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Genetic studies in spermatogenic failure
Visser, L.
Publication date
2014
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Final published version
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Citation for published version (APA):
Visser, L. (2014). Genetic studies in spermatogenic failure.
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Liesbeth Visser
genetic studies
in spermatogenic failure
genetic
studies
in spermatogenic failure
Liesbeth Visser
GENETIC STUDIES
in spermatogenic failure
Imprint
Visser, Liesbeth
Genetic studies in spermatogenic failure
[PhD Thesis] University of Amsterdam The Netherlands
Includes bibliographical references and summary © Liesbeth Visser, 2014
All rights reserved. No part of this publication may be reproduced or used in any form or by any means, graphic, electronic or mechanical, including photocopying, recording, typing or information storage and retrieval systems without written permission of the author
Support for the printing of this thesis was provided by the AMC Graduate School,
Stichting Gynaecologische Endocrinologie en Kunstmatige Humane Voortplanting (GEKHVO) and ABN Amro
Cover and lay-out: RheaLeneDesign Printing: Ipskamp Drukkers ISBN: 978-90-822849-0-4
GENETIC STUDIES
in spermatogenic failure
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus
prof. dr. D.C. van den Boom ten overstaan van een door het college voor promoties ingestelde commissie,
in het openbaar te verdedigen in de Agnietenkapel op woensdag 19 november 2014 te 10.00 uur door
Liesbeth Visser
geboren te WaddinxveenPromotiecommissie
Promotores: Prof. dr. S. Repping
Prof. dr. F. van der Veen
Co-promotor: Dr. A.M.M. van Pelt
Overige leden: Prof. dr. J.P.M. Geraedts Prof. dr. L.H.J. Looijenga Prof. dr. J.A.M. van der Post Prof. dr. H.R. Tournaye Prof. dr. D.G. de Rooij Faculteit der Geneeskunde
Contents
Chapter 1
8General introduction and outline of the thesis
Chapter 2
22A comprehensive gene mutation screen in men with asthenozoospermia
Fertility and Sterility 95: 1020-1024 (2011)
Chapter 3
48Y-chromosome gr/gr deletions are a risk factor for low semen quality
Human Reproduction 24: 2667-2673 (2009)
Chapter 4
66Unravelling the genetics of spermatogenic failure
Reproduction 139: 303-307 (2010)
Chapter 5
80Increase in DMRT1 copy number is associated with Sertoli Cell Only Syndrome
Submitted
Chapter 6
156High rate of novel copy number variants in men with Sertoli Cell Only Syndrome
Manuscript in preparation
Chapter 7
180General discussion and implications for future research
Chapter 8
198Summary Samenvatting
Dankwoord 216
About the author 219
List of publications 220
8
General introduction
and outline of the thesis
General introduction
Male subfertility
Subfertility, defined as the inability to conceive within one year of unprotected
intercourse, affects one in eight couples in the Western world1. In half of all
subfertile couples, the semen quality of the partner is below the standards of the
World Health Organisation and assumed to contribute to the subfertility1-4.
According to these standards, semen is assessed with respect to volume of the ejaculate and concentration, motility and morphology of the spermatozoa. According to current guidelines, the diagnosis of male subfertility is made when on at least two semen analyses, one or more sperm parameters are below the
WHO cutoff for normozoospermia3,5-7. However, the WHO criteria have never
been prospectively validated. Recently, high-level evidence has been published, demonstrating that the current WHO criteria do not discriminate between fertile and subfertile men. Instead, redefined sperm parameters, i.e. sperm
concentration <40×106/mL, total sperm count <200×106, and sperm morphology
<20% normal forms, were shown to have strong predictive value for the chances
of natural conception4. Using these definitions semen analysis can be used to
indicate if and to what extent semen quality contributes to subfertility. Spermatogenesis
Spermatogenesis encompasses the process of the development of mature, haploid spermatozoa, starting from diploid spermatogonial stem cells (SSCs), a special class of spermatogonia (figure 1A). SSCs secure the continuous production of spermatozoa throughout adult life. To this end, the process of spermatogenesis starts with the mitotic division of an SSC, resulting in either two new SSCs (self-renewal) or in the formation of two cells that are committed to differentiate into spermatozoa (SSC differentiation). SSC differentiation can be divided into three successive phases; a proliferative phase during which the spermatogonia undergo successive divisions to increase the number of germ cells, a meiotic phase during which the DNA of the diploid primary spermatocytes recombines and subsequently segregates to give rise to two haploid spermatids, and a spermiogenic phase during which the spermatids ripen into mature
spermatozoa that are capable of fertilization8,9.
Spermatogonia derive from primordial germ cells that migrate from their
origin at the base of the allantois to populate the genital ridge10,11. Here they
associate with somatic cells into the primitive sex cords that are the precursors for seminiferous tubules in the testes. Ultimately, within the testis, two
anatomical compartments can be distinguished; the interstitial compartment that
10
provides physical and chemical support, including androgen production by Leydig cells, and the tubular compartment, containing the seminiferous tubules where spermatogenesis takes place. Within the seminiferous tubules, SSCs reside on the basal membranes, enveloped by Sertoli cells whose tight junctions form the blood-testis barrier. Sertoli cells have a special role in nurturing and maintaining
Figure 1A. Schematic overview of spermatogenic cell types with DNA content
following cell division and/or differentiation
spermatogenesis that is brought about by the presence of this barrier and the differential expression and secretion of growth factors and other factors into the basal (stem cell) and adluminal (developing germ cell) seminiferous epithelium
compartment12. The behavior of SSCs in terms of self-renewal and differentiation
must be strictly regulated, both by intrinsic, germ cell-derived factors such as
primordial germ cells
2n spermatogonia 2n primary spermatocyte n n secondary spermatocytes 2n meiosis I n n n n meiosis II spermatids spermiogenesis spermatozoa 2n self-renewal (mitosis)
spermatogonial stem cell
mitosis differentiation
11
PLZF, as well as by extrinsic, mostly Sertoli cell-derived growth factors like GDNF and FGF2, to prevent SSC exhaustion from either too much differentiation or an
overproduction of SSCs9,13. It is assumed that regulation of SSCs occurs in the
specialized “niches”, where those cells resides, located on the basal membrane of those tubule areas that border on the interstitial tissue and in particular those
areas containing venules and arterioles9,13. It is thought that components of the
interstitial tissue may determine whether Sertoli cells produce factors inducing self-renewal or differentiation13.
In a cross-section of a seminiferous tubule with active spermatogenesis, germ cells in all phases of differentiation can be observed (figure 1B). After completion of the spermatogenic process, the mature spermatozoa are stored in the epididymis.
Figure 1B. Schematic representation of a cross section of (part of ) a seminiferous
tubule with active spermatogenesis
n n 2n 2n 2n adluminal compartment basal compartment 2n 2n 2n n n Blood-Testis Barrier 2n 2n n n Sertoli cell spermatogonia primary spermatocyte secondary spermatocytes spermatids spermatozoa blood vessel Leydig cell n n n basal membrane
peritubular myoid cells
n n 2n 2n 12 chapter 1
Clinical practice
The origin of poor semen quality can lie at the pre-testicular, testicular or post-testicular level2.
Hyperprolactinemia and hypogonadotrophic hypogonadism are examples of pre-testicular disorders that can be treated with exogenous hormones. Epididymal or vas deferens obstruction and erectile dysfunction are among the post-testicular disorders. In these conditions, epididymal and/or testicular microsurgery to obtain spermatozoa for IVF/ICSI is generally successful. At the testicular level, poor semen quality results from defects in the process of spermatogenesis at any of the stages described above and at any stage this can be a consequence of isolated or combined impairments in the germ cells, Sertoli cells or other (e.g. interstitial) cells involved. The most severe defect at the testicular level is Sertoli Cell Only Syndrome (SCOS), which is characterized by
the complete absence of germ cells at any stage of development14. In most cases,
the testicular phenotype is heterogeneous rather than homogenous such as in complete SCOS, with some tubules showing a Sertoli Cell Only phenotype, some meiotic arrest and some normal spermatogenesis, resulting in a wide variety of semen quality among different men.
There are currently no interventions to restore impaired spermatogenesis. At this time, the only intervention for couples with subfertility due to testicular spermatogenic failure is to retrieve spermatozoa from the ejaculate, from the epididymis through microsurgical sperm aspiration (MESA) or from the testis by testicular sperm extraction (TESE) for use in intracytoplasmic sperm injection
(ICSI)15. ICSI involves ovarian hyperstimulation, oocyte retrieval, fertilization by
injection of a single spermatozoon directly into the oocyte, embryo culture and
finally embryo transfer16,17. This makes ICSI a costly and burdensome treatment.
In addition, concerns have been raised about the health of children conceived through ICSI. Studies conducted over the past decade showed an increased risk of congenital malformations in ICSI-offspring, in particular that
of genital abnormalities in ICSI boys18. Recently a comprehensive study in a
large single population registry confirmed previous findings of an increased risk of birth defects among births conceived with ICSI as compared with births
from spontaneous conception19. It is thought that genetic or environmental
factors that lead to spermatogenic failure and thus the use of ICSI may underlie
this association20. The association between ICSI and epigenetic disturbances,
i.e. imprinting disorders, in ICSI-offspring remains controversial and its origin unclear. To what extent spermatogenic failure is transmitted to ICSI-offspring is of yet unknown but will become clear in the next decade as the first ICSI-offspring comes of reproductive age.
13
Genetic causes
Although our knowledge on the etiology of spermatogenic failure is limited, there is substantial evidence for a genetic background. This evidence comes from case reports and case control studies reporting familial clustering of spermatogenic
failure21-24. In addition, several animal models, including the worm Caenorhabditis
elegans, the fruitfly Drosophila melanogaster and the mouse Mus musculus have
suggested numerous genes whose disruption impairs male reproduction. As of
today there are over 400 mouse knockout models with an infertility phenotype25.
High-throughput transcriptome analyses of murine spermatogenesis have shown
that a few thousand genes are involved in the process26,27. In theory, mutations in
any of these genes may cause spermatogenic failure.
In men, only few genetic variants are known to cause testicular
spermatogenic failure. These include numerical and structural chromosomal alterations, monogenic disorders and Y-chromosome deletions. The most frequent numerical chromosomal abnormality is Klinefelter’s syndrome
(47, XXY), affecting approximately 1 in 750 men28. Monogenic disorders like
Kartagener syndrome, Kallmann syndrome and Noonan syndrome, are rare. Y-chromosome deletions, including AZFa, P5/proximal P1 (AZFb), P5/distal
P1 (AZFb+c) and b2/b4 (AZFc) deletions occur in up to 18% of men with
spermatogenic failure29. Still, in the majority of men no cause can be identified
and the spermatogenic failure is classified as idiopathic.
To identify genetic determinants of any given disease, several methods are available. A first method employs the segregation of a disease within a family (linkage analysis), but due to the nature of spermatogenic failure, large pedigrees are generally not available as the trait directly limits genetic transmission. A second method is genetic association analysis, which can be considered as an extended linkage analysis including the entire population as an extended pedigree. A third method involves the screening of candidate genes to search for mutations that are present in cases but not, or at lower frequency, in controls. Mutation analysis was previously assumed to be the only realistic method to
identify genes involved in spermatogenic failure30. Indeed, we and others have
applied this method for a number of candidate genes, which has led to the
identification of a small number of putative pathogenic mutations31-35.
Together with decisions on study design, choices have to be made on the type of genetic variation to be studied. Several types of genetic variants have been identified over the years, roughly moving from variation at a macroscopic level to variation at a single base pair level (SNPs) following technological advancements. Several years ago, by means of genomic microarrays, a novel class of genetic variation, so-called copy number variants (CNVs), was identified, in which
14
submicroscopic segments of DNA (1kb up to 3Mb in size) are either deleted or
duplicated36,37. Affecting gene dosage through gene disruption, gene fusion and
position effects, CNVs are associated with complex diseases38. CNVs can be
detected by array comparative genomic hybridization (aCGH) and SNP-tagged
arrays38,39 or through “next-generation sequencing” (NGS)36,37,39. The first two
are array-based techniques that make use of DNA hybridization and quantify the amount of labelled target DNA that hybridizes to synthetic array bound DNA oligonucleotides. In NGS, the sequencing process is parallelized allowing the production of millions of sequences concurrently, which subsequently can be aligned and analysed using specific algorithms to determine copy numbers of
specific DNA stretches40-43. A great advantage of NGS is that all types of variants,
from SNPs via small inversions and deletions to large CNVs can be assessed using the same technique. NGS emerged a few years after we conducted the CNV studies described in this thesis and has now become widely available.
Background of the research described in this thesis
In this thesis we consecutively employed some of the methodological designs outlined above to identify novel variants, at different levels of genetic variation, that are associated with spermatogenic failure.
First, we searched for mutations causing asthenozoospermia by means of candidate gene sequencing. In this study we included men with the well-defined phenotype of isolated asthenozoospermia and in contrast to previous studies, we did not focus on a single candidate gene, but screened all genes known at the time to cause isolated asthenozoospermia in mouse knockout models: AKAP4,
CATSPER1-4, ADCY10, SLC9A10, PLA2G6 and GAPDHS.
Second, we conducted a genotype driven cross-sectional cohort study to investigate the association between the Y-chromosome gr/gr deletion and spermatogenic failure. The effect of this deletion on semen quality was for a long time not clear, mostly due to methodological shortcomings of previous studies that all employed a case-control design. Compared to the case-control design, a cohort design is more powerful to identify genetic risk factors, as it avoids introduction of possible selection bias and does not require dichotomization of quantitative data. By assembling a large cohort of men with varying sperm counts and comparing the sperm counts of men with and without the gr/gr deletion, the effect of this genetic variant on the distribution of sperm counts was studied.
Third, we studied the role of CNVs in SCOS. We used a high-throughput genotyping SNP array to determine the precise breakpoints of an unbalanced translocation involving gain of 9p and loss of 11q in a man with SCOS. The 9p region contains the dosage sensitive DMRT1 gene that, when knocked out in
15
mice, results in SCOS. We studied the effect of DMRT1 upregulation in a rat spermatogonial stem cell line. To identify novel copy number variants associated with SCOS, SNP arrays were used to assess CNVs in 29 men with SCOS. By selecting only men with complete SCOS and no sperm on biopsies for testicular sperm extraction (TESE), we focused on a homogeneous phenotype to avoid confounding factors. CNVs in SCOS patients were compared to CNVs present in population samples to uncover novel SCOS specific CNVs. The gene content of these CNVs was analysed to identify potential candidate genes for SCOS.
Outline of the thesis
Chapter 2 describes a comprehensive candidate gene screening in 30 men with isolated asthenozoospermia. The genes AKAP4, CATSPER1-4, ADCY10,
SLC9A10, PLA2G6 and GAPDHS are screened for mutations. The putative effect
on gene function of identified mutations is evaluated. The frequency of missense mutations and base pair changes altering a splice site is determined in a control group of 90 normozoospermic men.
Chapter 3 reports a meta-analysis of previous case control studies and a novel cross-sectional cohort study into the effect of the Y-chromosome gr/gr deletion on semen quality. Pitfalls of previous case control studies are discussed and the two methods are compared.
Chapter 4 presents an overview of established genetic causes of spermatogenic failure, describes pitfalls in searching for novel genetic factors and discusses research opportunities for the future.
Chapter 5 presents a patient with complete SCOS carrying a large unbalanced translocation involving chromosome 9p (duplicated) and chromosome 11q (deleted), involving the dosage sensitive DMRT1 gene that is associated with SCOS in mice. Breakpoints are determined using a genome wide SNP array and DMRT1 duplication is validated with FISH and qPCR. In addition, DMRT1 copy numbers are determined in 30 men with SCOS and 90 normozoospermic controls. The impact of overexpression of DMRT1 is studied in vitro using the spermatogonial stem cell line GC-6spg.
Chapter 6 presents the role of CNVs in SCOS. Genome wide SNP arrays are used to identify CNVs in 29 men with SCOS. In silico analysis is conducted to
16
determine SCOS specific CNVs. The gene content of SCOS specific CNVs is studied to identify potential candidate genes for SCOS.
Chapter 7 aims to put the findings of the current thesis in a broader context and discusses implications and opportunities for future research.
Chapter 8 summarizes the results obtained in this thesis.
17
References
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semen. Geneva. 5th edition (2010)
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8. de Rooij, D.G. Proliferation and differentiation of spermatogonial stem cells.
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9. de Rooij, D.G. & Griswold, M.D. Questions about spermatogonia posed and answered since 2000. J Androl 33: 1085-1095 (2012).
10. Tarbashevich, K. & Raz, E. The nuts and bolts of germ-cell migration. Curr
Opin Cell Biol 22: 715-721 (2010).
11. Molyneaux, K. & Wylie, C. Primordial germ cell migration. Int J Dev Biol 48: 537-544 (2004).
12. Petersen, C. & Soder, O. The sertoli cell - a hormonal target and ‘super’ nurse for germ cells that determines testicular size. Horm Res 66: 153-161 (2006). 13. de Rooij, D.G. The spermatogonial stem cell niche. Microsc Res Tech 72:
580-585 (2009).
14. Ma, M. e.a. Sertoli cells from non-obstructive azoospermia and obstructive azoospermia patients show distinct morphology, Raman spectrum and biochemical phenotype. Hum Reprod 28: 1863-1873 (2013).
15. Palermo, G., Joris, H., Devroey, P. & Van Steirteghem, A.C. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340: 17-18 (1992).
16. Tournaye, H. e.a. Microsurgical epididymal sperm aspiration and
intracytoplasmic sperm injection: a new effective approach to infertility as a result of congenital bilateral absence of the vas deferens. Fertil Steril 61: 1045-1051 (1994).
17. Devroey, P. e.a. Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum
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Reprod 10: 1457-1460 (1995).
18. Bonduelle, M. e.a. A multi-centre cohort study of the physical health of 5-year-old children conceived after intracytoplasmic sperm injection, in vitro fertilization and natural conception. Hum Reprod 20: 413-419 (2005). 19. Davies, M.J. e.a. Reproductive technologies and the risk of birth defects. N
Engl J Med 366: 1803-1813 (2012).
20. Lie, R.T. e.a. Birth defects in children conceived by ICSI compared with children conceived by other IVF-methods; a meta-analysis. Int J Epidemiol 34: 696-701 (2005).
21. Lilford, R., Jones, A.M., Bishop, D.T., Thornton, J. & Mueller, R. Case-control study of whether subfertility in men is familial. Bmj 309: 570-573 (1994). 22. Gianotten, J. e.a. Partial DAZ deletions in a family with five infertile brothers.
Fertil Steril 79 Suppl 3: 1652-1625 (2003).
23. Tuerlings, J.H., van Golde, R.J., Oudakker, A.R., Yntema, H.G. & Kremer, J.A. Familial oligoasthenoteratozoospermia: evidence of autosomal dominant inheritance with sex-limited expression. Fertil Steril 77: 415-418 (2002). 24. Rolf, C., Gromoll, J., Simoni, M. & Nieschlag, E. Natural transmission of
a partial AZFb deletion of the Y chromosome over three generations: case report. Hum Reprod 17: 2267-2271 (2002).
25. Matzuk, M.M. & Lamb, D.J. The biology of infertility: research advances and clinical challenges. Nat Med 14: 1197-1213 (2008).
26. Chalmel, F. e.a. The conserved transcriptome in human and rodent male gametogenesis. Proc Natl Acad Sci USA 104: 8346-8351 (2007).
27. Schultz, N., Hamra, F.K. & Garbers, D.L. A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets. Proc Natl Acad Sci USA 100: 12201-12206 (2003). 28. Oates, R.D. The genetic basis of male reproductive failure. Urol Clin North
Am 35: 257-270, ix (2008).
29. Kuroda-Kawaguchi, T. e.a. The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men. Nat
Genet 29: 279-286 (2001).
30. Gianotten, J., Lombardi, M.P., Zwinderman, A.H., Lilford, R.J. & van der Veen, F. Idiopathic impaired spermatogenesis: genetic epidemiology is unlikely to provide a short-cut to better understanding. Hum Reprod Update 10: 533-539 (2004).
31. Gianotten, J., Schimmel, A.W., van der Veen, F., Lombardi, M.P. & Meijers, J.C. Absence of mutations in the PCI gene in subfertile men. Mol Hum Reprod 10: 807-813 (2004).
32. Westerveld, G.H. e.a. Heterogeneous nuclear ribonucleoprotein G-T (HNRNP
19
G-T) mutations in men with impaired spermatogenesis. Mol Hum Reprod 10: 265-269 (2004).
33. Westerveld, G.H., Repping, S., Leschot, N.J., van der Veen, F. & Lombardi, M.P. Mutations in the human BOULE gene are not a major cause of impaired spermatogenesis. Fertil Steril 83: 513-515 (2005).
34. Westerveld, G.H. e.a. Mutations in the testis-specific NALP14 gene in men suffering from spermatogenic failure. Hum Reprod 21: 3178-3184 (2006). 35. Westerveld, G.H., Repping, S., Lombardi, M.P. & van der Veen, F. Mutations
in the chromosome pairing gene FKBP6 are not a common cause of non-obstructive azoospermia. Mol Hum Reprod 11: 673-675 (2005).
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37. Iafrate, A.J. e.a. Detection of large-scale variation in the human genome. Nat
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38. Zhang, F., Gu, W., Hurles, M.E. & Lupski, J.R. Copy number variation in human health, disease, and evolution. Annu Rev Genomics Hum Genet 10: 451-481 (2009).
39. Feuk, L., Carson, A.R. & Scherer, S.W. Structural variation in the human genome. Nat Rev Genet 7: 85-97 (2006).
40. Alkan, C. e.a. Personalized copy number and segmental duplication maps using next-generation sequencing. Nat Genet 41: 1061-1067 (2009).
41. Tuzun, E. e.a. Fine-scale structural variation of the human genome. Nat Genet 37: 727-732 (2005).
42. Korbel, J.O. e.a. Paired-end mapping reveals extensive structural variation in the human genome. Science 318: 420-426 (2007).
43. Yoon, S., Xuan, Z., Makarov, V., Ye, K. & Sebat, J. Sensitive and accurate detection of copy number variants using read depth of coverage. Genome Res 19: 1586-1592 (2009).
20
21
A comprehensive gene mutation screen
in men with asthenozoospermia
Liesbeth Visser
G. Henrike Westerveld
Fang Xie
Saskia K.M. van Daalen
Fulco van der Veen
M. Paola Lombardi
Sjoerd Repping
Abstract
Objective
To find novel genetic causes of asthenozoospermia by comprehensively screening known candidate genes derived from mouse models.
Design
Case-control study. Setting
A fertility center based in an academic hospital. Patients
Thirty men with isolated asthenozoospermia. Interventions
Nine candidate genes were screened for mutations: ADCY10, AKAP4, CATSPER1,
CATSPER2, CATSPER3, CATSPER4, GAPDHS, PLA2G6, and SLC9A10. To account
for a possible effect of heterozygous mutations, we assessed imprinting of all candidate genes by studying the expression pattern of heterozygous SNPs in testis biopsies of five unrelated men.
Main outcome measures Mutations found in patients only. Results
We identified 10 heterozygous asthenozoospermia-specific mutations in ADYC10 (n=2), AKAP4 (n=1), CATSPER1 (n=1), CATSPER2 (n=1), CATSPER3 (n=1),
CATSPER4 (n=3) and PLA2G6 (n= 1). These mutations were distributed over six
patients. In silico analysis showed that eight of the ten mutations had a negative BLOSUM score, were located in conserved residues and/or were located in a functional domain. Expression analysis demonstrated that CATSPER1 and
CATSPER4 are imprinted.
Conclusions
Given their putative effect on protein structure, their location in conserved sequences or functional domains, and their absence in controls, the identified mutations may be a cause of asthenozoospermia in humans.
chapter 2
Introduction
Subfertility, i.e. the inability to conceive within one year of unprotected
intercourse, affects one in eight couples in the Western world1. It therefore
constitutes a significant clinical problem with psychological, social and economical consequences. In up to half of subfertile couples, semen quality is the only or a contributory etiologic factor. Low semen quality is diagnosed when the number of sperm cells that is produced, their morphology or their motility
is below the WHO cut offs for normal spermatogenesis2. Reduced motility, i.e.
asthenozoospermia, is found in approximately 18% of subfertile couples3,4 and is
the most important factor negatively affecting natural conception5,6.
Mouse models have provided a number of candidate genes for isolated asthenozoospemia, i.e. genes that in knockout models produce a phenotype
with no other abnormalities apart from asthenozoospermia7-14. These genes
include genes encoding sperm specific ion channels (CATSPER1 [NM_053054.2],
CATSPER2 [NM_172095.1], CATSPER3 [NM_178019.1], CATSPER4 [NM_198137.1], SLC9A10 [NM_183061.1]), enzymes (ADCY10 [NM_018417.4], GAPDHS
[NM_014364.4], PLA2G6 [NM_003560.2]) and a structural protein (AKAP4 [NM_003886.2]) (table S1).
The four homologous male-germ cell specific CATSPER (cation channel of sperm) genes, CATSPER1-4, appear to encode subunits of sperm calcium channels that are present in the principal piece of the sperm tail and are required for sperm cell hyperactivated motility and fertilization. It is currently unknown if and how the four CATSPER proteins interact. Catsper1-4 null sperm all lack the spermatozoal ICatsper calcium current that is present in wild-type animals15,16.
Interestingly, Catsper1-/- mice also lack Catsper2 protein, while Catsper1 protein
is also absent in Catsper2-/- mice, with knockout sperm showing identical
phenotypes17.
GAPDHS, PLA2G6 and ADCY10 encode sperm specific enzymes. In the
absence of Gapdhs, a glycolytic enzyme, sperm lack progressive motility18. Pla2g6
male knockouts, have greatly reduced fertility and produce spermatozoa with
impaired motility12. Bicarbonate ions present in the female genital tract activate
the adenylate cyclase Adcy10. Mice deficient for this enzyme are infertile because
of a severe sperm-motility defect13.
SLC9A10 is a member of the sodium-hydrogen exchanger (NHE) family. Slc9a10-null males have normal testis histology, normal sperm numbers and
morphology, but are completely infertile with severely diminished sperm motility14.
a comprehensive gene mutation screen in men with asthenozoospermia
The Adcy10-knockout and Slc9a10-null mice demonstrate a similar phenotype, with altered sperm motility, absence of hyperactivation and absence of tyrosine phosphorylation. In addition, for both models, sperm fertilizing potential can be restored by removing the oocyte zona pellucida, whereas motility can be
rescued by the addition of cell-permeable cAMP analogs19, suggesting that the
two proteins act together. Indeed, in vivo, Slc9a10 associates with the Adcy10,
forming a complex involved in bicarbonate signaling20.
AKAP4 encodes testis-specific fibrous sheath protein that functions as a
scaffold for signal transduction and enzymatic molecules. In mice its disruption results in infertility associated with reduced sperm motility, whereas sperm count
and morphology are unaffected7.
In humans, only few of these genes have been related to impaired sperm motility. The AKAP4 gene has been studied in relation to disrupted sperm motility caused by anatomical defects of the sperm tail known as dysplasia of the fibrous sheath (DFS). A single case report describes mutations in AKAP4 in a man with
DFS21. The CATSPER2 gene is one of two genes that are deleted in 15q15.3 deletion
syndrome [MIM:611102], characterized by deafness and male infertility resulting
from asthenoteratozoospermia22,23.
Because data on the genetic background of asthenozoospermia in humans are extremely limited, we set out to evaluate the association of nine candidate genes, ADCY10, CATSPER1, CATSPER2, CATSPER3, CATSPER4, GAPDHS,
SLC9A10, PLA2G6 with asthenozoospermia in a case control study including 30
men with isolated asthenozoospermia and 90 normozoospermic controls.
Materials and methods
Participants
As part of our ongoing research into genetic causes of low semen quality, we included men that attended our center and gave informed consent from January 1998 until September 2005. From January 1998 until January 2000 we included only men with low semen quality, i.e. one or more semen parameters below the WHO cut-offs, based on at least two semen analyses. From January 2000 until September 2005 we consecutively included all men, prior to semen analyses. Men with a history of orchitis, surgery of the vasa deferentia, bilateral orchidectomy, chemo- or radiotherapy, obstructive azoospermia, retrograde ejaculation, bilateral cryptorchidism, numerical or structural chromosome abnormalities and Y-chromosome deletions were excluded.
As cases, we included all men with isolated severe asthenozoospermia,
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defined as a normal sperm concentration (>20x106/ml) with less than 10% fast
progressive motile sperm (grade a) in at least two semen samples. For each case we randomly selected three controls from the same cohort that had a normal sperm concentration and more than 40% fast progressive motile sperm in two semen samples. The Institutional Review Board of the Academic Medical Center approved this study.
Mutation analysis
DNA was extracted from peripheral blood leucocytes according to standard procedures.
We amplified all coding exons and intron/exon boundaries of ADCY10,
AKAP4, CATSPER1-4, GAPDHS, PLA2G6, and SLC9A10. Primer pairs (table S2)
were designed with the aid of Primer3 using the available genomic sequence
information from NCBI24. In case several isoforms were known, we selected as
a reference sequence either the isoform that is expressed in the testis or else the longest known isoform. Large exons and promoter regions were covered by overlapping PCR products of maximally 500 bp. PCR was carried out in a total volume of 25 µl and contained 50 ng DNA, 3 µl of 10x PCR buffer (Roche, Woerden, the Netherlands), 0.2 mM dNTPs, 12 pmol forward and reverse primer, 2 mM MgCl2 and 0.5 U SuperTaq polymerase. We used a touchdown PCR program with a temperature range of 62-50°C with a 2°C decrement per cycle and 1 cycle increment per temperature step and a final amplification for 20 cycles at 94°C for 30s, 50°C for 30s and 72°C for 30s with a final extension at 72°C for 5 min.
Direct sequencing of both sense and antisense strands was performed using the same primers as those used for PCR, and an automated ABI Prism 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). All sequences were analyzed with the CodonCode Aligner software (CodonCode Corporation, Dedham, MA, USA).
For AKAP4, CATSPER1-3, SLC9A10, GAPDHS and PLA2G6, encountered mutations were analyzed using the Alamut mutation interpretation software (Interactive Biosoftware, Rouen, France). For CATSPER4 and ADCY10, which are not included in Alamut, mutations were evaluated using the following web-based interfaces. To predict the possible effect of silent exonic variants on splicing
activity, we used the ESE finder (Exonic Splicing Enhancer) program25. Similarly,
to predict the possible effect of intronic variants at the intron/exon boundaries and the branch site sequence on splicing activity, we used the Human Splicing
Finder26. We applied the BLOSUM62 Substitution Scoring Matrix to describe the
putative impact of identified amino acid changes27.
a comprehensive gene mutation screen in men with asthenozoospermia
To determine the conservation throughout species, the aligned amino acids of gene orthologues were analyzed using the ClustalW program (http://www.ebi. ac.uk/clustalw/index.html). Publicly available UniProt data were used to assess if variants were located in a functional protein domain (http://www.uniprot.org).
All missense mutations or SNPs as well as those intronic variants that were predicted to alter splice site activity were analyzed in controls.
Statistical analysis of genotype frequencies and distributions in patients and controls was performed using Chi-squared tests and R-code software (www.r-project.org). A p-value < 0.05 was considered statistically significant.
Imprinting analysis
Imprinting was verified by studying the expression pattern of heterozygous SNPs in testis biopsies from 5 unrelated men that underwent elective testicular surgery as part of the treatment for prostate cancer, or for suspected malignant testicular disease. Only tumor-free material was used.
Tissue samples were homogenized with the use of a Magna Lyser (Roche Diagnostics, Basel, Switzerland). DNA and RNA were extracted from biopsies by trizol digestion. For first-strand cDNA synthesis, we used M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, USA) with random primers on total RNA extracted from biopsies. At the DNA level, samples were screened for the presence of known SNPs. With the aid of Primer 3, intron-spanning primers were designed to evaluate the expression pattern of these SNPs in cDNA samples (table S3). DNA contamination of RNA samples was controlled for by incorporating a DNAse treatment in the extraction protocol as well as using intron-spanning primers for cDNA PCR.
In vitro functional assay
The effect of asthenozoospermia specific missense or splice site altering mutations in ADCY10 was assessed by mutant expression in a HEK293 cell system. Mutant human ADCY10 (A3542G, Asn1181Ser) was generated by QiukChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) using full-length human ADCY10 as template and primers CTCCATATCCATGTCGAGAAAAGCAGACACTTTCATTATGTG and CACATAATGAAAGTGTCTGCTTTTCTCGACATGGATATGGAG.
HEK293 cells were maintained in DMEM supplemented with 10% fetal calf serum. Transient transfections with either wild type or mutant human
ADCY10 were carried out using TransIT-LT1 (Mirus, Madison, WI) according
to the manufacturer’s instructions. Approximately 2 X106 HEK293 cells were
plated in 100-mm dishes 24 h before transfection. Ten micrograms of plasmid
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encoding V5-tagged full-length Human ADCY10 or V5-tagged mutant human ADCY10 was used to transfect the cells. Twenty-four hours after transfection, cells were harvested with lysis buffer containing 50 mM Tris–HCl, pH 7.5, 1 mM ß-mercaptoethanol, 1 mM EDTA, 0.32 M sucrose, and EDTA-free proteinase inhibitors (Roche Diagnostics, Notley, NJ) and disrupted by homogenization in a Dounce homogenizer by 20 strokes. Cell homogenates were centrifuged at 14,000 X g for 30 min at 4°C to obtain the soluble fraction.
Adenylyl cyclase activity was measured in the extracts (soluble fractions as described above) from cells expressing an empty plasmid (pcDNA3.1), or wild type and mutant human ADCY10 constructs. Briefly, enzyme preparations were incubated in a reaction buffer containing 40 mM Tris-HCl, pH 8.0, 5 mM MnCl2, or 5 mM MgCl2, 0.2 mM cAMP, and 10 mM phosphoenol pyruvate, 3 units of
pyruvate kinase, 10 µM GTP, 1 mM ATP, and 2 µCi of [32P] ATP for 20 min at 37°C.
Reaction was terminated with the addition of 20 µl of 2.2 N HCl containing [3H] cAMP (0.01 µCi) followed by boiling for 4 min and then cooling in an ice-water bath. Labeled cAMP was added to estimate and correct for recovery of the cyclic nucleotide during column chromatography. The cAMP generated was then separated from ATP by alumina column and eluted with 5 ml of 0.1 M ammonium acetate, pH 6.5 and quantitated by scintillation counting (Packard Instrument Co.).
Results
We included 30 men with isolated severe asthenozoospermia and 90 controls. Baseline characteristics of all men are summarized in table 1.
Table 1. Patient characteristics
a Based on at least 2 semen analyses per individual. Data are presented as mean ± SD or median
(25th-75th percentile) Cases (n=30) Controls (n=90) Age 36,1 ± 5,5 36,4 ± 6,0 Semen analysesa Volume 3,3 ± 1,3 3,9 ± 1,3 Concentration 32 (27-60) 87 (58-114) Fast progressive motility
(grade a) % 5 ± 3 49 ± 7 Normal morphology % 20 (15-32) 50 (46-56)
a comprehensive gene mutation screen in men with asthenozoospermia
Ta bl e 2. Va ria nt s f ou nd in p at ie nt s o nl y a rs11570606 Sample Gene Mutation Eff ect on trans -lation Predicted conse -quence Blosum score Conservation In functional domain 01.2007.3 AKAP4 c.887G>A p.Gly296Asn Substitution 0 moderate no 01.4007.3 CA TSPER4 c.247A>G p.Met83V al Substitution 1 yes yes D01/5080 CA TSPER4 c.157T>C p.T yr53His Substitution 2 no yes AMC0341 CA TSPER1 c.148G>A p.V al50Met Substitution 1 no no CA TSPER3 c.193T>C p.Phe65Leu Substitution 0 no yes D02/4481 CA TSPER2 c.1289C>G p.Thr430Arg Substitution -1 no no PLA2G6 c.187A>G a p.Arg63Gly Substitution -2 yes no AMC0536 CA TSPER4 c.992G>A p.Gly331Asn Substitution -1 no yes ADCY10 c.1020-40A>C Aberrant splicing ADCY10 c.3542A>G p.Asn1181Ser Substitution 1 yes no chapter 2 30
In total, we identified 135 variants in our case group. Seventy-five had not previously been annotated. A list of all variants and their distribution over the candidate genes is given in table S4. Thirty-eight variants were missense mutations, 33 were silent exonic mutations and 64 were intronic variants. In silico splice site analysis predicted that none of the silent exonic mutations would give rise to an intra-exonic splice site, while five of the intronic variants would alter an existing splice site.
Analysis of the thirty-eight missense mutations and 5 intronic splice site-altering variants in controls showed that ten of these, 9 missense mutations and one intronic splice site-altering mutation, were not present in controls
(table 2). Three missense mutations had a negative Blosum score, three missense mutations were located in highly conserved regions and six were located in functional domains.
The 10 asthenozoospermia-specific variants were distributed over 6 patients, with 1 patient carrying 3 mutations, 2 men carrying 2 mutations each and 3 men having one mutation (table 2). This distribution was not significantly different from expected as calculated with R-code (p= 0.09). Severity of the asthenozoospermic phenotype was independent of the number of mutations for each mutation carrier (table S5).
Comparison of DNA and RNA expression patterns revealed imprinting of
CATSPER1 and CATSPER4 (figure 1). CATSPER2, CATSPER3, GAPDHS, ADCY10
and SLC9A10 were not imprinted. No SNPs were available to determine imprinting of PLA2G6.
We were able to test one mutant (c.3542A>G) in a functional assay. HEK293 cells transfected with an empty vector (Control), wildtype ADCY10 or mutant
ADCY10 in the presence of 5 mM MnCl2, did not show a significant difference in
the recovered adenylyl cyclase activity with and without bicarbonate activation (figure 2).
a comprehensive gene mutation screen in men with asthenozoospermia
Figure 1. Imprinting status. Sequence analysis of the N18N and S210S polymorphisms in CATSPER1 and CATSPER4 respectively, on DNA and RNA extracted from testis tissue of a heterozygous carrier, shows expression of one allele only.
Discussion
We screened 30 asthenozoospermic men and 90 normozoospermic controls and identified 1 X-linked and 9 heterozygous mutations in AKAP4, CATSPER1-4,
ADCY10 and PLA2G6 that were present in asthenozoospermic cases only. Their
putative effect on protein structure, their location in conserved sequences or functional domains and their absence in controls, suggest that these mutations may be causative in the asthenozoospermia phenotype in humans.
The strength of this study is that we screened men with a single, strictly defined phenotype, i.e. severe asthenozoospermia without decreased sperm numbers, for mutations in nine established candidate genes for this specific phenotype and compared their prevalence with that in men with normozoospermia. The strictness of our inclusion criteria allowed us to identify 30 cases over seven years in a large fertility center. We were limited in testing the functional effects of encountered mutations, as only a functional assay for
ADCY10 is available.
The candidate genes screened in this study have not previously been studied in humans with isolated severe asthenozoospermia and normozoospermic controls. As mentioned earlier, the AKAP4 gene has been analyzed previously,
but in isolated small series of men with DFS21,28,29. As we were unable to study
sperm samples by means of electron microscopy, it remains unknown if the A C A C C A A T A A C G C A G A T A G G T T T DNA T C C T G C A G T C A G T G C C T G A C A G A C A C C A A T A A C G C A G A T A G G T T cDNA T C C T G C A G T C A G T G C C T G A C A chapter 2 32
Figures 2A, B. Expression of the recombinant human ADCY10 and mutant
human ADCY10 in HEK293 cells. Adenylyl cyclase activity was measured in the
cytosolic extracts from HEK293 cells transfected with an empty vector (Control), mutant human ADCY10, and wildtype human ADCY10 pCDNA3.1 plasmids in
the presence of (A) 5 mM MnCl2 and (B) Mg Cl2 with or without bicarbonate
activation. The data reported are representative of at least 3 different experiments.
Mg2+
Figure A
Control MuADCY10 ADCY10
0 1 2 3 4 5 6 7 8 9 10 -HCO3 +HCO3 AC ac tivi ty (p mo le s/ mi n/ mg pr ot ei n)
Control MuADCY10 ADCY10
0 10 20 30 40 - HCO3 +HCO3 AC act iv ity (p mo les/ mi n/ mg pr ot ei n) Mn2+ Figure B
a comprehensive gene mutation screen in men with asthenozoospermia
asthenozoospermic man with the AKAP4 mutation identified in our study had dysplasia of the fibrous sheath.
Mutations in CATSPER1 have recently been reported to cause male
infertility30. However, this study defined affected and unaffected family members
on the basis of (in)fertility, i.e. having or not fathered children, instead of
performing semen analysis in all male family members. As such, the authors may wrongfully claim an association between the described CATSPER1 mutations and male infertility due to sperm abnormalities.
One asthenozoospermia-specific mutation we found in PLA2G6 (rs11570606) is prevalent at a very low frequency in African populations (HapMap data). Our patient carrying this mutation was indeed of African descent, however, since we lack data on spermatogenic phenotype of the HapMap-YRI samples, it remains unclear whether this variant is neutral or asthenozoospermia-related.
For one of the two ADCY10 mutations we were able to assess its effect on protein function. Although we did not observe altered enzymatic function for the mutated ADCY10 protein, we cannot exclude the possibility that the ADCY10 mutation does affect protein function, for instance by altered binding to SLC9A10.
All heterozygous mutations may be causative in a number of ways. First, they may exert a dominant negative effect by binding to the wildtype protein and disabling its function, as previously described for mutations in the SYCP3 gene
and spermatogenesis31. Secondly, for mutations in CATSPER1 and -4, silencing of
the non-mutant allele through imprinting would allow for full penetrance of the mutated allele. Finally, these genes may be dosage sensitive in humans, requiring a specific amount of functional gene copy numbers.
Three patients carried heterozygous mutations in more than one gene. Although the distribution of 10 unique mutations over 6 patients was not unexpected from a statistical point of view, it might indicate that the
asthenozoospermia phenotype followed from a combined effect of the mutations identified. We did not observe an additive effect on semen quality of multiple mutations, although we can not exclude the possibility that some mutations may be more detrimental then others.
In conclusion we identified 10 putatively causative mutations for
asthenozoospermia in humans. Additional functional studies are required to assess if these mutations impair protein function in vivo.
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References
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2. WHO. WHO Laboratory manual for the examination of human semen and
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3. Curi, S.M. e.a. Asthenozoospermia: analysis of a large population. Arch
Androl 49: 343-349 (2003).
4. Thonneau, P. e.a. Incidence and main causes of infertility in a resident population (1,850,000) of three French regions (1988-1989). Hum Reprod 6: 811-816 (1991).
5. Hunault, C.C. e.a. Two new prediction rules for spontaneous pregnancy leading to live birth among subfertile couples, based on the synthesis of three previous models. Hum Reprod 19: 2019-2026 (2004).
6. van der Steeg, J.W. e.a. Pregnancy is predictable: a large-scale prospective external validation of the prediction of spontaneous pregnancy in subfertile couples. Hum Reprod 22: 536-542 (2007).
7. Miki, K. e.a. Targeted disruption of the Akap4 gene causes defects in sperm flagellum and motility. Dev Biol 248: 331-342 (2002).
8. Ren, D. e.a. A sperm ion channel required for sperm motility and male fertility. Nature 413: 603-609 (2001).
9. Quill, T.A. e.a. Hyperactivated sperm motility driven by CatSper2 is required for fertilization. Proc Natl Acad Sci U S A 100: 14869-14874 (2003).
10. Jin, J. e.a. Catsper3 and Catsper4 are essential for sperm hyperactivated motility and male fertility in the mouse. Biol Reprod 77: 37-44 (2007). 11. Miki, K. e.a. Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific
glycolytic enzyme, is required for sperm motility and male fertility. Proc Natl
Acad Sci USA 101: 16501-16506 (2004).
12. Bao, S. e.a. Male mice that do not express group VIA phospholipase A2 produce spermatozoa with impaired motility and have greatly reduced fertility. J Biol Chem 279: 38194-38200 (2004).
13. Esposito, G. e.a. Mice deficient for soluble adenylyl cyclase are infertile because of a severe sperm-motility defect. Proc Natl Acad Sci USA 101: 2993-2998 (2004).
14. Wang, D., King, S.M., Quill, T.A., Doolittle, L.K. & Garbers, D.L. A new sperm-specific Na+/H+ exchanger required for sperm motility and fertility. Nat Cell
Biol 5: 1117-1122 (2003).
15. Kirichok, Y., Navarro, B. & Clapham, D.E. Whole-cell patch-clamp
measurements of spermatozoa reveal an alkaline-activated Ca2+ channel.
a comprehensive gene mutation screen in men with asthenozoospermia
Nature 439: 737-740 (2006).
16. Qi, H. e.a. All four CatSper ion channel proteins are required for male fertility and sperm cell hyperactivated motility. Proc Natl Acad Sci USA 104: 1219-1223 (2007).
17. Carlson, A.E. e.a. Identical phenotypes of CatSper1 and CatSper2 null sperm. J
Biol Chem 280: 32238-32244 (2005).
18. Ford, W.C. Glycolysis and sperm motility: does a spoonful of sugar help the flagellum go round? Hum Reprod Update 12: 269-274 (2006).
19. Xie, F. e.a. Soluble adenylyl cyclase (sAC) is indispensable for sperm function and fertilization. Dev Biol 296: 353-362 (2006).
20. Wang, D. e.a. A sperm-specific Na+/H+ exchanger (sNHE) is critical for expression and in vivo bicarbonate regulation of the soluble adenylyl cyclase (sAC). Proc Natl Acad Sci USA 104: 9325-9330 (2007).
21. Baccetti, B. e.a. Gene deletions in an infertile man with sperm fibrous sheath dysplasia. Hum Reprod 20: 2790-2794 (2005).
22. Avidan, N. e.a. CATSPER2, a human autosomal nonsyndromic male infertility gene. Eur J Hum Genet 11: 497-502 (2003).
23. Zhang, Y. e.a. Sensorineural deafness and male infertility: a contiguous gene deletion syndrome. J Med Genet 44: 233-240 (2007).
24. Rozen, S. & Skaletsky, H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132: 365-386 (2000).
25. Cartegni, L., Wang, J., Zhu, Z., Zhang, M.Q. & Krainer, A.R. ESEfinder: A web resource to identify exonic splicing enhancers. Nucleic Acids Res 31: 3568-3571 (2003).
26. Desmet, F.O. e.a. Human Splicing Finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res 37: e67 (2009).
27. Henikoff, S. & Henikoff, J.G. Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci USA 89: 10915-10919 (1992).
28. Baccetti, B. e.a. Fluorescence in situ hybridization and molecular studies in infertile men with dysplasia of the fibrous sheath. Fertil Steril 84: 123-129 (2005).
29. Turner, R.M. e.a. Molecular genetic analysis of two human sperm fibrous sheath proteins, AKAP4 and AKAP3, in men with dysplasia of the fibrous sheath. J Androl 22: 302-315 (2001).
30. Avenarius, M.R. e.a. Human male infertility caused by mutations in the CATSPER1 channel protein. Am J Hum Genet 84: 505-510 (2009).
31. Miyamoto, T. e.a. Azoospermia in patients heterozygous for a mutation in SYCP3. Lancet 362: 1714-1719 (2003).
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Supplemental materials
Table S1. Candidate genes
Table S2 (1). PCR primers for DNA
Gene Protein product Location Function Knockout mouse AKAP4 A kinase
anchoring protein
Xp11.2 Scaffold for signal transduction and enzymatic molecules
Reduced motility, normal counts and morphology CATSPER1
Cation channel of sperm 1-4
11q12.1 Voltage gated calcium channel
Reduced sperm motility CATSPER2 15q14 Sperm capacitation No hyperactivated
motility CATSPER3 5q31.1 CATSPER4 1p35.5 GAPDHS Glyceraldehyde- 3-phosphate-dehydrogenase
19q13.1 Glycolysis Reduced sperm motility PLA2G6 Group VIa
phospholipase A2
22q13 Hydrolysis of glycerophospholipid
Reduced motility, mildly reduced total count ADCY10 Soluble adenylyl
cyclase
1q24 Convert ATP to cAMP Reduced sperm motility SLC9A10 Solute carrier 9,
family 10
3q13.2 Sodium/proton exchanger Reduced sperm motility
Gene Exon Forward primer (5'®3') Reverse primer (5'®3') Product size (bp) ADCY10 2 CCGCAGGTTAGAACAAAAC GGGCCACAAGCTATTTCCT 467
3 TGCCTTCCAGGGTAGTCCT ACATTTCATGGGGAAGAGG 307 4&5 TTGTCTGGGATGAGATCTTT GAGAGAGATGCCCCAGGAGT 610 5 AGGCTGAGGAATGCTTGATG TGTCCTGATGCCAAAGTTCA 383 6 ACCTGTGCTGCTGGGAAATA GCGTGCCCAGTGACTCTT 350 7 AAGTGTGCCAATCACTGTTCA CTGGTAAGGACCAGGAGCTG 335 8 AGCACTGCAGAGCCAAACTT AATTCCAGCTGTCCAGATCC 206 9 CATGCTGGCTCTGTCATGTT TCACCAATGTGCTTGTCCAT 404 10 GGCCAGTCCAGTTTAAAGCA CTGCATGCCCTCCCTACTTA 250
a comprehensive gene mutation screen in men with asthenozoospermia
Table S2 (2) 11 CCACCCTTCCCACTTTCTCT CAGGGCTCATGTCTCACTCA 252 12 ACTCCCTGCACATTTTGTGA TTGGTCTGGGGAAAGCATAG 504 13 CCGTGAGTCAGGGGATAGAC CCTGGTGTATTGCTATTTTTCT 345 14 CTCCCAAAGTGCTGGGATTA AAGGGTCATGGCATCCAATA 429 15 ACCATTGCGAGATAGCTTGG AGAGAGGATTGGGAGCCATT 471 16 CTAGTCCCTGGTGCCAAAAA TGGAAGGATGGAGGAGAATG 394 17 AAGCAAACCAGGAAGCTCAA GTGGGGCCAGGAAAGATACT 499 18 GCAATGCCTCAAAACTGAAA TCCCCAGAACCTTCTGACTC 403 19 CATCTCTGCCCAGTCTTCCT GGTGTGTGCCTGGCTAATTT 402 20 GATGGTTGTCCACCTGCTCT CGAAGCTGTTCCTCTTCACC 523 21 TGGAGTGACCCAATTTCCTC CCTCAAGATCCACACCCACT 497 22 CAACTCCGAAAAGGGAACAA CCAAAGTTTCCCACTTGCAT 293 23 GCTGGAATTACAGGCGTGAG CATGCTGGAGCTTCCTTCTG 412 24 GGATGGAAGTGGAGGTGGTA GAGCCCAGGGATTTGAGTTT 388 25 ATTATGCCAGGCACAGGAAG AATGGTCAGAGCCAAACAGG 453 26 ACCTGTGCTCCCCTAAATCA GAAGTGATCCTGGAGCCAAA 402 27 GGCCAAATGTCCAGCTCTAT GTTGGGATTCTGGAGCAGTG 450 28 CTGGATTTGGCCATTGAGTT CCTGGGAGACAGAGCAAGAC 378 30 TCTCGAACTCCTGACCTCGT AGCTCACACAACCAGCCTCT 371 31 AAACACAGGGCAATCCAGTC GACACCCTTAAGCTGTCTCAGG 411 32 CCAGGTAAAGGAAGCTGCAC GGGGAAGTGCCTTCAAAAAC 426 33 GTGGACCATGGTGGCTACTC CCAGAGCCTTGATCTTGGAC 569 34 TTTGGACAGCAAGATTCGTTT GCCACGGAGAAAGGTCAATA 376 AKAP4 1 GGCAGTCAAGGCTGTAGGAG TTTTCTGGGTATCCCAAATAAAAG 271 2 CCCCTCCAGAGATTCTTATCC CTTGGGCCCCAGGATATCTA 254 3 TTAATCCTCACAAGAACCCTCTG CCCCCAACATTTTCCATTCT 250 4 GGGGCAGCTTGGAGAAATA TTCAAATTGATGAGCCTGTTTG 281 5 CGTTCCCATACAAACTCCAAA TCTCCATCAGGGGAAATGAC 472 6 GGGCGAATATCACAGAGCAT CCACAGCTTCATAGGCCATT 407 7 AAAGTCCTTCAGCTCCTCCA TGCCACCTGATTTGCATAAA 458 8 CCAACAAGAGCAAAAGTGGAG GCCTTTGTCCCTCTCTTTCA 521 9 ATCCCAAATGCAGGAATCAG ACATGGAAGCTGCTTTGCTT 630 10 CTGGACTCCCAGAAAATGGA GAGCATGGGCACGTTAAACT 519 11 GGCAAATAAGCCCAATTTCA TGGTTCACCGTTTTTGTGTC 382 12 CTTCGTCCAGCAGGTCTTTC TTCACAGGCAACTGCTCAAG 358 CATSPER1 1I ATTGAGCTTGGCTCAGGAAA CGTTGGAGCTCATCATGGTA 455 1II CACCAATCTCACCACCACAG CACAGAGGAATGAGGGGAAA 534
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Table S2 (3)
1III ATCACCAAGTCCACCACCAT TTTTGGACATCTGGGTGACA 480 1IV CCCCTATCACGTAGCACACC GAACAAACACCTCATGGCCTA 378 2 CAGCTACTTGGGAGGCTGAG CATCTACTTCCCAGGGGTGA 505 3 TGTGAGGCTAATGCTGAGGA TGCATCAGCAAGAAGTCCAG 358 4 GCTGCGTGTGTTTAGTCTGG GTGCATGACAGAAGGCAGAA 400 5 TCACCCAAGTCACCAACAAG GCCAAACTCAGCTCTCCAGT 338 6 CCTGCCTCTGTATCCTGGTG GACCTGCTCCCTTCCCATAC 420 7&8 GTATGGGAAGGGAGCAGGTC GCTCCGTCAGTGAGTCTTCC 551 9 CCCAGCCTGGTGATTACTGT AGGTTAGGGGGATGGAGAGA 327 10 CCTGGCCAGAAACTCCATTA CTTCTAGAGGGGCAGGCAAT 282 11&12 AGGAGGCAGGCAAGAAGAG CGGACAATCATTCCAGCAG 484 CATSPER2 2 TGTTTCAAAACACCCTGAGTCC AATCATCCATGGCTAGTGGCTA 529 3 AGGGGAACCCATTACCTGAC GTGGAGTATGGGGAGCAAAA 351 4 TTTTGCTCCCCATACTCCAC CTGGTCTCCAACTCCTGAGC 305 5&6 CTAGAGAGGAAATTTGCATTCAGTT AAGCTCACCTGGCAATCC 1984 7 GTGCCATGGTTCAAAGATGA TTGGACAAGCTTCCTGGTCT 331 8&9 TCTCTGCTTCTGTCTATTCACATGT GAAAAGTGGGGTCGAGAAGT 617 10 TTCTCCCCAATTAGTTTTCTTCA ATTTCGGGTGCCACTAGTCA 305 11 CAGGGGCAGAACCAGAATAA GGGAAAGGGTGCATCAGTAA 530 12 TCCACCCACACCATAGGATT TGAGAAGCTGAGATGCCAGA 352 13 TGGCAAAGAGTACACAGAATCA ACGCCTGGCCTAGACACTTA 412 CATSPER3 1 GCAACAGAATGCCCAAGAGT TGAGGGAAGAGGCTGAGAGA 344 2 AGCAGAGCCTTGTCCCTACA GCACTCCAGCCTGACAGAGT 364 3 GACTTAGGAGCACCCCTTCC CTTAGTGTCTTGGGGGCAAA 410 4 AGCTGTGAACTGGGCAGAAT TCCCTAGGCCCAGATGAGTA 403 5 GTGGACTGGGAGGTGTTGAG CTGGAGACTTGCCCTACAGC 355 6 GAAAAGTGCTCCTGCTGAGG AGGCCACCTGCTCTCTACAC 340 7 GGTTCAGAAGGGGTCAGGAT CAGTCATCACCCCCTTCACT 413 8 CTGGCTCTGAAGTCCTTGCT CTGTCCAGGCCCAACTCTAA 371 CATSPER4 1 TCCAGGTTGGAATTCCTCAG GAGGGGCAGAGTTACCATGA 425 2 TGTCTCCATTCCATGATCCA GACCCTTGAAGAGCATCCAA 406 3 AAGGTTGCTGAGGACCAAGA TGTAGGGCCAGGAGTACAGG 315 4&5 AGGGTAACAGCAGTGGATGG ATGAACGCTGCTGAGGAAAT 672 6 CTGTTTCCCTTCCCTGAACA TCTACTGGGCCATCCTCATC 430 7 TCTGCACAACAGCAGTGACA TCTCCAGGCCCTTGATTATG 409 8 GACTCATCCTGAAGCCAAGC ATAAATGGTGAGGGCGTGAG 391
a comprehensive gene mutation screen in men with asthenozoospermia
Table S2 (4)
9 TTGGTGGAGGAACTGGAATC GGGGACCAGATCTTGGAATA 464 10 AAGCTGGTGAGACTGGAGGA GCCCAGGTATAAGGGAGAGG 260 GAPDHS 1 CCTCGGTAACATCACAGCAG CTCCCCACTACCCTTTCCAC 107 2 ACACTGGGCTAAATGGTTGG GTCGGAACGAAAGCATTGAT 413 3&4 GAGCCTCCTCTCCATTACCC AGAGGCTGGAATCCCATTTT 518 5&6 GACTATGAAGGTGGGGCTCA CCAAGCTTGTCCTCTGGAAG 503 7 CCCTCATCCTGACGTTTCAT TGAGGGTTGCACAGTGTTTT 355 8 CACACCTAGGCCACCAACTT CCTGTCAGCTTCCTGGAGAA 427 9 GGCCCTCAGTCCTTAAGAGG GACTTGGCACATTGAGAGCA 367 10&11 CATGGATGGGCGATAGAGTT GCAGCCAGATGCTGGAAC 429 PLA6G2 2 GGGAGTGATCTGGGTGTCTG AAGTGTGGAGTCTCGGAAGC 532
3 TCTGATTCCAGCAGGGATGT AGTGCTGGGACTGCGTTAGT 441 4 AAAGTCCGAGTTTCCGAGTG TGAGAGGCCTGAGAGTGACA 387 5 GCCTCCCAAAGTGCTAGGAT GGCCTAATGAGGCTTGTGAA 441 6 CTCCAAGTCCTGGCTTCATC GCTAGCCTCAGGGTCCTCTC 272 7 GGCCTCTCAGAGCAGAAGTG GGAGCAGCTGACGATAGGAG 388 8 GCTGGGTGAGTTGACAGGTT CTCCTCCTCGGTCCCTGTAT 274 9 TAGGAAGCTGCTTGGGATGT TAGGGATCCTGTTGCTTTGG 446 10 CTAGGGACCTCTGGGGTAGC GCTGTGAGGGTGCAGAGAGT 256 11 AGGGCTATGAGGGTGGATGT AGCCCTCCTCTACTCCTCCA 351 12 TTGCTTAGGCCTCGGTAAAC GCTCAGCAGGTGTCTGCAT 472 13 AATTGTGGGGAAAGGGAAAG GATGGCAAGTGCACGACTC 290 14 GATCTGGAGTGCATGGGTTT TGTGTAAAAGCCACCTGCTG 332 15 GACCTCTTGCCCTGTTCTCA GTCTCCTCCAACACCAAAGG 388 16 GCTCAGCCTGACTCGAAAGA GGGAAGGGAAAGTCCACACT 298 17 CCAGCCCTAGTGTGGACTTT CGGGGTTAGTGGGAGACAG 459 SLC9A10 2 TTAGAAGGCCACCAAGAAGG TTTGATTGAAAATATTGAGCTAAAGG 356 3 GCTGATAAGTGTGAGGGAGGA GGCCTGGCTCCTTTTTCTAC 415 4 GCCATGGCCAATTATTTGTA ATTTTTCCCCTGGGTCTTTG 499 5 TCTGCTGGTGGGTACTGTTG AACCCCCAATCCCTCACTAC 411 6 GGCCATAATTAATTTAGTGCCAAT CAAAATACACATCAGCTTTCAGA 364 7 TCCCAGGGATATCTGCACTC TGCCTGGTATGCATTTTTCA 388 8 GAAAAAGGACATGGGGTGGT TGGAGGATTCAAAGACACAGG 456 9 TCATCATTTTCAGCTACAAGTCAA TTTGCTGATGATCCTTGGAA 427 10 TGGAATACCTGCTGCCTTTC TTGCCAGTTGTTCCTCCTCT 501 11 CCCTGCCATGAGCATCTATT CCACGCCTGGCTAATTTTTA 371 12 TGCCTCACCTTTTGTGAGAA CCCCACAGTGATCATGAAGTT 542 chapter 2 40
Table S3. PCR primers for cDNA Table S2 (5) 13 AAAATTGCTTGTGGATTTTGC CAAACTTCCCTTTGCCTTTG 327 14 TCACACATTTTTCCCATCAAGA GGGAGCCAAGTTACCAGACA 278 15 CAACCATCATGGCACAAAAT GAATGAAGCACACCAACGTG 427 16 GTGGTGGCTGCTTTCAGAAT TTGTCTGTTGCTGGTGTTGA 412 17 TGAACTTGTTTGTTTGATTTTGGT TTTGCCAAAGGAAGCTAAAA 483 18 TTTCACATAGGTACCCCCAAA AAATGATGGATGAGGGCAAC 436 19 AATTCGAACCCCTGAAAACC AAAGGCCTTCATTGTTGCAT 418 20 CTCTGTGTTGAGGGCGGTAT GCTGCAGGAAGGAAACTGTC 433 21 GAGGTTGAAAATGTTCATGGAAG CAAAGCTGAAGTAGGCAGGAA 453 22 AGCAATTCTCCTTGGCTTGA AGGCTGAGGCAGGAGTATCA 434 23 TTGTTCAAACCAAAGTGGAAAA AGCTATTTTGCCAGGGCTTT 448 24 CATAGGGAAAGGACACACCA TGCATATTGTAGTTCCAATCCTGT 465 25 TGAATGGAATGGCATTAAAACA GGGAAAAGGGAGAAGATTGG 405 26 TTTTCCATTTTTCCCAGATACC GGCAGTTAAAAGGATTCCTCAA 361 27 CCACCAATTTCAGATCACAGG TCCACTGTACCCAGTGCTAAAA 340 28 TCCACCTTGCGAGAAATACC TGGCAGTATATAGAGAGGAAACCA 445 30 TGCTCAATTTCCTTCCTTGG CACACAGGATCCAACCTGAA 278
Gene Exon SNP* Forward primer (5'®3') Reverse primer (5'®3') Product size (bp) ADCY10 7 T234M CGGAGCATGATTGAAATTGA AGGGACATCTCCAGTTCAGG 200
11 G385G TGCTCTGGAATGTGCTATGG GACAGAGTCGCAGGTGACAA 208 17 I697V GTCCCTGTGTCCCTTCGTTA CCGTTTGTTGGAAAACGAGT 257 CATSPER1 1 N18N CAGAGTTCCCAGCACAGTCA CACCGAGATATTGGGGTCTG 1400
1 G133S CAGAGTTCCCAGCACAGTCA CACCGAGATATTGGGGTCTG 1400 2 R408R CCCAGGATATCTCCACCAAA TGTTGAGGCAGACAACGAAG 488 CATSPER2 3 V57I CTCTCTCATTGAGCATTTGCAA ACCTCCAAGGTCAGCTTCAA 366 CATSPER3 4 A169A/A215A AGCTCATGGGCAAACAGTTC CAAAGCAAATTCCCGATTGT 332 CATSPER4 2 Q77R/I108I ACCATTCACGAGTCCTAC AGCCAAGGAGAACCTCACAA 289 5 S210S CTACACTCTCAGGGCGCTTC TCATCATTTGCTCCAGGTTG 398 7 I293V CTACACTCTCAGGGCGCTTC TCATCATTTGCTCCAGGTTG 398 GAPDHS 6 V203V GGGAAGTGTGGAATTCAGGA AATCGCTCGTGGATGACTTT 336 11 Y392H AGGAGGCTGTAAAAGCAGCA AGGACCTTCCCGTTTCACTT 250 SLC9A10 8 I286V GGATGTCAACTGTTTTTGGTGA GCAGGAATTAGAAGTCCAAGAA 250 17 T705I CCTGGAACATATTCGAGTTAGCA TGACAGCAATTTCTGGGTGA 390 18 I768S CCTGGAACATATTCGAGTTAGCA TGACAGCAATTTCTGGGTGA 390
a comprehensive gene mutation screen in men with asthenozoospermia
Table S4 (1). All variants found
Patients (n=30) Controls (n=90)
Gene Varianta Location
Predicted effect
on translation WT HET HOMO WT HET HOMO pb
ADCY10 c.293-68A>C int4 29 1 0 ND ND ND
rs16859886 ex7 p.Thr234Met 24 6 0 76 14 0 0.58 c.739+30G>A int7 29 1 0 ND ND ND
c.1020+20T>A int9 29 1 0 ND ND ND
c.1020-40A>C int9 Alters splice site 29 1 0 90 0 0 0.25 rs203849 ex11 p.Gly385Gly 7 18 5 ND ND ND c.1216+26C>T int11 22 8 0 ND ND ND c.1216+35T>C int11 15 14 1 ND ND ND c.1406+36G>A int12 28 2 0 ND ND ND c.1463-31G>A int13 24 6 0 ND ND ND c.1718C>T ex14 p.Val511Val 22 7 1 ND ND ND c.1897-41G>A int16 21 7 2 ND ND ND c.1968T>C ex17 p.Phe656Phe 26 4 0 ND ND ND rs2071921 ex17 p.Val697Ile 3 10 17 13 50 27 0.04 c.2097A>G ex17 p.Ala699Ala 28 2 0 ND ND ND
c.2125A>G ex17 p.Ile709Val 29 1 0 89 1 0 0.44 rs203795 ex19 p.Gly799Gly 15 10 5 ND ND ND
c.2438-13C>T int19 24 6 0 ND ND ND c.3008-28C>T int21 29 1 0 ND ND ND c.3310-20A>G int23 27 3 0 ND ND ND
c.3542A>G ex25 p.Asn1181Ser 29 1 0 90 0 0 0.25 c.4141-37C>T int26 26 4 0 ND ND ND c.4353+44G>A int28 14 12 4 ND ND ND c.4058C>T ex29 p.Pro1354Leu 29 1 0 81 9 0 0.45 rs61745242 ex31 p.Ile1463Ile 29 1 0 ND ND ND chapter 2 42
Table S4 (2)
c.4482+33T>A int31 18 11 1 ND ND ND
AKAP4 c.887G>A ex6 p.Gly296Asn 29 na 1 90 na 0 0.25 CATSPER1 rs1893316 ex1 p.Asn18Asn 17 12 1 ND ND ND
c.144C>T ex1 p.His48His 29 1 0 ND ND ND
c.148G>A ex1 p.Val50Met 29 1 0 90 0 0 0.25 rs1203998 ex1 p.Gly133Ser 13 13 4 51 30 9 0.40 rs2845570 ex2 p.Arg408Arg 7 16 7 ND ND ND rs35484336 ex2 p.Thr430Thr 29 1 0 ND ND ND c.1467C>T ex3 p.Cys489Cys 26 4 0 ND ND ND rs34114713 int3 15 13 2 ND ND ND c.1884G>A ex6 p.Thr628Thr 29 1 0 ND ND ND rs3814747 ex7 p.Val652Ile 29 1 0 86 4 0 1.00 rs3814748 int8 29 1 0 ND ND ND c.2065-50G>C int8 29 1 0 ND ND ND rs3829937 ex9 p.Ala690Ala 23 6 1 ND ND ND
CATSPER2 rs8042868 ex3 p.Val57Ile 17 12 1 68 19 3 0.13 c.216G>A ex3 p.Gln72Gln 29 1 0 ND ND ND
c.1073G>A ex9 p.Arg358Gln 29 1 0 69 1 0 rs7169112 int10 25 5 0 ND ND ND rs7167634 int10 25 5 0 ND ND ND c.1179-3A>C int10 25 5 0 ND ND ND
rs7169097 int10 alters splice site 25 5 0 62 26 2 0.32 rs3853543 ex11 p.Thr425Arg 29 1 0 88 2 0 1.00 c.1289C>G ex11 p.Thr430Arg 29 1 0 90 0 0 0.25 c.1316-1317del2 ex11 p.Glu439ValfsX19 27 3 0 86 4 0 0.37 CATSPER3 c.193T>C ex2 p.Phe65Leu 29 1 0 90 0 0 0.25
rs10044060 ex4 p.Ala169Ala 10 13 7 ND ND ND
rs3896260 ex4 p.Asn204Lys 26 3 1 82 8 0 0.29 rs177252 ex4 p.Ala215Ala 16 11 3 ND ND ND
a comprehensive gene mutation screen in men with asthenozoospermia
Table S4 (3) c.676-35C>T int4 23 5 2 ND ND ND rs17167765 ex7 p.Tyr352Tyr 28 2 0 ND ND ND rs7719733 int7 28 2 0 ND ND ND c.1095-83G>T int7 29 1 0 ND ND ND rs7719874 ex8 p.His375His 28 2 0 ND ND ND
CATSPER4 rs41284333 ex1 p.His50Arg 18 10 2 59 27 4 0.76 c.157T>C ex1 p.Tyr53His 29 1 0 90 0 0 0.25 rs11247865 int1 17 11 2 ND ND ND
rs11247866 ex2 p.Gln77Arg 18 10 2 59 28 3 0.69 c.247A>G ex2 p.Met83Val 29 1 0 90 0 0 0.25 rs11247867 ex2 p.Ile109Ile 18 10 2 ND ND ND c.358+50G>A int2 29 1 0 ND ND ND rs12138368 ex3 p.Leu124Phe 25 5 0 76 13 1 0.83 c.459+16C>T int3 29 1 0 ND ND ND c.459+45A>C int3 29 1 0 ND ND ND c.557+6C>A int4 29 1 1 ND ND ND rs57653638 int4 18 9 3 ND ND ND rs61776651 ex5 p.Ser210Ser 21 9 0 ND ND ND c.679-28C>T int5 28 1 0 ND ND ND rs9970046 int5 23 7 0 ND ND ND c.813-27G>C int6 17 12 1 ND ND ND rs17257155 ex7 p.Ile293Val 17 12 1 60 28 2 0.47 c.987+5G>A int7 Alters splice site 20 10 0 57 27 6 0.47 c.988-11C>G int7 17 13 0 ND ND ND
c.988-17T>C int7 18 12 0 ND ND ND
c.992G>A ex8 p.Gly331Asp 29 1 0 90 0 0 0.25 c.1035T>C ex8 p.His345His 29 1 0 ND ND ND
c.1200-35G>A int8 25 3 2 ND ND ND c.1200-36A>C int8 25 3 2 ND ND ND
chapter 2
Table S4 (4)
rs17163674 ex9 p.Arg406Arg 22 5 3 ND ND ND rs41310785 ex9 p.Glu413Glu 27 3 0 ND ND ND c.1296G>A ex9 p.Thr432Thr 29 0 1 ND ND ND
rs6657616 ex9 p.Asp436Asn 24 3 3 62 24 4 0.09 GAPDHS c.68-5C>T int1 Alters splice site 29 1 0 89 1 0 0.44
c.165G>A ex2 p.Pro55Pro 29 1 0 ND ND ND c.245+7C>T int2 29 1 0 ND ND ND
c.393G>T ex3 p.Ala93Ser 29 1 0 84 6 0 0.68 rs2239945 ex6 p.Val203Val 21 8 1 ND ND ND
c.660-22G>A int6 20 8 2 ND ND ND
c.1272T>C ex11 p.Tyr386His 28 2 0 88 2 0 0.26 PLA2G6 rs2267369 ex2 p.Val29Val 26 4 0 ND ND ND
rs11570606 ex2 p.Arg63Gly 29 1 0 90 0 0 rs2267368 int2 26 4 0 ND ND ND rs11570647 int5 26 4 0 ND ND ND rs11089868 int5 23 7 0 ND ND ND c.894+35G>T int6 26 4 0 ND ND ND c.894+43C>T int6 15 13 2 ND ND ND rs11570679 ex7 p.Thr319Thr 26 4 0 ND ND ND rs11570680 ex8 p.Ala341Thr 29 1 0 85 5 0 1.00 rs2413502 ex9 p.Asn362Asn 26 4 0 ND ND ND c.1427+48T int10 29 1 0 ND ND ND c.1742+13G>A int12 29 1 0 ND ND ND c.1742+23C>T int12 29 1 0 ND ND ND rs2076114 int15 26 4 0 ND ND ND SLC9A10 c.190-36_ 190-29dup int3 26 4 0 ND ND ND rs12632408 int4 18 10 2 ND ND ND rs9809174 int4 29 0 1 ND ND ND
a comprehensive gene mutation screen in men with asthenozoospermia
