Figure 3 - uploaded by Thomas Duell
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Schematic representation of the human Y chromosome with the respective G-bands for the short (p) and long (q) arm indicated on the left; the yeast artificial chromosome (YAC) clones/ probes used for the hybridization experiments are displayed on the right, according to their order defined by Jones et al. (1994) and Kirsch et al. (1996); the correlation of YAC clones with the G- bands was enabled by the use of common markers in their maps and a map as published by Bardoni et al. (1991). Also indicated are the deletion intervals as defined by Vergnaud et al. (1986), according to a deletion map by O’Reilly et al. (1992), and the deleted regions as found in our case. ϩϩϩ ϭ represented; – – – ϭ absent.
Source publication
A constitutional de-novo deletion of the long arm of the Y chromosome was detected by standard cytogenetic analysis in a 38-year
old male who, except for small testes and cryptozoospermia, was phenotypically normal. The deletion was further characterized
by fluorescent in-situ hybridization (FISH) and digital image analysis using contigs of overlap...
Context in source publication
Context 1
... exact nature of a karyotypically abnormal, unusually small aberrant Y chromosome was studied. Figure 1 shows the G- banded karyotype obtained from a short term lymphocyte culture of the male individual. Figure 2A displays the aberrant chromosome after DAPI staining in size comparison with chromosomes 21 and X respectively. It is obvious that no brightly stained material is present along the small chromosome. We interpreted the small Y as a del(Y)(q11). In contrast, the person’s father, two brothers and nephew all exhibited a normal sized Y chromosome with a relatively large heterochromatic region. Figure 3 schematically displays the YAC clones used as probes and their location along a normal Y chromosome, according to the physical map by Jones et al. (1994) and Kirsch et al. (1996). It indicates the deletion according to the results obtained from our experiments with the aberrant Y. Also included are the deletion intervals as published by Vergnaud et al. (1986), defined according to markers given in different maps (Bardoni et al. , 1991; Jones et al. , 1994; Kirsch et al. , 1996). The clones were selected to provide a deep, continuous coverage of the entire euchromatin of the chromosome which should allow us to define the exact nature of the deletion. Table I lists the hybridization results on normal metaphase spreads and the aberrant Y chromosome, 328 and mapping positions as well as additional information contained in the Whitehead database. Using digital image analysis, some of the YACs consistently gave smaller and weaker signal domains on the del(Y) in comparison with the normal Y when looking at a number of chromosomes. The respective YACs (925D10, 786C10, 913B1) were located in the border regions of detected deletions. All YACs derived from the short arm were represented as expected from hybridization to a normal Y. Two YAC clones (858E6 and 758G1) did not give signals on the normal Y chromosome, but, cross-hybridized due to chimerism (or contamination), to chromosomes 4q and 14q respectively. A number of clones, deriving from the proximal long arm (900E7, 218F9, 361D10, 802D9, 951B1, 773E5, 908E8) were not represented on the aberrant chromosome, but resulted in visible signals on the cytogenetically normal Y. YAC 925D10 showed two domains on the normal Y q-arm, a stronger and more consistent distal one and a more proximal one which was not detectable on all analysed chromosomes. The same probe, when applied to the del(Y) however, gave only one comparatively weak, but consistent signal and was the first probe within the contig found to be retained distal to the deleted region on the long arm. Another more distally located clone (801F5) gave equally strong signals on either variant, in contrast to clones 786C10 and 913B1 which were found to be weaker on the aberrant Y. The most distally located 2 YAC clones, 750E11 and 870H10, which contain the most distal markers within the euchromatic region and, as determined by hybridization to the normal Y, heterochromatic repeats, were not represented on the deleted chromosome. These results allowed us to define one breakpoint on the proximal q-arm of the del(Y), most likely close to the centromere where no optimal coverage by our YAC contig was provided. Another breakpoint was obviously located distal to YAC 908E8, most likely within YAC 925D10, and the most distal break had occurred proximal to YAC 870H10, already containing heterochromatic repeats, and very likely within YACs 786C10 and 913B1, according to the observation of the reduced signal intensities. Besides the locus-specific probes, we also applied a PCR derived heterochromatin-specific repeat probe, showing a very strong signal on normal metaphase Y chromosomes and interphase nuclei. This probe failed to give a detectable hybridization signal on the aberrant Y, also supporting the additional terminal deletion. When co-hybridizing YAC probes mapping to the p- and q-arm on a normal Y, their signals were also found on different ends of the aberrant chromosome. Figure 2B shows an overview of images taken from hybridization experiments with the deleted Y in direct comparison with the normal sized variant. In summary, the normal Y exhibited all signals as expected according to the literature (Jones et al. , 1994; Kirsch et al. , 1996), except the two obviously chimeric clones. Only one clone (933A6) did not give any signals. We sought to characterize a major deletion of a Y chromosome in an infertile male individual by FISH. Contigs of overlapping YAC clones (Jones et al. , 1994; Kirsch et al. , 1996) were applied as probes, and, in comparison with the results on a karyotypically normal Y chromosome, the representation of these clones on the aberrant chromosome was tested. We were able to define a normal structure of the p-arm, whereas a large portion of the proximal q-arm and a more distal portion, including all of the heterochromatin, was missing. Since the Y chromosome structure of the proband’s relatives (father, two brothers, one nephew) were karyotypically normal, we concluded that the deleted Y chromosome was a de-novo constitutional aberration, and assumed that it was the cause of the cryptozoospermia and elevated FSH concentration. A more detailed characterization of the deletion by G-banding or other conventional means was impossible due to the small size of the aberrant chromosome. Using overlapping YAC clones as FISH probes and by detecting the presence and also intensity of FISH signals on the aberrant chromosome, we were able to define the deletion in detail. Digital image analysis ...
Citations
... Most AZFc transcriptional units have functional homologues on autosomes, which may explain the presence of some degree of spermatogenesis, albeit diminished, in many of these men. Numerous other laboratories have also detected AZFc microdeletions in variable percentages of azoospermic men depending upon the study design and specific patient population (Girardi et al., 1997; Pryor et al., 1997; Duell et al., 1998; Foresta et al., 1998; Grimaldi et al., 1998; Liow et al., 1998; Chang and Tsai, 1999; Kim et al., 1999) However, investigations describing the clinical characteristics of AZFcdeleted men are limited (Mulhall et al., 1997a; Silber et al., 1998; Kleiman et al., 1999; Page et al., 1999). Critically important questions need to be answered and can only be done so by looking at a large group of men with identical AZFc microdeletions. ...
Severe spermatogenic compromise may be the result of a Y-chromosomal deletion of the AZFc region. Prior studies are limited to relatively small numbers of AZFc-deleted men. In this study, we have fully characterized 42 infertile men with a Y chromosome microdeletion strictly confined to the AZFc region, and we report on 18 children conceived through the use of ICSI.
A total of 42 oligospermic or azoospermic men had AZFc deletions. History, physical examination, karyotype, FSH, LH, testosterone, testis histology and results of ICSI using ejaculated or testis sperm were retrospectively accumulated in two academic clinical practices.
All men were somatically healthy. Karyotypes were 46,XY in all but two men. FSH, LH, testosterone and testis histology could not differentiate those with oligospermia or azoospermia, nor could they predict whether sperm could be found in harvested testis tissue. Paternal age was not increased. Sperm production appeared stable over time. The results of ICSI were not affected by the AZFc deletion. All but one of the offspring were healthy. The sons inherited the AZFc deletion with no increase in length.
AZFc-deleted men are somatically healthy, will most likely have useable sperm, will have stable sperm production over time and will have a good chance to experience biological paternity, but their sons will also be AZFc-deleted.
... Numerous other laboratories have also detected AZFc microdeletions in variable percentages of azoospermic men depending upon the study design and specific patient population (Girardi et al., 1997;Pryor et al., 1997;Duell et al., 1998;Foresta et al., 1998;Grimaldi et al., 1998;Liow et al., 1998;Chang and Tsai, 1999;Kim et al., 1999) However, investigations describing the clinical characteristics of AZFcdeleted men are limited (Mulhall et al., 1997a;Silber et al., 1998;Kleiman et al., 1999;Page et al., 1999). ...
... As previously noted by us and other groups (Najmabadi et al., 1996a;Stuppia et al., 1996;Foresta et al., 1997Foresta et al., , 1998Girardi et al., 1997;Pryor et al., 1997;Duell et al., 1998;Rossato et al., 1998), PCR analysis frequently showed noncontiguous deletions. The Y chromosome seems to be highly unstable and prone to deletions, probably since it is rich in repetitive elements and repeats (Girardi et al., 1997;Yen et al., 1998), and interstitial double deletions may be explained by different hypotheses: (i) the PCR observations may reflect really separated microdeletions; (ii) some STS may be from repetitive sequences (as demonstrated for example for sY146, sY153 and sY155) (Yen et al., 1998); and (iii) a complex rearrangement (e.g. an inversion with subsequent interstitial deletion) in the father may be the cause. ...
Microdeletions in Yq11 overlapping three distinct 'azoospermia factors' (AZFa-c) represent the aetiological factor of 10-15% of idiopathic azoospermia and severe oligozoospermia, with higher prevalence in more severe testiculopathies, such as Sertoli cell-only syndrome. Using a PCR-based screening, we analysed Yq microdeletions in 180 infertile patients affected by idiopathic Sertoli cell-only syndrome and different degrees of hypospermatogenesis, compared with 50 patients with known causes of testicular alteration, 30 with obstructive azoospermia, and 100 normal fertile men. In idiopathic severe testiculopathies (Sertoli cell-only syndrome and severe hypospermatogenesis), a high prevalence of microdeletions (34.5% and 24.7% respectively) was found, while milder forms were not associated with Yq alteration. No deletions were found in testiculopathies of known aetiology, obstructive azoospermia, normal fertile men and male relatives of patients with deletions. Deletions in the AZFc region involving the DAZ gene were the most frequent finding and they were more often observed in severe hypospermatogenesis than in Sertoli cell-only syndrome, suggesting that deletions of this region are not sufficient to cause complete loss of the spermatogenic line. Deletions in AZFb involving the RBM gene were less frequently detected and there was no correlation with testicular phenotype, with an apparent minor role for such gene in spermatogenesis. The DFFRY gene was absent in a fraction of patients, making it a candidate AZFa gene. Our data suggest that larger deletions involving more than one AZF-candidate gene are associated with a more severe testicular phenotype.
Rapid incorporation of microarray analysis/aCGH
for studies of children with developmental disabilities and its endorsement as a first-tier test for these children [1] has yielded a vast number of subtle chromosome changes that were unseen by routine karyotyping.
To clarify whether cryptorchidism might be the expression of an intrinsic congenital testicular abnormality, we investigated the frequency of Y chromosome long arm (Yq) microdeletions in unilateral excryptorchid subjects manifesting an important bilateral testiculopathy. Microdeletion analysis of Yq was performed by polymerase chain reaction in the following subjects: 40 unilateral excryptorchid patients with azoospermia or severe oligozoospermia due to a bilateral severe testiculopathy (Sertoli cell-only syndrome or severe hypospermatogenesis); 20 unilateral excryptorchid men with moderate oligozoospermia and a normal testicular cytological picture in the contralateral testis; 110 patients affected by idiopathic severe primary testiculopathies; 20 patients affected by idiopathic moderate testiculopathy; and, as controls, 50 patients affected by known causes of testiculopathy and 100 fertile men. Eleven of 40 (27.5%) unilateral excryptorchid patients affected by bilateral testiculopathy and 28 of 110 (25.4%) patients affected by idiopathic severe primary testiculopathy showed Yq microdeletions, whereas no microdeletions were found in all the other subjects, nor in male relatives of patients with deletions. Microdeletions were located in different parts of Yq, including known regions involved in spermatogenesis (DAZ and RBM, AZFa, b, and c) and other loci still poorly defined. No difference in localization of deletions was evident between cryptorchid and idiopathic patients. Microdeletions in Yq may be responsible for severe bilateral testicular damage that could be phenotypically expressed by unilateral cryptorchidism, as well as by idiopathic infertility.
