A deletion map of the human Yq11 region: implications for the evolution of the Y chromosome and tentative mapping of a locus involved in spermatogenesis.

A deletion map of Yq11 has been constructed by analyzing 23 individuals bearing structural abnormalities (isochromosomes, terminal deletions and X;Y, Y;X, or A;Y translocations) in the long arm of the Y chromosome. Twenty-two Yq-specific loci were detected using 14 DNA probes, ordered in 11 deletion intervals, and correlated with the cytogenetic map of the chromosome. The breakpoints of seven translocations involving Xp22 and Yq11 were mapped. The results obtained from at least five translocations suggest that these abnormal chromosomes may result from aberrant interchanges between X-Y homologous regions. The use of probes detecting Yq11 and Xp22.3 homologous sequences allowed us to compare the order of loci within these two chromosomal regions. The data suggest that at least three physically and temporary distinct rearrangements (pericentric inversion of pseudoautosomal sequences and/or X-Y transpositions and duplications) have occurred during evolution and account for the present organization of this region of the human Y chromosome. The correlation between the patient' phenotypes and the extent of their Yq11 deletions permits the tentative assignment of a locus involved in human spermatogenesis to a specific interval within Yq11.23.     

Functional disomy of Xp22-pter in three males carrying a portion of Xp translocated to Yq

A number of Xp22;Yq11 translocations involving the transposition of Yq material to the distal short arm of the X chromosome have been described. The reciprocal product, i.e. the derivative Y chromosome resulting from the translocation of a portion of Xp to Yq, has never been recovered. We searched for this reciprocal product by performing dosage analysis of Xp22-pter loci in 9 individuals carrying a non-fluorescent Y chromosome. In three mentally retarded and dysmorphic patients, dosage analysis indicated the duplication of Xp22 loci. Use of the highly polymorphic probe CRI-S232 demonstrated the inheritance of paternal Xp-specific alleles in the probands. In situ hybridization, performed in one case, confirmed that 29CL pseudoautosomal sequences were present, in addition to Xpter and Ypter, in the telomeric portion of Yq. To our knowledge, these are the first cases in which the translocation of Xp material to Yq has been demonstrated. The X and Y breakpoints were mapped in the three patients by dosage and deletion analysis. The X breakpoint falls, in the three cases, in a region of Xp22 that is not recognized as sharing sequence similarities with the Y chromosome, thus suggesting that these translocations are not the result of a homologous recombination event.     

The human X-linked steroid sulfatase gene and a Y-encoded pseudogene: evidence for an inversion of the Y chromosome during primate evolution

The mammalian X and Y chromosomes are thought to have evolved from a common, nearly homologous chromosome pair. Although there is little sequence similarity between the mouse or the human X and Y, there are several regions in which moderate to extensive sequence homologies have been found, including, but not limited to, the so-called pseudoautosomal segment, in which X-Y pairing and recombination take place. The steroid sulfatase gene is in the pseudoautosomal region of the mouse, but not in man. We have cloned and characterized the human STS X-encoded locus and a pseudogene that is present on the long arm of the Y chromosome. Our data in humans and other primates suggest that there has been a pericentric inversion of the Y chromosome during primate evolution that has disrupted the former pseudoautosomal arrangement of these genes. These results provide additional insight into the evolution of the sex chromosomes and into the nature of this interesting portion of the human genome.     

The pseudoautosomal regions of the human sex chromosomes

In human females, both X chromosomes are equivalent in size and genetic content, and pairing and recombination can theoretically occur anywhere along their entire length. In human males, however, only small regions of sequence identity exist between the sex chromosomes. Recombination and genetic exchange is restricted to these regions of identity, which cover 2.6 and 0.4 Mbp, respectively, and are located at the tips of the short and the long arm of the X and Y chromosome. The unique biology of these regions has attracted considerable interest, and complete long-range restriction maps as well as comprehensive physical maps of overlapping YAC clones are already available. A dense genetic linkage map has disclosed a high rate of recombination at the short arm telomere. A consequence of the obligatory recombination within the pseudoautosomal region is that genes show only partial sex linkage. Pseudoautosomal genes are also predicted to escape X-inactivation, thus guaranteeing an equal dosage of expressed sequences between the X and Y chromosomes. Gene pairs that are active on the X and Y chromosomes are suggested as candidates for the phenotypes seen in numerical X chromosome disorders, such as Klinefelter's (47,XXY) and Turner's syndrome (45,X). Several new genes have been assigned to the Xp/Yp pseudoautosomal region. Potential associations with clinical disorders such as short stature, one of the Turner features, and psychiatric diseases are discussed. Genes in the Xq/Yq pseudoautosomal region have not been identified to date.     

Cytogenetic and molecular investigations of an abnormal Y chromosome: evidence for a pseudo-dicentric (Yq) isochromosome.

A derivative Y chromosome was found in a 55-year-old man with Lambert-Eaton paraneoplasic pseudomyastheniform disease. Small testicles, azoospermia were noticed and hormonal level values were as in the Klinefelter syndrome. A 45,X/46,XYp+ mosa?cism was described on peripheral blood lymphocytes. Cytogenetic investigations with R-G-C- and Q-banding have been performed. In situ hybridization with the GMGY 10 DNA probe showed two copies of proximal Yp sequences. Southern blot analyses were performed using the Y DNA probes 27a, 47z, 64a7, 50f2 disclosing specific Yp and Yq sequences from the pseudoautosomal boundary to the Yq proximal portion. The der(Y) has been defined as a dicentric isochromosome for the long arm with one active and one apparently suppressed centromere. The breakpoint leading to the der(Y), has been located in the pairing segment of the Y short arm (i.e. Yp11.32). So the der(Y) was interpreted as a psu dic(Y) (qter-->cen-->p11.32 ::p11.32-->qter). There was thus an almost complete duplication of the Y chromosome.     

An abnormal terminal X-Y interchange accounts for most but not all cases of human XX maleness

To determine if human XX maleness results from an abnormal chromosomal X-Y interchange, we studied the inheritance of the paternal pseudoautosomal region in nine patients. Those six patients in whom Y-specific DNA was found (Y(+)) inherited the entire pseudoautosomal region from the paternal Y chromosome and lost that of the paternal X chromosome. Moreover, in three Y(+) cases, we observed the deletion of a paternal Xp locus tightly linked to the pseudoautosomal region. These results definitively show that an abnormal and terminal X-Y interchange during paternal meiosis causes Y(+)XX maleness. In contrast, no abnormal X-Y interchange was observed in any of the three Y(-) cases analyzed, suggesting that maleness can occur in the absence of any Y-specific DNA.     

Analysis of complex Y chromosome aberrations using a single DNA probe (Y-367)

A specific cloned DNA sequence (Y-367) detects at least four loci in the euchromatic long arm and in the short arm of the human Y chromosome. Deletion mapping assigns one locus to the distal euchromatic long arm, another to a region close to the centromere on either Yq or Yp, and two additional loci to the Y short arm. Y-367 may thus be used for the rapid screening of even complex Y chromosome aberrations. This is exemplified in a 45,X male with Y chromosome material on the long arm of chromosome 10 by the detection of an inversion of a portion of Yp and by the confirmation of duplications and deletions in two individuals with duplications of part of the Y chromosome.     

Types of Y chromosome deletions and their frequency in infertile men

Deletions of Y chromosome AZF locus were analyzed during a large-scale andrological and genetic examination of 810 infertile men. The search for Yq microdeletions was carried out according to the standard EAA/EMQN guidelines. The breakpoints were mapped for the deletions in AZF locus. The Y chromosome macro- and microdeletions were detected in 61 (7.5%) infertile men. The frequencies of AZF deletions during azoospermia and severe oligozoospermia amounted to 12.2 and 8.1 %, respectively. On the whole, the frequencies of Yq microdeletions and the genophenotypic correlations characteristic of various AZF deletion types comply with the relevant published data. However, spermatozoids in the ejaculate sediment of men with completely deleted AZFa region or AZFb+c deletions (from solitary spermatozoids to several dozens) were detected for the first time. It was demonstrated that the breakpoints were localized between AZFa and AZFb regions proximally to AZFb+c microdeletions for the majority of cytogenetically detectable deletions in the Y chromosome long arm. This indicates that the mechanisms underlying Yq macro- and microdeletions are somewhat different. The issues related to the role of Y chromosome deletions in the origins of monosomy for X chromosome and X/XY mosaicism are discussed.     

Molecular characterization and population study of an X chromosome homolog of the Y-linked microsatellite DYS391

A novel microsatellite homologous to DYS391, a (GATA)(n) short tandem repeat on the human Y chromosome, was identified and characterized in the present work. Employing somatic cell hybrid and deletion panels in a PCR-based approach, we found out that the new microsatellite is located in Xp21.2-22.3, while its Y counterpart mapped to Yq11.21. This X-linked locus (provisionally called DXYS391) and its Y homolog constitute one more example of similarity outside the pseudoautosomal regions between the two human sex chromosomes. Sequencing data showed high levels of homology in the flanking regions of DXYS391 and DYS391 that differ primarily by the presence of a (GACA)(3) motif in the Y locus. Both loci were detected in chimpanzee DNA, suggesting that a putative transposition from the X to the Y occurred before the human/chimpanzee split. The allele frequencies of DYS391 and DXYS391 were investigated, respectively, in 271 Y and 337 X chromosomes from distinct human populations worldwide. DYS391 consistently displayed greater among-population component of the variance of the allele frequencies than DXYS391, as expected due to the three-times lower effective population size of Y chromosomes relative to the X. The intra-population diversity of DYS391, measured by Nei's locus diversity as well as by allele size variance, was lowest in Amerindians, while very low diversity of DXYS391 was seen in Africans. Since our African data are based on a small sample, further studies will be necessary to evaluate better this observation.     

An extremely polymorphic locus on the short arm of the human X chromosome with homology to the long arm of the Y chromosome.

A genomic DNA clone named CRI-S232 reveals an array of highly polymorphic restriction fragments on the X chromosome as well as a set of non-polymorphic fragments on the Y chromosome. Every individual has multiple bands, highly variable in length, in every restriction enzyme digest tested. One set of bands is found in all males, and co-segregates with the Y chromosome in families. These sequences have been regionally localized by deletion mapping to the long arm of the Y chromosome. Segregation analysis in families shows that all of the remaining fragments co-segregate as a single locus on the X chromosome, each haplotype consisting of three or more polymorphic fragments. This locus (designated DXS278) is linked to several markers on Xp, the closest being dic56 (DXS143) at a distance of 2 cM. Although it is outside the pseudoautosomal region, the S232 X chromosome locus shows linkage to pseudoautosomal markers in female meiosis. In determining the X chromosome S232 haplotypes of 138 offspring among 19 families, we observed three non-parental haplotypes. Two were recombinant haplotypes, consistent with a cross-over among the S232-hybridizing fragments in maternal meiosis. The third was a mutant haplotype arising on a paternal X chromosome. The locus identified by CRI-S232 may therefore be a recombination and mutation hotspot.     

Deletion of the pseudoautosomal region and lack of sex-chromosome pairing at pachytene in two infertile men carrying an X;Y translocation

Two males with a 46,Y,der(X),t(X;Y)(p22.3;q11) complement were referred independently for evaluation of sterility with azoospermia. Both patients exhibited minimal symptomatology, characterized only by psychological disturbances. Study of X-chromosome breakpoints with pseudoautosomal probes 68B (DXYZ2 elements), 113D (locus DXYS15), and 19B (locus MIC2) indicated in both patients that at least 97% of the X pseudoautosomal sequences are lost. Hybridization with Xp22.3-specific probes DXS283, DXS284, and DXS31 shows that these loci are retained on the rearranged chromosome. Thus, the X-chromosome breakpoints are located close to the proximal boundary of the pseudoautosomal region, between MIC2 and DXS284.     

A ZFY-negative 46,XX true hermaphrodite is positive for the Y pseudoautosomal boundary

Summary Two loci on the short arm of the human Y chromosome have recently been described as candidates for the testis determining factor (TDF); namely, ZFY, and a locus distal to ZFY, near the pseudoautosomal boundary. We have previously reported on seven 46,XX true hermaphrodites and one 45,X mixed gonadal dysgenesis case all presenting with testicular tissue in their gonads in the apparent absence of Y-specific DNA sequences. A reanalysis of these cases shows them all to lack ZFY, but one 46,XX true hermaphrodite carries sequences next to the Y pseudoautosomal boundary. This case provides further evidence for assigning the TDF locus very close to the pseudoautosomal region on Yp.     

Chromosome assignment of two cloned DNA probes hybridizing predominantly to human sex chromosomes

Summary In situ hybridization experiments were carried out with two clones, YACG 35 and 2.8, which had been selected from two genomic libraries strongly enriched for the human Y chromosome. Besides the human Y chromosome, both sequences strongly hybridized to the human X chromosome, with few minor binding sites on autosomes. In particular, on the X chromosome DNA from clone YACG 35 hybridized to the centromeric region and the distal part of the short arm (Xp2.2). On the Y chromosome, the sequence was assigned to one site situated in the border region between Yq1.1 and Yq1.2. DNA from clone 2.8 also hybridized to the centromeric region of the X and the distal part of the short arm (Xq2.2). On the Y, however, two binding sites were observed (Yp1.1 and Yq1.2). The findings indicate that sex chromosomal sequences may be localized in homologous regions (as suggested from meiotic pairing) but also at ectopic sites.     

Deletion mapping of DNA segments from the Y chromosome long arm and their analysis in an XX male

Twelve DNA segments have been localized to the long arm of the Y chromosome and were assigned to three intervals by deletion mapping. Of these segments, six were from distal Yq11.23, which is supposed to contain a spermatogenesis locus. The physical mapping information was used to analyze an XX male who is positive for DNA sequences both from distal Yp and from Yq. Two of the twelve sequences from Yq (Y-198 and Y-253) were detected in this patient along with two of six short-arm segments tested. Long-range physical mapping placed Y-198 and Y-253 on a common 1100-kb BssHII fragment. In this patient, the long-arm sequences were assigned to distal Xp by in situ hybridization. The data suggest that this XX male derived from an unequal interchange between an X and an inverted Y chromosome presumed to have been present in the patient's father.     

The minimal mammalian Y chromosome - the marsupial Y as a model system

The mammalian X and Y chromosomes are very different in size and gene content. The Y chromosome is much smaller than the X and consists largely of highly repeated non-coding DNA, containing few active genes. The 65-Mb human Y is homologous to the X over two small pseudoautosomal regions which together contain 13 active genes. The heterochromatic distal half of the human Yq is entirely composed of highly repeated non-coding DNA, and even the euchromatic portion of the differential region is largely composed of non-coding repeated sequences, amongst which about 30 active genes are located. The basic marsupial Y chromosome (about 10 Mb) is much smaller than that of humans or other eutherian mammals. It appears to include no PAR, since it does not undergo homologous pairing, synaptonemal complex formation or recombination with the X. We show here that the tiny dunnart Y chromosome does not share cytogenetically detectable sequences with any other chromosome, suggesting that it contains many fewer repetitive DNA sequences than the human or mouse Y chromosomes. However, it shares several genes with the human and/or mouse Y chromosome, including the sex determining gene SRY and the candidate spermatogenesis gene RBMY, implying that the marsupial and eutherian Y are monophyletic. This minimal mammalian Y chromosome might provide a good model Y in which to hunt for new mammalian Y specific genes.     

Homologies between X and Y chromosomes detected by DNA probes: localisation and evolution.

We have isolated and characterized DNA probes that detect homologies between the X and Y chromosomes. Clone St25 is derived from the q13-q22 region of the X chromosome and recognizes a 98% homologous sequence on the Y chromosome. Y specific fragments were present in DNAs from 5 Yq-individuals and from 4 out of 7 XX males analysed. An X linked TaqI RFLP is detected with the St25 probe (33% heterozygosity) which should allow one to establish a linkage map including other polymorphic X-Y homologous sequences in this region and to compare it to a Y chromosome deletion map. Probe DXS31 located in Xp223-pter detects a 80% homologous sequence in the Y chromosome. The latter can be assigned to Yq11-qter outside the region which contains the Y specific satellite sequences. ACT1 and ACT2, the actin sequences present on the X and Y chromosomes respectively, have been cloned. No homology was detected between the X and Y derived fragments outside from the actin sequence. ACT2 and the Y specific sequence corresponding to DXS31 segregate together in a panel of Y chromosomes aberrations, and might be useful markers for the region important for spermatogenesis in Yq. Various primate species were analysed for the presence of sequences homologous to the three probes. Sequences detected by St25 and DXS31 are found only on the X chromosome in cercopithecoidae. The sequences which flank ACT2 detect in the same species autosomal fragments but no male specific fragments. It is suggested that the Y chromosome acquired genetic material from the X chromosome and from autosomes at various times during primate evolution.     

Types of Y chromosome deletions and their frequency in infertile men

Y chromosome deletions in the AZF locus were analyzed during a large-scale andrological and genetic examination of 810 infertile men. The search for Yq microdeletions was carried out according to the standard EAA/EMQN guidelines. The breakpoints were mapped for the revealed AZF deletions. The Y chromosome macro-and microdeletions were detected in 61 (7.5%) infertile men. The frequencies of AZF deletions in patients with azoospermia and severe oligozoospermia amounted to 12.2 and 8.1%, respectively. On the whole, the frequencies of Yq microdeletions and the genophenotypic correlations characteristic of various AZF deletion types agree with the relevant published data. However, spermatozoa in the ejaculate sediment of men with completely deleted AZFa region or AZFb+c deletions (from solitary spermatozoa to several dozens) were detected for the first time. It was demonstrated that the breakpoints were localized between AZFa and AZFb regions proximally to AZFb+c microdeletions for the majority of cytogenetically detectable deletions of the Y chromosome long arm. This indicates that the mechanisms underlying Yq macro-and microdeletions are somewhat different. The issues related to the role of Y chromosome deletions in the origins of X chromosome monosomy and X/XY mosaicism are discussed.     

Comparative mapping of ZFY in the hominoid apes

Summary Within our project of comparative mapping of candidate genes for sex-determination/testis differentiation, we used a cloned probe from the human ZFY locus for comparative hybridization studies in hominoids. As in the human, the ZFY probe detects X- and Y-specific restriction fragments in the chimpanzee, the gorilla, the orangutan, and the gibbon. Furthermore, the X-specific hybridization site in the great apes resides in Xp21.3, the same locus defining ZFX in the human. The Y-specific locus of ZFY maps closely to the early replicating pseudoautosomal segment in the telomeric or subtelomeric position of the Y chromosomes of the great apes, again as found in the human. Thus, despite cytogenetically visible structural alterations within the euchromatic parts of the Y chromosomes of the human species and the great apes, a segment of the Y chromosome defined by the pseudoautosomal region and ZFY seems to be more strongly conserved than the rest of the Y chromosome.     

Strong linkage disequilibrium between the XY274 polymorphism and the pseudoautosomal boundary.

The pseudoautosomal boundary is defined by an Alu repeat element on the Y chromosome. The Alu element is found on all Y chromosomes and on no X chromosomes, establishing it as part of Y-specific sequences. Distal to the Alu element, sequences from the X and Y are strictly homologous, suggesting that the boundary is formed by an abrupt break in sequence homology. Further investigation of the function of the boundary has been undertaken by examining the population structure of an MspI restriction-site polymorphism (XY274), which is located 274 bp distal to the Alu insertion site. Southern blot and polymerase chain reaction analysis demonstrate fixation of the high allele (noncutting or AT base pair) of XY274 on the Y chromosome in most populations, while a full range of high allele frequencies is found on the X chromosomes of different populations. Two exceptions to fixation on the Y chromosome were found in African populations. The level of linkage disequilibrium suggests that the first few hundred base pairs of the pseudoautosomal region on the Y chromosome share a single common origin more recent than the origin of the species.