Molecular Human Reproduction, Vol. 9, No. 10, 587-592,
October 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Chromosomal region 11p15 is associated with male factor subfertility*
Submitted on June 9, 2003; accepted on June 26, 2003
1 Center for Reproductive Medicine, Department of Obstetrics and Gynaecology, 2 Department of Clinical Genetics and 3 Department of Clinical Epidemiology and Biostatistics, Academic Medical Center Amsterdam, 4 Department of Obstetrics and Gynaecology, Academic Hospital Maastricht, 5 Department of Obstetrics and Gynaecology and 6 Department of Human Genetics, University Hospital Nijmegen, The Netherlands
7 To whom correspondence should be addressed at: Center for Reproductive Medicine, Department of Obstetrics and Gynaecology, Academic Medical Center, Meibergdreef 9, H4-205, 1105 AZ Amsterdam, The Netherlands. e-mail: J.Gianotten{at}amc.uva.nl
*Partly presented orally at the 19th annual meeting of the European Society of Human Reproduction and Embryology in July 2003, Madrid.
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Abstract |
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The molecular aetiology of male factor subfertility, due to impaired spermatogenesis, is still unknown in the majority of cases. It is thought to be a complex disorder in which multiple genes are implicated. Cryptorchidism and reduced fecundity are symptoms in male Beckwith–Wiedemann patients and the ZNF214 and ZNF215 genes, localized on chromosomal region 11p15, are associated with this syndrome. We hypothesized that the ZNF214 and ZNF215 genes, which are predominantly expressed in the testis, could be involved in male factor subfertility in patients with idiopathic impaired spermatogenesis or in patients with impaired spermatogenesis due to cryptorchidism. Male partners of subfertile couples with idiopathic azoo- or severe oligozoospermia, male partners with azoo- or severe oligozoospermia and cryptorchidism in their medical history and men with normozoospermia were screened for nine single nucleotide polymorphisms in the ZNF214 and ZNF215 genes. An association study was performed based on allele and estimated haplotype frequencies. Statistically significant differences in allele frequencies and in estimated haplotype frequencies were found in both patient groups compared with controls. Thereafter, both genes were screened for mutations in all patients by PCR and single strand conformation polymorphism analysis. Aberrant patterns were confirmed by DNA sequencing. Mutation analysis in ZNF214 and ZNF215 revealed five new variants in the patients that were not present in the controls. At least three of these mutations were inherited from the mother. Our results suggest that chromosomal region 11p15 is associated with male factor subfertility due to impaired spermatogenesis with and without cryptorchidism.
Key words: genetics/impaired spermatogenesis/male subfertility/ZNF214/ZNF215
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The molecular aetiology of male factor subfertility due to impaired spermatogenesis is still unknown in the majority of cases. So far, it is known from cytogenetic analysis that the incidence of numerical and structural chromosomal abnormalities in men with azoospermia or severe oligozoospermia is 4–6% (Tuerlings et al., 1998; Hargreave, 2000; Dohle et al., 2002). These chromosomal aberrations have been shown to cause meiotic abnormalities, resulting in spermatogenic failure (Chandley, 1979; Quack et al., 1988). In addition, four classes of Y chromosome deletions cause spermatogenic failure: AZFa (Azoospermia Factor a), P5/proximal P1, P5/distal P1, and AZFc deletions (Reijo et al., 1995; Vogt et al., 1996; Repping et al., 2002). Deletions of the AZFc region are most frequent, as they occur in 6–12% of azoospermic or severely oligozoospermic men (Kuroda-Kawaguchi et al., 2001).
Thus, until now, impaired spermatogenesis can only be explained in a very small proportion of subfertile patients, which is not surprising as male factor subfertility is thought to be a complex disorder in which multiple genes are involved as suggested by gene targeting studies in Drosophila and mice (Grootegoed et al., 1998; Hackstein et al., 2000; Venables and Cook, 2000).
Recently we described a potential role of chromosomal region 11p15, and in particular of the Zinc Finger (ZNF) 214 and ZNF215 genes located in this region, in the aetiology of Beckwith–Wiedemann syndrome (BWS) (Alders et al., 2000), a congenital overgrowth disorder defined by a diversity of symptoms that can occur in various combinations. Cryptorchidism (Pettenati et al., 1986; MIM: Mendelian Inheritance in Man, http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=OMIM; Nowotny et al., 1994; Benacerraf and Bromley, 1998) and reduced fecundity (Moutou et al., 1992) are among the clinical findings described in male BWS patients. Interestingly, the ZNF214 and ZNF 215 are predominantly expressed in testis (Alders et al., 2000). In addition, a balanced translocation with a breakpoint at chromosome 11p15 has been described in a patient with severe oligozoospermia (Pernice et al., 2002).
On the basis of these observations, we hypothesized that the chromosomal region 11p15 might play a role in male factor subfertility due to impaired spermatogenesis, in the presence or absence of cryptorchidism in the patients’ medical history. To test this hypothesis we conducted an association study based on allele and estimated haplotype frequencies of common single nucleotide polymorphisms (SNP) of the ZNF214 and ZNF215 genes.
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Materials and methods |
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Patients
Male partners of subfertile couples from four Dutch fertility clinics (Academic Medical Center and the University Hospitals of Groningen, Maastricht and Nijmegen) were included in this study. Written informed consent was obtained from all participants and the Institutional Review Boards of all participating centres approved the study.
Male partners of subfertile couples with azoo- or severe oligozoospermia were divided in two patient groups. The first patient group consisted of patients with idiopathic azoo- or severe oligozoospermia. The second patient group consisted of patients with azoo- or severe oligozoospermia and cryptorchidism (uni- or bilateral, inguinal or abdominal) in their medical history.
Severe oligozoospermia was defined as a total sperm count of <20x106 in two consecutive semen samples. Semen analyses were performed according to the World Health Organization (1992). Patients with a history of orchitis, alcohol abuse, surgery of the vasa deferentia, bilateral orchidectomy, chemo- or radiotherapy, obstructive azoospermia (confirmed by testicular biopsy), and with numerical chromosomal abnormalities or microdeletions of the Y chromosome (Simoni et al., 1999) were excluded. Only patients of Caucasian origin were included in the study.
Male partners of subfertile couples with normozoospermia were included in the control group. Normozoospermia was defined as a total sperm count >40x106 with a progressive motility and normal morphology of 
40%, in two consecutive semen samples. Only controls of Caucasian origin were included in the study.
All patient data concerning levels of LH, FSH, prolactin, 17ß-estradiol and testosterone, and testicular volume (measured at physical examination using the Prader orchidometer) were retrieved from the patient’s medical records. Blood was drawn for DNA isolation from all patients and controls.
Genotyping of SNPs in the ZNF214 and ZNF215 genes
DNA was isolated from peripheral blood cells as described previously (Miller et al., 1988). The ZNF214 and ZNF215 genes were screened for the variants of nine SNPs (Table I). The cDNA sequence of ZNF214 (NM_013249) and ZNF215 (NM_013250) was used as the wild-type sequence. All variants tested have been previously detected in a mutation screening (data available on request) (Alders et al., 2000). Variants Y66C, L128H/R, I185R and R252C in the ZNF214 gene were detected by PCR and restriction enzyme digest analysis. The F292F variant in the ZNF214 gene and the M119V, G260G and L323V variants in the ZNF215 gene were detected by PCR and single strand conformation polymorphism (SSCP) analysis as described below.
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Mutation analysis and sequencing
The coding regions of ZNF214 and ZNF215 were amplified by PCR. The amplified fragments were analysed by SSCP analysis on 12.5% non-denaturating polyacrylamide gels (GeneGel Excel 12.5/24; Amersham Biosciences, Sweden). Gels were run at 5 and 15°C following the manufacturer’s recommendations and stained using the DNA Silver Staining Kit (Amersham Biosciences). PCR products presenting aberrant conformers were reamplified from genomic DNA and were sequenced in both directions by the fluorescent dideoxy chain-termination method on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, USA).
Statistical analysis
Allele frequencies were estimated by gene-counting, and departure from Hardy–Weinberg equilibrium within the study groups was tested using a 
2-test with 1 degree of freedom (d.f.) for the bi-allelic loci and 2 d.f. for the tri-allelic locus. Association between the phenotype and the alleles of the nine SNP was examined by comparing the allele frequencies between both patient groups and the control group using Fisher’s exact test.
Haplotype frequencies were estimated with an expectation-maximization (EM) algorithm as implemented in the Arlequin software package (Schneider et al., 2000). Differences in haplotype frequencies between both patient groups and the control group were examined using a log-likelihood ratio statistic (Zhao et al., 2000) that was computed from the estimated haplotype frequency log-likelihoods for a patient and the control group separately versus combined. This test statistic roughly follows a 2 distribution with k – 1 d.f., where k denotes the number of haplotypes considered. Individual haplotype odds ratios (OR) and 95% confidence intervals (CI) were estimated using a method described by Tanck et al. (2003) adapted for logistic regression. In short, haplotype effects and haplotype frequencies were jointly estimated using an EM algorithm in which individual haplotypes were handled as missing data. Throughout the analyses, P < 0.05 was considered as statistically significant.
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Results |
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Patients
In total, 62 men with idiopathic azoo- or oligozoospermia, 64 men with azoo- or oligozoospermia and cryptorchidism in their medical history and 72 men with normozoospermia were included in this study. A reduced testicular volume (<15 ml) was found in 63% of the patients with idiopathic impaired spermatogenesis and in 62% of the patients with cryptorchidism. Hormone levels showed an elevated FSH (>7.5 IU/l) in 54% of the patients with idiopathic impaired spermatogenesis and in 73% of patients with cryptorchidism. No other physical or hormonal abnormalities were identified. In the patient group with cryptorchidism, 51% had unilateral and 33% bilateral cryptorchidism, 16% of patients did not know whether the cryptorchidism was uni- or bilateral. None of the controls mentioned cryptorchidism in their medical history.
Allele frequencies of SNPs in the ZNF214 and ZNF215 genes
No significant deviations from Hardy–Weinberg proportions were observed at the SNP loci in the three study groups. In patients with idiopathic impaired spermatogenesis, significantly different allele frequencies were found for the L128H/R variant compared with the control group (P = 0.032) (Table I). In subfertile patients with cryptorchidism in their medical history, a significantly increased frequency was found for allele 1 of the Y66C variant compared with the control group (P = 0.019). All other variants showed no differences in allele frequencies between the two patient groups and the controls.
Haplotype analysis
Based on the observed individual genotypes, 27 haplotypes were estimated to be present in the three study groups (Table II). Haplotypes 21–24 were the most frequently observed haplotypes in the study groups, accounting for 50% of patients with idiopathic impaired spermatogenesis, for 44% of patients with impaired spermatogenesis and cryptorchidism and for 50% of the control group. The estimated haplotype frequencies differed significantly between the patients with idiopathic impaired spermatogenesis and the controls (P = 0.021; 25 d.f.). The difference in estimated haplotype frequencies between the patients with cryptorchidism and the controls was not significant (P = 0.089; 23 d.f.).
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Remarkable differences in individual haplotype frequencies were found for haplotypes 2 and 8, carrying both the 1 allele of the Y66C and the L128H/R variant. Haplotype 2 (1 1 1 1 1 1 2 1) was present in 11% of patients with idiopathic impaired spermatogenesis and in <1% of the controls [OR 13.3 (95% CI 2.0–88.9)]. Also in the group of patients with impaired spermatogenesis and cryptorchidism, the estimated frequency of haplotype 2 (4%) tended to be higher than that in the control group [OR 7.1 (95% CI 0.8–59.2)], but this difference was not significant. Haplotype 8 (1 1 2 1 1 1 2 1) occurred with an estimated frequency of 11% in the group of patients with impaired spermatogenesis and cryptorchidism whereas it had a frequency of 3% in the control group [OR 3.1 (95% CI 1.1–9.0)]. The frequencies of this haplotype in the idiopathic patients and the controls were not significantly different [OR 0.7 (95% CI 0.2–2.9)].
Mutation screening
Based on the association of chromosomal region 11p15 with impaired spermatogenesis observed both in patients with idiopathic impaired spermatogenesis and in patients with cryptorchidism, all patients were screened for mutations in the coding sequences and flanking intron sequences of ZNF214 and ZNF215. In addition to the known SNP, five sequence variants were identified in patients that were not present in the controls (Figure 1 and Table III). The first variant detected in the ZNF214 gene was a G
A transition that changes a cystine at amino acid position 224 into a tyrosine (C224Y). This variant was detected in a patient with cryptorchidism and was inherited from the mother (data not shown).
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In a patient with idiopathic impaired spermatogenesis we identified an in-frame insertion of three nucleotides, which leads to the insertion of an additional amino acid (asparagine) after glycine at amino acid position 271 in the first zinc finger domain of ZNF214 (816–817InsTAA). The parents were not available to study the inheritance pattern of this variant.
The third variant detected in a patient with idiopathic impaired spermatogenesis was a missense mutation at codon 408 in the ZNF214 gene. The CT transition replaces a histidine with a tyrosine residue. This patient also inherited the H408Y variant from his mother and this variant was not present in the patient’s brother who had a sperm count within the normal range (Figure 2).
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In the ZNF215 gene, an A
G transition was identified in a patient with idiopathic impaired spermatogenesis, resulting in a substitution of an isoleucine for a valine (I400V). Also in this case the variant was inherited from the mother.
The fifth variant detected was a missense mutation at codon 496 in the ZNF215 gene. A GT transversion results in a serine replacing an isoleucine at amino acid residue 496 (S496I). This variant was found in one patient with idiopathic impaired spermatogenesis as well as in a patient with cryptorchidism. In both cases the parents were not available for DNA analysis.
These five variants were not present in the controls and, apart from the common polymorphisms, were the only sequence variations identified throughout the coding and splice site consensus sequences of the ZNF214 and ZNF215 genes.
In ZNF215 we identified an additional GA transition occurring at the first nucleotide position of intron 6 and disrupting the invariant GT dinucleotide, which is part of the donor splice site sequence of intron 6. However, this variant was found in one control man as well as in two patients (Table III).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this paper we report an association between male factor subfertility due to severely impaired spermatogenesis, with and without cryptorchidism, and chromosomal region 11p15. This region harbours the ZNF214 and ZNF215 genes, originally investigated in relation to BWS and presumably acting as transcription factors.
Statistically significant differences in allele frequencies were found in patients with idiopathic impaired spermatogenesis and in patients with impaired spermatogenesis and cryptorchidism compared with men with normozoospermia. Estimated haplotype frequencies also differed significantly between the patients with idiopathic impaired spermatogenesis and the controls. Consistent with these findings, a number of mutations were found in both patients with idiopathic impaired spermatogenesis as well as in patients with impaired spermatogenesis and cryptorchidism. Taken together, these data validate our initial hypothesis and indicate that this region is associated with idiopathic impaired spermatogenesis as well as with impaired spermatogenesis due to cryptorchidism.
Cryptorchidism is one of the symptoms described in BWS patients. Both cryptorchidism and the BWS are embryonic developmental disorders, which might explain a possible involvement of chromosomal region 11p15 in both disorders.
In this study 50% of patients in whom a mutation was found reported a history of cryptorchidism. Studies on cryptorchidism do present with some intrinsic difficulties. First, the descent of the testes during childhood has not always been registered accurately. Second, the difference between cryptorchidism and retractile testes is not always clear, and formerly orchidopexy was performed to correct both conditions. For these reasons, cryptorchidism might be under- or over-diagnosed in our study population.
In this paper we describe six new variants occurring in the coding regions and intron–exon boundaries of the ZNF214 and ZNF215 genes. So far the specific biological function of the proteins coded by the ZNF214 and ZNF215 genes is not known, although it has been suggested that they might act as transcription factors through binding to specific DNA sequences (Alders et al., 2000). Therefore it is not yet possible to define the functional consequences of these mutations and firmly establish a causal relationship between the presence of the variants and the phenotype. However, two of the variants identified in ZNF214, 816–817InsTAA and H408Y occur in the conserved consensus sequence of the first and sixth zinc finger domain respectively. 816–817InsTAA results in an in-frame insertion of an asparagine residue, while at position 408 a neutral amino acid (tyrosine) replaces a basic (histidine) residue. Considering the high degree of conservation of the zinc finger motifs (Alders et al., 2000), these variants are likely to interfere with the functional properties of the ZNF214 protein.
Three additional missense mutations, C224H in ZNF214 and I400V and S496I in ZNF215, were found in zinc finger domains. Both the C224H and S496I variants result in non-conservative amino acid substitutions, which might hamper the DNA binding properties of the ZNF 214 and ZNF 215 proteins. In contrast, the isoleucine to valine substitution at position 400 of ZNF214 is a conservative change, which might not have functional consequences. Notably, all these variants were unique to the patient population. S496I was identified in two apparently unrelated subjects.
In the case of the ZNF215 712+1 GA variant, the predictable consequences at the molecular level are skipping of exon 6 and/or activation of cryptic splice sites up- or downstream of the original one. Skipping of exon 6 would result in an in-frame deletion of 31 amino acids of the zinc finger domain of ZNF215 (from alanine 206 to phenylalanine 237). The activation of cryptic splice sites is likely to produce greater functional consequences, with the disruption of the entire protein sequence downstream of exon 6. The same variant was identified in a control man with normozoospermia. This might indicate that the mutant shortened protein retains the functional properties of the normal one. An alternative explanation for this finding is provided by the fact that the ZNF215 gene is imprinted in a tissue-specific manner with preferential expression of the maternal allele in testis (Alders et al., 2000). Thus, for the 712+1 GA variant we can postulate that both patient carriers inherited the mutant allele from the mother, while the control individual with normozoospermia inherited the mutant allele from the father. As a consequence of the imprinting in this case, the disease phenotype would not be expressed. Unfortunately, DNA for family analysis was not available.
Of three patients in whom we found a mutation, DNA from their parents could be obtained and in all three cases the mutation was inherited from the mother. Inheritance of a genetic defect from the mother has not been described before in impaired spermatogenesis. At first sight, a genetic cause affecting reproductive fitness that segregates through a family might seem unlikely because of reproductive selection. However, an autosomal dominant defect of the maternal allele, as we found in our patients, could explain how male factor subfertility can be transmitted to the next generation.
In summary, the results of our study demonstrate a probable role of chromosomal region 11p15 in the aetiology of impaired spermatogenesis in patients with and without cryptorchidism. Additional functional studies are required to prove unequivocally the pathological role of the mutations identified in ZNF214 and ZNF215 in male infertility. Screening for mutations in other testis-specific genes present in this region (e.g. heterogeneous ribonucleoprotein G-T, HNRNPG-T) might reveal additional genes functionally responsible for impaired spermatogenesis.
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Acknowledgements |
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We thank Marja van Stralen for her technical support and Peter de Knijff for additional statistical advice and comments on the manuscript. We also thank Maas-Jan Heineman for including patients in this study.
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References |
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