A point mutation,R59G,within the HMG-SRY box in a female 45,X/46,X,psu dic(Y)(pter→q11::q11→pter)

We report a molecular and cytogenetic investigation of a psu dic(Yp) chromosome identified in blood and ovarian tissue from a female with mosaic karyotype 45,X/46,X,+ psu dic(Yp). FISH analysis showed that the psu dic(Yp) has two copies of the short arm, two centromeres and two copies of the proximal long arm. PCR analysis also confirmed the presence of the SRY gene and the Y centromere, and also confirmed the deletion of the Y-heterochromatic region. Because of the possibility of a mutation, a fragment of 609 bp of the SRY gene was sequenced from independent PCR products. The analysis of the sequence indicated the presence of two different copies of the gene: one presented a point mutation, R59G, within the HMG-box; the other had a sequence identical to that already published. Both sequences were found at a proportion of 1:1. The absence of a 46,XY cell line suggests that the rearrangement took place during gametogenesis or during the first division after fertilization. Also, the existence of different sequences of the SRYgene in the same Y chromosome suggests that the formation of the dicentric took place prior to the mutation of the SRY gene. To our knowledge, this is the first time that a mutation has been described in codon 59 within the HMG- SRY box, and also the first case of a psu dic(Yp) chromosome that displays two different copies of the SRY gene.     

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.     

Ambiguous genitalia by 9p deletion inherent to a dic(Y;9)(q12;p24)

We describe here a 3-month-old male infant with brachy-plagyocephaly, short neck, widely spaced nipples, mild hypertonia, and ambiguous external genitalia but with both testes in the scrotum and no Müllerian derivates. His karyotype was 45,X,der(Y;9)(q12;p24).ish der(Y;9)(DYZ3+,SRY+,9ptel-) de novo. This patient's impaired sex differentiation is consistent with gonadal dysgenesis and compares with the male-to-female sex reversal secondary to a partial 9p deletion in spite of an intact Yp or SRY locus documented in 24 patients including a sex-reversed girl with a (Y;9) dicentric derivative. As for the cytogenetic findings, this case represents the second instance of a de novo pseudodicentric (Y;9) chromosome with loss of both distal 9p and Yq12 regions, apparent intactness of SRY, and consistent or preferential inactivation of the Y centromere. In addition, the possible 9p23p-p22 duplication observed in this case evokes the concomitant 9p22-p21 duplication documented in the previous girl with a (Y;9) derivative. Hence, these striking similarities point to a nonrandom Y;9 rearrangement in patients with either sex reversal or gonadal dysgenesis. Even if the present pseudodicentric derivative had inactivated the Y centromere, the existence of some variant cells points to functional dicentricity as it has been documented in other Y;autosome dicentric derivatives.     

Centromeric association of a microchromosome Y in two male patients

We characterized two Y-ring microchromosomes (MC) found in an azoospermic patient with Turner stigmata (case A) and a male infant with hypospadias (case B). The karyotypes, as assessed by banding, FISH, and STRs/STSs analyses, were 46,X,r(Y).ish r(Y)(p11.3q11.222)(SRY+,DYZ3+) and 46,X,+r(Y)/45,X.ish r(Y)(p 11.2q11.2)(Xp/Yp-,SRY+,DYZ3+) respectively. In both cases, we evaluated the association of each MC with the centromere of the nearest and second nearest chromosomes in G-banded metaphases by means of measuring the intervening distance according to two criteria: < or =1 time or < or =3 times the size of the MC in each metaphase. The case A's MC was associated 84 times in 98 cells according to the latter or less strict criterion and two times in 98 cells according to the strict criterion; the corresponding values for case B were 84 and two in 95 cells respectively. The centromeric association appears to be related to centromeric attraction mediated by heterochromatin or centromere-specific proteins, the replication time, and the Rabl orientation.     

Deletion mapping of the testis determining locus with DNA probes in 46,XX males and in 46,XY and 46,X,dic(Y) females.

Eleven Y-specific DNA probes hybridizing with DNA from one or more 46,XX males were isolated from a recombinant phage DNA library constructed from flow sorted human Y chromosomes. Two probes hybridized with DNA from nine out of eleven, i.e. greater than 80% of these 46,XX males. The relative frequency of hybridization of the probes in the 46,XX males and in a 46,X,dic(Y) female, together with in situ hybridization data, allowed mapping of the probes on Yp in relation to a putative testis determining locus. Several of those probes were also absent in a 46,XY female, further refining a model for ordering the probes on Yp. The DNA of one XX male hybridized both with probes from Yp and probes from proximal Yq (excluding the pericentral region). This suggests that complex translocations may occur into the DNA of 46,XX males that involve not only parts of Yp but also parts of Yq.     

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.     

47,X,idic(Y),inv dup(Y): a non-mosaic case of a phenotypically normal boy with two different Y isochromosomes and neocentromere formation

A de novo aberrant karyotype with 47 chromosomes including 2 different-sized markers was identified during prenatal diagnosis. Fluorescence in situ hybridization (FISH) with a Y painting probe tagged both marker chromosomes which were supposed to be isochromosomes of the short and the long arm, respectively. A normal boy was born in time who shows normal physical and mental development. To characterize both Y markers in detail, we postnatally FISH-mapped a panel of Y chromosomal probes including SHOX (PAR1), TSPY, DYZ3 (Y centromere), UTY, XKRY, CDY, RBMY, DAZ, DYZ1 (Yq12 heterochromatin), SYBL1 (PAR2), and the human telomeric sequence (TTAGGG)(n). The smaller Y marker turned out to be an isochromosome containing an inverted duplication of the entire short arm, the original Y centromere, and parts of the proximal long arm, including AZFa. The bigger Y marker was an isochromosome of the rest of the Y long arm. Despite a clearly visible primary constriction within one of the DAPI- and DYZ1-positive heterochromatic regions, hybridization of DYZ3 detected no Y-specific alphoid sequences in that constriction. Because of its stable mitotic distribution, a de novo formation of a neocentromere has to be assumed.     

Organization of DYZ2 repetitive DNA on the human Y chromosome

The location of the human Y-specific repetitive DNA sequence DYZ2 with HaeIII cleavage sites spaced at 2.1 kb was reexamined. Previous reports had mapped the 2000 DYZ2 copies to the very distal end of the heterochromatic Yq12 band. In the present study, a cloned DYZ2 fragment (pHY2.1) was used for Southern and slot blot analyses of male DNA as well as for nonradioactive in situ hybridization to chromosomes. DNA and metaphase preparations from 79 individuals with polymorphic or aberrant Y chromosomes were examined. DYZ2 repeats are not confined to the distal tip of Yq12, but extend through the entire heterochromatin of Yq12. In the naturally occurring length polymorphisms of Yq, the amount of DYZ2 sequence varies in proportion to the measured sizes of band Yq12. Explanations are presented for the fact that previous studies restricted the location of DYZ2 to the telomeric end of Yq12.     

Dicentric Yp chromosome in a patient with the gonadal dysgenesis and gonadoblastoma.

A patient with a short stature, gonadal dysgenesis, and bilateral gonadoblastoma had 3 cell lines in the blood and in the skin: 46,X,dic (Y) (pter vector q 1 2 : : 1 2 vector pter) as a major cell line 46,X,del(Y) (q 1 2), and 45,X. The intensively flourescent distal part of the Yq was deleted in both the Y-dicentric and Yq--chromosome. Both parents had normal karyotypes.     

Heterogeneity of pericentric inversions of the human y chromosome

Pericentric inversions of the human Y chromosome (inv(Y)) are the result of breakpoints in Yp and Yq. Whether these breakpoints occur recurrently on specific hotspots or appear at different locations along the repeat structure of the human Y chromosome is an open question. Employing FISH for a better definition and refinement of the inversion breakpoints in 9 cases of inv(Y) chromosomes, with seemingly unvarying metacentric appearance after banding analysis, unequivocally resulted in heterogeneity of the pericentric inversions of the human Y chromosome. While in all 9 inv(Y) cases the inversion breakpoints in the short arm fall in a gene-poor region of X-transposed sequences proximal to PAR1 and SRY in Yp11.2, there are clearly 3 different inversion breakpoints in the long arm. Inv(Y)-types I and II are familial cases showing inversion breakpoints that map in Yq11.23 or in Yq11.223, outside the ampliconic fertility gene cluster of DAZ and CDY in AZFc. Inv(Y)-type III shows an inversion breakpoint in Yq11.223 that splits the DAZ and CDY fertility gene-cluster in AZFc. This inversion type is representative of both familial cases and cases with spermatogenetic impairment. In a further familial case of inv(Y), with almost acrocentric morphology, the breakpoints are within the TSPY and RBMY repeat in Yp and within the heterochromatin in Yq. Therefore, the presence of specific inversion breakpoints leading to impaired fertility in certain inv(Y) cases remains an open question.     

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.     

Selective protection of specific DNA sequences in the heterochromatin of C-banded human Y chromosomes.

The molecular basis of C-banding was investigated by in situ hybridization of human Y chromosome-derived repeated sequences, DYZ1 and DYZ2, to untreated or to alkaline-treated metaphases. Autoradiography of G-banded metaphases showed that both probes hybridized to the long arm of Y. Alkaline hydrolysis significantly reduced grain number for DYZ2 (58%-82%; P less than .05) but not for DYZ1 (P greater than .05). Similar results were observed for interphase nuclei. These findings demonstrated that the heterochromatin of the long arm contains at least two repetitive DNA fractions having two different sensitivities to alkaline hydrolysis. These observations support the notion that DYZ2 maps terminally on the Yq arm and may be nonheterochromatic.     

Sex reversal due to Xp disomy by t(X;Y)(p21;q11).

A 20-month-old infant exhibiting psychomotor retardation, dysmorphisms and ambiguous external genitalia was found to have a 46-chromosome karyotype including a normal X chromosome and a marker Y with most of Yq being replaced by an extra Xp21-->pter segment. The paternal karyotype (G and C bands) was 46,XY. The marker Y composition was verified by means of FISH with a chromosome X painting, an alphoid repeat and a DMD probe. Thus, the final diagnosis was 46,X,der(Y)t(X;Y)(p21;q11)de novo.ish der(Y)(wcpX+,DYZ3+,DMD+). The patient's phenotype is consistent with the spectrum documented in 13 patients with similar Xp duplications in whom sex reversal with female or ambiguous genitalia has occurred in spite of an intact Yp or SRY gene. A review of t(X;Y) identifies five distinct exchanges described two or more times: t(X;Y)(p21;q11), t(X;Y)(p22;p11), t(X;Y)(p22;q11-12), t(X;Y) (q22;q12), and t(X;Y)(q28;q12). These translocations probably result from a recombination secondary to DNA homologies within misaligned sex chromosomes in the paternal germline with the derivatives segregating at anaphase I.     

Pseudodicentric 22;Y translocation transmitted through four generations of a large family without phenotypic repercussion

The most frequent Y-autosome translocations involve an acrocentric autosome and they are frequently familial with neither phenotypic nor reproductive repercussion. However, different Y-autosome translocations have been related to infertility, due to abnormal pairing of the X and Y chromosomes at meiosis and an abnormal XY-body formation or by the disruption of the AZFs (Azoospermic Factor). Rare forms of Y-autosome translocations are those resulting in an unbalanced 45-chromosome karyotype that includes a dicentric Y+autosome chromosome. We describe a new case of a familial pseudodicentric 22;Y that is carried by 19 male members of a large family without phenotypic repercussion. Cytogenetic analysis, fluorescence in situ hybridisation (FISH) and subtelomeric Multiplex Ligation-dependent Probe Amplification (MLPA) assay have been performed. All male members of the family showed the karyotype 45,X,psu dic(22;Y)(p11.2;qter).ish psu dic(22;Y) (SRY+,DYZ3+,D14/D22Z1+). In conclusion, the presence of the dicentric chromosome in the male members of the family reported does not seem to interfere with the correct progression of spermatogenesis.     

Molecular detection of a translocation (Y;11)(q11.2;q24) in a 45,X male with signs of Jacobsen syndrome

Summary A 45,X karyotype was found in a boy with dysmorphic features, hypoglycaemia and pancytopenia. DNA analysis showed the presence of the Y-chromosomal DNA sequences SRY, ZFY, DYZ4, DYZ3 and DYS1. Using fluorescent in situ hybridization, we located DYZ4 and DYZ3 on chromosome llqter and concluded that a de novo translocation (Y;11)(q11.2;q24) with a deletion of 11q24qter and a deletion of Yq11.2Yqter were present; Jacobsen syndrome and azoospermia are associated with these deletions. Signs of Jacobsen syndrome were observed in the patient.     

Nonfluorescent Y chromosomes. Cytologic evidence of origin

Summary Twelve presumptive structurally altered Y chromosomes were studied with Q-, G-, G-11, C-, Cd, and lateral asymmetric banding techniques and were compared with normal X and Y chromosmes and with an abnormal [i(Yq)] Y chromosome that exhibited intact fluorescence. Significant to this work is the fact that the Y chromosome has a small block of Giemsa-11 heterochromatin adjacent to the centromere on the long arm, while the X chromosome does not, which allows a distinction between the X-and Y-derived chromosomes. Two of the twelve altered chromosomes of either X or Y origin are small nonfluorescent rings. Each ring has a G-11-positive band of heterochromatin at the centromere, confirming Y origin. Each of the normal-length nonfluorescent presumed Ys and a Y with a fluorescent band in the center have one G-11 band at the centromere and another at an equal distance from the end of the long arm, the bands also being Cd positive, indicating that these chromosomes are pseudodicentric. The likely mechanism of origin is a break at the distal bright heterochromatin/ euchromatin junction (or within the bright segment in the chromosome with the bright center band), fusion of the sister chromatids at the breakpoints, and loss of the distal segment.     

Combined cytogenetic techniques and non-fluorescent Y. Cytologic evidences of dic(Yp)(q11) in a previously interpreted 46,X,Yq-

mos 45,X/46,X,Y with no bright fluorescence was studied in 4 patients presenting variable phenotypes, from Turner's syndrome, with or without virilization, to ambiguous external genitalia, with combined cytogenetic techniques. G-11 staining demonstrated, in all cases, that the abnormal Y was a dic(Yp). Considerations about the possibility that some of the 46,X,Yq-males attending infertility clinics may be examples of dic(Yp) are made.     

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.     

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.     

Unbalanced translocation 8;Y (45,X,dic(Y;8)(q11.23;p23.1)): case report and review of terminal 8p deletions

A boy with a rare unbalanced de novo Y/autosome translocation is presented. Main clinical features in the boy comprised a psychomotor delay, talipes planus, a dolichocephalus, low set and retroverted ears, supraorbital fullness of subcutaneous tissue and a bulbous nasal tip. Chromosomal analysis on amniocytes showed a single X chromosome and a derivative 8p (Karyotype: 45,X,der(8)GTG). The following DAPI staining revealed the inactivated centromere of the chromosome Y located on 8p and the absence of heterochromatic material Yq. Microsatellite analysis on fetal blood DNA using markers between SRY on Yp and DYS 240 on Yq proved presence of the spermatogenetic relevant factors. A terminal deletion of 8p was confirmed by FISH postnatally. Molecular genetic reassessment revealed the monosomy 8p to be of maternal origin; the translocation can thus be proven to have occurred in the zygote. The breakpoint in 8p was localised distal to GATA4, a gene which is involved in heart development; the finding that our patient did not suffer from cardiac problems agrees with the disomic presence of GATA4. Only the application of FISH combined with microsatellite analysis allowed a precise correlation between clinical phenotype and a subtle deletion of terminal 8p; furthermore, a recurrence risk for the parents could be excluded.