Chromosome anomalies in human azoospermia

The anomalies of genome were found as a result of cytogenetic study of three azoospermic men. In two cases, the circular Y chromosome was revealed. Different methods of chromosome staining demonstrated complete loss of heterochromatic portion of the long arm of the Y chromosome in one case, and the absence of the euchromatic region in another. A balanced translocation among the chromosomes 1 and 15 was observed in the third case. A question concerning disturbances of spermatogenesis having chromosomal etiology is discussed.     

Centromeric alpha satellite DNA amplification and translocation in an unusually large chromosome 14p+ variant

Summary We report cytogenetic and molecular studies on a family that carries, in the father, an unusually large chromosome 14p+ variant [WSi-var(14)(p+)] and, in one of his children, a translocation [DSi-der(14)] involving the variant chromosome. Increase in the size of WSi-var(14)(p+) was estimated to be approximately 35% that of a normal chromosome 14. Presence of extra chromosomal material in this variant chromosome was demonstrated by G-banding using trypsin and staining with Leishman, G-banding using bromodeoxyuridine (BrdU) and Giemsa, and R-banding using BrdU and Giemsa. This material was positive using C-banding with BaOH and staining with Giemsa and negative in DAPI/distamycin staining, suggesting that it contained repetitive DNA but probably not of the types found in the heterochromatic regions of chromosomes 1, 9, 15, 16, and Y. Staining of the nucleolus organiser region (NOR) with AgNO₃ indicated the retention of the NOR in WSi-var(14)(p+) but not in DSi-der(14). In situ hybridisation of metaphase cells with an alpha satellite DNA probe specific for human acrocentric chromosomes demonstrated a significantly increased amount of centromeric alpha sequences in WSi-var(14)(p+). Most or all of the extra alpha sequences were retained in DSi-der(14), indicating translocation near the very distal end of the enlarged region. The extra alpha satellite DNA material may have originated through amplification of some centromeric segments. The possible role of the amplified DNA in chromosomal translocations is discussed.     

Amplification of satellite III DNA in an unusually large chromosome 14p+ variant

Summary A phenotypically normal male (WSm) was found to have an unusually large short arm of chromosome 14. Increase in the size of this variant chromosome [Wsm-var(14)] was estimated to be approximately 30% that of a normal chromosome 14 by G-banding using trypsin and staining with Leishman. The extra chromosomal material was positive in CBG staining (C-banding using BaOH and staining with Giemsa), suggesting the presence of repetitive DNA. In situ hybridisation using repetitive probes demonstrated this material to be strongly positive for satellite III DNA, and negative for Y-specific heterochromatic DNA. Hybridisation with an alpha DNA probe specific for human acrocentric chromosomes indicated the retention of the centromere, and the absence of alpha DNA in the extra chromosomal material. We propose the origin of the extra chromosomal material in WSm-var(14) to be a result of amplification of contiguous satellite III DNA that is normally present in the short arm of chromosome 14. This variant chromosome does not appear to be associated with the abnormal phenotype in WSm's daughter who is mentally retarded and carries a t(1;?)(q41;?) translocation of chromosome 1.     

The cytogenetics of 90 patients with idiopathic mental retardation/malformation syndromes and of 90 normal subjects

Summary A cytogenetic study, done on randomized coded slides, of 90 patients with idiopathic mental retardation and at least 3 other developmentally independent congenital anomalies and of 90 normal subjects is reported. Audiatorography, Q-banding and C-staining were used in the analysis of chromosomally abnormal cases. Eight patients were found to have chromosome abnormalities. Four had substantial chromosome aberrations that would be expected to cause abnormal phenotype. These were CD165 (46,18q-); CD25 (46,18q+) (partial trisomy of 10q); CD175 (46,4q+) and CD95 (46,mar22). In addition, 4 patients were found to have chromosomal anomalies that could not account for their conditions. Three of these were considered to have heterochromatic variants. Patient CD167 had an 9qh+ chromosome which had been inherited from her mother. Case CD137 had a No. 19 chromosome with additional centric heterochromatin. A similar chromosome was found in her mother, maternal grandmother and 2 of 3 half sibs. In patient CD125 a telocentric No. 13 was found. In addition, CD80 was shown to have an XYY constitution.In the normal subjects, no unbalanced chromosome rearrangements were found. Four persons, however, had minor chromosome anomalies. Three were considered to have heterochromatic variants. These were CD54 (46,22p+); CD149 (46,21p+) and CD19 (46,tel22). One normal subject (CD51) was found to be a balanced t(13q14q) carrier. The translocation chromosome had been inherited from his father.     

The Giemsa banding patterns of the standard and four reconstructed karyotypes of Vicia faba

The Giemsa banding patterns of the standard karyotype of Vicia faba and of four new karyotypes with easily interdistinguishable chromosomes due to interchanges and inversions are described and compared with the data of other authors on preferential Giemsa staining in Vicia faba. All karyotypes contain 14 easily reproducible marker bands which characterize chromosome segments known to be heterochromatic. It is shown that the preferential Giemsa staining of chromosome regions is a valuable tool for the localization of translocation and inversion points in the chromosomes of the reconstructed Vicia karyotypes. A close correlation exists between banding patterns, segment extension by incorporation into chromosomal DNA of azacytidine and mutagen-specific clustering of induced chromatid aberrations in the new karyotypes.     

A 45,X male with translocation of euchromatic Y chromosome material

Summary A phenotypically normal 32-year-old male with azoospermia was found to have a 45,X karyotype with presence of excess euchromatic material on 14p. The parents' karyotypes are normal. This observation is interpreted as a Y/14 translocation with loss of the heterochromatic Y chromosome material.     

Mapping of a single locus capable of complementing the defective heterochromatin phenotype of Roberts syndrome cells

Roberts syndrome (RS) is a developmental disorder characterized by tetraphocomelia and a broad spectrum of additional clinical features. Most patients with RS exhibit characteristic cytogenetic phenotypes, which include an abnormal appearance of pericentromeric heterochromatin on metaphase chromosomes, referred to as "heterochromatic repulsion." In the present study, we use complementation of this abnormal cytogenetic phenotype as a means to identify a specific region of the normal human genome capable of rendering phenotypic correction. We screened the entire human genome, using a transient chromosome-transfer assay, and demonstrated complementation exclusively after the transfer of proximal chromosome 8p, a result subsequently confirmed by stable microcell-mediated chromosome transfer. Additionally, homozygosity mapping was used to refine the interval of this complementing locus to 8p21. The results are consistent with the notion that the single gene defect responsible for heterochromatic splaying and developmental abnormalities maps to chromosome 8p21.     

Partial trisomy 14q due to maternal t(4;14)(p16;q32) in a dysmorphic newborn

Partial Trisomy 14q is a rare chromosomal disorder that mostly results from a parental translocation. We report here a newborn boy with partial trisomy 14q and dysmorphic features that are compatible with previously reported cases. Conventional cytogenetic analysis revealed an extra chromosomal segment at the end of the short arm of chromosome 4. In order to determine the origin of this chromosome region we used subtelomeric FISH technique. Based on the results of these cytogenetic studies and the physical examination, this dysmorphic case was diagnosed as partial trisomy of 14q and his karyotype determined as 46 XY, der(4)t(4;14)(p16;q32) resulting from a balanced maternal translocation identified as 46,XX, t(4;14)(p16;q32).     

Integrative mapping of Gossypium hirsutum L. by meiotic fluorescent in situ hybridization of a tandemly repetitive sequence (B77)

We determined the relative positions of the tandem-repeat molecular cytogenetic marker B77, translocation breakpoints, and telosome arms in Gossypium hirsutum cytogenetic stocks by fluorescence in situ hybridization (FISH) analysis of meiotic quadrivalents in 16 single and 2 double translocation heterozygotes and five monotelodisomics. Results delimited the B77 FISH locus to the right arm of the D-subgenome chromosome 14 (14R) and the short arm (14sh), respectively. By equating 14R with 14sh and 14L (left) with 14Lo (long), the findings established a unified nomenclature for the arms of chromosome 14. Previously reported chromosome 14 arm locations were confirmed for four of the five translocations involving chromosome 14, namely NT1L-14L (2780), NT2R-14R (2B-1), NT14L-23R (2777), and NT14R-24R (2781), whereas the location of breakpoint T6L-14L was not confirmed and was reassigned to arm 14R. When used as a probe on Southern blots, the B77 signal was associated with a terminus of the D-subgenome RFLP linkage group (LG) D04 by linkage analysis of an interspecific F(2) population, now known to be chromosome 20. However, additional codominant DNA marker information in the affected region excluded the B77 polymorphism detected by Southern blot hybridization from chromosome 20 and, indeed, from the remainder of the genome.     

Quinacrine Mustard—a Selective Fluorescent Stain for the Y Chromosome in Human Tissues for Routine Cytogenetic Screening

A simple sensitive and specific cytochemical method for detection of the Y chromosome in man (George 1970, Pearson et al. 1970) is based on staining by alkylating fluorochromes. The dye binds strongly to the heterochromatic regions of the Y chromosome, thus causing these regions to fluoresce very brightly under ultraviolet (uv) light. The Y chromosome could be identified easily at metaphase and also as a highly fluorescent body in interphase cells. Two bodies were seen by Pearson et at. in a subject having the XYY anomaly. In prophase cells, it appears as a fluorescing chromatin thread. Its presence has been demonstrable only in males; hence the usefulness of this technique for rapid cytogenetic screening is obvious. The constancy and reproducibility of the technique seems to rule out the possibility of unreliability caused by nonspecific binding of the dye. The present report gives techniques applied to tissues usually studied in cytogenetic screening     

Chromosomal in situ suppression hybridization of human gonosomes and autosomes and its use in clinical cytogenetics

Summary DNA libraries from sorted human gonosomes were used selectively to stain the X and Y chromosomes in normal and aberrant cultured human cells by chromosomal in situ suppression (CISS-) hybridization. The entire X chromosome was stained in metaphase spreads. Interphase chromosome domains of both the active and inactive X were clearly delineated. CISS-hybridization of the Y chromosome resulted in the specific decoration of the euchromatic part (Ypter-q11), whereas the heterochromatic part (Yq12) remained unlabeled. The stained part of the Y chromosome formed a compact domain in interphase nuclei. This approach was applied to amniotic fluid cells containing a ring chromosome of unknown origin (47,XY; +r). The ring chromosome was not stained by library probes from the gonosomes, thereby suggesting its autosomal origin. The sensitivity of CISS-hybridization was demonstrated by the detection of small translocations and fragments in human lymphocyte metaphase spreads after irradiation with ⁶⁰Co-gamma-rays. Lymphocyte cultures from two XX-males were investigated by CISS-hybridization with Y-library probes. In both cases, metaphase spreads demonstrated a translocation of Yp-material to the short arm of an X chromosome. The translocated Y-material could also be demonstrated directly in interphase nuclei. CISS-hybridization of autosomes 7 and 13 was used for prenatal diagnosis in a case with a known balanced translocation t(7;13) in the father. The same translocation was observed in amniotic fluid cells from the fetus. Specific staining of the chromosomes involved in such translocations will be particularly important, in the future, in cases that cannot be solved reliably by conventional chromosome banding alone.Dedicated to Professor Friedrich Vogel on the occasion of his 65th birthday     

Induction, processing and persistence of radiation-induced chromosomal aberrations involving hamster euchromatin and heterochromatin

Euchromatic and heterochromatic regions are easily distinguished in Chinese hamster sex chromosomes, hence offering the possibility of studying the role of chromatin structure in the induction, processing and persistence of radiation-induced chromosome damage. X-ray (4 Gy)-induced breaks in the euchromatic Xp and in the heterochromatic Xq were analysed immediately and 4h after irradiation by premature chromosome condensation (PCC) in combination with either FISH using chromosome arm-specific probes or Giemsa staining. The study, performed with female Chinese hamster splenocytes, was extended to a 34 h recovery followed by arm-specific FISH in metaphase. A significant over-involvement of the heterochromatic Xq in radiation-induced breakage was observed at all sampling times (p<0.001). However, the heterochromatic state had little effect on the processing of the induced lesions. In a second experiment, the persistence of radiation-induced chromosome aberrations (CAs) involving Xp, Xq and Y chromosome was studied with cultured Chinese hamster male splenocytes sampled 30, 56 and 96 h after irradiation (4 Gy). A higher involvement of the heterochromatic regions (Xq and Y) in radiation-induced CAs was again observed in the first sampling time (p<0.001), suggesting that Chinese hamster heterochromatin could be more radiosensitive than euchromatin. Cells with CAs involving heterochromatin were apparently less persistent than those with lesions involving euchromatin. This observation could be attributable to either the distribution of CA per cell or to the fraction of potentially stable exchanges.     

Molecular and classical cytogenetic analyses demonstrate an apomorphic reciprocal chromosomal translocation in Gorilla gorilla.

The existence of an apomorphic reciprocal chromosomal translocation in the gorilla lineage has been asserted or denied by various cytogeneticists. We employed a new molecular cytogenetic strategy (chromosomal in situ suppression hybridization) combined with high-resolution banding, replication sequence analysis, and fluorochrome staining to demonstrate that a reciprocal translocation between ancestral chromosomes homologous to human chromosome 5 and 17 has indeed occurred.     

Cytogenetic Analysis of the Second Chromosome Heterochromatin of Drosophila Melanogaster

This paper reports the cytogenetic characterization of the second chromosome heterochromatin of Drosophila melanogaster. High resolution cytological analysis of a sample of translocations, inversions, deficiencies and free duplications involving the pericentric regions of the second chromosome was achieved by applying sequential Hoechst 33258 and N-chromosome banding techniques to larval neuroblast prometaphase chromosomes. Heterochromatic rearrangements were employed in a series of complementation assays and the genetic elements previously reported to be within or near the second chromosome heterochromatin were thus precisely assigned to specific heterochromatic bands. The results of this analysis reveal a nonhomogeneous distribution of loci along the second chromosome heterochromatin. The l(2)41Aa, l(2)41Ab, rolled (l(2)41Ac) and l(2)41Ad loci are located within the proximal heterochromatin of 2R, while the nine remaining loci in the left arm and two (l(2)41Ae and l(2)41Ah) in the right arm map to h35 and to h46, respectively, the most distal heterochromatic regions. In addition, a common feature of these loci revealed by the cytogenetic analysis is that they map to specific heterochromatic blocks but do not correspond to the blocks themselves, suggesting that they are not as large as the Y fertility factors or the Rsp locus. Mutations of the proximal most heterochromatic loci, l(2)41Aa and rolled, were also examined for their phenotypic effects. Extensive cell death during imaginal disc development was observed in individuals hemizygous for either the EMS 31 and rolled mutations, leading to a pattern of phenotypic defects of adult structures.     

Mouse chromosome-specific painting probes generated from microdissected chromosomes

Using degenerate primer amplification of chromosomes microdissected from banded cytogenetic preparations, we constructed both whole chromosome painting probes for mouse Chromosomes (Chrs) 1, 2, 3, and 11 and a centromere probe that strongly paints most mouse centromeres. We also amplified a Robertsonian translocation chromosome microdissected from unstained preparations to construct a painting probe for Chrs 9 and 19. The chromosomes probes uniformly painted the respective chromosomes of origin. We demonstrated the utility of the Chr 11 probe in aberration analysis by staining mutants that we had previously identified as containing a Chr 11 translocation, and in some mutant cell lines we observed chromosome rearrangements not previously detected in stained cytogenetic preparations. The technology of microdissection and amplification applies to all mouse chromosomes or to specific subchromosomal regions and will be useful in mouse genetics, in aberration analysis, and for chromosome identification.     

Balanced translocation t(17p--; 23p+) in the chimpanzee]

A chimpanzee family was studied, in which the father had a balanced translocation t(17p--; 23p+). The mother showed the normal female chromosome complement. Their daughter had also the normal female karyotype, but with heteromorphic chromosomes 23. A cytogenetic analysis was made using G- and Q-banding techniques and in addition an alkaline silver method for NOR staining. A mechanism of the translocation inheritance is discussed.     

Supernumerary bisatellited chromosome in a family ascertained through a patient with Sturge-Weber syndrome.

The presence of a structurally abnormal extra chromosome in a patient with Sturge-Weber syndrome and several members of her family is described. With routine techniques the abnormal chromosome is slightly submetacentric, of the size of a G group chromosome and shows satellites on both arms. C-banding suggested the presence of 2 centromeric regions rather than one, and to explain this finding, in addition to the segregation of the abnormal chromosome through 3 generations and why only one centromere is visible with the usual cytogenetic technique, an hypothesis is advanced suggesting that it resulted from an unusual type of Robertsonian translocation, in which one of the breacks involved directly the centromere of an acrocentric producing a partially dicentric bisate-lited chromosome. The association of Sturge-Weber syndrome with the chromosome abnormality is thought to be fortuitous and the lack of clinical manifestations of all members of this family with the abnormal chromosome, including one with two extra ones, is explained by the fact that it was almost entirely formed by heterochromatic material. The usefulness of C-banding in the study of this patient is strongly emphasized.     

Use of fluorescent sequence-specific polyamides to discriminate human chromosomes by microscopy and flow cytometry

In this paper, we demonstrate the use of synthetic polyamide probes to fluorescently label heterochromatic regions on human chromosomes for discrimination in cytogenetic preparations and by flow cytometry. Polyamides bind to the minor groove of DNA in a sequence-specific manner. Unlike conventional sequence-specific DNA or RNA probes, polyamides can recognize their target sequence without the need to subject chromosomes to harsh denaturing conditions. For this study, we designed and synthesized a polyamide to target the TTCCA-motif repeated in the heterochromatic regions of chromosome 9, Y and 1. We demonstrate that the fluorescently labeled polyamide binds to its target sequence in both conventional cytogenetic preparations of metaphase chromosomes and suspended chromosomes without denaturation. Chromosomes 9 and Y can be discriminated and purified by flow sorting on the basis of polyamide binding and Hoechst 33258 staining. We generate chromosome 9- and Y-specific ‘paints’ from the sorted fractions. We demonstrate the utility of this technology by characterizing the sequence of an olfactory receptor gene that is duplicated on multiple chromosomes. By separating chromosome 9 from chromosomes 10–12 on the basis of polyamide fluorescence, we determine and differentiate the haplotypes of the highly similar copies of this gene on chromosomes 9 and 11.