Novel and recurrent genetic variants associated with male and female infertility

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Jankowska, Anna Kutkowska-Kazmierczak, Klaudia Ślusarczyk, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4711926/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Jan, 2025 Read the published version in Journal of Applied Genetics → Version 1 posted 5 You are reading this latest preprint version Abstract Recently, the knowledge of the genetic basis of fertility disorders has expanded enormously, mainly thanks to the use of next generation sequencing (NGS). However, the genetic cause of the infertility in majority patients is still undefined. Aim: The aim was to identify genetic variants associated with infertility disorders using targeting NGS technique and to describe phenotype - genotype correlation. Methods: We have enrolled 41 patients (36 males and 5 females) with infertility problems or delayed puberty including the patients with hypogonadism hypogonadotropic (HH, n = 12), primary hypogonadism (n = 15), abnormal semen parameters or gonadal dysgenesis (n = 11), androgen insensitivity (n = 3). Genetic tests were performed using NGS panel of 35 genes. Results: Overall, 14 pathogenic (P) or likely pathogenic (LP) variants including 3 novel and 11 recurrent variants were identified. Novel variants were found in genes associated with HH ( FGF8 , FGFR1, SEMA3 ). The genetic cause of the HH was determined in 58% (7/12) of the cases. Overall, The genetic testing enabled identification of the cause of the clinical phenotype in 26% (11/41) of the patients. Conclusions: Our study expands the knowledge of the genetic basis of the infertility disorders and highlights the importance of genetic testing for proper diagnosis making and genetic counselling. human infertility genetic variants genetic diagnostics Introduction Infertility, defined (according to WHO) as the failure of reproduction after one year of regular intercourse without the use of contraception, is one of the major global health and social problems that affects 10–15% of couples of reproductive age (in Poland: 20% of couples, 1.5 million) [ 1 , 2 ]. It is estimated that in 30–50% of cases, the causes of infertility are identified in women, 20–30% in men, while the problem affect both partners in approximately 20–30% of the couples [ 3 ]. Infertility is a multifactorial disease that can be caused by a number of reasons, including: abnormal gametogenesis, disorders of the structure and function of the reproductive system systemic diseases, including immunological disorders, infections, environmental factors, mental and genetic factors. Idiopathic infertility affects approximately 20–40% of couples. It is estimated that genetic disorders are the cause of approximately 50% of them, although the exact role of genetic factors in infertility is still unknown [ 3 ]. In both men and women, standard genetic tests towards infertility include karyotype assessment and, in the case of men with azoospermia/oligozoospermia, also analysis of the deletion of the AZF region of the Y chromosome and examination of pathogenic variants in CFTR gene [ 3 ]. Deletions of the AZF region of the Y chromosome are identified in 5–10% of men with azoospermia/oligozoospermia, while pathogenic variants of the CFTR gene in 6% of all infertile men and 60–70% of men with CBAVD [ 4 ]. However, according to literature data, about 30 genes are known to be associated with abnormal semen parameters in men (i.e. azoospermia, oligozoospermia, asthenozoospermia, or teratozoospermia) [ 5 ]. This number will certainly increase in the future, taking into account that approximately 2,000 genes are involved in the process of spermatogenesis, 600–900 of which are expressed in male sex cells exclusively [ 6 ]. Abnormal human spermatogenesis can be also caused by defects in androgen action and androgen insensitivity. Androgen Insensitivity Syndrome (AIS) is a disorder of male sexual development resulting in a wide range of clinical phenotypes and typically classified into three main types based on the degree of androgen receptor dysfunction and the resulting phenotypic characteristics. These types include complete androgen insensitivity syndrome (CAIS), partial androgen insensitivity syndrome (PAIS), and mild or minimal androgen insensitivity syndrome (MAIS). A variety of pathogenic variants have been described in the human androgen receptor gene ( AR ) associated with male infertility. Due to these molecular defects the androgen receptor becomes less responsive or completely unresponsive to androgens affecting sexual development and spermatogenesis [ 7 ]. The infertility can be also associated with endocrine disorders such as hypogonadism, in which the gonads do not function properly and produce inadequate amount of sex hormones There are two main categories of hypogonadism: hypogonadotropic and hypergonadotropic. Hypogonadotropic hypogonadism is characterized by low levels of sex hormones in males or females due to insufficient stimulation of the gonads by the pituitary gland and hypothalamus. Genetic causes of hypogonadotropic hypogonadism can result from mutations in genes involved in the development and functioning of these regulatory systems. Congenital hypogonadotropic hypogonadism (CHH) is a rare disorder (occurs in 1–10/100,000 infertile men) caused by the failure of the normal episodic gonadotropin-releasing hormone (GnRH) secretion, leading to delayed puberty and infertility [ 10 ]. Most of the genes mutated in CHH encode receptor-ligand pairs including GNRHR (gonadotropin-releasing hormone receptor) and GNRH1 (gonadotropin-releasing hormone 1), PROKR2 ( Prokineticin receptor2) and PROK2 (Prokineticin 2), KISS1R (KiSS1 receptor) and KISS1, TACR3 (Tachykinin Receptor 3) and TAC3 (Tachykinin 3), FGFR1 (Fibroblast Growth Factor Receptor 1) and FGF8 (Fibroblast Growth Factor 8) suggesting that multiple receptor-ligand-encoding gene networks are involved in the molecular pathology of the disease. Part of the cases with CHH demonstrate an impairment sense of smell, called also Kallmann syndrome, in which pathogenic variants in genes such as ANOS1 ( KAL1 ) (Anosmin 1), HS6ST1 (Heparan Sulfate 6-O-Sulfotransferase 1), SPRY4 (Sprouty RTK Signaling Antagonist 4), DUSP6 (Dual Specificity Phosphatase 6) are also identified [ 8 ]. The CHH is one of the few treatable causes of male infertility. Diagnosis of CHH is, however, challenging, especially in early adolescence where the clinical features mimics that of constitutional delay of growth and puberty. Timely diagnosis is essential for a proper treatment. Pathogenic variants, in more than 30 genes, has been associated with CHH, acting either alone or in combination [ 5 ]. Hypergonadotropic hypogonadism, also known as primary hypogonadism, is characterized by low levels of sex hormones accompanied by elevated levels of gonadotropins (FSH and LH). This condition can result from various genetic defects that directly affect the gonads. Some of the main genetic causes of hypergonadotropic hypogonadism involve mutations in genes such as FSHR (Follicle-Stimulating Hormone Receptor), LHR (Luteinizing Hormone Receptor), INSL3 (Insulin-Like 3), NR5A1 (Nuclear Receptor Subfamily 5 Group A Member 1) [ 5 ]. Identification of the causes of infertility is of paramount importance for each couple, due to several of the following reasons: the choice of therapy, genetic counselling, including determination of the risk of having offspring with cystic fibrosis (patients with CBAVD) or with deletion of the AZF region of the chromosome Y, improving mental comfort related to regaining hope for further effective treatment. Sometimes it may help to make decisions such as discontinue therapy and choosing alternative options (use of donor sperm, adoption, etc.).​ In recent years, thanks to the use of next generation sequencing (NGS), more and more genes have been described in the context of fertility disorders. However, still in only 4% of all infertile men the genetic cause is established, and majority of the cases are classified as unexplained. This is partly due to a delay to adopt NGS techniques in the field of diagnostics and the lack of genotype–phenotype correlations data [ 5 ]. Therefore, the objective of the study was to identify genetic variants associated with isolated infertility using NGS technique. Secondary aim was to describe genotype-phenotype correlations. Materials and Methods Patients The patients with fertility problems or delayed puberty were referred for genetic testing from Department of Endocrinology, Bielanski Hospital, Genetic counselling unit of the Department of Medical Genetics, Institute of Mother and Child, Oviclinic Infertility Center and nOvum Fertility Clinic in Warsaw. Women with endometriosis and PCOS were excluded. Phenotypical information was based on clinical interview, physical and laboratory examinations. All patients had karyotype analysis preformed before they were subjected to molecular analysis. All patients gave their informed consent for the genetic testing. The study was also approved by bioethical committee of the Institute of Mother and Child, Nr 22/2019. Genetic analysis AZF microdeletions analyses To exclude AZF microdeletions on the chromosome Y, all males were subjected to analysis of 6 loci in the AZF by PCR. The panel of analyzed loci, including AZF c (sY254, sY255), AZF b(sY134, sY127), AZF a(sY86,S84), is consistent with the recommendations of the European Molecular Quality Network: "EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions: state-of-the-art 2013”. NGS analysis Genetic tests were performed by targeted next generation sequencing. The NGS panel included 35 genes with well-established association with hypogonadotropic hypogonadism ( ANOS1, CHD7, DUSP6, FEZF1, FGF17, FGF8, FGFR1, FLRT3,FSHB, GNRH1, GNRHR, IL17RD, KISS1, KISS1R, LHB, NSMF, PROK2, PROKR2, SEMA3A, SOX10,SPRY4, WDR11, HS6ST1, TAC3 ) hypergonadotropic hypogonadism ( NR5A1 , INSL3 ,, LHCGR, LEP, GATA4, FSHR ), spermatogenesis disorders ( SPATA16, CFTR,) , androgen insensitivity (AR), ovarian dysgenesis ( BMP15 ) and sex reversal ( SRY ). Genes relevant to these phenotypes where included bases on OMIM and Human Phenotype Ontology (HPO) databases. The DNA library was prepared using the SureSelect kit from Agilent. Sequencing was preformed using The NextSeq (Illumina). Only the coding fragments of genes (exons) and exon-intron junctions +/- 15 nucleotides were analyzed. The sequences were mapped to the human reference genome version GRCh38/hg38. The results were interpreted using internal bioinformatics tools based on the VEP program (Ensembl), and in relation to the following databases: GnomAD v3, HGMD Professional, NCBI, Ensembl, OMIM, internal database of IMiD variants. The variants classification was preformed according to ACMG (American College of Medical Genetics) recommendations using Varsome or Franklin platform. The presence of the detected genetic variants was confirmed by Sanger sequencing (the primers available on the request). The detected variants were named according to HGVS nomenclature and canonical transcript (NCBI RefSeq). Results Patients The study group consisted of 41 patients with following clinical diagnosis: hypogonadism hypogonadotropic (n = 12), hypogonadism hypergonadotropic (n = 15), abnormal semen parameters (oligozoospermia, astenozoospermia, teratozoospermia or azoospermia) (n = 10), androgen insensitivity (n = 3), gonadal dysgenesis (n = 1). Genetic results No deletion in the AZF a,b,c regions of the Y chromosome was detected in the examined males. One of 41 patients showed karyotype abnormality. This was a patient with typical female external genitalia, however, the structures resembling testes were found during inguinal hernias surgery. The karyotype analysis revealed presence of XY indicating sex reversal in this patient. The rest of patient had normal karyotype results. The NGS gene panel analysis revealed 18 unique, missense or nonsense variants in 16/42 cases, including 11 known pathogenic (P) or likely pathogenic (LP) variants described in clinical databases (HGMD/ClinVar) and 4 novel or very rare variants (classified as LP or VUS according to ACMG recommendation), not reported in clinical databases and literature (Table 1 ). Table 1 Genotype- phenotype correlation in patients with infertility problems or delayed puberty. Patient Nr Sex Gene Inheritance OMIM Variant c.DNA Variant protein Zyg MAF gnomAD v.4.00 HGMD /ClinVar ACMG & Clinical diagnosis Other clinical features 1 M SPATA16 AR c.848G > A p.Arg283Gln het 0.0004 DM/P (Globozoospermia) LP nHH OAT, bilateral cryptorchidism, 6 toes on one foot 1 M FGF8 AD, Oligo c.77C > T p.Pro26Leu het 0.00115 DM/P (idiopathic HH) LP 2 M DUSP6 AD c.566A > G p.Asn189Ser het 0.00104 DM/P (Kallmann syndrome) LP nHH cryptozoospermiahypopituitarism (empty sella syndrome) 3 M SPRY4 AD, Oligo c.530A > G p.Lys177Arg het 0.00227 DM / Conflicting (Kallmann syndrome ) VUS/LP nHH OAT 4 M AR XLR c.2395C > G p.Gln799Glu hemi 0.0015 DM/Confliciting (AIS) P PAIS testicular hypoplasia, azooseprmia, decreased testosterone 5 F (k.XY) AR XLR c.2287C > G p.Leu763Val hemi 0 DM (AIS) P CAIS 6 M GNRHR AR, Oligo c.416G > A p.Arg139His het 0.00015 DM/P (HH) P nHH 7 M NR5A1 AD c.776C > T p.Pro259Leu het 0.00005 DM?( XY gonadal dysgenesis)/nd P Gonadal dysgenesis epididymal cysts, microcalcifications, varicocele 8 M DUSP6 AD c.215C > A p.Pro72Gln het 0.00013 nd/VUS VUS nHH tetratozoospermia (0% of normal sperm) 9 M HS6ST1 AD c.1132C > T p.Arg378Cys het 0.00003 nd/VUS VUS primary hypogonadism severe oligozoospermia, very high level of FSH 10 F FGFR1 AD c.1270C > T p.Arg424Cys het 0.00001 nd/VUS VUS nHH 11 M FGFR1 AD, Oligo c.246_247del p.Glu84GlyfsTer26 het 0 DM/ P (GnRH deficiency) P nHH 12 M FGFR1 AD, Oligo c.863A > T p.Gln288Leu het 0 nd/VUS LP Kallman syndrome azoospermia 12 M FGF8 AD, Oligo c.439G > A p.Ala147Thr het 0.00001 nd/nd LP Kallman syndrome azoospermia 13 M SEMA3 AD c.1630C > T p.Arg544Cys het 0.000001 nd/nd LP nHH tetrazoospermia 14 F FGFR1 AD c.420_423del p.Thr141IlefsTer10 het 0 nd/nd LP nHH delayed puberty, primary amenorrhea 15 M NSMF AD c.725A > G p.Tyr242Cys het 0.000001 nd/nd VUS (PM2) azoospermia high level of FSH but normal level of testosterone M - male; F - female; AD - autosomal dominant inheritance; AR - autosomal recessive inheritance; Oligo - oligogenic inheritance; nd - not described; Zyg -zygosity, MAF - allele minor frequency, DM - damaging, P -pathogenic, LP - likely pathogenic, VUS - variant of uncertain significance, PAIS - partial androgen insensitivity syndrome; AIS- androgen insensitivity, CAIS - complete androgen insensitivity syndrome; HH hypogonadotropic hypogonadism, nHH - normosomic hypogonadotropic hypogonadism; OAT - oligoasthenoteratozoospermia. k .-karyotype. & - ACMG classification according to .Brandt et al.2020, Genet Med. Bolded are novel variants (not found in clinical databases nor literature and absent/ very rare in gnomAD) The genotype-phenotype correlation is described in Table 1 . In patients with hypogonadotropic hypogonadism (HH), we have identified P/LP and VUS variants in 58% (7/12) and 25% (3/12) of the cases, respectively. One of these cases, diagnosed with Kallmann syndrome, harboured a very rare, heterozygous, LP variants in two different genes: p.Gln288Leu in FGFR1 and p.Ala147Thr in FGF8 . In two of the CHH cases without anosmia, we found novel p.Thr141IlefsTer10 (LP) variant and a very rare, heterozygous VUS, p.Arg424Cys in FGFR1 gene. In 1/15 cases with hypergonadotropic hypogonadism, we found one very rare VUS variant in HS6ST1 gene. In 2/3 of the cases with androgen insensitivity, we have detected a very rare, hemizygous, pathogenic variants in AR gene including p.Leu763Val identified in a patient with sex reversal (female phenotype but XY karyotype) and p.Leu763Val variant found in a male patient with partial androgen insensitivity (PAIS). One patient with gonadal dysgenesis harbored p.Arg139His, known pathogenic variant in one allele of the GNRHR gene. In 1/10 cases with abnormal semen parameters, we detected a very rare p.Tyr242Cys variant in NSMF gene classified as LP. Discussion In this study, we have found 11 known, pathogenic/likely pathogenic variants (P/PL), 3 novel, likely pathogenic variants (LP) and 4 variants of uncertain significance (including one novel) in the group of patients with infertility or delayed puberty. The genetic testing enabled identification of the cause of the infertility or delayed puberty in 26% of the patients. We have examined patients with different clinical features such as hypogonadotropic and hypergonadotropic hypogonadism, abnormal semen parameters, androgen insensitivity and gonadal dysgenesis. We have described observed genotype-phenotype correlations. Hypogonadism Genetic studies on congenital hypogonadotropic hypogonadism (CHH) have led to discovery of pathogenic variants in various genes implicated in specification and proliferation of GnRH neurons, their migration to the hypothalamus during embryonic development and the response of pituitary gonadotropes to GnRH stimulation [ 9 ]. The genetic complexity of the disease is reflected by different models of inheritance: X-linked, autosomal dominant and autosomal recessive as well as incomplete penetrance and variable phenotypic expression as well as oligogenic inheritance pattern in the certain cases [ 9 , 10 ]. Using our NGS panel, we identified P/LP in 58% (7/12) of the CHH (including one case of Kallmann syndrome) patients which is higher than in previous reports showing around 31–35% of the pathogenic variants detection rate [ 8 ]. Our results are consistent with the study by Cassatella D et al., which detected 51% of pathogenic variants in CHH patients using similar gene panel [ 11 ]. Multiple genes encoding components of the FGF pathway are known to be mutated in CHH and IHH eg. FGFR1 , FGF8 ,, HS6ST1 DUSP6 and SPRY4 [ 8 ]. In our CHH patients, FGFR1 and FGF8 were the most frequently mutated genes with the same frequency of 33% (4/12), each. These results are consistent with previous studies showing very important role of the two genes in the development of CHH [ 8 , 12 , 13 ]. In FGFR1 , we have detected one novel p.Thr141IlefsTer10 (LP) variant, one known pathogenic variant p.Glu84GlyfsTer26 and two very rare variants including p.Gln288Leu (LP) and p.Arg424Cys (VUS). The novel p.Thr141IlefsTer10 was found in a girl with hypogonadotropic hypogonadism, delayed puberty and primary amenorrhea. The p.Arg424Cys variant (VUS) was harboured by a girl with HH.Importantly, different amino acid substitution, Arg to His (p.Arg424His, c.1271G > A), was described in the same codon in a patient with HH (HGMD no.: CM2115069) suggesting that substations in this codon may be likely disruptive. In FGF8 , we found 2 variants including 1 know pathogenic p.Pro26Leu and 1 novel, LP p.Ala147Thr variant. Previously, Falardeau et al. described pathogenic variants in FGF8 , including p.Pro26Leu, in IHH probands with variable olfactory phenotypes. These patients exhibited varied degrees of GnRH deficiency, including the rare adult-onset form of hypogonadotropic hypogonadism [ 13 ]. Pathogenic variants in DUSP6 (OMIM #615269) and SPRY4 (OMIM#615266) gene cause CHH with normal smell or anosmia and both are inherited in autosomal dominant manner. In DUSP6 gene, we detected variants in 2 of the 12 cases with HH including a known pathogenic variant p.Asn189Ser and p.Pro72Gln classified as VUS. The p.Asn189Ser variant was previously reported in case with Kallman syndrome [ 8 ]. Our patient showed a normal sense of smell but had hypopituitarism (empty sella syndrome) and cryptozoospermia. The other case with p.Pro72Gln (VUS) had teterathozoospermia and HH. Pheotypic variability is described both in a group of unrelated patients and within members of one family with the same pathogenic variant in DUSP6 gene. In some patients, variable phenotypic expression, maybe related to the simultaneous occurrence of pathogenic variants in other genes associated with hypogonadotropic hypogonadism [ 14 ]. We also have identified known pathogenic variant p.Lys177Arg in SPRY gene in one patient with hypogonadism and oligoasthenospermia. Previously, Miraoui et al. identified heterozygous missense variants in the SPRY4 gene in 14 unrelated individuals with congenital CHH), including p.Lys177Arg variant which was detected in patients with Kallmann syndrome [ 8 ]. The genetic complexity and phenotypic variability of CHH is exemplified by oligogenesity and involvement of pleotropic genes implicated in the disease. In our study, oligogenic interactions defined as ≥ 2 genes mutated were found in 2 out of 12 CHH cases. In one patient with Kallman syndrome, we detected simultaneous occurrence of p.Gln288Leu in FGFR1 and a novel Ala147Thr variant in FGF8 . The pathogenic variants in FGFR1 and FGF8 genes are inherited in autosomal dominant manner, however, oligogenic interactions has been also reported [ 9 , 15 ]. For instance, study by Sykiotis GP et al , investigating a large cohort of 397 patients with idiopathic HH, found that 2.5% of the patients show oligogenic interactions including genes such as FGFR1 and FGF8 [ 9 ]. The other digenic interaction was observed in a male patients with hypogonadotropic hypogonadism, oligoasthenoteratozoospermia (OAT) and bilateral cryptorchidism. This patient harbored known pathogenic variants such as p.Pro26Leu and p.Arg283Gln in FGF8 and SPATA16 gene, respectively. Pathogenic variants in FGF8 gene are associated with hypogonadotropic hypogonadism with or without ansomia. The literature describes patients with the p.Pro26Leu variant in FGF8 who were diagnosed with Kalman syndrome with partial loss of smell (hyposomia) [ 13 , 16 ]. The phenotype is, however, variable even within members of one family with the same variant [ 13 ]. In our patient, no anosmia or hyposomia was present. Interestingly, this patient showed foot malformation (six toes) confirming a pleotropic role of the FGF8 gene [ 17 ]. Interestingly, FGFR1 pathogenic variants occur in 87% of patients with both CHH and split hand/food malformation [ 17 ]. Our patient also harbored heterozygous p.Arg283Gln variant in one allele of SPATA16 , known to disturb splicing [ 18 ]. The pathogenic variants SPATA16 are the cause of defects in spermatogenesis (including abnormal sperm morphology) inherited in an autosomal recessive manner. In this case, however, no variants in the second allele of SPATA16 gene was identified. NSMF encodes protein involved in guidance of olfactory axon projections and migration of luteinizing hormone-releasing hormone neurons. Defects in this gene are a cause of idiopathic hypogonadotropic hypogonadism (IHH) inherited in autosomal dominant manner (OMIM#614838). We have identified two variants in NSMF gene including a rare, LP, p.Pro213Leu and a novel p.Tyr242Cys (VUS) variant. The p.Pro213Leu variant, was present in one patient with hypogonadotropic hypogonadism and p.Tyr242Cys was found in a patient with azoospermia and high level of FSH but normal level of testosterone (not fulfilling criteria of hypogonadism). In our study, we studied 15 cases with hypergonadotropic hypogonadism (primary hypogonadyzm) but no P/LP was detected in this group using our NGS panel. We have detected one VUS - p.Arg378Cys variant - in HS6ST1 gene in a patient with hypergonadotropic hypogonadism and oligozoospermia. The low frequency of detected variants in this group of patients could be due to limited number genes associated with primary hypogonadism included in our panel. Androgen Insensitivity The androgen insensitivity syndrome (AIS) is a disorder of male sexual development resulting in a wide range of clinical phenotypes. It affects-XY males but phenotypically is characterized by a female phenotype. AIS is classified into two phenotypic forms: complete (CAIS) and partial (PAIS). We have detected a very rare hemizygous variant p.Leu763Val in AR gene in patient with XY karyotype, female phenotype and complete androgen insensitivity syndrome (CAIS). This patient with typical female external genitalia was referred to the genetic counselling unit because during inguinal hernias surgery, structures resembling testes were found. Ultrasound examination demonstrated presence of two structures corresponded to testes localized along iliac blood vessels in absence of müllerian structures as fallopian tubes and uterus and absence of extragenital abnormalities apart from 3 cafe-au-lait spots. Very recently the p.Leu763Val variant has been described in an Iranian family with two female patients with normal external genitalia and 46, XY karyotype and CAIS phenotype [ 19 ]. Thus, we have described the second case in the world with the p.Leu763Val variant in AR gene. A pathogenic variant in the same 763 codon leading to Phe amino acid substitution (p.Leu763Phe and causing the loss of androgen binding receptor function was described as well [ 20 ]. We also identified a known pathogenic p.Gln799Glu variant in AR gene in one patient with PAIS and clinical features such as hypogonadotropic hypogonadism, testicular hypoplasia and azoospermia. In the literature, the p.Gln799Glu variant was described in patients partial androgen insensitivity, hypospadia and oligoasthenoteratozoospermia [ 21 – 23 ]. Conclusions In summary, we have identified several new and recurrent pathogenic/likely pathogenic genetic variants associated with hypogonadism, androgen insensitivity and described observed genotype-phenotype correlations what expands the knowledge of the genetic basis of these disorders. Importantly, our results are in line with other studies. They provide further evidence for the heterogeneity of infertility disorders, especially in CHH, indicating that multigene panels are the best method of choice for genetic analysis in this group of patients. Further, this study highlights the importance of genetic testing for proper diagnosis making. Genetic diagnostics may also be very helpful in treating causes of infertility such as hypogonadism but requires the assistance of a clinician endocrinologist or andrologist, as well as a geneticist [ 24 ]. Moreover, the possibility of getting genetic counselling is also of high importance for the couple with infertility problems. Declarations Acknowledgements : We are grateful to all patients who participated in this study. Author’s Contributions: Conceptualization, AMR and KKJ; Methodology (Molecular Analysis): KŚ, AD, KD, JS; Formal analysis: AMR, KŚ, AD, KD, JS, KWT(molecular analysis), KKJ., AKK, JKW, JK, PL (clinical analysis); Writing – Original Draft Preparation: AMR.; Writing - Review & Editing KKJ, AKK, JKW, PL, KWT. Visualization, KŚ.; Supervision: AMR.; Funding Acquisition, AMR.”, All authors have read and agreed to the published version of the manuscript. Ethics approval This study complies with the latest Declaration of Helsinki and was approved by bioethical committee of the Institute of Mother and Child, Warsaw, Nr 22/2019. Consent to participate : Informed consent to participate was obtained from all subjects involved in the study. Consent for publication: Consent for publication was received from all the patients enrolled in the study. Competing interest: The authors declare no conflicts of interest. Funding: This research was funded by Ministry of Science and Higher Education, Poland , project nr 510-18-61 granted to Institute of Mother and Child ( A.M. 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J Clin Invest 117:457–463. 10.1172/JCI29884 Cassatella D, Howard SR, Acierno JS, Xu C, Papadakis GE, Santoni FA, Dwyer AA, Santini S, Sykiotis GP, Chambion C et al (2018) Congenital Hypogonadotropic Hypogonadism and Constitutional Delay of Growth and Puberty Have Distinct Genetic Architectures. Eur J Endocrinol 178:377–388. 10.1530/EJE-17-0568 Dodé C, Levilliers J, Dupont J-M, De Paepe A, Le Dû N, Soussi-Yanicostas N, Coimbra RS, Delmaghani S, Compain-Nouaille S, Baverel F et al (2003) Loss-of-Function Mutations in FGFR1 Cause Autosomal Dominant Kallmann Syndrome. Nat Genet 33:463–465. 10.1038/ng1122 Falardeau J, Chung WCJ, Beenken A, Raivio T, Plummer L, Sidis Y, Jacobson-Dickman EE, Eliseenkova AV, Ma J, Dwyer A et al (2008) Decreased FGF8 Signaling Causes Deficiency of Gonadotropin-Releasing Hormone in Humans and Mice. J Clin Invest 118:2822–2831. 10.1172/JCI34538 Fraietta R, Zylberstejn DS, Esteves SC (2013) Hypogonadotropic Hypogonadism Revisited. Clin (Sao Paulo) 68(Suppl 1):81–88. 10.6061/clinics/2013(sup01)09 Quaynor SD, Kim H-G, Cappello EM, Williams T, Chorich LP, Bick DP, Sherins RJ, Layman LC (2011) The Prevalence of Digenic Mutations in Patients with Normosmic Hypogonadotropic Hypogonadism and Kallmann Syndrome. Fertil Steril 96:1424–1430e6. 10.1016/j.fertnstert.2011.09.046 Kałużna M, Budny B, Rabijewski M, Kałużny J, Dubiel A, Trofimiuk-Müldner M, Wrotkowska E, Hubalewska-Dydejczyk A, Ruchała M, Ziemnicka K (2021) Defects in GnRH Neuron Migration/Development and Hypothalamic-Pituitary Signaling Impact Clinical Variability of Kallmann Syndrome. Genes 12:868. 10.3390/genes12060868 Villanueva C, Jacobson-Dickman E, Xu C, Manouvrier S, Dwyer AA, Sykiotis GP, Beenken A, Liu Y, Tommiska J, Hu Y et al (2015) Congenital Hypogonadotropic Hypogonadism with Split Hand/Foot Malformation: A Clinical Entity with a High Frequency of FGFR1 Mutations. Genet Sci 17:651–659. 10.1038/gim.2014.166 Dam AHDM, Koscinski I, Kremer JAM, Moutou C, Jaeger A-S, Oudakker AR, Tournaye H, Charlet N, Lagier-Tourenne C, van Bokhoven H et al (2007) Homozygous Mutation in SPATA16 Is Associated with Male Infertility in Human Globozoospermia. Am J Hum Genet 81:813–820. 10.1086/521314 Aghaei S, Parvizpour S, Farrokhi E, Molavi N, Hoseinzadeh M, Tabatabaiefar MA (2022) Characterization of a Novel Androgen Receptor Gene Variant Identified in an Iranian Family with Complete Androgen Insensitivity Syndrome (CAIS): A Molecular Dynamics Simulation Study. J Biomol Struct Dynamics 1–15. 10.1080/07391102.2022.2148125 Bevan CL, Hughes IA, Patterson MN (1997) Wide Variation in Androgen Receptor Dysfunction in Complete Androgen Insensitivity Syndrome. J Steroid Biochem Mol Biol 61:19–26. 10.1016/s0960-0760(97)00001-0 Hellmann P, Christiansen P, Johannsen TH, Main KM, Duno M, Juul A (2012) Male Patients with Partial Androgen Insensitivity Syndrome: A Longitudinal Follow-up of Growth, Reproductive Hormones and the Development of Gynaecomastia. Arch Dis Child 97:403–409. 10.1136/archdischild-2011-300584 Lorenzi D, Fernández C, Bilinski M, Fabbro M, Galain M, Menazzi S, Miguens M, Perassi PN, Fulco MF, Kopelman S et al (2020) First Custom Next-Generation Sequencing Infertility Panel in Latin America: Design and First Results. JBRA Assist Reprod. 10.5935/1518-0557.20190065 Kalfa N, Philibert P, Werner R, Audran F, Bashamboo A, Lehors H, Haddad M, Guys JM, Reynaud R, Alessandrini P et al (2013) Minor Hypospadias: The Tip of the Iceberg of the Partial Androgen Insensitivity Syndrome. PLoS ONE 8:e61824. 10.1371/journal.pone.0061824 Jankowska KK, Kutkowska-Kazmierczak A, Rygiel AM Hypogonadism — When Does Genetic Diagnosis Help in Therapy? Ginekol Pol 2023, VM/OJS/J/97327, 10.5603/gpl.97327 Cite Share Download PDF Status: Published Journal Publication published 15 Jan, 2025 Read the published version in Journal of Applied Genetics → Version 1 posted Editorial decision: Major Revisions Needed 08 Sep, 2024 Reviewers agreed at journal 20 Jul, 2024 Reviewers invited by journal 13 Jul, 2024 Editor assigned by journal 11 Jul, 2024 First submitted to journal 09 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Rygiel","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-3838-2900","institution":"Institute of Mother and Child","correspondingAuthor":true,"prefix":"","firstName":"Agnieszka","middleName":"Magdalena","lastName":"Rygiel","suffix":""}],"badges":[],"createdAt":"2024-07-09 12:04:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4711926/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4711926/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13353-024-00935-3","type":"published","date":"2025-01-15T15:56:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":74284447,"identity":"3672d98a-b549-4022-8e43-335d04f7fc53","added_by":"auto","created_at":"2025-01-20 16:05:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":824482,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4711926/v1/2c6436b0-857e-433e-9889-d589bcf5fb93.pdf"}],"financialInterests":"","formattedTitle":"Novel and recurrent genetic variants associated with male and female infertility","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInfertility, defined (according to WHO) as the failure of reproduction after one year of regular intercourse without the use of contraception, is one of the major global health and social problems that affects 10\u0026ndash;15% of couples of reproductive age (in Poland: 20% of couples, 1.5\u0026nbsp;million) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is estimated that in 30\u0026ndash;50% of cases, the causes of infertility are identified in women, 20\u0026ndash;30% in men, while the problem affect both partners in approximately 20\u0026ndash;30% of the couples [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Infertility is a multifactorial disease that can be caused by a number of reasons, including: abnormal gametogenesis, disorders of the structure and function of the reproductive system systemic diseases, including immunological disorders, infections, environmental factors, mental and genetic factors. Idiopathic infertility affects approximately 20\u0026ndash;40% of couples. It is estimated that genetic disorders are the cause of approximately 50% of them, although the exact role of genetic factors in infertility is still unknown [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn both men and women, standard genetic tests towards infertility include karyotype assessment and, in the case of men with azoospermia/oligozoospermia, also analysis of the deletion of the AZF region of the Y chromosome and examination of pathogenic variants in \u003cem\u003eCFTR\u003c/em\u003e gene [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Deletions of the AZF region of the Y chromosome are identified in 5\u0026ndash;10% of men with azoospermia/oligozoospermia, while pathogenic variants of the \u003cem\u003eCFTR\u003c/em\u003e gene in 6% of all infertile men and 60\u0026ndash;70% of men with CBAVD [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, according to literature data, about 30 genes are known to be associated with abnormal semen parameters in men (i.e. azoospermia, oligozoospermia, asthenozoospermia, or teratozoospermia) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This number will certainly increase in the future, taking into account that approximately 2,000 genes are involved in the process of spermatogenesis, 600\u0026ndash;900 of which are expressed in male sex cells exclusively [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAbnormal human spermatogenesis can be also caused by defects in androgen action and androgen insensitivity. Androgen Insensitivity Syndrome (AIS) is a disorder of male sexual development resulting in a wide range of clinical phenotypes and typically classified into three main types based on the degree of androgen receptor dysfunction and the resulting phenotypic characteristics. These types include complete androgen insensitivity syndrome (CAIS), partial androgen insensitivity syndrome (PAIS), and mild or minimal androgen insensitivity syndrome (MAIS). A variety of pathogenic variants have been described in the human androgen receptor gene (\u003cem\u003eAR\u003c/em\u003e) associated with male infertility. Due to these molecular defects the androgen receptor becomes less responsive or completely unresponsive to androgens affecting sexual development and spermatogenesis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe infertility can be also associated with endocrine disorders such as hypogonadism, in which the gonads do not function properly and produce inadequate amount of sex hormones There are two main categories of hypogonadism: hypogonadotropic and hypergonadotropic. Hypogonadotropic hypogonadism is characterized by low levels of sex hormones in males or females due to insufficient stimulation of the gonads by the pituitary gland and hypothalamus. Genetic causes of hypogonadotropic hypogonadism can result from mutations in genes involved in the development and functioning of these regulatory systems. Congenital hypogonadotropic hypogonadism (CHH) is a rare disorder (occurs in 1\u0026ndash;10/100,000 infertile men) caused by the failure of the normal episodic gonadotropin-releasing hormone (GnRH) secretion, leading to delayed puberty and infertility [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Most of the genes mutated in CHH encode receptor-ligand pairs including \u003cem\u003eGNRHR\u003c/em\u003e (gonadotropin-releasing hormone receptor) and \u003cem\u003eGNRH1\u003c/em\u003e (gonadotropin-releasing hormone 1), \u003cem\u003ePROKR2 (\u003c/em\u003eProkineticin receptor2) and \u003cem\u003ePROK2\u003c/em\u003e (Prokineticin 2), \u003cem\u003eKISS1R\u003c/em\u003e (KiSS1 receptor) and \u003cem\u003eKISS1, TACR3\u003c/em\u003e (Tachykinin Receptor 3) and \u003cem\u003eTAC3\u003c/em\u003e (Tachykinin 3), \u003cem\u003eFGFR1\u003c/em\u003e (Fibroblast Growth Factor Receptor 1) and \u003cem\u003eFGF8\u003c/em\u003e (Fibroblast Growth Factor 8) suggesting that multiple receptor-ligand-encoding gene networks are involved in the molecular pathology of the disease. Part of the cases with CHH demonstrate an impairment sense of smell, called also Kallmann syndrome, in which pathogenic variants in genes such as \u003cem\u003eANOS1\u003c/em\u003e (\u003cem\u003eKAL1\u003c/em\u003e) (Anosmin 1), \u003cem\u003eHS6ST1\u003c/em\u003e (Heparan Sulfate 6-O-Sulfotransferase 1), \u003cem\u003eSPRY4\u003c/em\u003e (Sprouty RTK Signaling Antagonist 4), \u003cem\u003eDUSP6\u003c/em\u003e (Dual Specificity Phosphatase 6) are also identified [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The CHH is one of the few treatable causes of male infertility. Diagnosis of CHH is, however, challenging, especially in early adolescence where the clinical features mimics that of constitutional delay of growth and puberty. Timely diagnosis is essential for a proper treatment. Pathogenic variants, in more than 30 genes, has been associated with CHH, acting either alone or in combination [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHypergonadotropic hypogonadism, also known as primary hypogonadism, is characterized by low levels of sex hormones accompanied by elevated levels of gonadotropins (FSH and LH). This condition can result from various genetic defects that directly affect the gonads. Some of the main genetic causes of hypergonadotropic hypogonadism involve mutations in genes such as \u003cem\u003eFSHR\u003c/em\u003e (Follicle-Stimulating Hormone Receptor), \u003cem\u003eLHR\u003c/em\u003e (Luteinizing Hormone Receptor), \u003cem\u003eINSL3\u003c/em\u003e (Insulin-Like 3), \u003cem\u003eNR5A1\u003c/em\u003e (Nuclear Receptor Subfamily 5 Group A Member 1) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIdentification of the causes of infertility is of paramount importance for each couple, due to several of the following reasons: the choice of therapy, genetic counselling, including determination of the risk of having offspring with cystic fibrosis (patients with CBAVD) or with deletion of the AZF region of the chromosome Y, improving mental comfort related to regaining hope for further effective treatment. Sometimes it may help to make decisions such as discontinue therapy and choosing alternative options (use of donor sperm, adoption, etc.).​\u003c/p\u003e \u003cp\u003eIn recent years, thanks to the use of next generation sequencing (NGS), more and more genes have been described in the context of fertility disorders. However, still in only 4% of all infertile men the genetic cause is established, and majority of the cases are classified as unexplained. This is partly due to a delay to adopt NGS techniques in the field of diagnostics and the lack of genotype\u0026ndash;phenotype correlations data [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, the objective of the study was to identify genetic variants associated with isolated infertility using NGS technique. Secondary aim was to describe genotype-phenotype correlations.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eThe patients with fertility problems or delayed puberty were referred for genetic testing from Department of Endocrinology, Bielanski Hospital, Genetic counselling unit of the Department of Medical Genetics, Institute of Mother and Child, Oviclinic Infertility Center and nOvum Fertility Clinic in Warsaw. Women with endometriosis and PCOS were excluded. Phenotypical information was based on clinical interview, physical and laboratory examinations. All patients had karyotype analysis preformed before they were subjected to molecular analysis. All patients gave their informed consent for the genetic testing. The study was also approved by bioethical committee of the Institute of Mother and Child, Nr 22/2019.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eGenetic analysis\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eAZF microdeletions analyses\u003c/h2\u003e \u003cp\u003eTo exclude AZF microdeletions on the chromosome Y, all males were subjected to analysis of 6 loci in the AZF by PCR. The panel of analyzed loci, including AZF c (sY254, sY255), AZF b(sY134, sY127), AZF a(sY86,S84), is consistent with the recommendations of the European Molecular Quality Network: \"EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions: state-of-the-art 2013\u0026rdquo;.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eNGS analysis\u003c/h2\u003e \u003cp\u003eGenetic tests were performed by targeted next generation sequencing. The NGS panel included 35 genes with well-established association with hypogonadotropic hypogonadism (\u003cem\u003eANOS1, CHD7, DUSP6, FEZF1, FGF17, FGF8, FGFR1, FLRT3,FSHB, GNRH1, GNRHR, IL17RD, KISS1, KISS1R, LHB, NSMF, PROK2, PROKR2, SEMA3A, SOX10,SPRY4, WDR11, HS6ST1, TAC3\u003c/em\u003e) hypergonadotropic hypogonadism (\u003cem\u003eNR5A1\u003c/em\u003e, \u003cem\u003eINSL3\u003c/em\u003e,, \u003cem\u003eLHCGR, LEP, GATA4, FSHR\u003c/em\u003e), spermatogenesis disorders (\u003cem\u003eSPATA16, CFTR,)\u003c/em\u003e, androgen insensitivity (AR), ovarian dysgenesis (\u003cem\u003eBMP15\u003c/em\u003e) and sex reversal (\u003cem\u003eSRY\u003c/em\u003e). Genes relevant to these phenotypes where included bases on OMIM and Human Phenotype Ontology (HPO) databases.\u003c/p\u003e \u003cp\u003eThe DNA library was prepared using the SureSelect kit from Agilent. Sequencing was preformed using The NextSeq (Illumina). Only the coding fragments of genes (exons) and exon-intron junctions +/- 15 nucleotides were analyzed. The sequences were mapped to the human reference genome version GRCh38/hg38. The results were interpreted using internal bioinformatics tools based on the VEP program (Ensembl), and in relation to the following databases: GnomAD v3, HGMD Professional, NCBI, Ensembl, OMIM, internal database of IMiD variants. The variants classification was preformed according to ACMG (American College of Medical Genetics) recommendations using Varsome or Franklin platform. The presence of the detected genetic variants was confirmed by Sanger sequencing (the primers available on the request). The detected variants were named according to HGVS nomenclature and canonical transcript (NCBI RefSeq).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eThe study group consisted of 41 patients with following clinical diagnosis: hypogonadism hypogonadotropic (n\u0026thinsp;=\u0026thinsp;12), hypogonadism hypergonadotropic (n\u0026thinsp;=\u0026thinsp;15), abnormal semen parameters (oligozoospermia, astenozoospermia, teratozoospermia or azoospermia) (n\u0026thinsp;=\u0026thinsp;10), androgen insensitivity (n\u0026thinsp;=\u0026thinsp;3), gonadal dysgenesis (n\u0026thinsp;=\u0026thinsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGenetic results\u003c/h2\u003e \u003cp\u003eNo deletion in the AZF a,b,c regions of the Y chromosome was detected in the examined males. One of 41 patients showed karyotype abnormality. This was a patient with typical female external genitalia, however, the structures resembling testes were found during inguinal hernias surgery. The karyotype analysis revealed presence of XY indicating sex reversal in this patient. The rest of patient had normal karyotype results.\u003c/p\u003e \u003cp\u003eThe NGS gene panel analysis revealed 18 unique, missense or nonsense variants in 16/42 cases, including 11 known pathogenic (P) or likely pathogenic (LP) variants described in clinical databases (HGMD/ClinVar) and 4 novel or very rare variants (classified as LP or VUS according to ACMG recommendation), not reported in clinical databases and literature (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenotype- phenotype correlation in patients with infertility problems or delayed puberty.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"19\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c17\" colnum=\"17\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c18\" colnum=\"18\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c19\" colnum=\"19\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003ePatient Nr\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eInheritance OMIM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eVariant\u003c/p\u003e \u003cp\u003ec.DNA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eVariant\u003c/p\u003e \u003cp\u003eprotein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003eZyg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eMAF gnomAD v.4.00\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e \u003cp\u003eHGMD\u003c/p\u003e \u003cp\u003e/ClinVar\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e \u003cp\u003eACMG \u0026amp;\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c17\" namest=\"c16\"\u003e \u003cp\u003eClinical\u003c/p\u003e \u003cp\u003ediagnosis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c19\" namest=\"c18\"\u003e \u003cp\u003eOther clinical features\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eSPATA16\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ec.848G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep.Arg283Gln\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003ehet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e0.0004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e \u003cp\u003eDM/P (Globozoospermia)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c16\" namest=\"c15\"\u003e \u003cp\u003eLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c18\" namest=\"c17\" rowspan=\"2\"\u003e \u003cp\u003enHH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOAT, bilateral cryptorchidism, 6 toes on one foot\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eFGF8\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAD, Oligo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ec.77C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep.Pro26Leu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003ehet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e0.00115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eDM/P (idiopathic HH)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003eLP\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eDUSP6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ec.566A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep.Asn189Ser\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003ehet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e0.00104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eDM/P (Kallmann syndrome)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003eLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003enHH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003ecryptozoospermiahypopituitarism (empty sella syndrome)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eSPRY4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAD,\u003c/p\u003e \u003cp\u003eOligo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ec.530A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep.Lys177Arg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003ehet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e0.00227\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eDM /\u003c/p\u003e \u003cp\u003eConflicting (Kallmann syndrome )\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003eVUS/LP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003enHH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003eOAT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAR\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eXLR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ec.2395C\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep.Gln799Glu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003ehemi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e0.0015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eDM/Confliciting (AIS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003ePAIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003etesticular hypoplasia, azooseprmia, decreased testosterone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003cp\u003e(k.XY)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eAR\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eXLR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ec.2287C\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep.Leu763Val\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003ehemi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eDM (AIS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003eCAIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eGNRHR\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAR, Oligo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ec.416G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep.Arg139His\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003ehet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e0.00015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eDM/P (HH)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003enHH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eNR5A1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ec.776C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep.Pro259Leu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003ehet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e0.00005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eDM?( XY gonadal dysgenesis)/nd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003eGonadal dysgenesis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003eepididymal cysts, microcalcifications, varicocele\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eDUSP6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ec.215C\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep.Pro72Gln\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003ehet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e0.00013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003end/VUS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003eVUS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003enHH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003etetratozoospermia (0% of normal sperm)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eHS6ST1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ec.1132C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep.Arg378Cys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003ehet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e0.00003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003end/VUS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003eVUS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003eprimary\u003c/p\u003e \u003cp\u003ehypogonadism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003esevere oligozoospermia, very high level of FSH\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eFGFR1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ec.1270C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep.Arg424Cys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003ehet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e0.00001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003end/VUS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003eVUS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003enHH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eFGFR1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAD, Oligo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ec.246_247del\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep.Glu84GlyfsTer26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003ehet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eDM/ P (GnRH deficiency)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003enHH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eFGFR1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAD, Oligo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ec.863A\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep.Gln288Leu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003ehet\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003end/VUS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003eLP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003eKallman syndrome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003eazoospermia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003eM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eFGF8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eAD, Oligo\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cb\u003ec.439G\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003ep.Ala147Thr\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e\u003cb\u003ehet\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e\u003cb\u003e0.00001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cb\u003end/nd\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003e\u003cb\u003eLP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003e\u003cb\u003eKallman syndrome\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e\u003cb\u003eazoospermia\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003eM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eSEMA3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eAD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cb\u003ec.1630C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003ep.Arg544Cys\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e\u003cb\u003ehet\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e\u003cb\u003e0.000001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cb\u003end/nd\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003e\u003cb\u003eLP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003e\u003cb\u003enHH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e\u003cb\u003etetrazoospermia\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003eF\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eFGFR1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eAD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cb\u003ec.420_423del\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003ep.Thr141IlefsTer10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e\u003cb\u003ehet\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cb\u003end/nd\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003e\u003cb\u003eLP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003e\u003cb\u003enHH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e\u003cb\u003edelayed puberty, primary amenorrhea\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cb\u003eM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eNSMF\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eAD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cb\u003ec.725A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003ep.Tyr242Cys\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e\u003cb\u003ehet\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003e\u003cb\u003e0.000001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cb\u003end/nd\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c16\" namest=\"c14\"\u003e \u003cp\u003e\u003cb\u003eVUS\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(PM2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c18\" namest=\"c17\"\u003e \u003cp\u003e\u003cb\u003eazoospermia\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c19\"\u003e \u003cp\u003e\u003cb\u003ehigh level of FSH but normal level of testosterone\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"19\"\u003e\u003cb\u003eM\u003c/b\u003e - male; \u003cb\u003eF\u003c/b\u003e- female; \u003cb\u003eAD\u003c/b\u003e - autosomal dominant inheritance; \u003cb\u003eAR\u003c/b\u003e - autosomal recessive inheritance; \u003cb\u003eOligo\u003c/b\u003e - oligogenic inheritance; \u003cb\u003end\u003c/b\u003e - not described; \u003cb\u003eZyg\u003c/b\u003e -zygosity, \u003cb\u003eMAF\u003c/b\u003e- allele minor frequency, \u003cb\u003eDM\u003c/b\u003e- damaging, \u003cb\u003eP\u003c/b\u003e-pathogenic, \u003cb\u003eLP\u003c/b\u003e- likely pathogenic, \u003cb\u003eVUS\u003c/b\u003e- variant of uncertain significance, \u003cb\u003ePAIS\u003c/b\u003e - partial androgen insensitivity syndrome; AIS- androgen insensitivity, \u003cb\u003eCAIS\u003c/b\u003e - complete androgen insensitivity syndrome; \u003cb\u003eHH\u003c/b\u003e hypogonadotropic hypogonadism, \u003cb\u003enHH\u003c/b\u003e - normosomic hypogonadotropic hypogonadism; \u003cb\u003eOAT\u003c/b\u003e- oligoasthenoteratozoospermia. \u003cb\u003ek\u003c/b\u003e.-karyotype. \u003cb\u003e\u0026amp;\u003c/b\u003e - ACMG classification according to .Brandt et al.2020, Genet Med. \u003cb\u003eBolded\u003c/b\u003e are novel variants (not found in clinical databases nor literature and absent/ very rare in gnomAD)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe genotype-phenotype correlation is described in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In patients with hypogonadotropic hypogonadism (HH), we have identified P/LP and VUS variants in 58% (7/12) and 25% (3/12) of the cases, respectively. One of these cases, diagnosed with Kallmann syndrome, harboured a very rare, heterozygous, LP variants in two different genes: p.Gln288Leu in \u003cem\u003eFGFR1\u003c/em\u003e and p.Ala147Thr in \u003cem\u003eFGF8\u003c/em\u003e. In two of the CHH cases without anosmia, we found novel p.Thr141IlefsTer10 (LP) variant and a very rare, heterozygous VUS, p.Arg424Cys in \u003cem\u003eFGFR1\u003c/em\u003e gene.\u003c/p\u003e \u003cp\u003eIn 1/15 cases with hypergonadotropic hypogonadism, we found one very rare VUS variant in \u003cem\u003eHS6ST1\u003c/em\u003e gene. In 2/3 of the cases with androgen insensitivity, we have detected a very rare, hemizygous, pathogenic variants in \u003cem\u003eAR\u003c/em\u003e gene including p.Leu763Val identified in a patient with sex reversal (female phenotype but XY karyotype) and p.Leu763Val variant found in a male patient with partial androgen insensitivity (PAIS). One patient with gonadal dysgenesis harbored p.Arg139His, known pathogenic variant in one allele of the \u003cem\u003eGNRHR\u003c/em\u003e gene. In 1/10 cases with abnormal semen parameters, we detected a very rare p.Tyr242Cys variant in \u003cem\u003eNSMF\u003c/em\u003e gene classified as LP.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we have found 11 known, pathogenic/likely pathogenic variants (P/PL), 3 novel, likely pathogenic variants (LP) and 4 variants of uncertain significance (including one novel) in the group of patients with infertility or delayed puberty. The genetic testing enabled identification of the cause of the infertility or delayed puberty in 26% of the patients. We have examined patients with different clinical features such as hypogonadotropic and hypergonadotropic hypogonadism, abnormal semen parameters, androgen insensitivity and gonadal dysgenesis. We have described observed genotype-phenotype correlations.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHypogonadism\u003c/h2\u003e \u003cp\u003eGenetic studies on congenital hypogonadotropic hypogonadism (CHH) have led to discovery of pathogenic variants in various genes implicated in specification and proliferation of GnRH neurons, their migration to the hypothalamus during embryonic development and the response of pituitary gonadotropes to GnRH stimulation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The genetic complexity of the disease is reflected by different models of inheritance: X-linked, autosomal dominant and autosomal recessive as well as incomplete penetrance and variable phenotypic expression as well as oligogenic inheritance pattern in the certain cases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Using our NGS panel, we identified P/LP in 58% (7/12) of the CHH (including one case of Kallmann syndrome) patients which is higher than in previous reports showing around 31\u0026ndash;35% of the pathogenic variants detection rate [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Our results are consistent with the study by Cassatella D et al., which detected 51% of pathogenic variants in CHH patients using similar gene panel [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMultiple genes encoding components of the FGF pathway are known to be mutated in CHH and IHH eg. \u003cem\u003eFGFR1\u003c/em\u003e, \u003cem\u003eFGF8\u003c/em\u003e,, \u003cem\u003eHS6ST1 DUSP6\u003c/em\u003e and \u003cem\u003eSPRY4\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In our CHH patients, \u003cem\u003eFGFR1\u003c/em\u003e and \u003cem\u003eFGF8\u003c/em\u003e were the most frequently mutated genes with the same frequency of 33% (4/12), each. These results are consistent with previous studies showing very important role of the two genes in the development of CHH [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eFGFR1\u003c/em\u003e, we have detected one novel p.Thr141IlefsTer10 (LP) variant, one known pathogenic variant p.Glu84GlyfsTer26 and two very rare variants including p.Gln288Leu (LP) and p.Arg424Cys (VUS). The novel p.Thr141IlefsTer10 was found in a girl with hypogonadotropic hypogonadism, delayed puberty and primary amenorrhea. The p.Arg424Cys variant (VUS) was harboured by a girl with HH.Importantly, different amino acid substitution, Arg to His (p.Arg424His, c.1271G\u0026thinsp;\u0026gt;\u0026thinsp;A), was described in the same codon in a patient with HH (HGMD no.: CM2115069) suggesting that substations in this codon may be likely disruptive.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eFGF8\u003c/em\u003e, we found 2 variants including 1 know pathogenic p.Pro26Leu and 1 novel, LP p.Ala147Thr variant. Previously, Falardeau et al. described pathogenic variants in \u003cem\u003eFGF8\u003c/em\u003e, including p.Pro26Leu, in IHH probands with variable olfactory phenotypes. These patients exhibited varied degrees of GnRH deficiency, including the rare adult-onset form of hypogonadotropic hypogonadism [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePathogenic variants in \u003cem\u003eDUSP6\u003c/em\u003e (OMIM #615269) and \u003cem\u003eSPRY4\u003c/em\u003e (OMIM#615266) gene cause CHH with normal smell or anosmia and both are inherited in autosomal dominant manner. In \u003cem\u003eDUSP6\u003c/em\u003e gene, we detected variants in 2 of the 12 cases with HH including a known pathogenic variant p.Asn189Ser and p.Pro72Gln classified as VUS. The p.Asn189Ser variant was previously reported in case with Kallman syndrome [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Our patient showed a normal sense of smell but had hypopituitarism (empty sella syndrome) and cryptozoospermia. The other case with p.Pro72Gln (VUS) had teterathozoospermia and HH. Pheotypic variability is described both in a group of unrelated patients and within members of one family with the same pathogenic variant in \u003cem\u003eDUSP6\u003c/em\u003e gene. In some patients, variable phenotypic expression, maybe related to the simultaneous occurrence of pathogenic variants in other genes associated with hypogonadotropic hypogonadism [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. We also have identified known pathogenic variant p.Lys177Arg in \u003cem\u003eSPRY\u003c/em\u003e gene in one patient with hypogonadism and oligoasthenospermia. Previously, Miraoui et al. identified heterozygous missense variants in the \u003cem\u003eSPRY4\u003c/em\u003e gene in 14 unrelated individuals with congenital CHH), including p.Lys177Arg variant which was detected in patients with Kallmann syndrome [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genetic complexity and phenotypic variability of CHH is exemplified by oligogenesity and involvement of pleotropic genes implicated in the disease. In our study, oligogenic interactions defined as \u0026ge;\u0026thinsp;2 genes mutated were found in 2 out of 12 CHH cases. In one patient with Kallman syndrome, we detected simultaneous occurrence of p.Gln288Leu in \u003cem\u003eFGFR1\u003c/em\u003e and a novel Ala147Thr variant in \u003cem\u003eFGF8\u003c/em\u003e. The pathogenic variants in \u003cem\u003eFGFR1\u003c/em\u003e and \u003cem\u003eFGF8\u003c/em\u003e genes are inherited in autosomal dominant manner, however, oligogenic interactions has been also reported [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. For instance, study by Sykiotis GP \u003cem\u003eet al\u003c/em\u003e, investigating a large cohort of 397 patients with idiopathic HH, found that 2.5% of the patients show oligogenic interactions including genes such as \u003cem\u003eFGFR1\u003c/em\u003e and \u003cem\u003eFGF8\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The other digenic interaction was observed in a male patients with hypogonadotropic hypogonadism, oligoasthenoteratozoospermia (OAT) and bilateral cryptorchidism. This patient harbored known pathogenic variants such as p.Pro26Leu and p.Arg283Gln in \u003cem\u003eFGF8\u003c/em\u003e and \u003cem\u003eSPATA16\u003c/em\u003e gene, respectively. Pathogenic variants in \u003cem\u003eFGF8\u003c/em\u003e gene are associated with hypogonadotropic hypogonadism with or without ansomia. The literature describes patients with the p.Pro26Leu variant in \u003cem\u003eFGF8\u003c/em\u003e who were diagnosed with Kalman syndrome with partial loss of smell (hyposomia) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The phenotype is, however, variable even within members of one family with the same variant [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In our patient, no anosmia or hyposomia was present. Interestingly, this patient showed foot malformation (six toes) confirming a pleotropic role of the \u003cem\u003eFGF8\u003c/em\u003e gene [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Interestingly, \u003cem\u003eFGFR1\u003c/em\u003e pathogenic variants occur in 87% of patients with both CHH and split hand/food malformation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Our patient also harbored heterozygous p.Arg283Gln variant in one allele of \u003cem\u003eSPATA16\u003c/em\u003e, known to disturb splicing [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The pathogenic variants \u003cem\u003eSPATA16\u003c/em\u003e are the cause of defects in spermatogenesis (including abnormal sperm morphology) inherited in an autosomal recessive manner. In this case, however, no variants in the second allele of \u003cem\u003eSPATA16\u003c/em\u003e gene was identified.\u003c/p\u003e \u003cp\u003e \u003cem\u003eNSMF\u003c/em\u003e encodes protein involved in guidance of olfactory axon projections and migration of luteinizing hormone-releasing hormone neurons. Defects in this gene are a cause of idiopathic hypogonadotropic hypogonadism (IHH) inherited in autosomal dominant manner (OMIM#614838). We have identified two variants in \u003cem\u003eNSMF\u003c/em\u003e gene including a rare, LP, p.Pro213Leu and a novel p.Tyr242Cys (VUS) variant. The p.Pro213Leu variant, was present in one patient with hypogonadotropic hypogonadism and p.Tyr242Cys was found in a patient with azoospermia and high level of FSH but normal level of testosterone (not fulfilling criteria of hypogonadism).\u003c/p\u003e \u003cp\u003eIn our study, we studied 15 cases with hypergonadotropic hypogonadism (primary hypogonadyzm) but no P/LP was detected in this group using our NGS panel. We have detected one VUS - p.Arg378Cys variant - in \u003cem\u003eHS6ST1\u003c/em\u003e gene in a patient with hypergonadotropic hypogonadism and oligozoospermia. The low frequency of detected variants in this group of patients could be due to limited number genes associated with primary hypogonadism included in our panel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAndrogen Insensitivity\u003c/h2\u003e \u003cp\u003eThe androgen insensitivity syndrome (AIS) is a disorder of male sexual development resulting in a wide range of clinical phenotypes. It affects-XY males but phenotypically is characterized by a female phenotype. AIS is classified into two phenotypic forms: complete (CAIS) and partial (PAIS). We have detected a very rare hemizygous variant p.Leu763Val in \u003cem\u003eAR\u003c/em\u003e gene in patient with XY karyotype, female phenotype and complete androgen insensitivity syndrome (CAIS). This patient with typical female external genitalia was referred to the genetic counselling unit because during inguinal hernias surgery, structures resembling testes were found. Ultrasound examination demonstrated presence of two structures corresponded to testes localized along iliac blood vessels in absence of m\u0026uuml;llerian structures as fallopian tubes and uterus and absence of extragenital abnormalities apart from 3 cafe-au-lait spots. Very recently the p.Leu763Val variant has been described in an Iranian family with two female patients with normal external genitalia and 46, XY karyotype and CAIS phenotype [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thus, we have described the second case in the world with the p.Leu763Val variant in \u003cem\u003eAR\u003c/em\u003e gene. A pathogenic variant in the same 763 codon leading to Phe amino acid substitution (p.Leu763Phe and causing the loss of androgen binding receptor function was described as well [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We also identified a known pathogenic p.Gln799Glu variant in \u003cem\u003eAR\u003c/em\u003e gene in one patient with PAIS and clinical features such as hypogonadotropic hypogonadism, testicular hypoplasia and azoospermia. In the literature, the p.Gln799Glu variant was described in patients partial androgen insensitivity, hypospadia and oligoasthenoteratozoospermia [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, we have identified several new and recurrent pathogenic/likely pathogenic genetic variants associated with hypogonadism, androgen insensitivity and described observed genotype-phenotype correlations what expands the knowledge of the genetic basis of these disorders. Importantly, our results are in line with other studies. They provide further evidence for the heterogeneity of infertility disorders, especially in CHH, indicating that multigene panels are the best method of choice for genetic analysis in this group of patients. Further, this study highlights the importance of genetic testing for proper diagnosis making. Genetic diagnostics may also be very helpful in treating causes of infertility such as hypogonadism but requires the assistance of a clinician endocrinologist or andrologist, as well as a geneticist [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Moreover, the possibility of getting genetic counselling is also of high importance for the couple with infertility problems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: We are grateful to all patients who participated in this study. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Conceptualization, AMR and KKJ; Methodology (Molecular Analysis): KŚ, AD, KD, JS; \u0026nbsp;Formal analysis: AMR, KŚ, AD, KD, JS, KWT(molecular analysis), KKJ., AKK, JKW, JK, PL (clinical analysis); Writing \u0026ndash; Original Draft Preparation: AMR.; Writing - Review \u0026amp; Editing KKJ, AKK, JKW, PL, KWT. Visualization, KŚ.; Supervision: AMR.; Funding Acquisition, AMR.\u0026rdquo;, All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study complies with the latest Declaration of Helsinki and was approved by bioethical committee of the Institute of Mother and Child, Warsaw, Nr 22/2019.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent \u0026nbsp; to participate was obtained from all subjects involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for \u0026nbsp;publication:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for \u0026nbsp;publication was received from all the patients enrolled in the study.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Ministry of Science and Higher Education, Poland , project nr 510-18-61 granted to Institute of Mother and Child ( A.M. Rygiel)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZegers-Hochschild F, Adamson GD, de Mouzon J, Ishihara O, Mansour R, Nygren K, Sullivan E, Vanderpoel S, International Committee for Monitoring Assisted Reproductive Technology; World Health Organization International Committee for Monitoring Assisted Reproductive (2009) Technology (ICMART) and the World Health Organization (WHO) Revised Glossary of ART Terminology, \u003cem\u003eFertil Steril\u003c/em\u003e 2009, \u003cem\u003e92\u003c/em\u003e, 1520\u0026ndash;1524, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.fertnstert.2009.09.009\u003c/span\u003e\u003cspan address=\"10.1016/j.fertnstert.2009.09.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuczyński W, Kurzawa R, Oszukowski P, Pawelczyk L, Poreba R, Radowicki S, Szamatowicz M, Wołczyński S (2012) Polish Gynecological Society and Polish Society for Reproductive Medicine [Polish Gynecological Society and Polish Society for Reproductive Medicine recommendations for the diagnosis and treatment of infertility]. 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PLoS ONE 8:e61824. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0061824\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0061824\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJankowska KK, Kutkowska-Kazmierczak A, Rygiel AM Hypogonadism \u0026mdash; When Does Genetic Diagnosis Help in Therapy? \u003cem\u003eGinekol Pol\u003c/em\u003e 2023, VM/OJS/J/97327, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.5603/gpl.97327\u003c/span\u003e\u003cspan address=\"10.5603/gpl.97327\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joag","sideBox":"Learn more about [Journal of Applied Genetics](https://www.springer.com/journal/13353)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/joag/default.aspx","title":"Journal of Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"human infertility, genetic variants, genetic diagnostics","lastPublishedDoi":"10.21203/rs.3.rs-4711926/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4711926/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRecently, the knowledge of the genetic basis of fertility disorders has expanded enormously, mainly thanks to the use of next generation sequencing (NGS). However, the genetic cause of the infertility in majority patients is still undefined.\u003c/p\u003e \u003cp\u003eAim: The aim was to identify genetic variants associated with infertility disorders using targeting NGS technique and to describe phenotype - genotype correlation.\u003c/p\u003e \u003cp\u003eMethods: We have enrolled 41 patients (36 males and 5 females) with infertility problems or delayed puberty including the patients with hypogonadism hypogonadotropic (HH, n\u0026thinsp;=\u0026thinsp;12), primary hypogonadism (n\u0026thinsp;=\u0026thinsp;15), abnormal semen parameters or gonadal dysgenesis (n\u0026thinsp;=\u0026thinsp;11), androgen insensitivity (n\u0026thinsp;=\u0026thinsp;3). Genetic tests were performed using NGS panel of 35 genes.\u003c/p\u003e \u003cp\u003eResults: Overall, 14 pathogenic (P) or likely pathogenic (LP) variants including 3 novel and 11 recurrent variants were identified. Novel variants were found in genes associated with HH (\u003cem\u003eFGF8\u003c/em\u003e, \u003cem\u003eFGFR1, SEMA3\u003c/em\u003e). The genetic cause of the HH was determined in 58% (7/12) of the cases. Overall, The genetic testing enabled identification of the cause of the clinical phenotype in 26% (11/41) of the patients.\u003c/p\u003e \u003cp\u003eConclusions: Our study expands the knowledge of the genetic basis of the infertility disorders and highlights the importance of genetic testing for proper diagnosis making and genetic counselling.\u003c/p\u003e","manuscriptTitle":"Novel and recurrent genetic variants associated with male and female infertility","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-07 03:48:26","doi":"10.21203/rs.3.rs-4711926/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2024-09-08T17:28:56+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-07-20T07:41:33+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-13T20:49:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-12T00:23:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Applied Genetics","date":"2024-07-09T08:03:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joag","sideBox":"Learn more about [Journal of Applied Genetics](https://www.springer.com/journal/13353)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/joag/default.aspx","title":"Journal of Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4ae58af9-b555-4c6a-a1be-5ec7f7e3d65f","owner":[],"postedDate":"August 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-20T15:58:36+00:00","versionOfRecord":{"articleIdentity":"rs-4711926","link":"https://doi.org/10.1007/s13353-024-00935-3","journal":{"identity":"journal-of-applied-genetics","isVorOnly":false,"title":"Journal of Applied Genetics"},"publishedOn":"2025-01-15 15:56:56","publishedOnDateReadable":"January 15th, 2025"},"versionCreatedAt":"2024-08-07 03:48:26","video":"","vorDoi":"10.1007/s13353-024-00935-3","vorDoiUrl":"https://doi.org/10.1007/s13353-024-00935-3","workflowStages":[]},"version":"v1","identity":"rs-4711926","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4711926","identity":"rs-4711926","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}