Centromeric inactivation in a dicentric human Y;21 translocation chromosome

A de novo dicentric Y;21 (q11.23;p11) translocation chromosome with one of its two centromeres inactive has provided the opportunity to study the relationship between centromeric inactivation, the organization of alphoid satellite DNA and the distribution of CENP-C. The proband, a male with minor features of Down’s syndrome, had a major cell line with 45 chromosomes including a single copy of the translocation chromosome, and a minor one with 46 chromosomes including two copies of the translocation chromosome and hence effectively trisomic for the long arm of chromosome 21. Centromeric activity as defined by the primary constriction was variable: in most cells with a single copy of the Y;21 chromosome, the Y centromere was inactive. In the cells with two copies, one copy had an active Y centromere (chromosome 21 centromere inactive) and the other had an inactive Y centromere (chromosome 21 centromere active). Three different partial deletions of the Y alphoid array were found in skin fibroblasts and one of these was also present in blood. Clones of single cell origin from fibroblast cultures were analysed both for their primary constriction and to characterise their alphoid array. The results indicate that (1) each clone showed a fixed pattern of centromeric activity; (2) the alphoid array size was stable within a clone; and (3) inactivation of the Y centromere was associated with both full-sized and deleted alphoid arrays. Selected clones were analysed with antibodies to CENP-C, and staining was undetectable at both intact and deleted arrays of the inactive Y centromeres. Thus centromeric inactivation appears to be largely an epigenetic event. Received: 30 January 1997; in revised form: 3 April 1997 / Accepted: 8 May 1997     

Extensive cytogenetic analysis of a stable dicentric isochromosome 21, idic(21), formed by fusion of the terminal long arms

The dicentric isochromosome 21 described in this paper was formed by fusion of the terminal parts of the long arms of two chromosomes 21. No interstitial telomeric AGGGTT repeats could be detected at the fusion point, but G-banding, comparative genomic hybridization, and fluorescence in situ hybridization with painting probes for 21qter revealed no loss of other terminal DNA sequences at the fusion point. Thus, only the telomeric repeats seem to have been lost prior to, or as a consequence of, isochromosome formation. Both short arms of the isochromosome were intact with complete NORs, and staining for alpha-satellite DNA showed that the DNA content of the two centromeres was the same. Antibody staining for the centromeric proteins CENP-C and CENP-E and for topoisomerase IIalpha and IIbeta demonstrated that these proteins were localized predominantly or exclusively at the centromere in the primary constriction. A novel functional in situ assay for topoisomerase activity in vivo similarly demonstrated enzyme activity exclusively at the primary constriction centromere.     

An unusual dicentric Y chromosome with a functional centromere with no detectable alpha-satellite

We describe an unusual marker chromosome Y. This marker is present in 5% of the lymphocytes of a dysgenetic woman showing a mosaic karyotype 45,X/46,XY/ 47,XY+mar. Q-banding revealed that the marker was morphologically identical to the Y chromosome of the patient but presented the primary constriction in the heterochromatic region. C-banding confirmed that the heterochromatic region was C-positive; furthermore, it showed two spots in the euchromatic region in a position corresponding to that of the centromere in the normal Y Fluorescence in situ hybridization with the centromere-specific probe pDP 97 and the pancentromeric alpha-satellite probe 2730 failed to detect any signal at the primary constriction site. To improve the characterization of the marker chromosome, hybridization was performed using pDP 105, a probe located on the short arm of the Y chromosome, together with chromosome-Y- specific paint-hybridizing to the single sequence spanning the Y short arm. In both cases, positive signals telomeric to the inactive centromere were observed. Possible mechanisms resulting in the formation of the marker chromosome are discussed.     

Two dicentric Y isochromosomes,one without the Yqh heterochromatic segment

Summary Two women with primary amenorrhoea and few other stigmata of Turner's syndrome were found to be chromosome mosaics: 45,X/46,X,idic(Y). In Case 1, the dicentric isochromosome Y was found to have a long-arm breakpoint of formation. This structure was interpreted as containing two Y short arms and centromeres separated by a region derived from the proximal Y long arm. One of the centromeres in the Case 1 —idic(Y) was suppressed in 80% of cells in blood, and in these cells it appeared as a regular Y-shaped chromosome. In Case 2 the idic(Y) was derived by a short-arm breakpoint of formation. In all the dicentrics of this case with one primary constriction (functional monocentrics) there was a single Cd band. In the 10% of dicentrics with two primary constrictions, there were two Cd bands. It is argued that the instability of sex isochromosomes is due to this functional dicentricity in some cells. These cases are compared with 42 other Y isochromosomes with various short- and long-arm breakpoints of formation. It is suggested that some of the nonheterochromatic, nonfluorescent Y chromosomes previously reported may be explained as dicentric i(Y) with proximal long-arm breakpoints of formation and one suppressed centromere.     

True vs. false inv(Y)(p11q11.2): a familial instance concurrent with trisomy 21

A boy with Down syndrome due to a free trisomy 21 also had a metacentric Y chromosome with an arm euchromatic and the other heterochromatic inherited from his phenotypically normal father. This chromosome was mitotically stable and hybridized with the DYZ3 probe precisely at its primary constriction; in addition, a subtelomeric Xp/Yp probe gave the expected signal near the end of the euchromatic arm. So, the proband's karyotype was 47,X,inv(Y)(p11q11.2),+21. Given the high frequency of both chromosome anomalies, we regard its concurrence as a mere coincidence. This observation, along with previous reports, allows us to classify the apparent pericentric inversions of the Y chromosome into two types: "true" inversions characterized by an alphoid single centromere and mitotic stability, and "false" inversions in which a nonalphoid centromere has taken over the usual alphoid centromere; indeed, these chromosomes are dicentric and mitotically unstable. Finally, the inv(Y) polymorphism in man compares with that documented in other mammal species, in which the rearranged Y chromosome neither impairs the fertility nor has other phenotypical consequences.     

Tetrasomy 18p de novo: Identification by FISH with conventional and microdissection probes and analysis of parental origin and formation by short sequence repeat typing

We report a de novo supernumerary isochromosome 18p in a child with tetrasomy 18p, analyzed by a straightforward combination of cytogenetic and molecular cytogenetic methods. The diagnostic procedure consisted of standard banding techniques and fluorescence in situ hybridization (FISH) with centromere and library DNA probes for chromosome 18, and 18p-specific FISH probes prepared by chromosome microdissection and in vitro amplification. The maternal origin as well as the most probable cell stages of formation of the supernumerary isochromosome were determined by typing of short sequence repeats (SSRs). The pattern of allelic distribution suggests a nondisjunction during meiosis followed by a centromeric misdivision in an early postzygotic mitosis as the most probable mode of isochromosome 18p formation. The combination of the applied methods represents a powerful tool to investigate the nature and the origin of de novo marker chromosomes. Received: 28 August 1995 / Revised: 3 November 1995; 20 December 1995     

Organizational variation of DYZ1 repeat sequences on the human Y chromosome and its diagnostic potentials

The long arm of the human Y chromosome is flecked with various fractions of repetitive DNA. DYZ1 is one such fraction, which is organized tandemly as an array of a 3.4-kb repeat ranging from 2000-4000 copies in normal males. We have studied the organizational variation of the DYZ1 fraction on the human Y chromosome using DNA samples from CEPH family members and the random population employing the RFLP approach, fluorescence in situ hybridization (FISH), and conducted a similarity search with GenBank sequences. Typing of genomic DNA using DYZ1 as a probe showed an allele length and copy number variations even between two male siblings. Hybridization of DNA from monochromosome hybrids with this probe showed its presence on chromosome 15 in addition to the Y chromosome. Fluorescence in situ hybridization of metaphase chromosomes from an apparently normal male showed DYZ1 sequences in the proximal region of chromosome 11 in addition to the long arm of the Y chromosome. Typing of sets of semen and blood DNA samples from the same human individuals showed discernible allelic variation between the two samples, indicating tissue-specific programmed sequence modulation. DYZ1 seems to be the first probe having the unique potential to discriminate unequivocally the difference between the DNA originating from semen and blood samples, and may be exploited in forensic cases. This probe may also be used as a diagnostic tool to ascertain Y chromosome mosaicism in patients (e.g., Turner), its aberrant status in somatic cells, and possible sequence modulation/rearrangement in the germline samples. Additionally, this can be used to uncover sequence polymorphism in the human population.     

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.     

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.     

A human moderately repeated Y-specific DNA sequence is evolutionarily conserved in the Y chromosome of the great apes.

Evolutionary conservation of the human-derived moderately repeated Y-specific DNA sequence Y-190 (DYZ5) was investigated in the chimpanzee, orangutan, and gorilla. Southern blot analysis showed the presence of the sequence in the Y chromosome of all great apes. Pulsed-field gel electrophoresis and in situ hybridization revealed that the repeat is organized in one major block and confined to a small region of the Y chromosome of the three species. DYZ5 was assigned to the proximal short arm of the Y chromosome of the chimpanzee and orangutan and to the long arm of the Y chromosome of the gorilla. In light of its evolutionary conservation, DYZ5 may have an as yet undetermined structural function in the Y chromosome.     

Molecular cloning and mapping of 10 new probes on the human Y chromosome

We have developed a novel positive cloning vector whose use precludes the cloning of any fragments less than 0.8 kb as well as 3.4-kb EcoRI fragments of DYZ1, the largest repeating-DNA family on the long arm of the human Y chromosome. Using this vector, we subcloned inserts of a Y-chromosome-specific phage library constructed from EcoRI-digested flow-sorted Y-chromosome DNA. Ten novel Y-specific fragments were obtained. Their localization on the Y chromosome was determined by deletion mapping using clinical samples with structurally abnormal Y chromosomes. The long arm of the Y chromosome was divided into 12 segments by the novel probes in combination with established probes. The amelogenin-like sequence, mapped on the long arm in Human Gene Mapping 10, has been mapped on the short arm.     

Characterization of a maize isochromosome 8S*8S.

An isochromosome was found in the maize HiII Parent B line during somatic karyotyping with a multiprobe fluorescence in situ hybridization (FISH) system. Cytological analyses showed that it pairs with the short arm of chromosome 8 during the pachytene stage of meiosis. The chromosome 8 short arm origin of this isochromosome was also confirmed by FISH at mitotic metaphase. Knob heterochromatin signals were present at the short arms of chromosome 8 when subjected to prolonged exposure and also observed at both ends of the isochromosome. This isochromosome can be a univalent or a trivalent by pairing with the normal chromosome 8 short arms during meiosis. At anaphase and telophase, the isochromosome lagged behind other chromosomes. It had a transmission rate of 17%-20% from both male and female gametes. One plant homozygous for the isochromosome contained 2 isochromosomes that differed in the quantity of their CentC centromere repeat sequence. Both variations of the isochromosome were transmitted to the next generation. Because the 2 isochromosomes should be identical by descent, these observations document a radical change in copy number of the centromere repeat array within 1 generation. Plants with 1 isochromosome were not normal as compared with the original HiII Parent B plants. Those that contained a pair of this isochromosome (6 total copies of 8S) were even more abnormal and had reduced fertility. The results indicate the ability of the somatic karyotyping system to recognize and characterize chromosomal aberrations.     

The role of the sex-determining region of the Y chromosome (SRY) in the etiology of 46,XX true hermaphroditism

Summary The syndrome of 46,XX true hermaphroditism is a clinical condition in which both ovarian and testicular tissue are found in one individual. Both Mullerian and Wolffian structures are usually present, and external genitalia are often ambiguous. Two alternative mechanisms have been proposed to explain the development of testicular tissue in these subjects: (1) translocation of chromosomal material encoding the testicular determination factor (TDF) from the Y to the X chromosome or to an autosome, or (2) an autosomal dominant mutation that permits testicular determination in the absence of TDF. We have investigated five subjects with 46,XX true hermaphroditism. Four individuals had a normal 46,XX karyotype; one subject (307) had an apparent terminal deletion of the short arm of one X chromosome. Genomic DNA was isolated from these individuals and subjected to Southern blot analysis. Only subject 307 had Y chromosomal sequences that included the pseudoautosomal boundary, SRY (sex-determining region of Y), ZFY (Y gene encoding a zinc finger protein), and DXYS5 (an anonymous locus on the distal short arm of Y) but lacked sequences for DYZ5 (proximal short arm of Y) and for the long arm probes DYZ1 and DYZ2. The genomic DNA of the other four subjects lacked detectable Y chromosomal sequences when assayed either by Southern blotting or after polymerase chain reaction amplification. Our data demonstrate that 46,XX true hermaphroditism is a genetically heterogeneous condition, some subjects having TDF sequences but most not. The 46,XX subjects without SRY may have a mutation of an autosomal gene that permits testicular determination in the absence of TDF.     

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.     

石貂的染色体研究

本文对分布在我国的石貂北方亚种染色体进行了较详细的研究。结果表明２ｎ＝３８,核型为１４（Ｍ）＋４（ＳＭ）＋１８（ＳＴ）,ＸＹ（Ｍ,Ａ）。Ｃ－带显示该亚种的一些染色体着丝粒区域结构异染色质弱化或消失。Ｎｏ,９染色体的短臂完全异染色质化;Ｘ染色体长臂丰出现插入杂色质带;Ｙ为完全结构异染色质组成。     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

Y;autosome translocations and mosaicism in the aetiology of 45,X maleness: assignment of fertility factor to distal Yq11

Summary Three 45,X males have been studied with Y-DNA probes by Southern blotting and in situ hybridization. Southern blotting studies with a panel of mapped Y-DNA probes showed that in all three individuals contiguous portions of the Y chromosome including all of the short arm, the centromere, and part of the euchromatic portion of the long arm were present. The breakpoint was different in each case. The individual with the largest portion (intervals 1–6) is a fertile male belonging to a family in which the translocation is inherited in four generations. The second adult patient, who has intervals 1–5, is an azoospermic, sterile male. These phenotypic findings suggest the existence of a gene involved in spermatogenesis in interval 6 in distal Yq11. The third case, a boy with penoscrotal hypospadias, has intervals 1–4B. In situ hybridization with the pseudoautosomal probe pDP230 and the Y chromosome specific probe pDP105 showed that Y-derived DNA was translocated onto the short arm of a chromosome 15, 14, and 14, respectively. One of the patients was a mosaic for the 14p+ translocation chromosome. Our data and those reported by others suggest the following conclusions based on molecular studies in eight 45,X males: The predominant aetiological factor is Y;autosome translocation observed in seven of the eight cases. As the remaining case was a low-grade mosaic involving a normal Y chromosome, the maleness in all cases was due to the effect of the testis determing factor, TDF. There is preferential involvement of the short arm of an acrocentric chromosome (five out of seven translocations) but other autosomal regions can also be involved. The reason why one of the derivative translocation chromosomes becomes lost may be that it has no centromere.     

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.     

白颊长臂猿染色体的研究

本文对我国云南南部的白须长臂猿（Ｈ．ｌｅｕｃｏｇｅｎｙｓ）染色体的Ｇ带、Ｃ带、晚复制带及Ａｇ－ＮＯＲｓ进行了较为详细的研究。它的２ｎ＝５２,核型公式为４４（Ｍ或ＳＭ）＋６（Ａ）,ＸＹ（Ｍ,Ａ）。Ｃ带表明一些染色体着丝点Ｃ带弱化;有的染色体出现插入的和端位的Ｃ带;Ｘ染色体两臂有端位Ｃ带,Ｙ染色体是Ｃ带阳性和晚复制的。Ａｇ－ＮＯＲｓ的数目,雌体有４个,雄体有５个,Ｙ染色体上具ＮＯＲ。本文对白颊长臂猿与其它长臂猿间的亲缘关系、核型进化的可能途径进行了讨论。