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From the * Department of Medical Genetics, State
Key Laboratory of Biotherapy, West China Hospital, West China Medical School,
Sichuan University, Chengdu, Sichuan, People's Republic of China; and the
Reproductive Medicine Center, West China
Second Hospital, Sichuan University, Chengdu, Sichuan, People's Republic of
China.
| Correspondence to: Dr Yuan Yang, Department of Medical Genetics, State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Gaopeng Street, Keyuan Road 4, Chengdu, Sichuan 610041, People's Republic of China (e-mail: yangyuan{at}scu.edu.cn). |
| Received for publication December 8, 2008; accepted for publication March 5, 2009. |
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Abstract |
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Key words: Y chromosome, DAZ, ancestral gene, spermatogenesis
DAZ (deleted in azoospermia) is the most important gene related to spermatogenesis in the AZFc region, and deletion copies of the gene family present strong candidates for spermatogenic impairment leading to male infertility (Reijo et al, 1995; Kuroda-Kawaguchi et al, 2001). DAZL (DAZ-like) and BOULE (also known as BOLL) genes are 2 autosomal ancestral genes of DAZ and are believed to play a role in spermatogenesis (Eberhart et al, 1996; Yen et al, 1996; Xu et al, 2003; Yen, 2004; Reynolds et al, 2007). Because 2 common partial AZFc deletions, gr/gr and b2/b3, always involve 2 DAZ copies (Repping et al, 2003, 2004), it is reasonable to investigate association of the spermatogenic phenotype of partial AZFc-deleted men with the DAZL and BOULE variations. In the present study, a total of 15 known mutations and single-nucleotide polymorphisms (SNPs) of the 2 genes were detected in 57 normozoospermic and 157 azoo-/oligozoospermic men—both groups with partial AZFc deletions—and allele, genotype, and haplotype frequencies between the 2 groups were compared. The subjects were selected from 634 men with normozoospermia and 1286 men with azoo-/oligozoospermia, and the methodological details for the detection of partial AZFc deletions are described in the literature (Yang et al, 2008).
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Materials and Methods |
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Genotyping of DAZL and BOULE Variations
Genomic DNA was extracted from peripheral blood lymphocytes using DNA
isolation kits (TaKaRa Co, Ostu, Japan). All subjects were analyzed for 7
known DAZL variations, P6H, N10C, T12A, I37A, T54A, I71V, and R115G
(Teng et al, 2002;
Thangaraj et al, 2006;
Tung et al, 2006,
Tung et al, 2006), and 2
BOULE variations, Q2E and S9Y, in their coding regions
(Lepretre et al, 2004;
Westerveld et al, 2005),
wherein P6H, N10C, T12A, and I37A were detected by DNA sequencing directly
with polymerase chain reaction (PCR) product, considering all in DAZL
exon 2. Primers for the PCR were 5'-TCCTGAGCCTGAACTAACTT-3'
(sense) and 5'-ACCTATGGGTCAAATGTAAA-3' (anti-sense), and the
product was 413 bp in length. The rest were detected with PCR restriction
fragment length polymorphism (PCR-RFLP) analysis, and their PCR primers were
designed with Primer Premier 5.0 according to the genomic sequence of
DAZL (GenBank NM_001351.2) and BOULE (GenBank NC_000002.10).
The sequences of primers, the lengths of PCR products, the restriction
endonucleases, and the lengths of the digested products are shown in
Supplemental Table 1 (available online at
www.andrologyjournal.org).
The different alleles observed in PCR-RFLP analysis were confirmed with DNA
sequencing on an ABI377A DNA sequencer (Applied Biosystems, Foster City,
California) according to the Dye Terminator method.
Moreover, in noncoding regions of the 2 genes, 3 DAZL SNPs, c.888+1853 G>C (dbSNP accession no. rs2347312), c.570+356 C>T (rs4234537), and c.294+222 C>T (rs2217204), and 3 BOULE SNPs, c.852+1595 A>G (rs2272166), c.828+7055 A>C (rs700642), and c.221+68 C>T (rs700655), in NM_033030 were detected. Genotyping of the 6 SNPs was performed by PCR-RFLP with corresponding restriction enzymes shown in Supplemental Table 1. After restriction enzyme digestion, the products were separated on 3% agarose gel stained with ethidium bromide and observed with the Gel Doc1000 system (Bio-Rad, Hercules, California). For each SNP, 2 wild homozygotes, mutation heterozygotes, and homozygotes were sequenced to confirm the results of PCR-RFLP analysis.
Statistical Analysis
The deviation of DAZL and BOULE SNPs from the
Hardy-Weinberg equilibrium was evaluated with SNPAlyze Version 6.0 (Dynacom
Co, Ltd, Kanagawa, Japan). The SNP allele and genotype frequencies were
compared between partial AZFc-deleted men with normozoospermic men
and men with spermatogenic failure by the 
2 test with SPSS
11.0 software. The pairwise linkage disequilibrium and haplotype were analyzed
with Haploview 4.0 software (Mark Daly's Lab, Broad Institute of MIT and
Harvard, Cambridge, Massachusetts; Barrett
et al, 2005). The comparison of haplotype distribution between the
groups was performed with the use of an exact test of population
differentiation with Arlequin software ver.3.11 (CMPG, University of Berne,
Berne, Switzerland; Raymond and Rousset,
1995; Excoffier et al,
2005). P < .05 was regarded as statistically
significant.
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Results |
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Furthermore, in 6 intron SNPs of the 2 genes, BOULE c.852+1595 A>G was not observed in all subjects. Because the rare allele frequencies of the other 5 SNPs were not 1% or less of the population, they were included in the analyses of allele and genotype frequencies, and the SNP frequency data in men with spermatogenic impairment and normozoospermia are shown in Table 1. The statistical analyses with HWE software showed that the SNP allele and genotype distributions followed Hardy-Weinberg equilibrium in both the patient and control groups. At 5 SNPs, no significant differences in allele and genotype frequencies were observed between different partial AZFc-deleted men with spermatogenic impairment and normozoospermia (Tables 1 and 2).
The pairwise linkage disequilibrium (LD) coefficients (D') between alleles at DAZL SNPs was calculated with Haploview 4.0 software (Supplemental Table 2). For men with spermatogenic impairment, DAZL T12A was observed to have strong LD with c.888+1853G>C (D' = .898) and moderate linkage disequilibrium with c.570+356C>T (D' = .654), whereas for controls with normozoospermia, the DAZL SNP c.888+1853G>C was found to be in complete LD with c.294+222C>T (D' = 1.000), which showed that the LD map of DAZL might be different between partial AZFc-deleted men with spermatogenic impairment and normozoospermic men. Moreover, the measures of LD between alleles at 2 BOULE SNPs were estimated, and no LD was found in both groups. Furthermore, the haplotype analysis was performed with 4 DAZL SNPs, T12A, c.294+222C>T, c.570+356C>T, and c.888+1853G>C, and 2 BOULE SNPs, c.221+68C>T and c.828+7055A>C, in order (Table 3). For DAZL, a total of 11 haplotypes with a frequency of more than 1% in men with spermatogenic failure or normozoospermia were estimated with Haploview 4.0, whereas no significant difference in haplotype distribution was observed between the 2 groups after carrying out an exact test of population differentiation with Arlequin ver.3.11 (P = .179 ± .017). Similarly, the distribution of BOULE haplotypes were not significantly different between men with impaired spermatogenesis and normozoospermic men (P = .524 ± .022). Furthermore, all 15 DAZL and BOULE haplotype frequencies were similar between the 2 groups according to the P values corrected by permutation tests.
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Discussion |
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Recently in DAZL, 4 novel missense mutations—P6H, N10C, I37A, and R115G—were observed in an infertile population and the homozygous N10C mutation was found in an azoospermic man. The association of the mutations with male infertility was suggested because of their putative contribution to the loss of gene function, in that they are close to or in the RNA binding domain of the protein (Tung et al, 2006, Tung et al, 2006). In the present study, however, the alleles carrying the mutations were not found in the group of men with normozoospermia nor those with azoo-/oligozoospermia, indicating that because of rarity, the mutations do not significantly influence the spermatogenic phenotype of partial AZFc-deleted men in the population.
T54A in DAZL, a non-synonymous SNP found first in a Taiwan Chinese population and having significant association with male infertility (Teng et al, 2002), was not observed in all 214 subjects, which is consistent with findings in Italian, German, Indian, and Japanese men (Bartoloni et al, 2004; Becherini et al, 2004; Tschanter et al, 2004; Yang et al, 2005; Thangaraj et al, 2006). To our knowledge, this is the first report about the distribution of T54A in a non-Taiwan Chinese population. Our results suggest that the mutation reported previously could be an incorrect result, and its existence remains doubtful. In this study, we found only 1 sY1191/DAZ3/DAZ4-deleted man with azoospermia heterozygous for I71V, and 2 exonic SNPs, Q2E and S9Y, in BOULE were not observed in men with spermatogenic failure. Taken together, the results showed that the known variations in the DAZL and BOULE coding regions were rare in the population, also suggesting that they might have no significant influence in susceptibility to spermatogenic impairment of men with partial AZFc deletions.
In recent years, some studies on the association of DAZL polymorphisms and spermatogenesis have suggested that specific DAZL haplotypes arising from partly different SNP might correlate with sperm count or spermatogenic failure (Teng et al, 2006; Tung et al, 2006, Tung et al, 2006). In the present study, we compared the distribution of the DAZL haplotypes between partial AZFc-deleted men with normozoospermic men and men with spermatogenic failure after observing the difference in the DAZL LD map between the 2 groups. As a result, no population differences in haplotype distribution were found between the 2 groups. The BOULE haplotype analysis also showed similar results. All together, these results suggest that the variations in the 2 DAZ ancestral genes on the Y chromosome might be independent of the spermatogenic phenotype of men with partial AZFc deletions. Because of potential genetic differences in the factors influencing the spermatogenic phenotype among populations of different ethnic origins, the need remains for further genetic study in more populations.
Our results suggest that the DAZL and BOULE genetic variants might not influence significantly the spermatogenic phenotype of men with partial AZFc deletion in the Chinese population. Taking into account the co-deletion of other gene families in partial AZFc deletions, the contribution of their autosomal homologues, such as CDYL, to the partial AZFc deletion phenotype should be further investigated.
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Footnotes |
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