Diagram of the Y chromosome microdeletions. The solid box ( j ) indicates presence, and dashed lines (– – –) indicate absence of a sequence-tagged site (STS). 

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Context 1

... microdeletions were made available. DNA probes specific for Y chromosome loci (Vergnaud et al ., 1986) or polymerase chain reaction (PCR) amplification of sequence-tagged sites (STS) (Vollrath et al ., 1992) have allowed the identification of microdeletions in Yq regions known not to pair or recombine with the X chromosome. Up to now, three non-overlapping loci have been identified on Yq11; and their deletion is associated with sterility (azoospermia or severe oligospermia) (Vogt et al ., 1996). Two have been localized to interval 6 of Yq11 and are called AZFb and AZFc , and one is more proximal to the centromere in Yq11 and is called AZFa . In interval 6 of Yq11, two genes have been identified that are deleted in a number of infertile men; one is the Y chromosome RNA- recognition motif ( YRRM/RBM ) identified by Ma et al . (1993), the other is the Deleted in AZoospermia gene ( DAZ ) identified by Reijo et al . (1995). The first gene RBM , has 1116 been tentatively linked with AZF (Ma et al ., 1993) and is a member of an RNA-binding protein gene family, which is represented in multiple copies in different regions of the Y chromosome, and at least one copy is localized to the AZFb region (Elliott et al ., 1997). The second gene, DAZ , is at present the strongest candidate for AZF , and its deletion has been found to be associated with a wide range of spermatogenic defects from bona fide Sertoli cell-only (SCO) syndrome to reduced spermatogenesis resulting in severe oligozoospermia (Reijo et al ., 1995, 1996). In the proximal region of Yq11 the gene DFFRY has been recently linked to AZFa . This gene is the Y-linked homologue of the DFFRX ( Drosophila fat-facets related X gene), and it has been shown to be deleted in three azoospermic males (Brown et al ., 1998). However it must be stressed that the rare deletions in this locus (from 1–5% in different surveys) generally associate with a SCO phenotype (Ma et al ., 1992; Vogt et al. , 1996), with the exception of one published case showing reduced spermatogenesis (Qureshi et al. , 1996), thus suggesting that this locus is important for early steps of spermatogenesis, possibly in prenatal development. As a whole the data available would indicate that AZF is a multi- gene complex preferentially located in Yq11, whose members might act at specific differentiative steps of spermatogenesis. In the present report we analysed 67 azoospermic and severe oligozoospermic patients for the presence of microdeletions in Yq11 using 18 STS and two DNA probes. We found four subjects with deletions in AZFc , including the DAZ gene, and one patient with deletions in AZFa . The 67 patients with azoospermia or severe oligozoospermia, selected as described in Methods, were screened by PCR amplification and Southern blot analysis for potential deletions of 20 loci on Yq11. STSs and Y chromosome DNA probes used in this study, and their localization on Yq11 are shown in Figure 1. Most of the STS map to interval 6 of Yq11, which includes the DAZ gene in AZFc locus (sY254, sY277, sY283). Four STS (sY83, sY84, sY86, sY87) map to AZFa , and another STS (sY117) maps to AZFb . We detected deletions on Yq11 in five patients by PCR amplification. Figure 2 shows the Y-specific STS found deleted: patients SP8, FO3, FA16, and FA45 showed deletions in 1117 AZFc involving the DAZ locus and patient FA14 showed microdeletions in AZFa . Patients SP8 and FO3 showed deletions with the same extension including STS sY152, sY242, sY254, sY277 and sY283 but differed for STS sY155, which was deleted in FO3 and present in SP8. Patient FA16 showed a slightly shorter deleted region including STS from sY155 to sY277. Patient FA45 has a unique pattern of deletion in our screening, limited to the DAZ locus (sY277, sY254, sY283). The absence of the DAZ locus was confirmed in patients SP8, FO3 and FA16 by Southern blot analysis, using as a probe the same PCR product found deleted. Figure 3A shows a representative Southern blot experiment using STS sY254, sY277, sY283 as probes. DNA from the same patients was also analysed by a hybridization technique using the Y chromosome DNA probes p49f and p50f2 (see map in Figure 1), showing deletions of both loci DYS1 (detected by p49f) and DYS7C (detected by p50f2) (Figure 3B,C). The absence, in these patients, of DYS1 sequences, previously demonstrated to be 1118 the DAZ gene cluster (Saxena et al ., 1996), agrees with PCR data. The available DNA from the other DAZ -deleted patient FA45 was not sufficient for Southern analysis. Three more patients showed microdeletions of a single STS in AZFc by PCR amplification, being sY283 negative in two cases and sY157 in the third one. Although the PCR amplification was repeatedly negative in separate reactions, we demonstrated that they were false negatives, by a Southern blot study using as probe the same PCR product found deleted. The FA14 patient showed microdeletions only in AZFa , including the STS sY84, sY86, sY87, and sY83 (Figure 2). All other STS analysed in patient FA14 were normally amplified. Southern blot analysis using these STS as probes did not give conclusive results due to cross-reactivity with autosomal DNA. The clinical parameters of the five patients with microdeletions are summarized in Table I. Four patients were azoospermic and only the DAZ -deleted FO3 patient showed very rare and aberrant spermatozoa. Testicular histology was available in three deleted patients. FA14, carrying microdeletions in AZFa , had Sertoli cell-only syndrome with complete lack of germ cells. Patients carrying deletion of the DAZ gene (SP8 and FA45) showed different spermatogenic defects including Sertoli cell-only syndrome and maturation arrest. Testicular histology of patient FA45 carrying a DAZ deletion was associated with a SCO phenotype and is shown in Figure 4. Spermatogenesis is a complex event leading to the formation of mature spermatozoa, occurring under the control of a wide variety of factors acting from prenatal to adult life (Vogt, 1997). An important role in the regulation of spermatogenesis is certainly played by the genetic programming of germ cell itself based both on autosomal and sex chromosomal genes. A group of idiopathic infertility has been, in fact, associated with genetic causes linked to the Y chromosome and many studies have recently shown deletions on Y chromosome long arm in idiopathic azoospermic males. The frequencies of deletions of Yq, reported in different studies, range between 3 and 18% of males with non-obstructive azoospermia or severe oligozoospermia (Reijo et al ., 1995; Stuppia et al. , 1996; Vogt et al ., 1996; Girardi et al ., 1997; Pryor et al ., 1997; Qureshi et al ., 1997; Simoni et al. , 1997; Van der Ven et al ., 1997). In our screening of 67 infertile males we have found five patients carrying microdeletions, corresponding to a frequency of 7.5%, that perfectly agrees with the statistical value obtained averaging all the surveys reported to date (Simoni et al ., 1998). The frequency of microdeletions reported here is the result of a correction of the PCR data, made on the basis of Southern blot analyses. In fact, we could exclude three more patients showing a single STS deletion by PCR amplification, not confirmed by Southern blotting, thus lowering the microdeletion rate from ~12% to 7.5%. Even though it is not possible from our result to evaluate statistically whether some STS are more prone to give false negatives, or whether this technical problem might be relevant in explaining the higher frequency of microdeletions found by others, in our opinion Southern blot analysis has to be performed at least in those cases where PCR reaction fails to amplify a single STS. However, we cannot exclude the possibility that the failure of PCR amplification in Southern blot positive cases is due to mutations or small deletions occurring in the genomic sequence matching the STS primers that we used. This should be investigated by sequence analysis of wider genomic regions spanning the involved STS. We could not obtain control DNA of fathers or brothers of the patients carrying Yq microdeletions, thus the possibility that some of the deletions that we have identified simply reflect polymorphisms cannot be excluded. However, larger studies have shown that most of Yq microdeletions are not paternally inherited (Reijo et al ., 1995), therefore they must occur de novo at some stage of paternal germ cell differentiation (at stages compatible with completion of sperm cell differentiation and fertilization even in the lack of the affected gene) or possibly in fertilized eggs (Edwards and Bishop, 1997). As for the mechanism of the appearance of microdeletions, it has been suggested the occurrence of an intrachromosomal recombination event between repeated sequences flanking the affected gene, causing a loop that is deleted (Edwards and Bishop, 1997). If this is the case, the possibility exists of more such events in the same chromosome causing discontinuous deletions, e.g. the one observed in patient SP8. Many efforts have been made to identify the genes on Yq that are important for a normal spermatogenesis (Lahn and Page, 1997), but up to now the gene/s that are absolutely required for germ cell development have not been characterized. The first genes identified on Yq that could be involved in the control of spermatogenesis belong to the RBM gene family, and include up to 30 copies of genes and pseudogenes, spread over both arms of the Y chromosome (Ma et al ., 1993). RBM genes encode RNA-binding proteins and are specifically expressed in spermatogonia and early spermatocytes (Chandley and Cooke, 1994). The RBM1 gene has been mapped to the AZFb region of the Yq (Vogt. et al ., 1996; Elliott et al. , 1997), but the precise role of this gene in spermatogenesis is not clear, since it is present in many azoospermic males. The other candidate gene for AZF is DAZ which, similar to the RBM1 gene, encodes a ...

Context 2

... infertility in subjects with azoospermia or severe oligozoospermia with unknown aetiology was first associated with a possible genetic cause by Tiepolo and Zuffardi (1976), in a report of six azoospermic patients carrying a deletion of the distal portion of Y chromosome long arm. On the basis of this finding they proposed the existence of a spermatogenesis gene, the ‘azoospermia factor’ ( AZF ) on Yq. Confirmatory data for the actual presence of genes of spermatogenesis in Yq had to wait until molecular maps of Y chromosome and molecular tools for mapping microdeletions were made available. DNA probes specific for Y chromosome loci (Vergnaud et al ., 1986) or polymerase chain reaction (PCR) amplification of sequence-tagged sites (STS) (Vollrath et al ., 1992) have allowed the identification of microdeletions in Yq regions known not to pair or recombine with the X chromosome. Up to now, three non-overlapping loci have been identified on Yq11; and their deletion is associated with sterility (azoospermia or severe oligospermia) (Vogt et al ., 1996). Two have been localized to interval 6 of Yq11 and are called AZFb and AZFc , and one is more proximal to the centromere in Yq11 and is called AZFa . In interval 6 of Yq11, two genes have been identified that are deleted in a number of infertile men; one is the Y chromosome RNA- recognition motif ( YRRM/RBM ) identified by Ma et al . (1993), the other is the Deleted in AZoospermia gene ( DAZ ) identified by Reijo et al . (1995). The first gene RBM , has 1116 been tentatively linked with AZF (Ma et al ., 1993) and is a member of an RNA-binding protein gene family, which is represented in multiple copies in different regions of the Y chromosome, and at least one copy is localized to the AZFb region (Elliott et al ., 1997). The second gene, DAZ , is at present the strongest candidate for AZF , and its deletion has been found to be associated with a wide range of spermatogenic defects from bona fide Sertoli cell-only (SCO) syndrome to reduced spermatogenesis resulting in severe oligozoospermia (Reijo et al ., 1995, 1996). In the proximal region of Yq11 the gene DFFRY has been recently linked to AZFa . This gene is the Y-linked homologue of the DFFRX ( Drosophila fat-facets related X gene), and it has been shown to be deleted in three azoospermic males (Brown et al ., 1998). However it must be stressed that the rare deletions in this locus (from 1–5% in different surveys) generally associate with a SCO phenotype (Ma et al ., 1992; Vogt et al. , 1996), with the exception of one published case showing reduced spermatogenesis (Qureshi et al. , 1996), thus suggesting that this locus is important for early steps of spermatogenesis, possibly in prenatal development. As a whole the data available would indicate that AZF is a multi- gene complex preferentially located in Yq11, whose members might act at specific differentiative steps of spermatogenesis. In the present report we analysed 67 azoospermic and severe oligozoospermic patients for the presence of microdeletions in Yq11 using 18 STS and two DNA probes. We found four subjects with deletions in AZFc , including the DAZ gene, and one patient with deletions in AZFa . The 67 patients with azoospermia or severe oligozoospermia, selected as described in Methods, were screened by PCR amplification and Southern blot analysis for potential deletions of 20 loci on Yq11. STSs and Y chromosome DNA probes used in this study, and their localization on Yq11 are shown in Figure 1. Most of the STS map to interval 6 of Yq11, which includes the DAZ gene in AZFc locus (sY254, sY277, sY283). Four STS (sY83, sY84, sY86, sY87) map to AZFa , and another STS (sY117) maps to AZFb . We detected deletions on Yq11 in five patients by PCR amplification. Figure 2 shows the Y-specific STS found deleted: patients SP8, FO3, FA16, and FA45 showed deletions in 1117 AZFc involving the DAZ locus and patient FA14 showed microdeletions in AZFa . Patients SP8 and FO3 showed deletions with the same extension including STS sY152, sY242, sY254, sY277 and sY283 but differed for STS sY155, which was deleted in FO3 and present in SP8. Patient FA16 showed a slightly shorter deleted region including STS from sY155 to sY277. Patient FA45 has a unique pattern of deletion in our screening, limited to the DAZ locus (sY277, sY254, sY283). The absence of the DAZ locus was confirmed in patients SP8, FO3 and FA16 by Southern blot analysis, using as a probe the same PCR product found deleted. Figure 3A shows a representative Southern blot experiment using STS sY254, sY277, sY283 as probes. DNA from the same patients was also analysed by a hybridization technique using the Y chromosome DNA probes p49f and p50f2 (see map in Figure 1), showing deletions of both loci DYS1 (detected by p49f) and DYS7C (detected by p50f2) (Figure 3B,C). The absence, in these patients, of DYS1 sequences, previously demonstrated to be 1118 the DAZ gene cluster (Saxena et al ., 1996), agrees with PCR data. The available DNA from the other DAZ -deleted patient FA45 was not sufficient for Southern analysis. Three more patients showed microdeletions of a single STS in AZFc by PCR amplification, being sY283 negative in two cases and sY157 in the third one. Although the PCR amplification was repeatedly negative in separate reactions, we demonstrated that they were false negatives, by a Southern blot study using as probe the same PCR product found deleted. The FA14 patient showed microdeletions only in AZFa , including the STS sY84, sY86, sY87, and sY83 (Figure 2). All other STS analysed in patient FA14 were normally amplified. Southern blot analysis using these STS as probes did not give conclusive results due to cross-reactivity with autosomal DNA. The clinical parameters of the five patients with microdeletions are summarized in Table I. Four patients were azoospermic and only the DAZ -deleted FO3 patient showed very rare and aberrant spermatozoa. Testicular histology was available in three deleted patients. FA14, carrying microdeletions in AZFa , had Sertoli cell-only syndrome with complete lack of germ cells. Patients carrying deletion of the DAZ gene (SP8 and FA45) showed different spermatogenic defects including Sertoli cell-only syndrome and maturation arrest. Testicular histology of patient FA45 carrying a DAZ deletion was associated with a SCO phenotype and is shown in Figure 4. Spermatogenesis is a complex event leading to the formation of mature spermatozoa, occurring under the control of a wide variety of factors acting from prenatal to adult life (Vogt, 1997). An important role in the regulation of spermatogenesis is certainly played by the genetic programming of germ cell itself based both on autosomal and sex chromosomal genes. A group of idiopathic infertility has been, in fact, associated with genetic causes linked to the Y chromosome and many studies have recently shown deletions on Y chromosome long arm in idiopathic azoospermic males. The frequencies of deletions of Yq, reported in different studies, range between 3 and 18% of males with non-obstructive azoospermia or severe oligozoospermia (Reijo et al ., 1995; Stuppia et al. , 1996; Vogt et al ., 1996; Girardi et al ., 1997; Pryor et al ., 1997; Qureshi et al ., 1997; Simoni et al. , 1997; Van der Ven et al ., 1997). In our screening of 67 infertile males we have found five patients carrying microdeletions, corresponding to a frequency of 7.5%, that perfectly agrees with the statistical value obtained averaging all the surveys reported to date (Simoni et al ., 1998). The frequency of microdeletions reported here is the result of a correction of the PCR data, made on the basis of Southern blot analyses. In fact, we could exclude three more patients showing a single STS deletion by PCR amplification, not confirmed by Southern blotting, thus lowering the microdeletion rate from ~12% to 7.5%. Even though it is not possible from our result to evaluate statistically whether some STS are more prone to give false negatives, or whether this technical problem might be relevant in explaining the higher frequency of microdeletions found by others, in our opinion Southern blot analysis has to be performed at least in those cases where PCR reaction fails to amplify a single STS. However, we cannot exclude the possibility that the failure of PCR amplification in Southern blot positive cases is due to mutations or small deletions occurring in the genomic sequence matching the STS primers that we used. This should be investigated by sequence analysis of wider genomic regions spanning the involved STS. We could not obtain control DNA of fathers or brothers of the patients carrying Yq microdeletions, thus the possibility that some of the deletions that we ...