Human chromosome 15 confers partial complementation of phenotypes to xeroderma pigmentosum group F cells.

Microcell-mediated transfer of a single human chromosome from repair-proficient human cells to genetic complementation group F cells from the hereditary disease xeroderma pigmentosum (XP) results in partial complementation of repair-defective phenotypes. The complementing chromosome was identified by cytogenetic and molecular analysis as human chromosome 15. Transfer of this chromosome to XP-F cells restores approximately 20% of the resistance of wild-type cells to killing by UV radiation or by the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), as well as partial repair synthesis of DNA measured as unscheduled DNA synthesis. Additionally, complemented XP-F cells have an enhanced capacity for reactivation of the plasmid-borne E. coli cat gene following its inactivation by UV radiation. Phenotypic complementation of XP cells by chromosome 15 is specific to genetic complementation group F; no effect on the UV sensitivity of XP-A, XP-C, or XP-D cells was detected. The observation that phenotypic complementation is partial is open to several interpretations and does not allow the definitive conclusion that the XP-F locus is carried on chromosome 15.     

Unusual primitive heteromorphic ZZ/ZW sex chromosomes in Proceratophrys boiei (Anura, Cycloramphidae, Alsodinae), with description of C-Band interpopulational polymorphism

We performed cytogenetic analyses on specimens from three population samples of Proceratophrys boiei from southeastern and northeastern Brazil. We stained chromosomes of mitotic and meiotic cells with Giemsa, C-banding and Ag-NOR methods. All specimens of P. boiei presented a karyotype with a full chromosome complement of 2n=22, metacentric and submetacentric. We observed the secondary constriction within the short arm of pair 8, which was in the same position of the nucleolus organizer region (NOR). NOR heteromorphism was observed within two specimens from the municipality of Mata de S?o Jo?o (northeastern Bahia State). The C-banding evidenced an unusual heterochromatic pattern in the genome of P. boiei. In the southern most population samples (S?o Paulo State), we observed large blocks of heterochromatin in the centromeric regions of all chromosomes, whereas the northernmost samples (Bahia State) presented a small amount of constitutive heterochromatin. We suppose that this geographic variation in heterochromatin quantities could be due to heterochromatinization of some chromosome regions in the genome of the S?o Paulo samples. Furthermore, females from S?o Paulo presented, within chromosome pair 1 from C-banded karyotypes, one homologous chromosome almost heterochromatic, whereas males had heterochromatin restricted to the centromeric region. This unusual heterochromatic arrangement led us to assume that P. boiei owns a ZZ/ZW type of sexual determination system. This finding is very important, as this is the first record of ZZ/ZW sex chromosomes within Cycloramphidae. We believe that the cytogenetic differences found between southeastern and northeastern Brazilian population samples of P. boiei strongly supports the existence of a species complex under the name P. boiei, and the requirement of taxonomic and systematic reviews by morphological, bioacoustical, molecular, and cytogenetic data could define this taxonomic issue in the future.     

Genetic Mapping Using Microcell-Mediated Chromosome Transfer Suggests a Locus for Nijmegen Breakage Syndrome at Chromosome 8q21-24

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by microcephaly, short stature, immunodeficiency, and a high incidence of cancer. Cultured cells from NBS show chromosome instability, an increased sensitivity to radiation-induced cell killing, and an abnormal cell-cycle regulation after irradiation. Hitherto, patients with NBS have been divided into the two complementation groups V1 and V2, on the basis of restoration of radioresistant DNA synthesis, suggesting that each group arises from a different gene. However, the presence of genetic heterogeneity in NBS has been considered to be controversial. To localize the NBS gene, we have performed functional complementation assays using somatic cell fusion between NBS-V1 and NBS-V2 cells, on the basis of hyper-radiosensitivity, and then have performed a genomewide search for the NBS locus, using microcell-mediated chromosome transfer followed by complementation assays based on radiosensitivity. We found that radiation resistance was not restored in the fused NBS-V1 and NBS-V2 cells and that only human chromosome 8 complements the sensitivity to ionizing radiation, in NBS cell lines. In complementation assays performed after the transfer of a reduced chromosome, merely the long arm of chromosome 8 was sufficient for restoring the defect. Our results strongly suggest that NBS is a homogeneous disorder and that the gene for NBS is located at 8q21-24.     

Familial transmission of a translocation Y/14

Summary A translocation of heterochromatic material, brightly fluorescent after actinomycin D-DAPI staining, to the short arm of chromosome 14 was prenatally detected during cytogenetic examination of cells obtained by amniocentesis on the indication of advanced maternal age. Besides this abnormal chromosome, 43 autosomes and two X chromosomes were present. Silver staining made clear that an active nucleolus-organizing region was included in the translocation product. Both the intense fluorescence and the size of the translocated extra heterochromatic block were indicative of a Yq origin. Upon cytogenetic investigation of the parents, the mother appeared to carry the same t(Y;14) chromosome. Therefore, we expected a normal girl to be born. This was confirmed after birth.     

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.     

Unstable transmission of the RS447 human megasatellite tandem repetitive sequence that contains the <Emphasis Type="Italic">USP17</Emphasis> deubiquitinating enzyme gene

The RS447 megasatellite DNA, which maps to human chromosome 4p16.1, is a highly polymorphic conserved tandem repetitive sequence containing a functional deubiquitinating enzyme gene, USP17. To characterize the hypervariability seen in RS447 fully, we have conducted a pedigree analysis of RS447 transmission by high-resolution pulsed-field gel electrophoresis. We have identified 44 distinct alleles in 74 unrelated chromosomes containing 20-103 copies of the 4.7-kb RS447 unit. Five of 60 parent-to-offspring transmissions clearly show changes in copy number, indicating a high frequency (approximately 8.3%) of meiotic instability. Evidence for somatic mosaicism has also been observed. Searches of the database have revealed the presence of minor RS447 sequences mapping to chromosome 8p23, raising the possibility of a rearrangement or transposition of RS447 within the human genome. These results suggest that the unstable nature of RS447 megasatellite DNA gives rise to its hypervariability and may contribute to the structural dynamics of this repetitive DNA in the genome.     

Genomic structure and paralogous regions of the inversion breakpoint occurring between human chromosome 3p12.3 and orangutan chromosome 2

Intrachromosomal duplications play a significant role in human genome pathology and evolution. To better understand the molecular basis of evolutionary chromosome rearrangements, we performed molecular cytogenetic and sequence analyses of the breakpoint region that distinguishes human chromosome 3p12.3 and orangutan chromosome 2. FISH with region-specific BAC clones demonstrated that the breakpoint-flanking sequences are duplicated intrachromosomally on orangutan 2 and human 3q21 as well as at many pericentromeric and subtelomeric sites throughout the genomes. Breakage and rearrangement of the human 3p12.3-homologous region in the orangutan lineage were associated with a partial loss of duplicated sequences in the breakpoint region. Consistent with our FISH mapping results, computational analysis of the human chromosome 3 genomic sequence revealed three 3p12.3-paralogous sequence blocks on human chromosome 3q21 and smaller blocks on the short arm end 3p26-->p25. This is consistent with the view that sequences from an ancestral site at 3q21 were duplicated at 3p12.3 in a common ancestor of orangutan and humans. Our results show that evolutionary chromosome rearrangements are associated with microduplications and microdeletions, contributing to the DNA differences between closely related species.     

High-resolution single-copy gene fluorescence in situ hybridization and its use in the construction of a cytogenetic map of maize chromosome 9

High-resolution cytogenetic maps provide important biological information on genome organization and function, as they correlate genetic distance with cytological structures, and are an invaluable complement to physical sequence data. The most direct way to generate a cytogenetic map is to localize genetically mapped genes onto chromosomes by fluorescence in situ hybridization (FISH). Detection of single-copy genes on plant chromosomes has been difficult. In this study, we developed a squash FISH procedure allowing successful detection of single-copy genes on maize (Zea mays) pachytene chromosomes. Using this method, the shortest probe that can be detected is 3.1 kb, and two sequences separated by approximately 100 kb can be resolved. To show the robust nature of this protocol, we localized nine genetically mapped single-copy genes on chromosome 9 in one FISH experiment. Integration of existing information from genetic maps and the BAC contig-based physical map with the cytological structure of chromosome 9 provides a comprehensive cross-referenced cytogenetic map and shows the dramatic reduction of recombination in the pericentromeric heterochromatic region. To establish a feasible mapping system for maize, we also developed a probe cocktail for unambiguous identification of the 10 maize pachytene chromosomes. These results provide a starting point toward constructing a high-resolution integrated cytogenetic map of maize.     

Human supernumeraries: are they B chromosomes?

In humans, the presence of supernumerary chromosomes is an unusual phenomenon, which is often associated with developmental abnormalities and malformations. In contrast to most animal and plant species, the extensive knowledge of the human genome and the ample set of molecular and cytogenetic tools available have permitted to ascertain not only that most human supernumerary chromosomes (HSCs) derive from the A chromosome set, but also the specific A chromosome from which most of them arose. These extra chromosomes are classified into six types on the basis of morphology and size. There are both heterochromatic and euchromatic HSCs, the latter being more detrimental. Most are mitotically stable, except some producing individual mosaicism. No information is available on the HSC transmission rate since extensive familial studies are not usually performed generally because of death of the relatives or lack of cooperation. The main B chromosome property failing in HSCs seems to be their population spread as polymorphisms, since most HSCs seem to correspond to extra A chromosomes or centric fragments spontaneously arisen in the analysed individual or one of his/her parents. However, we cannot rule out at this moment, that more intensive studies on population distribution and frequency of those HSCs most closely resembling B chromosomes (i.e. those heterochromatic and thus less detrimental) would reveal possible HSCs polymorphisms. Although HSCs cannot be considered B chromosomes, some of them might be a source for future B chromosomes. The best candidates would be heterochromatic HSCs, which might manage to drive in either sex. To ascertain this possibility, research on inheritance and population studies would be very helpful in combination with the powerful cytogenetic and molecular tools available for our species.     

Molecular cytogenetic characterization of eight small supernumerary marker chromosomes originating from chromosomes 2, 4, 8,18, and 21 in three patients

Small supernumerary marker chromosomes (sSMCs) are a morphologically heterogeneous group of additional structurally abnormal chromosomes that cannot be identified unambiguously by conventional banding techniques alone. Molecular cytogenetic methods enable detailed characterization of sSMCs; however, in many cases interpretation of their clinical significance is problematic. The aim of our study was to characterize precisely sSMCs identified in three patients with dysmorphic features, psychomotor retardation and multiple congenital anomalies. We also attempted to correlate the patients' genotypes with phenotypes by inclusion of data from the literature. The sSMCs were initially detected by G-banding analysis in peripheral blood lymphocytes in these patients and were subsequently characterized using multicolor fluorescence in situ hybridization (M-FISH), (sub)centromere-specific multicolor FISH (cenM-FISH, subcenM-FISH), and multicolor banding (MCB) techniques. Additionally, the sSMCs in two patients were also studied by hybridization to whole-genome bacterial artificial chromosome (BAC) arrays (array-CGH) to map the breakpoints on a single BAC clone level. In all three patients, the chromosome origin, structure, and euchromatin content of the sSMCs were determined. In patient RS, only a neocentric r(2)(q35q36) was identified. It is a second neocentric sSMC(2) in the literature and the first marker chromosome derived from the terminal part of 2q. In the other two patients, two sSMCs were found, as M-FISH detected additional sSMCs that could not be characterized in G-banding analysis. In patient MK, each of four cell lines contained der(4)(:p11.1-->q12:) accompanied by a sSMC(18): r(18)(:p11.2-->q11.1::p11.2-->q11.1:), inv dup(18)(:p11.1-->q11.1::q11.1-->p11.1:), or der(18) (:p11.2-->q11.1::q11.1-->p11.1:). In patient NP, with clinical features of trisomy 8p, three sSMCs were characterized: r(8)(:p12-->q11.1::q11.1-->p21:) der(8) (:p11.22-->q11.1::q11.1-->p21::p21-->p11.22:) and der(21)(:p11.1-->q21.3:). The BAC array results confirmed the molecular cytogenetic results and refined the breakpoints to the single BAC clone resolution. However, the complex mosaic structure of the marker chromosomes derived from chromosomes 8 and 18 could only be identified by molecular cytogenetic methods. This study confirms the usefulness of multicolor FISH combined with whole-genome arrays for comprehensive analyses of marker chromosomes.     

Two new cases of the Christchurch (Ch1) chromosome 21: evidence for clinical consequences of de novo deletion 21P-

We performed an investigation of two unrelated cases with extremal variants of chromosome 21 without visible materials of the short arms (Christchurch or Ch1 chromosome). In the first case chromosome 21p- was initially detected during routine cytogenetic amniocentesis. Chromosomal variant was inherited from phenotypically normal father to phenotypically normal fetus (phenotypically normal boy after the birth). The second case of chromosome 21p- was detected in 7 years old boy, referred to cytogenetic analysis due to mental retardation and mild congenital malformation, including prenatal hypoplasia, microcephaly, low-set dysplastic ears, short nose, micrognatia, short neck. Molecular characterization of 21p-variant chromosomes was performed by the use of FISH with DNA probes specific to the short arm and centromeric region of chromosome 21 (telomeric, beta-satellite, ribosomal, classical satellite and alphoid DNA probes). Chromosomes 21p-hybridized positively only with telomeric DNA at both chromosomal ends and alphoid DNA probes at centromeric region of the first patient. In second case (de novo deletion of 21p), the Ch1 was associated with clinical phenotype and loss of telomeric and subtelomeric DNA in the p-arm of chromosome 21. Therefore, the complete absent of the short arm of chromosome 21 may be considered as abnormal. We propose that de novo deletion 21p- could have negative consequences due to absence of large portion of chromosomal DNA from the p-arm (telomeric, satellite or ribosomal DNAs) and following imbalance in organization and functioning of genome.     

Characterization of the first supernumerary tricentric ring chromosome 1 mosaicism by conventional and molecular cytogenetic techniques

We report on the conventional cytogenetic and fluorescence in situ hybridization (FISH) results obtained for a 3.5-year-old girl with developmental and language delay and a supernumerary ring chromosome mosaicism in 8% of T-lymphocytes analyzed. Using different conventional and molecular cytogenetic techniques as YAC hybridization and comparative genomic hybridization, we could show that the extra tricentric ring chromosome consists of three heterochromatic blocks with inserted euchromatic material. Additionally, chromosome microdissection followed by FISH analysis demonstrated that the small tricentric ring chromosome consisted of material from the pericentromeric region of chromosome 1q21. Thus, the patient has a mosaic of normal cells and cells with partial pentasomy of the pericentromeric region of chromosome 1. So far, 19 cases with single supernumerary marker chromosome 1 have been published, but no tricentric ring chromosome 1 is, to our knowledge, reviewed in the literature. In this study, we compare the clinical features of our patient with cytogenetically comparable cases described in the literature. We introduce a hypothesis for the formation of a tricentric ring chromosome: starting with a monocentric ring, sister chromatid exchange leading to the formation of a tetracentric ring, which underwent intrastrand recombination generating the tricentric ring.     

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.     

Genomic homology of the domestic ferret with cats and humans

We used chromosome paints from both the domestic cat and humans to directly establish chromosomal homology between the genome of these species and the domestic ferret. The chromosome painting data indicate that the ferret has a highly conserved karyotype closer to the ancestral carnivore karyotype than that of the cat. The cat chromosome paints revealed 22 homologous autosomal regions in the ferret genome: 16 ferret chromosomes were hybridized by a single cat paint, while 3 ferret chromosomes were hybridized by two cat paints. In situ hybridization combined with banding showed that ferret Chromosome (Chr) 1 = cat A2p/C2, Chr 2 = F2/C1q, and Chr 3 = A2q/D2. Five ferret chromosomes are homologous to single arms of cat chromosomes: ferret 4 = A1q, 5 = B1q, 6 = C1p, 10 = A1p, and 12 = B1p. The human chromosome paints revealed 32 + XY homologous regions in the ferret genome: 9 ferret chromosomes were each hybridized by a single human paint, 7 by two paints, 3 by three paints. The 10 ferret chromosomes hybridized by multiple human paints produced the following associations: ferret 1 = human 19/3/21, 2 = 8q/2q, 3 = 10/7, 5 = 8/4, 8 = 15/14, 9 = 10/12/22, 11 = 20/2, 12 = 8/4, 14 = 12/22/18, 18 = 19/16. We present an index of genomic diversity, Z, based on the relative number of conserved whole chromosome and chromosome segments as a preliminary statistic for rapid comparison between species. The index of diversity between human-ferret (Z = 0.812) is slightly less than human-cat (Z = 0.843). The homology data presented here allow us to transfer gene mapping data from both cats and humans to the ferret. Received: 21 December 1999 / Accepted: 30 May 2000     

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.     

Intraspecific crosses resulting in the first occurrence of eight and nine B chromosomes in Prochilodus lineatus (Characiformes, Prochilodontidae)

B chromosomes are supernumerary elements present in about 15% of eukaryotic species and are most frequently heterochromatic, behave parasitically, show a transmission rate higher than standard (A) chromosomes, and can provoke harmful effects on carriers. In the current work, Prochilodus lineatus individuals carrying eight and nine B chromosomes were obtained by induced crossing performed involving breeders with different B chromosome numbers in their cells. The high B chromosome numbers found in the offspring were recorded for the first time in this species. The use of cytogenetic techniques applied in the present study revealed that regardless of the increase in number of B chromosomes in the genome of these individuals, those elements did not presented active genes, and showed their normal heterochromatic characteristic.     

M-FISH applications in clinical genetics

Until recently, presence of de novo marker or derivative chromosomes was quite problematic for genetic counseling especially in prenatal diagnosis, because characterization of marker and derivative chromosomes by conventional cytogenetic techniques was nearly impossible. However, recently developed molecular cytogenetic technique named Multicolor Fluorescence in Situ Hybridization (M-FISH) which paints all human chromosomes in 24 different colors allows us to characterize marker and derivative chromosomes in a single hybridization. In this study, we applied M-FISH to determine the origin of 3 marker and 3 derivative chromosomes. Marker chromosomes were found to originate from chromosome 15 in two postnatal and one prenatal case. Of these, one of the postnatal cases displayed clinical findings of inv dup (115) syndrome and the other of infertility, and the prenatal case went through amniocentesis due to the triple test results. Karyotypes of the patients with derivative chromosomes were designated as 46,XY,der (21)t(1;21)(q32;p11), 46,XX,der(8)t(8;9)(p23;p22) and 46,XX,der(18)t(18;20)(q32;p11.2) according to cytogenetic and M-FISH studies. All of the M-FISH results were confirmed with locus specific or whole chromosome painting probes. The case with der (8)t(8;9) had trisomy 9(p22-pter) and monosomy 8(p23-pter) due to this derivative chromosome. The case with der(18)t(18;20) had trisomy 20(p11.2-pter) and monosomy 18(q32-qter). Parental origins of the derivative chromosomes were analyzed using microsatellite markers located in the trisomic chromosomal segments. Patients' clinical findings were compared with the literature.     

Extended cytogenetic maps of sheep chromosome 1 and their cattle and river buffalo homoeologues: comparison with the OAR1 RH map and human chromosomes 2, 3, 21 and 1q

Cytogenetic maps are useful tools for several applications, such as the physical anchoring of linkage and RH maps or genome sequence contigs to specific chromosome regions or the analysis of chromosome rearrangements. Recently, a detailed RH map was reported in OAR1. In the present study, we selected 38 markers equally distributed in this RH map for identification of ovine genomic DNA clones within the ovine BAC library CHORI-243 using the virtual sheep genome browser and performed FISH mapping for both comparison of OAR1 and homoeologous chromosomes BBU1q-BBU6 and BTA1-BTA3 and considerably extending the cytogenetic maps of the involved species-specific chromosomes. Comparison of the resulting maps with human-identified homology with HSA2q, HSA3, HSA21 and HSA1q reveals complex chromosome rearrangements differentiating human and bovid chromosomes. In addition, we identified 2 new small human segments from HSA2q and HSA3q conserved in the telomeric regions of OAR1p and homoeologous chromosome regions of BTA3 and BBU6, and OAR1q, respectively. Evaluation of the present OAR1 cytogenetic map and the OAR1 RH map supports previous RH assignments with 2 main exceptions. The 2 loci BMS4011 and CL638002 occupy inverted positions in these 2 maps.     

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.