Skip to main content
NCBI home page
UKPMC Funders Author Manuscripts logoLink to UKPMC Funders Author Manuscripts
. Author manuscript; available in PMC: 2023 Jul 26.
Published in final edited form as: Hum Fertil (Camb). 2021 Jun 30;25(5):939–946. doi: 10.1080/14647273.2021.1946173

Screening by single-molecule Molecular Inversion Probes targeted sequencing panel of candidate genes of infertility in azoospermic infertile Jordanian males

Osamah Batiha 1,*,#, George J Burghel 2,#, Ayesha Alkofahi 1, Emad Alsharu 3, Hannah Smith 4, Bilal Alobaidi 4, Mohammad Al-Smadi 3, Nour Awamlah 5, Lama Hussein 5, Amid Abdelnour 5, Harsh Sheth 4,6, Joris A Veltman 4
PMCID: PMC7614817  EMSID: EMS181577  PMID: 34190021

Abstract

Infertility is a common health problem that affects around 1 in 6 couples in the United States, where half of these cases are attributed to male factors. Genetics play an important role in infertility and it is estimated that up to 50% of cases are due to genetic factors. Despite this, many male infertility cases are still idiopathic. This study aimed to identify the presence of possibly pathogenic rare variants in a set of candidate genes related to azoospermia in a Jordanian cohort composed of 69 cases using a next-generation sequencing-based panel covering more than a hundred male infertility related genes. A total of 9 variants were found and validated. Among them, two variants included reported pathogenic variants in CFTR and one novel pathogenic variant in the USP9Y gene. We also report the detection of 6 other variants with uncertain significance in other genes. Interestingly, male cases with CFTR variants did not show the expected cystic fibrosis phenotypes except for infertility. This work helps to uncover the contribution of additional genetic factors to the etiology of male infertility and highlights the importance to obtain more reliable information about the presence of genetic variation in the Jordanian population.

Keywords: Male infertility, Azoospermia, single molecule Molecular Inversion Probes, next generation sequencing, CFTR mutations

Introduction

Infertility is a worldwide problem, defined as a disease of the reproductive system, results in the failure to achieve a pregnancy after 12 months of regular unprotected sexual intercourse (World Health Organization, 2018). Fertility care is a reproductive right, health equity, and gender equality issue. Approximately, 1 in every six couples in the United States are infertile, and among them, male factor infertility accounts for approximately 50% of causes (Thoma et al., 2013; Zorrilla & Yatsenko, 2013). Despite the high burden, couples who desire but are unable to achieve and maintain a pregnancy, have needs that are not being addressed, especially in lower resource settings worldwide. Yet, the field of reproductive medicine and endocrinology is rapidly growing (Agarwal et al., 2015; World Health Organization, 2018).

Male infertility is a multifactorial disease encompassing a wide variety of disorders and can be initially diagnosed by semen fluid analysis (Poongothai et al., 2009). Genetic factors play a major role in idiopathic male infertility (Mazhar S. Al Zoubi et al., 2020; Mazhar Salim Al Zoubi et al., 2020; Plaseska-Karanfilska et al., 2012). The main genetic cause of male infertility is chromosomal abnormalities, which accounts for ~5% of infertile males, and the prevalence increases to 15% in the azoospermic males (Krausz & Riera-Escamilla, 2018; Zorrilla & Yatsenko, 2013). Men with non-obstructive azoospermia have a high prevalence of aneuploidy, particularly in their sex chromosomes. The second most common genetic cause of male infertility is Y chromosome microdeletions affecting the azoospermia factor (AZF) region (Batiha et al., 2012; Zorrilla & Yatsenko, 2013). Microdeletions in this region cause defects in spermatogenesis that lead to the development of azoospermia and oligozoospermia (Krausz & Riera-Escamilla, 2018). There are also male infertility cases caused by defects in single genes including CFTR, DDX3Y, SYCP3, TEX11, AURKC, and DPY19L2, but the number of genes confidently linked to male infertility remains very low (Oud et al., 2019). The poor genetic diagnosis makes a large proportion of infertile males falling in the "idiopathic infertility" category with no obvious reasons explaining their infertility problem.

Recent advances in molecular biology technologies such as next-generation sequencing (NGS) has enabled rapid and relatively cost-effective whole-exome and whole-genome sequencing. This, in turn, allowed rapid, sensitive, and efficient detection of the genetic etiologies of many diseases (Gilissen et al., 2011). The development of such technologies is promising to revolutionize the hunt for new genetic markers for many diseases, including male infertility.

Recently, we conducted a study on a group of azoospermic Jordanian infertile males and analyzed them for common mutations in the Y chromosome, in addition to the androgen receptor (AR) gene (Batiha et al., 2018). We detected microdeletions in the Y chromosome in only 5% of samples and found no significant difference of a common mutation in the AR gene (Batiha et al., 2018).

To further characterize and identify additional causative genetic variants we sequenced these cases using single molecule Molecular Inversion Probes (smMIPs) NGS-based panel covering more than a hundred male infertility-related genes (Oud et al., 2017).

Materials and methods

Patients

The cohort was described previously (Batiha et al., 2018). In summary, 142 unrelated, idiopathic, azoospermic Jordanian Arab males who were previously tested for AZF deletion/duplications and AR-CAG gene repeats were included. Samples with congenital bilateral absence of the vas deferens (CBAVD) was excluded when possible. Patients’ age ranges from 20 and 50 years, with an average of 32.4 ± 6 years. The Institutional Review Board (IRB) at Jordan University of Science and Technology and the ethical committee at King Hussein Medical Center approved the study. Participants were informed about the goal of the study, and signed a written consent form. Samples with Y-chromosome microdeletions, and samples with low DNA concentration and/or poor quality were excluded. The remaining 69 DNA samples were qualified for smMIP sequencing.

smMIP based targeted sequencing

smMIPs targeting 134 genes were previously described (Oud et al., 2017), with modifications to add recently published infertility genes (Oud et al., 2019). All 69 samples were sequenced in three runs on the Illumina NextSeq platform at an average 1200x depth per amplicon/sample. Genomic regions of interest were captured in a reaction containing a molecule ratio between patients’ gDNA and smMIP of 1:1000. The conditions of smMIP were: for denaturation of DNA ten minutes at 95 ° C, then incubation period for 23 hours, next all non-circular targets were amplified with primers containing barcoded reverse primers by the next PCR conditions: denaturation at 98° c for 30 sec, then 17 cycles of 10 sec at 98 °c, 30 sec at 60 °c, and 30 sec at 72 ° c and finally 2 min. at 72° c.

Bioinformatic analysis

Data was analyzed as previously described using an in-house smMIP-pipeline (Oud et al 2017). The files produced via the pipeline were run through a custom script in RStudio v3.5.1 (RStudio Team. (2015). RStudio: Integrated Development Environment for R. Boston, MA. Retrieved from http://www.rstudio.com/) which filtered for only variants which showed more than 10 reads at the potential variant loci; this was to ensure reliability in the results (Oud et al., 2017). These variants were then segregated into two separate files based on their inheritance pattern, those with 20% to 80% of all the reads at a given locus being mutated were sorted into the heterozygous file and those with > 80% were then classed as homozygous. These files were then passed through a custom Linux script. This filtered only variants with an allele frequency in the general population of less than 1% (ExAC, gnomAD, and 1000 genomes). The remaining variants were only kept when they occurred in exonic regions such as frameshifts, missense, and stop gained as well as splice site donor and acceptor region mutations.

Variant interpretation

The filtered variants were prioritized using Alamut® Visual V.2.11 (Interactive Biosoftware, Rouen, France) based on pathogenicity scores provided in the annotated files and additional information on the affected gene itself. The pathogenicity scores highlight how damaging the change in an amino acid is to the overall protein function (Sorts Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen)). The Combined Annotation Dependent Depletion, Phred scale (CADD Phred) score measures deleteriousness of the variant using the observed variant frequency as the basis for its calculation, the score ranges from 1 to 100 with scores over 10 being classed as being the 10% most deleterious substitutions and scores > 20 being in the top 1% (Rentzsch et al., 2019). All identified variants were also interpreted using the American College of Medical genetics (ACMG) recommendations (Richards et al., 2015).

Sanger sequencing

University of California, Santa Cruz (UCSC) genome browser, Primer 3, OligoCalc and UCSC in silico PCR tools were all used to design the primers. Sanger sequencing was used to validate all variants classified as pathogenic, likely pathogenic and of uncertain significance.

Results

DNA samples from 69 azoospermic infertile Jordanian males were analyzed using SmMIP targeted sequencing NGS panel for 134 known and candidate male infertility genes. After filtering for rare likely pathogenic variants, a total of 9 variants were prioritized. Three variants were classified as pathogenic and likely pathogenic (Table 1), and 6 variants were classified as variants of uncertain clinical significance (Table 2). All detected variants were confirmed by Sanger sequencing.

Table 1. Summary of the variants highlighted and validated as being Pathogenic or Likely pathogenic in 3 patients.

Sample ID Gene Variant Protein Change Consequence Zygosity SIFT PolyPhen CADD Phred ACMG-AMP classification Gnomad Population frequency ClinVar
J34 CFTR c.3909C>G p.(Asn1303Lys) Missense Homozygous 0 1.00 27.9 Class 5: Pathogenic - known CFTR mutation 1.39x10^-4 10 pathogenic entries
SI008 CFTR c.3454G>C p.(Asp1152His) Missense Homozygous 0 0.80 31.0 Class 4: Likely pathogenic 3.75x10^-4 15 path/LP entries on ClinVar
J93 USP9Y* c.6537T>A p.(Tyr2179Ter) Stop gained Hemizygous - - - Class 5: Pathogenic Absent Absent
Open in a new tab

Table 2. Summary of the 6 variants highlighted and validated as being Uncertain in significance in 5 patients.

Sample ID Gene name Variant Protein Change Consequence Zygosity SIFT PolyPhen CADD Phred ACMG-AMP classification Gnomad ClinVar
SI002 MCM8 c.997G>A p.(Gly333Arg) Missense Homozygous 0 0.99 31.0 Class 3: Uncertain significance Absent Absent
J53 KDM5D c.4402C>T p.(Arg1468Trp) Missense Hemizygous 0 0.80 - Class 3: Uncertain significance 2/57279 (South Asian) Absent
SI004 MAST2 c.3039delC p.(Thr1014GlnfsTer34) Frameshift deletion Heterozygous - - - Class 3: Uncertain significance Absent Absent
SI004 MEIOB c.1098delC p.(Leu367TrpfsTer13) Frameshift deletion Heterozygous - - - Class 3: Uncertain significance Absent Absent
SI008 UTP14C c.268C>T p.(Leu90Phe) Missense Heterozygous 0 0.99 24.9 Class 3: Uncertain significance Absent Absent
SI010 DNAH6 c.2837T>C p.(Leu946Pro) Missense Heterozygous 0 0.99 26.0 Class 3: Uncertain significance Absent Absent
Open in a new tab

We detected a homozygous c.3909C>G p.(Asn1303Lys) variant in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in one of the samples (J34). This is a previously reported pathogenic variant in CFTR (https://www.ncbi.nlm.nih.gov/clinvar/RCV000007556/). The patient is 34 years old and does not show any cystic fibrosis phenotype. Both endo-rectal and scrotal ultrasonography performed did not show any abnormality or congenital bilateral absence of vas deferens (CBAVD). We also detected a homozygous c.3454G>C p.(Asp1152His) variant in the CFTR gene in another sample (SI008). This is also a known and previously reported pathogenic variant in CFTR. The patient history did not indicate a cystic fibrosis phenotype, but we could not re-examine the patient to exclude CBAVD, however, it was a recruitment criterion. Additionally, a novel hemizygous c.6537T>A p.(Tyr2179Ter) likely pathogenic nonsense variant in the ubiquitin-specific protease 9 Y-linked (USP9Y) gene was detected in case J93.

Homozygous variants in the minichromosome maintenance 8 homologous recombination repair factor (MCM8) and the lysine-specific demethylase 5D (KDM5D) genes were detected but with uncertain pathogenicity. Both are missense mutations with a predicted p.(Gly333Arg) and p.(Arg1468Trp) amino acid substitutions, respectively. Four other heterozygous variants were detected in the microtubule-associated serine/threonine kinase 2 (MAST2), a meiosis-specific protein with OB domains (MEIOB), UTP14C small subunit processome component (UTP14C) and dynein axonemal heavy chain 6 (DNAH6) genes with uncertain pathogenicity. These variants are predicted to cause either missense or frameshift deletions in the encoded proteins.

Discussion

Infertility is a major health problem that harms both social and economic levels. In this study, a total of 69 DNA samples from azoospermic infertile Jordanian men were analyzed by MIPs with 6014 probes targeting 134 genes associated with male infertility. A total of 9 variants were found using MIPs and confirmed by Sanger sequencing, these variants include both reported pathogenic and novel variants.

Three pathogenic and likely pathogenic variants were found in CFTR and USP9Y genes. Pathogenic variants in the CFTR gene have been associated with different forms of male infertility (Chen et al., 2012). In this study, two homozygous variants in the CFTR gene have been found in two different patients; N1303K and D1152H, none of the patients had cystic fibrosis (CF). CFTR c.3909C>G p.(Asn1303Lys) has been reported to be a pathogenic variant causing CF and is linked to CBAVD (De Braekeleer & Ferec, 1996; Van Hoorenbeeck et al., 2007). However, the patient in this study was normal and confirmed to have the vas deferens by ultrasonography, and none of his family members had CF. Consistent with this, some studies have shown that CFTR pathogenic variants may also cause other non-CBAVD Azoospermia (Chen et al., 2012; Dohle, 2002; Smits et al., 2019), in addition, phenotypic heterogeneity of CF patients with N1303K variant could be explained by the presence of specific haplotypes (Cordovado et al., 2012; Osborne et al., 1992). The contribution of CFTR variants to male infertility has not yet been assessed in Jordan, moreover, the N1303K mutation and its association with cystic fibrosis have not been studied yet in the Jordanian population.

On the other hand, D1152H (3454G>C) has been initially linked to CBAVD and CF (Feldmann et al., 2003; Highsmith et al., 2005). Recently, it has shown that this mutation is associated with pancreatitis but not CF (LaRusch et al., 2014), while the CFTR2 database classifies the D1152H as a mutation with variable penetrance (http://www.http.com//www.cftr2).

The third patient had a pathogenic novel mutation in the USP9Y gene (c.6537T>A) with unknown inheritance pattern. This mutation causes a premature stop codon in the USP9Y gene located in the azoospermia factor a region (AZFa) of Y-chromosome which is known to cause azoospermia when deletions or premature stop codons occurs (Luddi et al., 2009; Online Mendelian Inheritance in Man, 2019). Furthermore, USP9Y is linked to spermatogenic failure and azoospermia (Krausz et al., 2006; Sun et al., 1999). Early reports have suggested a role for USP9Y in spermatogenic failure associated with azoospermia and male infertility, where point mutations in USP9Y were found in males with spermatogenic failure and were absent in their fertile male siblings or in the control fertile group (Brown et al., 1998; Hall et al., 2003; Sun et al., 1999), however recent papers have challenged this view (Krausz et al., 2006; Luddi et al., 2009). Deletions in USP9Y were found in a normozoospermic male, and both in his fertile father and brother (Luddi et al., 2009), while another paper found that USP9Y does not perform an essential function during spermatogenesis (Krausz et al., 2006), opposite to what has been suggested before. The homozygous substitution mutation –found in this study- could explain the azoospermic phenotype manifested in the patient, or this phenotype could be linked to other genetic or non-genetic factors.

In addition to the previously mentioned variants, six novel variants with uncertain pathogenicity have been found, two homozygous missense mutations in MCM8 and KDM5D, two heterozygous frameshift mutations in MAST2 and MEIOB, and two heterozygous missense mutations in UTP14C and DNAH6.

MCM8 is crucial for gametogenesis and gonadal development (Tenenbaum-Rakover et al., 2015). Mutations in MCM8 have been shown to cause gonadal and spermatogenesis failure (Lutzmann et al., 2012; Tenenbaum-Rakover et al., 2015). KDM5D is one of the AZFb region genes on Y-chromosome, similar to USP9Y, it has been linked to azoospermia and spermatogenic failure (Rastegar et al., 2015; Yu et al., 2015).

The heterozygous frameshift mutations have been found in MAST2 and MIEOB. MAST2 functions in spermatids maturation (PubChem database. National Center for Biotechnology Information, 2016). Mutations in MAST2 have is associated with non-obstructive azoospermia (Huang et al., 2015). MIEIOB has been linked to spermatogenic failure and male infertility associated with oligospermia or azoospermia (GeneCards. The human gene database)(Gershoni et al., 2017). A recent study linked a frameshift mutation in MIEOB with azoospermia (Gershoni et al., 2019).

The last two heterozygous missense mutations are found in UTP14C and DNAH6. UTP14C is essential for spermatogenesis (GeneCards.The human gene database). Mutations in UTP14C gene have been linked to spermatogenic arrest and male infertility (Rohozinski et al., 2006). Furthermore, mutations in DNAH6 were shown to be associated with spermatogenic abnormalities and male infertility (Gershoni et al., 2017; Li et al., 2018; Tu et al., 2019).

We predict that heterozygous variants have a dominant pathogenic pattern of inheritance with reduced penetrance and could be inherited maternally, or had arisen from denovo mutations in the germline cells.

Finally, it is interesting to note that the mutation pick-up rate in this study was significantly lower than the original cohort where this panel was developed and validated (Oud et al., 2017), which shows that genetic variants responsible for infertility in the Jordanian populations may be very different.

In conclusion, this work provided the first insight into monogenic causes of male infertility in Jordan and highlighted a different spectrum of genotype-phenotype correlation of known pathogenic CFTR variants in the Jordanian population. For clinical implementation, it is important to obtain more reliable information about the presence of genetic variation in the Jordanian population. Also, it is clear that these kinds of genetic studies should go hand in hand with detailed clinical phenotyping to be able to interpret these findings in a clinical setting.

Acknowledgment

The authors would like to thank all volunteers who participated in this study. Many thanks to the Royal Medical services for approving and participating in the study. This study was funded by the deanship of research at Jordan University of science and technology (grant # 12/2019). J.A.V. is supported by grants from The Netherlands Organization for Scientific Research (918-15-667) as well as an Investigator Award in Science from the Wellcome Trust (209451).

Biographies

Author biography

Osamah Batiha: assistant professor of cell & developmental genetics, Jordan University of science and technology, Irbid, Jordan

George J Burghel: Principal Clinical Scientist in cancer pharmacogenetics, Manchester Centre for Genomic Medicine, Manchester University Hospital, Manchester, UK

Emad Alsharu: Senior specialist IVF /OBGYN, Royal medical services, Amman, Jordan

Joris A Veltman: Jacobson chair of Personalized Medicine, Dean Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom

Harsh Sheth: Assistant Professor and Head of Advanced Genomic Technologies Division at FRIGE's Institute of Human Genetics

Footnotes

Disclosure Statement

The authors report no conflict of interest

References

  1. Agarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. Reproductive Biology and Endocrinology. 2015 doi: 10.1186/s12958-015-0032-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Al Zoubi Mazhar S, Al-Batayneh K, Alsmadi M, Rashed M, Al-Trad B, Al Khateeb W, Aljabali A, Otoum O, Al-Talib M, Batiha O. 4,977-bp human mitochondrial DNA deletion is associated with asthenozoospermic infertility in Jordan. Andrologia. 2020 doi: 10.1111/and.13379. [DOI] [PubMed] [Google Scholar]
  3. Al Zoubi MazharSalim, Bataineh H, Rashed M, Al-Trad B, Aljabali AAA, Al-Zoubi RM, Al Hamad M, Issam AbuAlArjah M, Batiha O, Al-Batayneh KM. CAG Repeats in the androgen receptor gene is associated with oligozoospermia and teratozoospermia in infertile men in Jordan. Andrologia. 2020 doi: 10.1111/and.13728. [DOI] [PubMed] [Google Scholar]
  4. Batiha O, Al-Ghazo MA, Elbetieha AM, Jaradat SA. Screening for deletions in the AZF region of Y chromosome in infertile Jordanian males. Journal of Applied Biological Sciences. 2012;6(2) [Google Scholar]
  5. Batiha O, Haifawi S, Al-Smadi M, Burghel GJ, Naber Z, Elbetieha AM, Bodoor K, Sumadi A, Al, Swaidat S, Jarun Y, Abdelnour A. Molecular analysis of CAG repeat length of the androgen receptor gene and Y chromosome microdeletions among Jordanian azoospermic infertile males. Andrologia. 2018 doi: 10.1111/and.12979. [DOI] [PubMed] [Google Scholar]
  6. Brown GM, Furlong RA, Sargent CA, Erickson RP, Longepied G, Mitchell M, Jones MH, Hargreave TB, Cooke HJ, Affara NA. Characterisation of the coding sequence and fine mapping of the human DFFRY gene and comparative expression analysis and mapping to the Sxrb interval of the mouse Y chromosome of the Dffry gene. Human Molecular Genetics. 1998 doi: 10.1093/hmg/7.1.97. [DOI] [PubMed] [Google Scholar]
  7. Chen H, Ruan YC, Xu WM, Chen J, Chan HC. Regulation of male fertility by CFTR and implications in male infertility. Human Reproduction Update. 2012;18(6):703–713. doi: 10.1093/humupd/dms027. [DOI] [PubMed] [Google Scholar]
  8. Cordovado SK, Hendrix M, Greene CN, Mochal S, Earley MC, Farrell PM, Kharrazi M, Hannon WH, Mueller PW. CFTR mutation analysis and haplotype associations in CF patients ☆. Molecular Genetics and Metabolism. 2012;105(2):249–254. doi: 10.1016/j.ymgme.2011.10.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. De Braekeleer M, Ferec C. Mutations in the cystic fibrosis gene in men with congenital bilateral absence of the vas deferens. Molecular Human Reproduction. 1996;2(9) doi: 10.1093/molehr/2.9.669. [DOI] [PubMed] [Google Scholar]
  10. Dohle GR. Genetic risk factors in infertile men with severe oligozoospermia and azoospermia. Human Reproduction. 2002;17(1):13–16. doi: 10.1093/humrep/17.1.13. [DOI] [PubMed] [Google Scholar]
  11. Feldmann D, Couderc R, Audrezet MP, Ferec C, Bienvenu T, Desgeorges M, Claustres M, Mittre H, Blayau M, Bozon D, Malinge MC, et al. CFTR genotypes in patients with normal or borderline sweat chloride levels. Human Mutation. 2003 doi: 10.1002/humu.9183. [DOI] [PubMed] [Google Scholar]
  12. GeneCards. The human gene database. MEIOB. (n.d.) [Google Scholar]
  13. GeneCards. The human gene database. UTP14C Gene. (n.d.) [Google Scholar]
  14. Gershoni M, Hauser R, Barda S, Lehavi O, Arama E, Pietrokovski S, Kleiman SE. A new MEIOB mutation is a recurrent cause for azoospermia and testicular meiotic arrest. Human Reproduction. 2019;34(4):666–671. doi: 10.1093/humrep/dez016. [DOI] [PubMed] [Google Scholar]
  15. Gershoni M, Hauser R, Yogev L, Lehavi O, Azem F, Yavetz H, Pietrokovski S, Kleiman SE. A familial study of azoospermic men identifies three novel causative mutations in three new human azoospermia genes. Genetics in Medicine. 2017;19(9):998–1006. doi: 10.1038/gim.2016.225. [DOI] [PubMed] [Google Scholar]
  16. Gilissen C, Hoischen A, Brunner HG, Veltman JA. Unlocking Mendelian disease using exome sequencing. Genome Biology. 2011 doi: 10.1186/gb-2011-12-9-228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hall NM, Brown GM, Furlong RA, Sargent CA, Mitchell M, Rocha D, Affara NA. Usp9y (ubiquitin-specific protease 9 gene on the Y) is associated with a functional promoter and encodes an intact open reading frame homologous to Usp9x that is under selective constraint. Mammalian Genome. 2003 doi: 10.1007/s00335-002-3068-4. [DOI] [PubMed] [Google Scholar]
  18. Highsmith WE, Friedman KJ, Burch LH, Spock A, Silverman LM, Boucher RC, Knowles MR. A CFTR mutation (D1152H) in a family with mild lung disease and normal sweat chlorides [1] Clinical Genetics. 2005 doi: 10.1111/j.1399-0004.2005.00459.x. [DOI] [PubMed] [Google Scholar]
  19. Huang N, Wen Y, Guo X, Li Z, Dai J, Ni B, Yu J, Lin Y, Zhou W, Yao B, Jiang Y, et al. A Screen for Genomic Disorders of Infertility Identifies MAST2 Duplications Associated with Nonobstructive Azoospermia in Humans1. Biology of Reproduction. 2015;93(3) doi: 10.1095/biolreprod.115.131185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Krausz C, Degl’Innocenti S, Nuti F, Morelli A, Felici F, Sansone M, Varriale G, Forti G. Natural transmission of USP9Y gene mutations: A new perspective on the role of AZFa genes in male fertility. Human Molecular Genetics. 2006 doi: 10.1093/hmg/ddl198. [DOI] [PubMed] [Google Scholar]
  21. Krausz C, Riera-Escamilla A. Genetics of male infertility. Nature Reviews Urology. 2018 doi: 10.1038/s41585-018-0003-3. [DOI] [PubMed] [Google Scholar]
  22. LaRusch J, Jung J, General IJ, Lewis MD, Park HW, Brand RE, Gelrud A, Anderson MA, Banks PA, Conwell D, Lawrence C, et al. Mechanisms of CFTR Functional Variants That Impair Regulated Bicarbonate Permeation and Increase Risk for Pancreatitis but Not for Cystic Fibrosis. PLoS Genetics. 2014;10(7) doi: 10.1371/journal.pgen.1004376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li L, Sha Y-W, Xu X, Mei L-B, Qiu P-P, Ji Z-Y, Lin S-B, Su Z-Y, Wang C, Yin C, Li P. DNAH6 is a novel candidate gene associated with sperm head anomaly. Andrologia. 2018;50(4):e12953. doi: 10.1111/and.12953. [DOI] [PubMed] [Google Scholar]
  24. Luddi A, Margollicci M, Gambera L, Serafini F, Cioni M, De Leo V, Balestri P, Piomboni P. Spermatogenesis in a man with complete deletion of USP9Y. New England Journal of Medicine. 2009 doi: 10.1056/NEJMoa0806218. [DOI] [PubMed] [Google Scholar]
  25. Lutzmann M, Grey C, Traver S, Ganier O, Maya-Mendoza A, Ranisavljevic N, Bernex F, Nishiyama A, Montel N, Gavois E, Forichon L, et al. MCM8- and MCM9-Deficient Mice Reveal Gametogenesis Defects and Genome Instability Due to Impaired Homologous Recombination. Molecular Cell. 2012;47(4):523–534. doi: 10.1016/j.molcel.2012.05.048. [DOI] [PubMed] [Google Scholar]
  26. Online Mendelian Inheritance in Man, O. OMIM: 415000. 2019 [Google Scholar]
  27. Osborne L, Santis G, Schwarz M, Klinger K, Dörk T, McIntosh I, Schwartz M, Nunes V, Macek M, Reiss J. Incidence and expression of the N1303K mutation of the cystic fibrosis (CFTR) gene. Human Genetics. 1992;89(6):653–658. doi: 10.1007/bf00221957. [DOI] [PubMed] [Google Scholar]
  28. Oud MS, Ramos L, O’Bryan MK, McLachlan RI, Okutman Ö, Viville S, de Vries PF, Smeets DFCM, Lugtenberg D, Hehir-Kwa JY, Gilissen C, et al. Validation and application of a novel integrated genetic screening method to a cohort of 1,112 men with idiopathic azoospermia or severe oligozoospermia. Human Mutation. 2017 doi: 10.1002/humu.23312. [DOI] [PubMed] [Google Scholar]
  29. Oud MS, Volozonoka L, Smits RM, Vissers LELM, Ramos L, Veltman JA. A systematic review and standardized clinical validity assessment of male infertility genes. Human Reproduction (Oxford, England) 2019 doi: 10.1093/humrep/dez022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Plaseska-Karanfilska D, Noveski P, Plaseski T, Maleva I, Madjunkova S, Moneva Z. Genetic causes of male infertility. Balkan Journal of Medical Genetics. 2012 doi: 10.2478/v10034-012-0015-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Poongothai J, Gopenath TS, Manonayaki S, Poongothai S. Genetics of human male infertility. Singapore Medical Journal. 2009 [PubMed] [Google Scholar]
  32. PubChem database. National Center for Biotechnology Information. MAST2 - microtubule associated serine/threonine kinase 2 (human) 2016. https://pubchem.ncbi.nlm.nih.gov/gene/MAST2/human .
  33. Rastegar DA, Tabar MS, Alikhani M, Parsamatin P, Sahraneshin Samani F, Sabbaghian M, Sadighi Gilani MA, Mohammad Ahadi A, Mohseni Meybodi A, Piryaei A, Ansari-Pour N, et al. Isoform-level gene expression profiles of human y chromosome azoospermia factor genes and their X chromosome paralogs in the testicular tissue of non-obstructive azoospermia patients. Journal of Proteome Research. 2015;14(9):3595–3605. doi: 10.1021/acs.jproteome.5b00520. [DOI] [PubMed] [Google Scholar]
  34. Rentzsch P, Witten D, Cooper GM, Shendure J, Kircher M. CADD: Predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Research. 2019 doi: 10.1093/nar/gky1016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K, et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in Medicine. 2015 doi: 10.1038/gim.2015.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rohozinski J, Lamb DJ, Bishop CE. UTP14c Is a Recently Acquired Retrogene Associated with Spermatogenesis and Fertility in Man1. Biology of Reproduction. 2006;74(4):644–651. doi: 10.1095/biolreprod.105.046698. [DOI] [PubMed] [Google Scholar]
  37. Smits RM, Oud MS, Vissers LELM, Lugtenberg D, Braat DDM, Fleischer K, Ramos L, D’Hauwers KWM. Improved detection of CFTR variants by targeted next-generation sequencing in male infertility: a case series. Reproductive BioMedicine Online. 2019 doi: 10.1016/j.rbmo.2019.08.005. [DOI] [PubMed] [Google Scholar]
  38. Sun C, Skaletsky H, Birren B, Devon K, Tang Z, Silber S, Oates R, Page DC. An azoospermic man with a de novo point mutation in the Y-chromosomal gene USP9Y. Nature Genetics. 1999 doi: 10.1038/70539. [DOI] [PubMed] [Google Scholar]
  39. Tenenbaum-Rakover Y, Weinberg-Shukron A, Renbaum P, Lobel O, Eideh H, Gulsuner S, Dahary D, Abu-Rayyan A, Kanaan M, Levy-Lahad E, Bercovich D, et al. Minichromosome maintenance complex component 8 (MCM8) gene mutations result in primary gonadal failure. Journal of Medical Genetics. 2015;52(6):391–399. doi: 10.1136/jmedgenet-2014-102921. [DOI] [PubMed] [Google Scholar]
  40. Thoma ME, McLain AC, Louis JF, King RB, Trumble AC, Sundaram R, Buck Louis GM. Prevalence of infertility in the United States as estimated by the current duration approach and a traditional constructed approach. Fertility and Sterility. 2013 doi: 10.1016/j.fertnstert.2012.11.037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tu C, Nie H, Meng L, Yuan S, He W, Luo A, Li H, Li W, Du J, Lu G, Lin G, et al. Identification of DNAH6 mutations in infertile men with multiple morphological abnormalities of the sperm flagella. Scientific Reports. 2019;9(1) doi: 10.1038/s41598-019-52436-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Van Hoorenbeeck K, Storm K, van den Ende J, Biervliet M, Desager KN. N1303K and IVS8-5T, clinical presentation within a family with atypical cystic fibrosis. Journal of Cystic Fibrosis. 2007;6(3):220–222. doi: 10.1016/j.jcf.2006.10.002. [DOI] [PubMed] [Google Scholar]
  43. World Health Organization. Infertility definitions and terminology. WHO; 2018. https://doi.org/http://www.who.int/reproductivehealth/topics/infertility/definitions/en/ [Google Scholar]
  44. Yu XW, Wei ZT, Jiang YT, Zhang SL. International Journal of Clinical and Experimental Medicine. 9. Vol. 8. E-Century Publishing Corporation; 2015. Y chromosome azoospermia factor region microdeletions and transmission characteristics in azoospermic and severe oligozoospermic patients; pp. 14634–14646. [PMC free article] [PubMed] [Google Scholar]
  45. Zorrilla M, Yatsenko AN. The Genetics of Infertility: Current Status of the Field. Current Genetic Medicine Reports. 2013;1(4):247–260. doi: 10.1007/s40142-013-0027-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES