: http://exac.broadinstitute.org) now exclude these variants. Another point mutation has been described in homozygosis in the AURKC gene, which is expressed in the testis and has been implicated in cytokinesis, meiosis, and mitosis. The single-nucleotide variant p.L49 W has been related with abnormal spermatogenesis, as well as polyploid and morphologically abnormal spermatozoa. Other mutations in the SPATA16 gene, involved in the formation of the sperm acrosome, have been reported in patients with impaired spermatogenesis showing diverse phenotypes (e.g., asthenozoospermia, see later). A relevant point in the nonobstructive azoospermia has been the analysis of the X chromosome and autosomal genes in consanguineous families. Recent data (reviewed in



Genes and Male Factor Infertility

57

Ref. [17]) indicate the relevance of the TEX11 gene. This gene is expressed in the testis and is a regulator of crossover during meiosis, involved in initiation and/or maintenance of chromosome synapsis, as well as the formation of crossovers. The variants p.Asp435Leufs*10, c.1838-1G > A, c.792 + 1G > A, c.1837 + 1G > C, and p.Thr218_Lys296del have been found in azoospermic patients, whereas they have not been found in controls in the ExAC database. On the contrary, the study of recessive forms of NOA has generated an enormous interest. Recessive causative genes of NOA have been described in autosomes. In this context nonsense, splice and missense variants have been reported in azoospermic patients. Examples include p.Leu50fs*30 in the ZMYND15 gene (chr. 17), p.Arg611* in the TAFA4B gene (chr.18); c.198-2A > G in the SYCE1 gene (chr. 10), c.1954-1G in the MCM8 gene (chr. 20), p.Y710* in the TEX15 gene (chr. 8), and p.Pro455Ala in the NPAS2 gene (chr. 2) (data obtained from review of Ref. [17]). The confirmation of these data is crucial and functional studies are necessary. Nevertheless, complete exome and genome approaches have increased new discoveries for these phenotypes exponentially. It is important to note that heterozygous variants, found in particular patients, have been refuted when population frequencies from thousands of complete exomes and genomes have been published. This fact has occurred in variants found in genes such as USP26, NANOS1, MTHFR, GSTM1, FSHB, or NR5A1 [17]. For this reason, “candidate genes” sequencing strategies have been reconsidered and new massive strategies are strongly recommended. A clear example has been proposed by Quaynor and coworkers [27] who described 18 new genes, which are probably implicated with hypogonadotropic hypogonadism and Kallmann syndrome, by using targeted NGS techniques. Finally, other frequent sperm abnormalities, beyond sperm quantity, are astenozoospermia and teratozoospermia. Genetic causes of these motility and morphological sperm disorders are still unknown. Deletions in the AKAP3 and AKAP4 genes were described as possibly being responsible for sperm motility affectation due to fibrous sheath dysplasia (reviewed in Ref. [18]). Ben Khelifa and coworkers [50] coined the acronym MMAF (Multiple Morphological Abnormalities of sperm Flagella), in order to identify the nonsyndromic sperm motility affectation and pathogenic variants in the DNAHI gene, which encodes a protein in the axonemal dynein cluster related with microtubule motor activity. Recently, three point variants in the DNAHI gene have been associated with MMAF phenotype: c.8626-1G > A, Val1287Gly, and Asp1293Asn [28] and thus concluding that this gene is one of the main genes involved in the phenotype. In regards to morphological abnormalities, it is difficult to detect a “pure” phenotype. Usually, several abnormalities of sperm coexist in the same sample with varying grades of severity (e.g., round or abnormal heads or multiflagellation). In addition, abnormal sperm shapes could appear jointly with reduced sperm count or limited motility. In this scenario with a multiplicity of phenotypes, it is difficult to establish a clear phenotype-genotype correlation. However, a monomorphic teratozoospermia with round heads and acrosome-lacked spermatozoa exists. This is the globozoospermia, which is a quasipure phenotype. Three genes have been described as responsible for the phenotype: SPATA16, PRKCA1, and DPY19L2 (reviewed in Ref. [29]). The homozygous point variant c.848G > A in the SPATA16 gene has been clinically associated with the abnormal phenotype. The codified protein is expressed in the Golgi apparatus and is involved in the formation of the sperm acrosome, which explains its potential role in spermatogenesis and sperm-egg fusion.

58

3.  New Genetic Point Mutations in Male Infertility

Single-Nucleotide Polymorphism A critical aspect that has been focused upon in the study of male factor infertility researcher is the probable concomitant function of specific polymorphic changes on the infertile phenotype. The role of these genome variants has been associated with a “genetic susceptibility” or “genetic predisposition” to infertility. In this sense, it is interesting to review the role of specific single-nucleotide polymorphisms (SNPs) in particular genes implicated in meiosis, spermatogenic function and regulation. Krausz and coworkers [16] reviewed a large set of metaanalysis, case-control, and GWAs studies performed on more than 370,000 SNPs in key genes related with cell maintenance (general cell functions), apoptotic process, DNA repair, response to reactive oxygen species, etc. in regards to expression in germ cells. The intensive comparison of studies concluded that only a few number of SNPs have been clearly correlated with infertility phenotypes. An interesting point is the crucial role of ethnography in the phenotypic expression. Concretely, a positive association has been reported for five genes: MTHFR, ESR1/ESR2, NOS3, DAZL, MSH5, SIRPA/SIRPG, and HLA-DRA. The point polymorphic variants A222V (c.677C > T) and E429A (c.1298C > A) in the MTHFR gene play a role in processing amino acids in folate metabolism, and have been associated with azoospermia and oligoastenoteratozoospermia. Variable penetrance has been reported and related to folate intake. This information could be relevant in relation with the response to folate supplementation in certain patients. Other SNPs in the estrogen receptor genes ESR1 and ESR, which are highly expressed in testicular germ cells, are c.453-397 T > C, c.453-351A > G in the ESR1 gene, and c.984G > A in the ESR2 gene. Once again, its modulatory effect has been reported depending on geographic background. In the same sense, clinical relevancy has been conferred to SNPs c.-786C > T and 4a4b (27 base pairs tandem repeats) in the NOS3 gene, which are responsible for nitric oxide (NO) production. The missense point variant c.160A > G (p.Thr54Ala) in the DAZ gene codifies RNA-binding proteins that are key in spermatogenesis, and predispose carriers to spermatogenic failure. Concordant results between studies have been reported for the c.85C > T (p.Pro29Ser) SNP in the MSH5 gene, which has been implicated in DNA repair and apoptosis. A significant susceptibility for oligozoospermia and NOA has been reported for c.*273G > T in the SIRPA gene and c.*223 T > G in the SIRPG gene, with both of them playing a key role in intracellular signaling during synaptogenesis and in synaptic function. Finally, three point polymorphic variants in the HLA-DRA gene participating in the immune system show significant association to male infertility. Concretely, c.82 + 926A > C, c.*63G > A, and c.724 T > G have been defined as susceptibility factors for autoimmune diseases. Probably these nucleotide changes may have a role in inflammatory responses of patients with antecedents of urogenital inflammation. The continuous updating and publication of new SNPs, sequence variations, uncertain significance changes in DNA, etc. exceed the review capability of this chapter. New metaanalysis and case-control studies investigating the probable biological functions of specific SNPs are being published constantly. In the future, the sharing of massive databases in the context of big data platforms will offer a whole and systemic perspective of the biological pathways involved in the etiopathogenesis of idiopathic male factor. In this new scenario, possible gene interactions will probably help to elucidate the role of genome variability and will expand the diagnosis potential.

REFERENCES 59

FINAL COMMENTS The reproductive male failure is an extremely complex trait that includes overlapping phenotypes. This circumstance complicates the study of its etiology. The reproductive function in males is a highly coordinated process. Thousands of genes are working together beginning in the first stages of embryo development. In addition, the spermatogenic function is a continuous and prolonged process (with duration close to 3 months) and, for this reason, it is highly susceptible to environmental, genetic, or epigenetic changes. Genetically speaking, hundreds of studies have been published during the past years in order to look for candidate genes. Several studies propose new genes or new mutations in key genes. However, a great limitation of the majority of studies is the inclusion of specific patients in particular families, and that the extrapolation to other phenotypically similar patients is not clear. Moreover, the ethnicity and geographical background is crucial in these phenotypes, and the sequence-based phylogenetic analysis has revealed complex mechanisms of selection (positive/negative selection) of particular traits. In addition, in some cases, a clear clinical correlation failed to be proved (obviously, the “infertility” trait implies the impossibility of natural transmission of the phenotype). Other milestones in order to elucidate the clear genetic contribution to the idiopathic male fertility disorders are reduced penetrance, locus heterogeneity, and multigenic (digenic or trigenic) inheritance detected in particular features. In this imbricated scenario, the contribution of new massive technologies is highly encouraging. The exome or whole genome sequencing approaches are allowing for the discovery of thousands of single-­ nucleotide variants. In the majority of cases, these variants have been described previously as disease-causing variants or frequent nonpathogenic population polymorphisms. However, the new found variants should be described as new polymorphisms, new pathogenic variants or accidental sequencing errors. This is the real challenge of these new approaches. For this purpose, it is extremely important to filter and cure the data concerning these new findings, depending on factors such as inheritance pattern of a particular trait in our experimental population (considering de novo appearance), family history, variable expressivity, sample size, and other confounding factors (e.g., eventual iatrogenic or environmental factors). In this sense, general databases should be used with caution because they may be “contaminated” by nonclearly defined variants. The next widespread use of these techniques for clinical diagnosis or screening might accomplish strict criteria concerning accurate sequence assembly, annotating of the new variants, implementing of comprehensive collection of functionally validated datasets and new precise function predictor algorithms. Most likely, these premises will help with the systemic understanding of the male reproductive process.

References [1] WHO/World Bank, World Report on Disability. http://www.who.int/disabilities/world_report/2011/ report/en/. [2] A. Jungwirth, T. Diemer, Z. Kopa, C. Krausz, H. Tournaye, B. Kelly, R. Pal, European Association of Urology Guidelines, http://uroweb.org/guideline/male-infertility/, 2016. [3] R. Hochstenbach, J.H. Hackstein, The comparative genetics of human spermatogenesis: clues from flies and other model organisms, Results Probl. Cell Differ. 28 (2000) 271–298. [4] N. Schultz, F.K. Hamra, D.L. Garbers, A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets, Proc. Natl. Acad. Sci. USA 100 (21) (2003) 12201–12206.

60

3.  New Genetic Point Mutations in Male Infertility

[5] K.I. Aston, Genetic susceptibility to male infertility: news from genome-wide association studies, Andrology 2 (3) (2014) 315–321. [6] S.H. Song, K. Chiba, R. Ramasamy, D.J. Lamb, Recent advances in the genetics of testicular failure, Asian J. Androl. 18 (3) (2016) 350–355. [7] S. Gunes, M.A. Arslan, G.N.T. Hekim, R. Asci, The role of epigenetics in idiopathic male infertility, J. Assist. Reprod. Genet. 33 (5) (2016) 553–569. [8] X. Chen, X. Li, J. Guo, P. Zhang, Z. Wenxian, The roles of microRNAs in regulation of mammalian spermatogenesis, J. Anim. Sci. Biotechnol. 8 (2017) 35. [9] A. Salas-Huetos, J. Blanco, F. Vidal, M. Grossmann, M.C. Pons, N. Garrido, E. Anton, Spermatozoa from normozoospermic fertile and infertile individuals convey a distinct miRNA cargo, Andrology 4 (6) (2016) 1028–1036. [10] K. O'Brien, A. Varghese, A. Agarwal, The genetic causes of male factor infertility: a review, Fertil. Steril. 93 (1) (2010) 1–12. [11] A. Ferlin, F. Raicu, V. Gatta, D. Zuccarello, G. Palka, C. Foresta, Male infertility: role of genetic background, Reprod. BioMed. Online 14 (6) (2007) 734–745. [12] F.T. Neto, P.V. Bach, B.B. Najari, P.S. Li, M. Goldstein, Genetics of male infertility, Curr Urol Rep 17 (10) (2016) 70. [13] X.  Vendrell, M.  Ferrer, E.  García-Mengual, P.  Muñoz, J.C.  Triviño, C.  Calatayud, V.Y.  Rawe, M.  Ruiz-Jorro, Correlation between aneuploidy, apoptotic markers and DNA fragmentation in spermatozoa from normozoospermic patients, Reprod. BioMed. Online 28 (4) (2014) 492–502. [14] P.H. Vogt, A. Edelmann, S. Kirsch, O. Henegariu, P. Hirschmann, F. Kiesewetter, F.M. Köhn, W.B. Schill, S. Farah, C.  Ramos, M.  Hartmann, W.  Hartschuh, D.  Meschede, H.M.  Behre, A.  Castel, E.  Nieschlag, W.  Weidner, H.J. Gröne, A. Jung, W. Engel, G. Haidl, Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11, Hum. Mol. Genet. 5 (7) (1996) 933–943. [15] C.  Krausz, L.  Hoefsloot, M.  Simoni, F.  Tüttelmann, European Academy of Andrology, European Molecular Genetics Quality Network, EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions: state-of-the-art 2013, Andrology 2 (1) (2014) 5–19. [16] C. Krausz, A.R. Escamilla, C. Chianese, Genetics of male infertility: from research to clinic, Reproduction 150 (5) (2015) R159–R174. [17] M.J. Mitchell, C. Metzler-Guillemain, A. Toure, C. Coutton, C. Arnoult, P.F. Ray, Single gene defects leading to sperm quantitative anomalies, Clin. Genet. 91 (2) (2017) 208–216. [18] M.M. Matzuk, D.J. Lamb, The biology of infertility: research advances and clinical challenges, Nat. Med. 14 (11) (2008) 1197–1213. [19] A.  Mahmoud, F.  Comhaire, Systemic causes of male infertility, in: W.-B.  Schill, F.  Comhaire, T.B.  Hargreave (Eds.), Andrology for the Clinician, Springer-Verlag, Berlin; Heidelberg, 2006, pp. 57–63. [20] A. Bashamboo, C. Eozenou, S. Rojo, K. McElreavey, Anomalies in human sex determination provide unique insights into the complex genetic interactions of early gonad development, Clin. Genet. 91 (2) (2017) 143–156. [21] A. Bashamboo, K. McElreavey, Gene mutations associated with anomalies of human gonad formation, Sex Dev. 7 (1–3) (2013) 126–146. [22] A.  Bashamboo, K.  McElreavey, Human sex-determination and disorders of sex-development (DSD), Semin. Cell Dev. Biol. 45 (2015) 77–83. [23] J. Dhanoa, C. Mukhopadhyay, J. Arora, Y-chromosomal genes affecting male fertility: a review, Vet. World 9 (7) (2016) 783–791. [24] C. Krausz, E. Casamonti, Spermatogenic failure and the Y chromosome, Hum. Genet. 136 (5) (2017) 637–655. [25] T.  Miyamoto, G.  Minase, K.  Okabe, H.  Ueda, K.  Sengoku, Male infertility and its genetic causes, J. Obstet. Gynaecol. Res. 41 (10) (2015) 1501–1505. [26] A. Geisinger, R. Benavente, Mutations in genes coding for synaptonemal complex proteins and their impact on human fertility, Cytogenet. Genome Res. 150 (2) (2016) 77–85. [27] S.D.  Quaynor, M.E.  Bosley, C.G.  Duckworth, K.R.  Porter, S.H.  Kim, H.G.  Kim, L.P.  Chorich, M.E.  Sullivan, J.H.  Choi, R.S.  Cameron, L.C.  Layman, Targeted next generation sequencing approach identifies eighteen new candidate genes in normosmic hypogonadotropic hypogonadism and Kallmann syndrome, Mol. Cell. Endocrinol. 437 (2016) 86–96. [28] A. Amiri-Yekta, C. Coutton, Z.E. Kherraf, T. Karaouzène, P. Le Tanno, M.H. Sanati, M. Sabbaghian, N. Almadani, M.A. Sadighi Gilani, S.H. Hosseini, S. Bahrami, A. Daneshipour, M. Bini, C. Arnoult, R. Colombo, H. Gourabi, P.F. Ray, Whole-exome sequencing of familial cases of multiple morphological abnormalities of the sperm flagella (MMAF) reveals new DNAH1 mutations, Hum. Reprod. 31 (12) (2016) 2872–2880.

REFERENCES 61

[29] H. Ghédir, S. Ibala-Romdhane, O. Okutman, G. Viot, A. Saad, S. Viville, Identification of a new DPY19L2 mutation and a better definition of DPY19L2 deletion breakpoints leading to globozoospermia, Mol. Hum. Reprod. 22 (1) (2016) 35–45. [30] T. Svingen, P. Koopman, Building the mammalian testis: origins, differentiation, and assembly of the component cell populations, Genes Dev. 27 (22) (2013) 2409–2426. [31] I.A. Hughes, C. Houk, S.F. Ahmed, P.A. Lee, Lawson Wilkins Pediatric Endocrine Society/European Society for Paediatric Endocrinology Consensus Group, Consensus statement on management of intersex disorders, J. Pediatr. Urol. 2 (3) (2006) 148–162. [32] J.F. Hughes, S. Rozen, Genomics and genetics of human and primate y chromosomes, Annu. Rev. Genomics Hum. Genet. 13 (2012) 83–108. [33] P. Navarro-Costa, Sex, rebellion and decadence: the scandalous evolutionary history of the human Y chromosome, Biochim. Biophys. Acta 1822 (12) (2012) 1851–1863. [34] N.B.  Phillips, J.  Racca, Y.S.  Chen, R.  Singh, A.  Jancso-Radek, J.T.  Radek, N.P.  Wickramasinghe, E.  Haas, M.A. Weiss, Mammalian testis-determining factor SRY and the enigma of inherited human sex reversal: frustrated induced fit in a bent protein-DNA complex, J. Biol. Chem. 286 (42) (2011) 36787–36807. [35] T.  Wagner, J.  Wirth, J.  Meyer, B.  Zabel, M.  Held, J.  Zimmer, J.  Pasantes, F.D.  Bricarelli, J.  Keutel, E.  Hustert, U. Wolf, N. Tommerup, W. Schempp, G. Scherer, Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9, Cell 79 (6) (1994) 1111–1120. [36] A.  Bashamboo, B.  Ferraz-de-Souza, D.  Lourenço, L.  Lin, N.J.  Sebire, D.  Montjean, J.  Bignon-Topalovic, J. Mandelbaum, J.P. Siffroi, S. Christin-Maitre, U. Radhakrishna, H. Rouba, C. Ravel, J. Seeler, J.C. Achermann, K. McElreavey, Human male infertility associated with mutations in NR5A1 encoding steroidogenic factor 1, Am. J. Hum. Genet. 87 (4) (2010) 505–512. [37] A.  Bashamboo, P.A.  Donohoue, E.  Vilain, S.  Rojo, P.  Calvel, S.N.  Seneviratne, F.  Buonocore, H.  Barseghyan, N.  Bingham, J.A.  Rosenfeld, S.N.  Mulukutla, M.  Jain, L.  Burrage, S.  Dhar, A.  Balasubramanyam, B.  Lee, Members of UDN, M.C. Dumargne, C. Eozenou, J.P. Suntharalingham, K. de Silva, L. Lin, J. Bignon-Topalovic, F. Poulat, C.F. Lagos, K. McElreavey, J.C. Achermann, A recurrent p.Arg92Trp variant in steroidogenic factor-1 (NR5A1) can act as a molecular switch in human sex development, Hum. Mol. Genet. 25 (16) (2016) 3446–3453. [38] M.  Igarashi, K.  Takasawa, A.  Hakoda, J.  Kanno, S.  Takada, M.  Miyado, T.  Baba, K.I.  Morohashi, T.  Tajima, K. Hata, K. Nakabayashi, Y. Matsubara, R. Sekido, T. Ogata, K. Kashimada, M. Fukami, Identical NR5A1 missense mutations in two unrelated 46,XX individuals with testicular tissues, Hum. Mutat. 38 (1) (2017) 39–42. [39] R. Werner, I. Mönig, J. August, C. Freiberg, R. Lünstedt, B. Reiz, L. Wünsch, P.M. Holterhus, A. Kulle, U. Döhnert, S.A. Wudy, A. Richter-Unruh, C. Thorns, O. Hiort, Novel insights into 46,XY disorders of sex development due to NR5A1 gene mutation, Sex Dev. 9 (5) (2015) 260–268. [40] D. Lourenço, R. Brauner, M. Rybczynska, C. Nihoul-Fékété, K. McElreavey, A. Bashamboo, Loss-of-function mutation in GATA4 causes anomalies of human testicular development, Proc. Natl. Acad. Sci. USA 108 (4) (2011) 1597–1602. [41] A.  Bashamboo, R.  Brauner, J.  Bignon-Topalovic, S.  Lortat-Jacob, V.  Karageorgou, D.  Lourenco, A.  Guffanti, K. McElreavey, Mutations in the FOG2/ZFPM2 gene are associated with anomalies of human testis determination, Hum. Mol. Genet. 23 (14) (2014) 3657–3665. [42] R.  Werner, H.  Merz, W.  Birnbaum, L.  Marshall, T.  Schröder, B.  Reiz, J.M.  Kavran, T.  Bäumer, P.  Capetian, O. Hiort, 46,XY gonadal dysgenesis due to a homozygous mutation in desert hedgehog (DHH) identified by exome sequencing, J. Clin. Endocrinol. Metab. 100 (7) (2015) E1022–E1029. [43] P. Callier, P. Calvel, A. Matevossian, P. Makrythanasis, P. Bernard, H. Kurosaka, A. Vannier, C. Thauvin-Robinet, C. Borel, S. Mazaud-Guittot, A. Rolland, C. Desdoits-Lethimonier, M. Guipponi, C. Zimmermann, I. Stévant, F. Kuhne, B. Conne, F. Santoni, S. Lambert, F. Huet, F. Mugneret, J. Jaruzelska, L. Faivre, D. Wilhelm, B. Jégou, P.A. Trainor, M.D. Resh, S.E. Antonarakis, S. Nef, Loss of function mutation in the palmitoyl-transferase HHAT leads to syndromic 46,XY disorder of sex development by impeding Hedgehog protein palmitoylation and signaling, PLoS Genet. 10 (5) (2014) e1004340. [44] C.E. Flück, A.V. Pandey, Steroidogenesis of the testis new genes and pathways, Ann. Endocrinol. (Paris) 75 (2) (2014) 40–47. [45] J.  Yu, Z.  Chen, Y.  Ni, Z.  Li, CFTR mutations in men with congenital bilateral absence of the vas deferens (CBAVD): a systemic review and meta-analysis, Hum. Reprod. 27 (1) (2012) 25–35. [46] E.H. Stephen, A. Chandra, Declining estimates of infertility in the United States: 1982–2002, Fertil. Steril. 86 (2006) 516–523.

62

3.  New Genetic Point Mutations in Male Infertility

[47] M.M. Matzuk, D.J. Lamb, Genetic dissection of mammalian fertility pathways, Nat. Cell Biol. 4 (Suppl (10)) (2002) s41–s49. [48] P. Navarro-Costa, C.E. Plancha, J. Gonçalves, Genetic dissection of the AZF regions of the human Y chromosome: thriller or filler for male (in)fertility? J Biomed Biotechnol 2010 (2010) 1–18. [49] M.A. Jobling, C. Tyler-Smith, Human Y-chromosome variation in the genome-sequencing era, Nat. Rev. Genet. 18 (8) (2017) 485–497. [50] M. Ben Khelifa, C. Coutton, R. Zouari, T. Karaouzène, J. Rendu, M. Bidart, S. Yassine, V. Pierre, J. Delaroche, S.  Hennebicq, D.  Grunwald, D.  Escalier, K.  Pernet-Gallay, P.S.  Jouk, N.  Thierry-Mieg, A.  Touré, C.  Arnoult, P.F. Ray, Mutations in DNAH1, which encodes an inner arm heavy chain dynein, lead to male infertility from multiple morphological abnormalities of the sperm flagella, Am. J. Hum. Genet. 94 (1) (2014) 95–104.