Hum. Reprod. Advance Access originally published online on November 17, 2005
Human Reproduction 2006 21(3):755-759; doi:10.1093/humrep/dei388
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Single nucleotide polymorphisms of the gonadotrophin-regulated testicular helicase (GRTH) gene may be associated with the human spermatogenesis impairment
1 Department of Medical Genetics, West China Hospital, Sichuan University, and Division of Human Morbid Genomics, National Key Laboratory of Biotherapy, Chengdu, 610041, 2 Department of Biology and Chemistry, Dali College, Dali, 671000, China
3 To whom correspondence should be addressed at: Department of Medical Genetics, West China Hospital, Sichuan University, Renmin Nanlu Section 3, No. 17, Chengdu, 610041, China. E-mail: szzhang{at}vip.163.com; sizhongzhang{at}hotmail.com
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Abstract |
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BACKGROUND: Gonadotropin-regulated testicular RNA helicase (GRTH) is a testis-specific RNA helicase that is essential for completion of spermatogenesis and is involved in pathogenesis of impaired spermatogenesis in mouse. It is therefore reasonable to postulate that human GRTH gene may also play a role in impaired spermatogenesis in humans. To test this hypothesis, we investigated the possible association between the variations of the GRTH gene and human spermatogenesis impairment. METHODS: Mutation screening of exons and intron/exon boundaries of GRTH gene was carried out by denaturing high-performance liquid chromatography (DHPLC) in 347 infertile patients with idiopathic azoospermia and severe oligozoospermia as well as 201 fertile men. RESULTS: Four single nucleotide polymorphisms (SNP), namely IVS6+55G
T, ISV8+10AC, c.852CT and c.927GA, were identified. Among them, significant differences in polymorphism frequencies were observed at the polymorphic IVS6+55GT and c.852CT loci between the patients and controls, and a significant association between haplotypes of these two loci and male infertility with impaired spermatogenesis was detected. CONCLUSIONS: Results of the present study indicate that SNP IVS6+55GT and c.852CT of GRTH gene may be associated with male infertility with azoospermia or severe oligozoospermia, suggesting that variations in GRTH gene may contribute to susceptibility to spermatogenic impairment in humans.
Key words: GRTH gene/single nucleotide polymorphism/spermatogenesis impairment
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementReferences |
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Approximately 50% of infertile couples can be traced to male infertility (De Kretser and Baker, 1999
). A significant proportion of male infertility is accompanied by idiopathic azoospermia or severe oligozoospermia, which is generally assumed to be the result of genetic alterations (Rucker et al., 1998; Cram et al., 2001). In recent years, several genetic factors which may play an important role in the spermatogenesis impairment have been identified and characterized, including some numerical and structural chromosomal abnormalities, Y-chromosome microdeletions and mutations of some known spermatogenesis-related genes. However, these genetic alterations account for only 
15% of the cases of impaired spermatogenesis; the majority of genetic causes including gene mutations still remain to be deciphered (Foresta et al., 2002).
Gonadotrophin-regulated testicular RNA helicase (GRTH) belongs to the DEAD-box (Asp-Glu-Ala-Asp) protein family of RNA helicases. Members of this family play a regulatory role in many aspects of RNA functions including translation, nuclear transcription and pre-mRNA splicing (Schmid and Linder, 1992; De la Cruz et al., 1999). As the first member of the family that has been found to be regulated by HCG, GRTH is predominantly expressed in the testis of rat, mouse and human, in both Leyding cells and germ cells (spermatocytes and round spermatids). It possesses ATPase and RNA helicase activities and increases translation in vitro (Tang et al., 1999). The gene of the helicase is remarkably up-regulated by HCG and shows a cell- and stage-specific expression during the germ cell development, suggesting that GRTH is likely related to the regulation of spermatogenesis (Tang et al., 1999; Sheng et al., 2003). Furthermore, in GRTH gene knockout mice, the male mice are infertile with azoospermia resulting from a complete arrest of spermiogenesis at step 8 of round spermatids and failure to elongate, which provide evidences that GRTH gene is essential for completion of spermatogenesis in mouse (Tsai-Morris et al., 2004). Therefore, it is reasonable to postulate that human GRTH gene may also play a role in impaired spermatogenesis in humans.
Since there has been no study on the relationship between human GRTH gene and spermatogenesis, we performed a mutation screening of all exons and intron/exon boundaries of the gene in 347 infertile patients with idiopathic azoospermia or severe oligozoospermia and compared with those in 201 fertile controls using denaturing high-performance liquid chromatography (DHPLC) in order to explore the possible association between variations of the gene and impaired spermatogenesis in humans.
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Materials and methods |
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Subjects
The patient group included 228 infertile males with idiopathic azoospermia and 119 infertile males with severe oligozoospermia (semen count < 5x106/ml) aged from 25 to 38 years. They were recruited from the Department of Urology and Department of Androlgy, West China Hospital, Sichuan University between November 2001 and January 2005. After examination by specialists, who excluded a history of orchitis, maldescensus of testis, varicocoele, obstruction of vas deferens, numerical or structural chromosomal abnormalities and microdeletions of AZF region on Y chromosome, all patients underwent at least two semen analyses according to World Health Organization (1999)
guidelines. The control group included 201 fertile men aged 26 – 40 years who had fathered at least one child without assisted reproductive measures and had normal spermatogenesis with average semen count of 60.07 ± 24.56x106/ml (range 24 x 106 to 138 x 106/ml). All participants in the study were of Han nationality that makes up >90% of the Chinese population and informed consent was obtained from all of them.
PCR amplification
Genomic DNA was extracted from the peripheral blood leukocytes of patients and controls using a salting out procedure (Miller et al., 1988). According to the alignments of the mRNA (GenBank accession No. NM_013264
[GenBank]
) and human genomic sequence, 10 pairs of primers were designed and synthesized to amplify all 12 exons and adjacent intron/exon boundaries of the GRTH gene. The sequences of primers and the lengths of the PCR products analysed are shown in Table I. PCR amplification was carried out in a total volume of 30 µl containing 3 µl of 10 x PCR buffer, 100 ng of genomic DNA, 200 µmol/l dNTP, 8 pmol of each primer, 1.5 mmol/l MgCl2 and 1 IU Taq polymerase (Takara, Shiga, Japan). The reaction profile was: pre-denaturation at 94°C for 5 min followed by 94°C for 30 s, annealing between 53 and 63°C for 30 s (Table I), extension at 72°C for 30 s (except exons 2, 3 and exon 5, 6 for 40 s) for 35 cycles, with a final extra extension at 72°C for 5 min.
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Denaturing high performance liquid chromatography analysis (DHPLC) and DNA sequencing
Mutation screening of GRTH gene was carried out using a transgenomic automated denaturing high-performance liquid chromatography system (WAVE) (Transgenomic Inc., Omaha, USA). The PCR products were denatured at 95°C for 5 min and allowed to cool at 65°C for 30 min, then analysed on WAVE according to the melting temperature shown in Table I and the gradient conditions recommended by the WAVEMAKE Software Version 4.1 for 7 min. The elution profiles of heterozygous fragments were represented as multiple peaks and the homozygous fragments as single peaks. Heterozygous fragments were reamplified and purified with a QIAquick PCR purification Kit (Qiagen, Chatsworth, USA), then sequenced in both directions on an ABI377A DNA sequencer using the Big-dye terminator cycle sequencing Kit (Sangon Co., Shanghai, China) to determine the locations and chemical nature of the sequence changes.
Genotyping
All participants were genotyped for single nucleotide polymorphisms (SNP) identified in present study. For the SNP resulting in change of restriction site (SNP IVS6+55GT, c.852CT and c.927GA), genotyping was performed by digestion with corresponding restriction enzymes (VspI, HhaI and MspI) (MBI, Vilnius, Lithuania). After restriction enzyme digestion, the products were electrophoresed on a 3% agarose gel and observed with Gel Doc 1000 system (Bio-Rad, Hercules, CA, USA). For the SNP without affecting restriction site, genotyping was performed by re-DHPLC analysis of the mixed amplicons of homozygous samples demonstrated by previous DHPLC analysis with the reference sample of known sequence. For the samples identical with the known sequence, a single elution peak was predicted, while for the samples different from the known sequence, an elution profile of multiple peaks was predicted (Colosimo et al., 2002).
Statistical analysis
The allele and genotype frequencies of the patient and control groups were calculated by counting. The Hardy–Weinberg equilibrium was tested using HWE software (Terwilliger and Ott, 1994). The differences in allelic and genotypic frequencies of SNP identified in the present study between patients with azoospermia or severe oligozoospermia and controls were evaluated by 
2-test. Linkage disequilibrium among the SNP was estimated by |D/| calculation using the 2LD program (http://www.iop.kcl.ac.uk/iop/Departments/PsychMed/GEpiBSt/software.shtml). Haplotypes of SNP were assessed using PHASE Software (http://www.stat.washington.edu/stephens/software.html).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementReferences |
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Using PCR amplification with primers flanking each exon followed with DHPLC, we screened the nucleotide sequence variations in a total of 12 exons and adjacent intron/exon boundaries in GRTH gene of 347 infertile men with azoospermia or severe oligozoospermia. The candidate SNP found during DHPLC analysis were confirmed by DNA sequencing. Four SNP were identified, namely a G
T change at the position plus 55 in intron 6 (IVS6+55GT), a AC change at the position plus 10 in intron 8 (IVS8+10AC), a CT change in exon 10 at the cDNA sequence position 852 (c.852CT) and a GA change in exon 11 at the cDNA sequence position 927 (c.927GA) (GenBank accession No. NM_013264
[GenBank]
). Although the SNP c.852CT and c.927GA are located in the coding region of the gene, they do not change the amino acid sequence of GRTH protein. The two SNP IVS8+10 AC and c.927GA were newly discovered (Figure 1).
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The allele and genotype frequencies of the SNP in patient and control groups are reported in Table II. The distributions of genotypes of the four SNP followed Hardy–Weinberg equilibrium in both the patient and control groups (data not shown). The frequencies of genotype GT [25.6 versus 15.9%, P = 0.008, odds ratio (OR) 1.822, 95% confidence interval (CI) 1.163–2.852], the individuals with allele T (GT+TT) (26.5 versus 16.9%, P = 0.010, OR 1.772, 95% CI 1.142–2.749) and allele T (13.7 versus 9.0%, P = 0.02, OR 1.688, 95% CI 1.075–2.418) were significantly higher in patient group than those in the control group at ISV6+55GT locus. At c.852CT, the frequency of allele T (31.4% versus 25.4%, P = 0.034, OR 1.347, 95% CI 1.002–1.775) was also significantly higher than that in controls. At ISV8+10AC and c.927GA loci, no significant differences in frequencies of allele and genotype frequencies were observed between patients and controls, and the two SNP were not included in subsequent analysis because their rare allele frequencies were not 
1%.
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A moderate linkage disequilibrium was detected between SNP IVS6+55GT and c.852CT in controls (|D/| = 0.675). The PHASE case–control test revealed a significant association between haplotypes of these two SNP loci and male infertility with azoospermia or severe oligozoospermia. Haplotype TC (12.90 versus 7.98%, P = 0.011, OR 1.723, 95% CI 1.128–2.632) was significantly increased, whereas haplotype GC (55.8 versus 65.6%, P = 0.0003, OR 0.623, 95% CI 0.483–0.805) was significantly decreased in patients compared with controls (Table III).
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Discussion |
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Spermatogenesis is a complex process that requires highly regulated expressions of a series of genes, and many genetic aetiological factors may be involved in male infertility with impaired spermatogenesis. However, to date, only a few genetic defects have been confirmed to result in abnormal spermatogenesis (Matzuk and Lamb, 2002
). Recently, with the introduction of gene targeting techniques that disrupt specific genes in animal models, hundreds of candidate genes related to spermatogenesis have been identified in animals (Lamb, 1999; Maduro and Lamb, 2002). Whether these candidate genes are also related to human male infertility remains to be proven.
GRTH is a male-specific gene involved in impaired spermatogenesis in mouse (Tsai-Morris et al., 2004). To date, 85 SNP have been reported in the gene in dbSNP and only 16 have frequencies reported. The three most frequent SNP are rs485878, rs603675 and rs603675, which are all in introns. In this study, we screened the nucleotide variations of exons and intron/exon boundaries in GRTH gene, and found four SNP, namely IVS6+55GT, ISV8+10AC, c.852CT and c.927GA. Of these, ISV8+10AC and c.927GA have not been reported previously. Since the rare alleles of them were <1% and their distributions were similar between patients with azoospermia or severe oligozoospermia and fertile controls, they should be scarce in the population investigated and may not be disease-associated variations.
The other two SNP, IVS6+55GT (NCBI SNP CLUSTER ID rs551373) and c.852CT (NCBI SNP CLUSTER ID rs683155), are known SNP. The frequency of c.852CT is similar to that reported in dbSNP, but the frequency of IVS6+55GT is lower than that presented in dbSNP. At IVS6+55GT locus of GRTH gene, significant differences in frequencies of allele T, genotype GT and carriers with allele T (GT+TT) between patients with azoospermia or severe oligozoospermia and fertile controls were observed, indicating that there is an association of this polymorphism with impaired spermatogenesis. As IVS6+55GA is an intronic SNP that is not close to the site of pre-RNA splicing, we applied the NetGene2 program (www.cbs.dut.dk/services/NetGene2) to evaluate its possible effect on the branch site sequence in intron 6, thereby affecting splicing activity. The result of NetGene2 analysis showed that this SNP does not change any conserved nucleotides of the branch site in intron 6 and affect splicing activity. Therefore, it could be presumed that IVS6+55GA might be in linkage disequilibrium with other mutations or variations that could play a role in impaired spermatogenesis in GRTH gene or other genes.
The frequencies of allele T were also significantly higher in patient groups with azoospermia or severe oligozoospermia than in the control group at c.852CT locus, suggesting that allele T may increase the risk of impairment of spermatogenesis. Although the CT polymorphism at codon 284 does not change amnio acid of GRTH protein, it is just 7 bp away from the donor site of pre-RNA splice. We therefore used the ESEfinder (Exonic Splicing Enhancer) program (http://exon.cshl.org/ESE; Cartegni et al., 2003) to predict the potential effect of c.852CT variation on splicing activity. It was shown that this polymorphism is located in one of the binding motifs of splicing factor 2 (SF2/ASF) (CGCCCGA), and SF2/ASF is an important member of serine/arginine-rich (SR) protein family that plays a key role in pre-RNA splicing (Krainer et al., 1991; Graveley, 2000; Cartegni et al., 2002; Sanford et al., 2005). According to the analysis with ESEfinder, allele T apparently may decrease the activity of pre-RNA splicing compared to allele C, hence reduce the expression of GRTH gene. As a binding protein and a component of mRNP, GRTH could play an important role in the translation of some crucial genes at a specific stage during spermatogenesis and influence the expression of sets of genes determining the progression of spermatogenesis (Tsai-Morris et al., 2004). Therefore the decreased expression of GRTH gene may affect the expression of important genes required in spermatogenesis leading even to spermatogenesis failure. This may explain why the allele T of c.852CT polymorphism of GRTH gene increases the risk of impaired spermatogenesis. However, it could not be excluded that the polymorphism may be in linkage disequilibrium with other loci susceptible to spermatogenesis failure.
Since the linkage disequilibrium of ISV6+55GT and c.852CT is not very strong, we performed further haplotype analysis of them in the patient and control groups. The results showed that haplopyte TC significantly increased whereas the haplotype GC significantly decreased in patients compared with controls, suggesting that haplopyte TC might be a risk factor for impaired spermatogenesis and haplotype GC might have some protection effect from impaired spermatogenesis. These findings again provided evidence that GRTH gene may be involved in spermatogenesis impairment.
To our knowledge, present study is the first mutation analysis of GRTH gene in male infertility with azoospermia or severe oligozoospermia. Our findings indicated that GRTH gene may be associated with impaired spermatogenesis and male infertility. However, more studies in different populations as well as further investigation and functional analysis of the variants are needed in order to elucidate the role of GRTH gene in human spermatogenesis and its pathology.
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Acknowledgement |
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This work was supported by the National High Technology Research and Development Program of China (863 Program), Grant Numbers 2001AA224021-03 and 2002BA711A08.
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Submitted on July 19, 2005; resubmitted on October 15, 2005; accepted on October 21, 2005.
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