Human Molecular Genetics, 2000, Vol. 9, No. 8 1161-1169
© 2000 Oxford University Press
Deletion and expression analysis of AZFa genes on the human Y chromosome revealed a major role for DBY in male infertility
University of Padova, Department of Medical and Surgical Sciences, Clinica Medica 3, 35128 Padova, Italy
Received 6 January 2000; Revised and Accepted 13 March 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Three distinct regions, designated AZFa, b and c from proximal to distal Yq, are required for normal spermatogenesis in humans. Deletions involving AZFa (deletion interval 5C/D) seem to occur less frequently in infertile men and to be associated with a more severe testicular phenotype, with almost complete absence of germ cells. AZFa contains three genes, named USP9Y, DBY and UTY, and presents high homology with the mouse
Sxrb interval, deletion of which causes a severe spermatogenic impairment. However, the specific role of these genes in human spermatogenesis is still unknown and it is not clear which of them is responsible for the AZFa phenotype. Here we describe a complete sequence map of the AZFa region, the genomic structure of AZFa genes and their deletion analysis in a large number of infertile men characterized by well-defined spermatogenic alterations. Both USP9Y and DBY may cause severe testiculopathies, but DBY appears to be the major AZFa candidate. DBY is frequently deleted in infertile patients and its absence produces severe spermatogenic damage leading to a significant reduction of germ cells or even to their complete absence. Expression analysis of AZFa genes and their X-homologues revealed ubiquitous expression for all of them except DBY; this gene produces a long transcript which is ubiquitously expressed in addition to a shorter transcript which is only expressed in the testis, suggesting a specific role for DBY in the spermatogenic process. This hypothesis is further supported by the high similarity of DBY to other DEAD box proteins belonging to the PL10 subclass. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Three distinct regions, designated azoospermia factor (AZF) a, b and c, from proximal to distal Yq are required for normal spermatogenesis in humans (1–3). Deletions responsible for male infertility occur in the AZFc region more frequently than in AZFb and AZFa (2,4), probably reflecting the current knowledge on the structure and function of these regions. Indeed, it seems sufficiently clear that the DAZ (deleted in azoospermia) gene family (5) is responsible for the AZFc phenotype, since a number of studies have reported a high incidence of deletion of this gene in azoospermic and severely oligozoospermic patients (1,5–18). Furthermore, DAZ is specifically expressed in germ cells (19–22) and is highly homologous to the Drosophila sterility gene, boule (23). Similarly, the germ-cell-specific RBMY (RNA-binding motif, Y chromosome) gene family (24) is considered the candidate for the AZFb region (25), even if it has not yet been clearly demonstrated that loss of RBMY actually causes the testiculopathy in AZFb-deleted patients. The lack of such a demonstration is probably due to the multicopy nature of this gene, which has several copies dispersed across the short and long arms of the Y chromosome (26).
In contrast, it is still debatable whether the AZFa phenotype is caused by the loss of one or more genes. The first gene identified in AZFa and subsequently shown to be absent in a fraction of infertile patients was USP9Y (ubiquitin-specific protease 9, Y chromosome), previously known as DFFRY (Drosophila fat facets related Y) (27,28). This gene substantially differs from the other AZF-candidate genes DAZ and RBMY, as it does not encode an RNA-binding protein, but seems to function as ubiquitin C-terminal hydrolase and, more importantly, it is ubiquitously expressed rather than testis specific (28). However, USP9Y occupies only a small part of the AZFa interval (29) while the majority of infertile males carrying AZFa deletions show the absence of this entire interval (1,2,9,10,14–16,18,29). These findings suggest that other gene(s) in this region may be responsible, either singly or in combination with USP9Y, for the spermatogenic disruption observed in AZFa deleted patients. Subsequently, two other genes, DBY (DEAD/H box polypeptide, Y chromosome) and UTY (ubiquitously transcribed tetratricopeptide repeat gene, Y chromosome), were precisely mapped in the AZFa interval (deletion interval 5C/D) (27,29), suggesting that they may have a role in human spermatogenesis. This hypothesis was further supported by the syntenic homology between the human AZFa region and the mouse Sxrb interval (29), deletion of which causes a severe spermatogenic impairment (30) very similar to that observed in patients with an AZFa deletion.
Recently, an additional anonymous expressed sequence tag (AZFaT1) was mapped proximal to USP9Y, and it has been suggested that the absence of USP9Y and/or AZFaT1 is associated with an oligozoospermia phenotype, while a more severe testiculopathy (Sertoli cell-only syndrome) may reflect the additional loss of DBY (31). While preparing this manuscript, another group reported the first evidence for a de novo mutation in USP9Y (4-bp deletion in a splice-donor site) causing azoospermia, supporting the critical role of this gene in the AZFa phenotype (32).
The existence in the GenBank of several BAC clones located in the AZFa region, allowed us to assemble a complete sequence map of this region, clearly locate the genes and develop new STS markers. To clarify the relative role of AZFa genes in human spermatogenesis, we performed a detailed deletion analysis of USP9Y, DBY and UTY in a large number of highly selected infertile patients. Even if USP9Y may have a role in human spermatogenesis, our results strongly suggest that DBY is the major AZFa candidate. This hypothesis is further strengthened by expression analysis of AZFa genes and their homologues on the X chromosome, showing that DBY has testis-specific transcripts.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Physical map of the AZFa region and genomic structure of USP9Y, DBY and UTY
Nine clones available from the GenBank overlapped in the AZFa region resulting in 1084 kb of contiguous sequence (Fig. 1). This contig includes the complete sequence of USP9Y and DBY and the 3' region of UTY, and therefore covers the whole AZFa region. The 5' end of UTY is contained in clone NH0386L03. Therefore, the gap between clone 494G17 and clone NH0386L03 includes the middle portion of UTY. A comparison of the cDNA sequences of USP9Y and DBY with these clones allowed us to determine the complete genomic structure of these genes. USP9Y covers 159 kb of DNA and it is composed of 46 exons, varying from 80 bp for exon 4 to 751 bp for exon 35 (Fig. 2). The 5' UTR of the USP9Y transcript described by Lahn and Page (27) is somewhat different from the BAC clone sequence, where it is much longer, probably reflecting cloning artefacts. DBY extends for 15.5 kb and consists of 17 coding exons (Fig. 3a). The length of the exons varies from 45 bp for exon 1 to 182 bp for exon 13, and the sizes of the introns vary from 74 bp for intron 13 to 2.5 kb for intron 1. We have also directly sequenced the amplification product for DBY1 and DBY2 from genomic DNA, confirming the reported sequences of the BAC clone and cDNA (but 33 bp in exon 17 are lacking from the sequence of the clone). The poly(A) tail for alternative transcript 1 is located 258 bp downstream from the stop codon, while alternative transcript 2 has an additional 2.1 kb 3'-UTR (Fig. 3b). The 5' end of DBY is located ~45 kb distal to the 3' end of USP9Y and the 3' end of UTY is located 330 kb downstream of the 3' end of DBY (Fig. 1). The assembly of the AZFa clones allowed us to precisely map all the markers previously defined as anonymous AZFa STSs (Fig. 1), including GY6 (33) that was found to amplify 1161 bp covering the 5'-end of DBY and exon 1 (Figs 1 and 3a). The location of the markers is shown in Figures 1, 2 and 3.
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AZFa deletion analysis in infertile patients
Semen analysis, bilateral testicular fine needle aspiration cytology (34) and PCR analysis of Yq euchromatin using 30 STSs (15) allowed us to select 173 well-characterized infertile patients, who satisfied the following criteria: they were affected either by azoospermia or oligozoospermia of unknown origin (idiopathic infertility) with a testicular cytological picture of Sertoli cell-only syndrome (complete absence of spermatogenic cells) or hypospermatogenesis (quantitative reduction of these cells) (35,36), and they had normal Yq constitution outside AZFa. As control subjects, we studied 100 normal fertile men and 80 infertile men with known causes of testiculopathy or obstruction of the seminiferous pathways (Table 1). A total of 22 STS markers were considered suitable for the screening of the AZFa region by PCR on genomic DNA and allowed us to find deletions of portions of AZFa in nine patients (9/173, 5.2%) (Table 1 and Fig. 1). No deletions were detected in the control groups, even when the sperm output was < 5 x 106/ml (moderate oligozoospermia). Deletions were clearly more frequent in the more severe testiculopathies, reaching 9.7% in Sertoli cell-only syndrome. One patient had a deletion involving USP9Y (patient 327, ref. 15), while the other eight patients presented deletions involving DBY, either alone or in association with USP9Y (patient 576) or UTY (patient 303). Therefore, in six men the deletion involved DBY only, and no specific deletion of UTY was found. There was no clear correlation between the size and localization of deletions and the testicular phenotype, as identical deletions were associated with different spermatogenic damage (Figs 1 and 4). Most of the deletion breakpoints in DBY-deleted patients were clustered and involved no additional markers outside this gene (Fig. 1). Therefore, the proximal breakpoint was between sY87 and the 5' end of DBY (a region of 7.5 kb), while the distal breakpoint was between the 3' end of DBY and marker 475Tel (a region of 74 kb). No additional genes or sequences with homology to known expressed sequence tags have been found in these two regions by using GRAIL (37), Genscan (38) and BLASTN software (39).
These findings suggest a predominant role for DBY in spermatogenesis and indicate this gene as the major AZFa candidate. To further support this hypothesis we have performed Southern blot analysis for DBY and USP9Y in all patients with deletions, with the exception of patient 327 for whom insufficient DNA was available (Fig. 5). This analysis confirmed the PCR results. Furthermore, fathers or brothers of six out of nine patients with deletions (Fig. 5) were analysed and found to carry all markers by PCR and normal hybridization on Southern blot. Therefore, all such deletions were of de novo origin, strengthening their pathogenic role in determining the spermatogenic disruption.
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Expression analysis of AZFa genes and their X-homologues
Expression analysis was performed by PCR on cDNA libraries using primers specific for DBY, UTY and USPY9 and for their X-homologues DBX/DDX3 (27,40), UTX (27) and USP9X (41). Furthermore, DBY has two different alternative transcripts (27), which differ only in the length of the 3'-UTR. RT–PCR analysis with primers specific for alternative transcript 2 (long 3'-UTR) has been performed previously and showed ubiquitous expression (27); we have therefore designed PCR assays specific for DBY alternative transcript 1 (short 3'-UTR) (Figs 3 and 6). USP9Y, USP9X, UTY and UTX showed ubiquitous expression, and DBX/DDX3 was clearly present in all tissues tested. In contrast, only testis-specific expression was detected for DBY alternative transcript 1 (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The actual candidate gene(s) for the AZFa phenotype remained debatable until very recently, since the major part of the deletions detected in infertile men using anonymous STS markers covered the entire region (1,2,9,10,14–16,18,29). We have reported previously a patient with a deletion removing USP9Y (15) and two recent reports have further focused attention on this gene (31,32). However, a possible involvement also of DBY could not be excluded. To clarify this aspect and better understand the role of AZFa genes, we therefore carried out a mapping of deletion breakpoints in a large number of well-characterized infertile patients. We first assembled the complete nucleotide sequence of the region containing USP9Y, DBY and UTY (deletion interval 5C/D), and we determined the genomic sequence of these genes. Therefore, this map allowed us to precisely locate STS markers previously assigned to AZFa and to design new primers. The deletion screening was then performed on selected patients affected by azoospermia or oligozoospermia and well-defined testicular damage, histologically defined as Sertoli cell-only syndrome or hypospermatogenesis, respectively.
We found AZFa deletions in nine out of 173 idiopathic infertile patients (5.2%); such deletions were more prevalent in more severe testiculopathies (9.7% in Sertoli cell-only syndrome), whereas no deletion was found in moderately oligozoospermic men, nor in the control subjects. Seven patients had deletions involving a single gene: in one case USP9Y only and in six cases DBY only. Two patients had larger deletions involving the block USP9Y–DBY or DBY–UTY. No deletions involving UTY only were detected. In six out of nine cases we confirmed the pathogenic role of such deletions showing that they were of de novo origin. Such results suggested that both USP9Y and DBY may be responsible for the AZFa phenotype, excluding any involvement of UTY. Therefore, we confirmed the study by Sargent et al. (31) and by Sun et al. (32) on the critical role of USP9Y. However, our findings focused attention on DBY as a fundamental gene for human spermatogenesis.
Patient 327 with deletion of USP9Y has a testicular phenotype identical to that of patient SAYER described by Sargent et al. (31), even if he retained AZFaT1 (marker sY84). Therefore, we can suppose that the oligozoospermia in SAYER is the result of the deletion of only USP9Y. This is also consistent with the 4-bp deletion in USP9Y observed by Sun et al. (32) in an infertile male (WHT 2780). The testicular phenotype of this patient is again hypospermatogenesis, and therefore we can assume that the effect of the loss of USP9Y on spermatogenesis is a reduction of germ cells. However, the high frequency of deletion of DBY detected in our study raises the question as to whether the deletion of USP9Y may downregulate expression of DBY, making it possible that DBY is the only causative gene. We had no possibility to examine the expression of DBY in patient 327 nor had any analysis been performed on patient WHT2780 (32). However, in SAYER it has been clearly shown that the expression levels of both DBY and UTY were unaffected by the adjacent deletion removing AZFaT1 and USP9Y (31). Furthermore, database analysis of the 45 kb sequence between USP9Y and DBY revealed no additional genes, and the distal deletion breakpoint in patient 327 is even more proximal than that of SAYER. These data allow us to conclude that the loss of USP9Y undoubtedly represents the aetiological factor for the testiculopathy of patient 327. However, it has to be noted that the prevalence of deletions of USP9Y in male infertile patients with azoo–severe oligozoospermia is very low: 1/143 in this study and 1/576 in the study by Sun et al. (32).
The most intriguing finding of our deletion screening of AZFa genes is the high prevalence of deletions specifically removing DBY (4.2% in severe idiopathic testiculopathies). These results suggest a crucial role for DBY in spermatogenesis and seem to indicate this gene as the major AZFa candidate. The analysis of male relatives of patients with DBY deletions confirmed their de novo origin, further strengthening their pathogenic role in determining the spermatogenic disruption. The absence of DBY was associated both with azoospermia (3/6 patients) and severe oligozoospermia (3/6 patients) and therefore there was no clear relation between genotype and testicular phenotype. The gap between DBY and UTY may contain further contributory genes. However, the distal deletion breakpoint in DBY-deleted patients is no more than 74 kb downstream of the 3' end of DBY and in this interval no genes were found by database analysis. The phenotypic variation in patients with a DBY deletion is therefore unclear. One possible explanation is that differences in genetic background or environment exist. The discovery of point mutations in DBY would allow us to better understand the genotype–phenotype relation. Anyway, the alteration of spermatogenesis caused by the loss of DBY may be explained by a possible function of DBY during the first phases of the spermatogenic process and a very similar tubular damage is observed in the mouse in the presence of the Sxrb deletion (30). Patient 576 had both DBY and USP9Y absent and his testicular phenotype is Sertoli cell-only. This is in agreement with patients JOLAR, ELTOR and AZ539 (31), whose deletions are very similar to our patient. Therefore, the combined deficiency of DBY and USP9Y seems unequivocally to determine the full AZFa phenotype (Sertoli cell-only).
Nevertheless, two data seemed to be against a fundamental and specific role of DBY (as well as of USP9Y) in human spermatogenesis: it was thought to be ubiquitously expressed (27) and has an X-homologue, initially named DBX (27) and subsequently DDX3 (40) located in Xp11.3–p11.23 (42), which escapes X-inactivation (27) and has 92% sequence identity at the protein level. These data raise the question as to whether the Y- and X-linked genes have the same biological function in males and females or the differences in amino acid sequence may lead to subtle differentiation of the biological activity, altering for example substrate specificity. Northern blot analysis revealed that DBY is expressed as a single transcript in all tissues except testis which contained an additional smaller mRNA (27); furthermore, two different alternative transcripts for this gene are described (27) which differ only in the length of the 3'-UTR. RT–PCR analysis with primers specific for alternative transcript 2 (long 3'-UTR) has been performed previously and showed ubiquitous expression (27). Given the predominant role of DBY in determining the AZFa phenotype, we tried to clarify these aspects by performing expression analysis on cDNA libraries from different tissues using PCR assays specific for DBX/DDX3 and for DBY alternative transcript 1 (short 3'-UTR). DBX/DDX3 was clearly ubiquitously expressed, while only testis-specific expression was detected for DBY alternative transcript 1. These results suggest that DBY may have a different function to its X-homologue and may act specifically during the spermatogenic process or, in other words, DBY appears to have both housekeeping and testis-specific functions. On the contrary, USP9Y, USP9X, UTY and UTX showed ubiquitous expression. Therefore, we could not determine whether USP9Y and USP9X perform the same function in germ cell development or whether the Y-linked copy has a role in spermatogenesis not provided by the X-linked one.
The finding that Y-linked genes (DBY and USP9Y) with homologues on the X chromosome may play a substantial role in spermatogenesis is an important observation, since it was thought that this function was peculiar mainly to Y-specific genes (for example DAZ ) (27). However, the identification of a testis-specific transcript for DBY and the recent identification of a X-homologue gene also for the AZFb candidate gene RBMY (43,44) suggest that also X/Y homologous genes are required for male-specific functions such as normal spermatogenesis.
To support a major role for DBY in human spermatogenesis, we analysed in more detail possible homologies with other known genes and proteins. The protein encoded by DBY (DDXY) belongs to the DEAD box proteins (45), which are putative ATP-dependent RNA helicases (45,46) with a characteristic Asp-Glu-Ala-Asp (DEAD) box as one of eight highly conserved sequence motifs (Fig. 5c). Alignment searches in different data banks revealed that DDXY has a high amino acid identity with mouse PL10 (47) and other members of this subclass, such as mouse DEAD3/Ddx3 (48): DBY has 89% amino acid identity with PL10 and 91% with DEAD3/Ddx3, and the central core of these proteins (containing the most conserved motifs characteristic of DEAD box proteins) has even greater similarity. Interestingly, both PL10 and DEAD3/Ddx3, as well as other DEAD box proteins in other species, have very similar expression patterns to that of the human DBY product, showing testis-specific transcripts in addition to ubiquitous transcripts (47,48). In particular, PL10 is male germ- cell-specific and developmentally regulated during spermatogenesis, with higher expression in spermatocytes that are in prophase of the first meiotic division. Northern blot analysis with PL10 cDNA on RNAs from human tissues showed a larger transcript ubiquitously expressed and a shorter transcript expressed only in the testis (47). The most intriguing data is that PL10 was identified by screening a mouse testis cDNA library with a genomic sequence, called 12f3, from a region of the human Yq that corresponds to AZFa (47,49,50). We therefore propose that DBY is the human homologue of mouse PL10, especially concerning their role in spermatogenesis.
In conclusion, our results suggest that the AZFa region contains two genes whose deletion cause male infertility: USP9Y and DBY. This latter gene appears to represent the major AZFa candidate and may be responsible for testicular damage causing severe male infertility. This role awaits confirmation by detection of point mutation in the gene inducing similar phenotype, and expression analysis to determine its eventual germ cell specificity.
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MATERIALS AND METHODS |
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Physical map of the AZFa region and genomic structure of USP9Y, DBY and UTY
Ten BAC clones available from the GenBank (accession nos AC004772, AC005942, AC002992, AC004617, AC004810, AC002531, AC004474, AC006565, AC005820, AC006376) have been assembled to construct a high resolution map of the AZFa region. We identified BAC clones containing the STSs previously mapped to this interval and USP9Y, DBY and UTY cDNA sequences. Nine clones overlapped and gave a 1084 kb sequence, which covers the entire AZFa region (deletion interval 5C) and includes the complete sequence of USP9Y and DBY and the 3' region of UTY. Between this sequence and clone NH0386L03 (accession no. AC006376), which covers the 5' region of UTY, a gap exists. The genomic structure of DBY was determined by comparing the sequence of the BAC clone 475I1 (accession no. AC004474), which extends from the 3'-end of USP9Y through DBY, with DBY cDNA sequences (accession no. AF000984 and AC000985) (27). Furthermore, DNA fragments obtained by PCR on genomic DNA with primers DBY1 and DBY2F (see below) were directly sequenced using an automated DNA sequencer (Applied Biosystems, Model 373A). DNA fragments were sequenced twice on both strands and sequences were compared with sequences of DBY cDNA and of the BAC clone covering DBY. The genomic structure of USP9Y was determined by comparison of cDNA sequence (accession no. AF000986, Y13618 and Y13619) (27,28) with BAC clone 486O2 (accession no. AC002531). Partial genomic sequence of UTY was determined comparing cDNA sequence (accession no. AF000994, AF000995, AF000996) (27) with clones 494G17 (accession no. AC005820) and NH0386L03 (accession no. AC006376).
Patient selection
We selected 173 infertile patients affected by idiopathic azoospermia or oligozoospermia (sperm count < 20 x 106/ml) in whom bilateral testicular fine needle aspiration cytology (34,35) showed Sertoli cell-only syndrome (n = 41), severe hypospermatogenesis (n = 92) or moderate hypospermatogenesis (n = 40). As control subjects we have studied 100 normal fertile men, 30 azoospermic men with congenital or acquired obstruction of the seminal tract, and 50 azoospermic or severely oligozoospermic patients (sperm count < 5 x 106/ml) with known causes of testicular damage (previous chemo-radiotherapy, orchi-epididymitis or testicular trauma).
All subjects carried a normal 46,XY karyotype and an intact Yq euchromatin outside the AZFa region, as shown by PCR analysis using 30 STSs, as previously described (15). Details of the testicular fine needle aspiration technique and analysis have been previously given (34–36); briefly, Sertoli cell-only syndrome is characterized by the complete absence of spermatogenic cells in both testes, while hypospermatogenesis shows a reduction in the absolute number of these cells, which are however in normal relative proportions, i.e. no maturation disturbances are present.
PCR and Southern blot analysis of the AZFa region
PCR was performed on genomic DNA extracted from peripheral blood using STS markers sY82, sY83, sY86, sY85, sY84, sY87, sY88 (51), GY6 (33), 264Tel, 475Tel, 494Cen, 494.146K, 494-130K (31), and primers specifically amplifying USP9Y, DBY and UTY genes (27–29,52). Since some of the previously described primers for these genes (27,52) gave unreliable results, we designed PCR assays specific for these genes in order to amplify both cDNA and genomic DNA: DBY1F 5'-TATTGGCAATCGTGAAAGAC-3' and DBY1R 5'-TGCCGGTTGCCTCTACTGGT-3' (accession no. G49468); DBY2F 5'-ATCGACAAAGTAGTGGTTCC-3' and DBY2R 5'-AGATTCAGTTGCCCCACCAG-3' (accession no. G49469) (Fig. 5); UTY1F 5'-GTGTCCTCTTTTCGCATTTG-3' and UTY1R 5'-CCGCTGGTTGGTTGATGGTC-3' (accession no. G49471).
Thermocycling conditions were 35 cycles of 1 min at 94°C, 1 min at 57–60°C and 1 min at 72°C. Reactions were performed using 1 U Taq polymerase (Pharmacia, Milan, Italy) with the supplier’s buffer (1x = 10mM Tris–HCl pH 8.0, 50 mM KCl, 1.5 mM MgCl2), 200 µM each dNTP and 1 mM each primer, in a final volume of 20 µl. Detection of amplification products was performed by electrophoresis on agarose gel transilluminated with UV. Negative results (no amplification) were considered only after three amplification failures, eventually repeating the experiments on new DNA extracted from a second blood collection, in the presence of normal amplification of the other Yq STSs and confirmed by the co-amplification of the SRY gene (sY14) (51) by multiplex PCR. STS–PCR analysis of Yq euchromatin outside AZFa was performed as previously described (15).
PCR amplification failures for DBY and USP9Y were confirmed by Southern blot using 20 µg of genomic DNA digested with different enzymes and 32P-labelled probes prepared from PCR products for DBY1 and DF3.1 by the random primed method (Boehringer Mannheim, Milan, Italy). Prehybridization and subsequent hybridization with the probe (60°C, 2 h) was done in Rapid-hyb buffer (Amersham, Milan, Italy); washing was done several times in 2x SSC and 0.1% SDS at room temperature, and in 0.1x SSC and 0.1% SDS for 30 min at 50°C. Blots were exposed to Kodak X-Omatic film at –70°C for at least 72 h. When available, fathers or brothers of deleted patients were also investigated, both with PCR and Southern blot analyses.
Expression analysis
Human cDNA libraries from different tissues (Human MTC Panel II, Clontech, Palo Alto, CA) were screened by PCR for each AZFa gene and its X-homologue with the following primers: DFFRX1F 5'-TCCATGAAGACTTCATTCAG-3' and DFFRX1R 5'-CATCTGCAGGATCTATCAGC-3' for USP9X (accession no. G49472); DFFRY J/D (28) for USP9Y; UTX1F 5'-CACTGGAGAGACACCTAACA-3' and UTX1R 5'-TATCCAACAAAATGCTACTG-3' for UTX (accession no. G49474); UTY1 (see above) for UTY; DBX1F 5'-TTGGCAGCCGTGGTGACAGA-3' and DBX1R 5'-ACTCAAGATGGGCAACAGAA-3' for DBX/DDX3 (accession no. G49473). For DBY analysis we used a primer (DBYRev: 5'-TTTTTTTTTTGGGGTGGCAC-3'), specifically designed to amplify alternative transcript 1 (short) together with DBY2F (accession no. G49470). PCR conditions were as listed above with the exception of a more stringent annealing temperature (61°C) for DBY2F/DBYRev.
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ACKNOWLEDGEMENTS |
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The financial support of Telethon-Italy (grant no. E.C0988) and MURST 1999 is gratefully acknowledged.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +39 049 8212639; Fax: +39 049 657391/851888; Email forestac@protec.it
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REFERENCES |
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