Further cytologic evidence for Xp-Yp translocation in XX males using in situ hybridization with Y-derived probe

Summary Chromosome preparations from seven subjects with aberrations of sex chromosomes were utilized for in situ hybridization studies with the tritium-labeled Y-derived probe p50f. Two subjects had a pseudodicentric chromosome consisting of two copies of Yp and a portion of Y long arm; two were XX males [46,XX,t(Xp;Yp)], one was missing part of the Y short arm, and another had t(5p;Yq); in addition cells from an XYY male as well as a normal 46,XY male, and a 46,XX female, were hybridized with the same probe. The hybridization technique of Harper and Saunders (1981) was used. There was excess labeling of the Yp/paracentromeric regions in the cases with the normal Y, the XYY, the pseudodicentric Y, and the 5/Y translocation. No significant label was seen on metaphases from the normal 46,XX female or the female with the partially missing Y short arm. Excess label was present on the X short arm in the cases of the XX males; there were 8% and 9.5% of cells with label. The combined cytogenetic and hybridization data indicate that one X short arm in these XX males has undergone a translocation with Yp, and that genes for sex determination probably reside on the distal half of the Y short arm.     

Mapping the testis determinants by an analysis of Y-specific sequences in males with apparent XX and XO karyotypes and females with XY karyotypes.

A number of patients with paradoxical sex chromosome complements (so-called XY females, XX and XO males) have been investigated with a series of 19 Yp and 4 Yq DNA probes to establish which region of the Y is essential for male sexual differentiation. Of the 23 XX males, 18 possessed one or more Yp probe sequences with only 5 lacking such sequences. Of 9 XY females examined, only one showed evidence of a deletion in Yp occurring either as a result of X-Y interchange or interstitial deletion. This suggests that the majority of XY females are not commonly deleted for those Y sequences which are found to be transferred to the X in XX males. The DNA of two XO males both contained different portions of the Y. From a comparison of the patterns of Yp sequences in these patients, it has been possible to elaborate a model of Yp in terms of the order of probe sequences and to suggest a location for the testis determining region in distal Yp.     

Three cases of 45,X/46,XYnf mosaicism

Summary Three patients with 45,X/46,XYnf mosaicism were investigated by Southern hybridization using both X- and Y-specific DNa probes. Our patients seem to be hemizygous for the X chromosomal loci tested. Single-copy and low-copy repeated Y chromosomal sequences assigned to the short arm, centromere, and euchromatin of the long arm have been detected in our patients, suggesting the Y chromosomal origin of the marker chromosome both in male and female cases studied. Densitometry of autoradiographs revealed a double dose of Yp-specific fragments of the DXYS1 locus. None of the patients tested showed either the 3.4- or the 2.1-kb Hae III malespecific repeated DNa sequences. It seems likely that the Ynf is a pseudodicentric chromosome with duplication of Yp and euchromatic Yq sequences, the Yq heterochromatin being lost. Our findings indicate structural heterogeneity of the marker chromosome and in addition provide further information on the relative position of DNa sequences detectected by DNA probes 50f2, M1A, and pDP105.     

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.     

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.     

Dynamic increase of a 45,X cell line in a patient with multicentric ring Y chromosomes

Because ring Y chromosomes are unstable during cell division most reported patients are mosaics, usually including a 45,X cell line. The phenotype varies from normal males or females with streak gonads to sexual ambiguities. We present here the case of a 23-year-old man who was referred at 11 years for growth delay. The GTG-banded karyotypes of lymphocytes revealed two cell lines: 46,X,dic r(Y) seen in 76% of the metaphases analyzed and 45,X (24%). Karyotypes and FISH were performed eight years later with the following probes: DYZ3 (Y centromere), SRY (sex-region of the Y), DYZ1 (Yq heterochromatin), CEPX/Y (X centromere and Yq heterochromatin), TelVysion Xp/Yp, Xq/Yq (X and Y subtelomeres), pan-telomeric, cosmid clones LLycos130G04 and LLycos37C09 (PARII), and BAC clone RP11-5C5 (Yq11.223). The results showed an increase in the 45,X cell line (60%) and a reduction in the 46,X,dic r(Y) cell line (36.4%). The use of Yq probes showed that the ring Y chromosome was dicentric. In addition, other ring Y structures were observed. The breakpoints occurred in proximal Yp11.32 or in Yp11.31 distal to SRY and in Yq12 distal to the PARII region. Therefore, most of the Y remained intact and all genes, with the exception of those in PARI, are present in double dosage in the dic r(Y). The level of mosaicism was important in defining the phenotype.     

Heteromorphic X chromosomes in 46,XX males: Evidence for the involvement of X-Y interchange

Summary G- and R-banded chromosome preparations from eight of twelve 46,XX males, with no evidence of mosaicism or a free Y chromosome, were distinguished in blind trials from preparations from normal 46,XX females by virtue of heteromorphism of the short arm of one X chromosome. Photographic measurements on X chromosomes and on chromosome pair 7 in cells from twelve 46,XX males, eight 46,XX females, and four 46,XY males revealed a significant increase in the size of the p arm of one X chromosome in the group of XX males, independently characterised as being heteromorphic for Xp. No such differences were observed between X chromosomes of normal males and females or between homologues of chromosome pair 7 in all groups. The heteromorphism in XX males is a consequence of an alteration in shape (banding profile) and length of the tip of the short arm of one X chromosome, and the difference in size of the two Xp arms in these 46,XXp+ males ranged from 0.4% to 22.9%. From various considerations, including the demonstration of a Y-specific DNA fragment in DNA digests from nuclei of one of three XX males tested, it is concluded that the Xp+ chromosome is a product of Xp-Yp exchange. These exchanges are assumed to originate at meiosis in the male parent and may involve an exchange of different amounts of material. The consequences of such unequal exchange are considered in terms of the inheritance of genes located on Yp and distal Xp. No obvious phenotypic difference was associated with the presence or absence of Xp+. Thus, some males diagnosed as 46,XX are mosaic for a cryptic Y-containing cell line, and there is now excellent evidence that maleness in others may be a consequence of an autosomal recessive gene. The present data imply that in around 70% of 46,XX males, maleness is a consequence of the inheritance of a paternal X-Y interchange product.     

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.     

Isolation and characterization of Y chromosome DNA probes.

A sorted, cloned Y chromosome phage library was screened for unique Y chromosome sequences. Of the thousands of plaques screened, 13 did not hybridize to radiolabeled 46,XX total chromosomal DNA. Three plaques were characterized further. Clone Y1 hybridized to multiple restriction enzyme fragments in both male and female DNA with more intense bands in male DNA. Clone Y2, also found in female and male DNA, is probably located in the pseudosutosomal region because extra copies of either the X or Y chromosomes increased Y2 restriction enzyme fragment intensity in total cellular DNA. Clone Y5 was male specific in three of four restriction enzyme digests although in the fourth a light hybridizing band was observed in both male and female DNA. Clone Y5 was sublocalized to band Yq 11.22 by hybridization to a panel of cellular DNA from patients with Y chromosome rearrangements. Clone Y5 can be used to test for retention of the proximally long arm Y suggested to cause gonadal cancer in carrier females. The long series of GA repeats in Y5, anticipated to be polymorphic, may provide a sensitive means to follow Y chromosome variation in human populations.     

Absence of Y-specific DNA sequences in human 46,XX true hermaphrodites and in 45,X mixed gonadal dysgenesis

Summary A search for Y-specific DNA sequences has been performed in a sample of seven 46,XX true hermaphrodites and one 45,X mixed gonadal dysgenesis case and compared with a sample of 11 XX males. Using six Y-specific DNA probes no hybridization signal was obtained in the hermaphrodite group; in contrast, all XX males gave a positive signal with at least one probe. This difference is statistically highly significant. We conclude that the aetiology of true hermaphroditism is different from that of the XX male syndrome. As all cases of the hermaphrodite group are positive for the serological sex-specific antigen (Sxs) it is concluded that this antigen can be present even in the absence of Y-specific DNA.     

Regional assignment of Y-linked DNA probes by deletion mapping and their homology with X-chromosome and autosomal sequences.

A series of Y recombinants have been isolated from Y-specific DNA libraries and regionally located on the Y chromosome using a Y deletion panel constructed from individuals carrying structural abnormalities of the Y chromosome. Of twenty recombinants examined twelve have been assigned to Yp and eight to Yq. Five of the Yp recombinants map between Yp11.2 and Ypter and one can only be assigned to Yp. Of the former, four detect homologies on the X chromosome between Xq13 and Xq24 and the latter one between Xp22.3 and Xpter. The sixth recombinant detects autosomal homologous sequences. The six remaining Yp probes are located between Ycen and Yp11.2. One of these detects a homology on the X chromosome at Xq13-Xq24 and a series of autosomal sequences, two detect uniquely Y-specific sequences and three a complex pattern of autosomal homologies. The remaining eight recombinants have been assigned to three intervals on Yq. Of three recombinants located between Ycen and Yq11.21 two detect only Y sequences and one additional autosomal homologies. Two recombinants lie in the interval Yq11.21-Yq11-22, one of which detects only Y sequences and the other an Xp homology between Xp22.3 and Xpter. Finally, the three remaining Yq recombinants all detect autosomal homologies and are located between Yq11.22 and Yq12. The divergence between homologies on different chromosomes has been examined for three recombinants by washing Southern Blots at different levels of stringency. Additionally, Southern analysis of DNA from flow sorted chromosomes has been used to identify autosomes carrying homologies to two of the Y recombinants.     

Molecular characterization of a Y;15 translocation segregating in a family

Summary We have used Y-specific and Y-derived DNA probes for in situ hybridization and Southern blotting analysis to characterize a Y;15 translocation showing normal Mendelian inheritance in a family. Cytogenetically there appeared to be an unbalanced translocation of Yqh to 15p; this translocation may be considered as a prototype of those translocations between Yq and the short arm of an acrocentric chromosome which have a population incidence of approximately 1 in 2,000. Our molecular studies showed that, in all probability, the breakpoints were near the border between Yq11.23 and Yq12, and in 15p11, respectively; the translocation is abbreviated t(Y;15)(q12;p11). Using the Y-specific probe pY431 in a quantitative Southern hybridization assay, normal females had no hybridization, female carriers and normal men had the same amount, and male carriers had twice that amount. Cytogenetic analysis and quantitative in situ hybridization using probes pY431 and pY3.4 were consistent with the hypothesis that the portion of Yq translocated to 15p comprised all of Yq12 and none of Yq11. The absence of Southern hybridization with probes specific for Yp and Yq11 confirmed this observation. Even though the family was ascertained through two brothers who both had schizophrenia and were carriers of the translocation, the clinical evaluation of a total of nine individuals with the translocation and five without it did not suggest its association with an abnormal phenotype.     

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.     

Molecular analysis of aberrations of Xp and Yq

Summary Three cases of Y chromosomal aberrations were studied using a panel of Y-specific DNA sequences from both Yp and euchromatic Yq. One case was a phenotypic male fetus with a Y-derived marker chromosome. The short arm of this chromosome was intact, but most of its long arm was missing. The second case had a 46,Xyq- karyotype with portions of euchromatic Yq, including the spermatogenesis region, missing. The third case was a phenotypic female with a 46,XXp+ karyotype. The extra material on the Xp+ chromosome was derived from the heterochromatic, and part of the euchromatic, portion of Yq. Application of X-specific DNA sequences demonstrated that the distal portion of the short arm of the translocation X chromosome was deleted (Xpter—p22.3). The three examples demonstrate the importance of diagnostic DNA analysis in cases of marker chromosomes, and X and Y chromosomal aberrations. In addition, the findings in the patients facilitate further deletion mapping of euchromatic Yq.     

The sole presence of the testis-determining region of the Y chromosome (SRY) in 46,XX patients is associated with phenotypic variability.

Four cases of XX patients with testis development are reported. The aim of this study was to describe their clinical features and to see if there was any relationship between phenotypes and the presence of Y material. Several human Y-derived sequences including the SRY probe were used to analyze the DNA of the patients. Yp material including the pseudo-autosomal region and SRY was detected. The cases reported in this study confirm that XX true hermaphrodites cannot be distinguished from XX males on the basis of their genotypes. There is no relationship between clinical and anatomical phenotypes and the presence of Y material. SRY does not warrant a complete and normal testis differentiation. Although similar in some features with Klinefelter's syndrome patients, XX males exhibit specific clinical manifestations due to the lack of Y-specific genes.     

Ring Y chromosome: molecular characterization by DNA probes

A young male with a karyotype of 46,X,+ mar is described. Physical mapping of the marker chromosome by using Y-specific single-copy or moderately repeated DNA sequences as molecular probes showed that, in addition to the heterochromatic part of the Yq, a considerable portion of the euchromatin in both Yp and Yq had been lost. These findings suggest that the marker chromosome is a ring Y, for the generally accepted model of ring formation implies breakages in both chromosome arms. The clinical features of the patient correlated well with the phenotypic changes expected from the loss of genetic material from the Y.     

Breakage of the human Y-chromosome short arm between two blocks of tandemly repeated DNA sequences

Y-chromosomal rearrangements, a common cause of sex reversal in man, frequently occur between two blocks of repeated DNA. Both blocks are composed of 20-kb tandemly repeated Y-chromosome-specific DNA sequences. They are located in the proximal portion of the Y short arm on a NotI restriction fragment of approximately 5.3 Mb and on an MluI fragment of approximately 5.5 Mb. Chromosome breaks positioned between the two blocks were detected in two of three 46,XY females with deletions of Yp and in five of six 46,XX males positive for the repeat sequences. The rearranged NotI fragments in the 46,XX males were 4.4 Mb and the MluI fragments were 2.0 Mb in length. This indicates that breaks occur within a small region of Yp defined by the two blocks of specific repeated DNA sequences. The region between the two blocks thus appears to be a focus of structural lability in the human Y chromosome.     

Different chromosome Y abnormalities in Turner syndrome.

A 17-year-old phenotypically female girl was referred for evaluation because of short stature and primary amenorrhea. Cytogenetic analysis showed a mosaic 46,XY/45,X/47,XYY/46,X,idic(Yq)/47,XY,idic(Yq)/48,XXY,idic(Yq)/46,X,t(C;Y) karyotype. Conventional cytogenetic results were supplemented with fluorescence in situ hybridization (FISH) techniques to ensure a better characterization of abnormalities. By using FISH, a supernumerary marker chromosome derived from chromosome Y which could not be detected by conventional cytogenetics was revealed. Furthermore, additional abnormalities and their frequencies were highlighted by the application of DNA probes specific for X and Y chromosomes. Thus, FISH proved useful in determining low frequency cell lines which would need analysis of a large number of good quality metaphase spreads by conventional cytogenetic techniques: it helped in identifying the nature and the origin of unknown markers and rearrangements which have important implication in sexual differentiation and development of gonadal tumours.     

Variable transfer of Y-specific sequences in XX males.

A series of twelve XX males and their relatives have been examined by Southern blot analysis with fourteen different Y recombinants. The pattern of Y sequences present shows considerable variation between XX males. Furthermore, on the basis of the terminal transfer model, anomalous patterns of Y sequences are evident in certain XX males in that sequences located as proximal Yp by means of a Y deletion panel are found to be present in the absence of distal sequences. These anomalies can be resolved by proposing that the order of Yp sequences varies in the population in the form of inversion polymorphisms in the Y chromosomes of normal males. Alternatively, it is necessary to invoke multiple recombination events between the X and Y chromosomes to explain the patterns of Y sequences in these XX males. Southern analysis on DNA prepared from flow sorted X chromosomes of XX males indicates that the Y sequences in these patients are linked to X chromosomes.     

A 45,X male with a Yp/18 translocation

Summary A patient described as a 45,X male (Forabosco et al. 1977) was examined for the presence of Y-specific DNA by using various probes detecting restriction fragments from different regions of the Y chromosome. Positive hybridization signals were obtained for Yp fragments only. In situ hybridization with two different probes, pDP31 and the pseudoautosomal probe 113F, led to a clear assignment of the Yp sequences to the short arm of one chromosome 18. Cytogenetically, the presence of all of Yp including the Y centromere on 18p could be demonstrated replacing a segment of similar size of 18p. Thus, the Y/18 translocation chromosome is dicentric structurally, but it was shown to be monocentric functionally with the no. 18 centromere active. Gene dosage studies with the probe B74 defining a sequence at 18p11.3 demonstrated a single dose of this sequence in the patient. In agreement with these observations, the patient shows clinical signs of the 18p-syndrome. It is concluded that in XO males in general, the X is of maternal origin while the maleness is due to a de novo Y/autosome translocation derived from the father. Depending on the nature of the autosomal deficiency caused by the Y/autosome translocation, the patient may have congenital malformations.