Chromosome Banding in Amphibia. XXXIII. Demonstration of 5-Methylcytosine-Rich Heterochromatin in Anura.

An experimental approach using monoclonal anti-5-methylcytosine (5-MeC) antibodies and indirect immunofluorescence was elaborated for detecting 5-MeC-rich chromosome regions in anuran chromosomes. This technique was applied to mitotic metaphases of 6 neotropical frog species belonging to 6 genera and 4 families. The hypermethylation patterns were compared with a variety of banding patterns obtained by conventional banding techniques. The hypermethylated DNA sequences are species-specific and located exclusively in constitutive heterochromatin. They are found in centromeric, pericentromeric, telomeric, and interstitial positions of the chromosomes and adjacent to nucleolus organizer regions. 5-MeC-rich DNA sequences can be embedded both in AT- and GC-rich repetitive DNA. The experimental parameters that have major influence on the reproducibility and quality of the anti-5-MeC antibody labeling are discussed.

Reporting of Diagnostic Cytogenetic Results.

This appendix, developed by the staff at the Center for Advanced Molecular Diagnostics in the Department of Pathology at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a useful reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding, FISH, or chromosomal microarray). Multi-specimen usage macros are included that can be applied to two or more specimen types.

Murine multicolor banding.

Multicolor banding approach, first introduced for human chromosomes only, was established as an optimal approach for karyotyping of murine chromosomes. Here we present the established mcb probe sets for all murine autosomes and the X-chromosome and review their potential application.

Standardized reference ideogram for physical mapping in the saltwater crocodile (Crocodylus porosus).

Basic cytogenetic data, such as diploid number and general chromosome morphology, are available for many reptilian species. Here we present a detailed cytogenetic examination of the saltwater crocodile (Crocodylus porosus) karyotype, including the creation of the first fully annotated G-band standard ideogram for any crocodilian species. The C. porosus karyotype contains macrochromosomes and has a diploid number of 34. This study presents a detailed description of each chromosome, permitting unambiguous chromosome identification. The fully annotated standardized C. porosus ideogram provides the backbone to a standard nomenclature system which can be used to accurately identify specific band locations. Seven microsatellite containing fosmid clones were fluorescently labeled and used as fluorescent in situ hybridization (FISH) probes for physical localization. Chromosome locations for each of these FISH probes were successfully assigned, demonstrating the utility of the fully annotated ideogram for genome mapping.

Cytogenetic heteromorphisms: survey results and reporting practices of giemsa-band regions that we have pondered for years.

CONTEXT: Cytogenetic heteromorphisms (normal variants) pose diagnostic dilemmas. Common Giemsa-band heteromorphisms are not described in the literature, although Giemsa-banding is the method most frequently used in cytogenetic laboratories. OBJECTIVE: To summarize the responses from more than 200 cytogeneticists concerning the definition and reporting of cytogenetic heteromorphisms, to offer these responses as a reference for use in clinical interpretations, and to provide guidance for interpretation of newly defined molecular cytogenetic heteromorphisms. DESIGN: The Cytogenetics Resource Committee of the College of American Pathologists and the American College of Medical Genetics administered a proficiency testing survey in 1997 to 226 participant cytogenetic laboratories. Supplemental questions asked whether participants considered particular Giemsa-banded chromosomal features to be heteromorphisms and if these would be described in a cytogenetic clinical report. RESULTS: Responses were obtained from 99% of participants; 61% stated they would include selected heteromorphism data in a clinical report. More than 90% considered prominent short arms, large or double satellites, or increased stalk length on acrocentric chromosomes to be heteromorphisms; 24% to 36% stated that they would include these in a clinical report. Heterochromatic regions on chromosomes 1, 9, 16, and Y were considered heteromorphisms by 97% of participants, and 24% indicated they would report these findings. Pericentric inversions of chromosomes 1, 2, 3, 5, 9, 10, 16, and Y were considered heteromorphisms with more than 75% of respondents indicating they would report these findings. CONCLUSIONS: Responses were not unanimous, but a clear consensus is presented describing which Giemsa-band regions were considered heteromorphisms and which would be reported.

Comparison between conventional banding analysis and FISH screening with an AML-specific set of probes in 260 patients.

Fluorescence in situ hybridization (FISH) is becoming popular in the diagnosis of clonal chromosomal abnormalities. We set up a fast FISH procedure using an extensive set of specific probes. Conventional banding analysis (CBA) and FISH were compared in 260 newly diagnosed acute myeloid leukemia (AML) patients. For FISH the following probes were used: MLL, CBF-beta/MYH11, ETV-6/AML1; AML1/ETO, BCR/ABL, PML/RAR, c-MYC, TP53, RB1, 5q31/5p15.2, 5q33-34, 7q31/CEP7, 20q13; CEP 4, X, Y. Result time was 96 h for CBA versus 5 h for FISH from direct harvest. CBA showed clonal abnormalities in 41% (n=105/260), normal karyotype in 39% (n=102/260) and failed in 20% (n=53/260). FISH screened all patients and detected abnormalities in 39% (n=102/260); CBA and FISH together identified abnormalities in 49% (n=128/260). In six patients with normal CBA and in eight patients with clonal karyotype, it detected further cryptic abnormalities. CBA showed clonal abnormalities in 13% of patients negative at FISH (n=21/158). FISH screening does not add relevant information to CBA, but is the quickest method for detecting major genetic abnormalities in AML. The speed of FISH is very valuable in AML-M3/M3v because PML/RAR+ patients require specific therapy. Furthermore, we suggest FISH screening in failed, complex or suboptimal quality chromosome and specific FISH analysis for 5q, 7q, 12p, 17p, inv(16), t(11q23) in order to implement CBA accuracy.

Development and assignment of bovine-specific PCR systems for the Texas nomenclature marker genes and isolation of homologous BAC probes.

In 1996, Popescu et al. published the Texas standard nomenclature of the bovine karyotype in which 31 marker genes, already mapped in man, were chosen to permit unambiguous identification and numbering of each bovine chromosome. However, specific PCR systems were not available for each marker gene thus preventing the assignment of part of these markers by somatic cell hybrid analysis. In addition, some difficulties remained with the nomenclature of BTA25, BTA27 and BTA29. In this work, specific PCR systems were developed for each of the marker genes except VIL1 (see results), from either existing bovine or human sequences, and a bovine BAC library was screened to obtain the corresponding BAC clones. These PCR systems were used successfully to confirm the assignment of each marker gene (except for LDHA, see results) by analysis on the INRA hamster-bovine somatic cell hybrid panel. The difficulties observed for LDHA and VIL1 are probably due to the fact that these genes belong to large gene families and therefore suggest that they may not be the most appropriate markers for a standardisation effort. This panel of BACs is available to the scientific community and has served as a basis for the establishment of a revised standard nomenclature of bovine chromosomes.

Localization by FISH of the 31 Texas nomenclature type I markers to both Q- and R-banded bovine chromosomes.

A series of 31 marker genes (one per chromosome) were localized precisely to both Q- and R-banded bovine chromosomes by fluorescence in situ hybridization (FISH), as a contribution to the revised chromosome nomenclature of the three major domestic bovidae (cattle, sheep and goat). All marker genes except one (LDHA) are taken from the Texas Nomenclature of the cattle karyotype published in 1996. Homologous probes for each marker gene were obtained by screening a bovine BAC library by PCR with specific primer pairs. After labeling with biotin, each probe preparation was divided into two fractions and hybridized to bovine chromosomes identified either by Q or R banding. Clear signals and good quality band patterns were observed in all cases. Results of the two series of hybridizations are totally concordant both for Q and R band chromosome numbering and precise band localization. This work permits an unambiguous correlation between the Q/G- and R-banded 31 bovine chromosomes, including chromosomes 25, 27 and 29 which remained unresolved in the Texas Nomenclature (1996). Hybridization of the chromosome 29 marker gene to metaphase spreads from a 1;29 Robertsonian translocation bull carrier showed a positive signal on the short arm of this rearranged chromosome, confirming that the numbering of this long-known translocation in cattle is correct when referring to the Texas Nomenclature (1996). Taking into account that cattle, goat and sheep have very similar banded karyotypes, the data presented here will help to establish a definite and complete reference chromosome nomenclature for these species.

Twenty-four-color spectral karyotyping reveals chromosome aberrations in cytogenetically normal acute myeloid leukemia.

Multicolor spectral karyotyping allows simultaneous visualization of all human chromosomes and screening for chromosomal rearrangements without a priori knowledge of any abnormalities involved. Based on this potentially increased sensitivity, we investigated, in a preliminary manner, whether spectral karyotyping could detect cytogenetic aberrations in karyotypically normal leukemia. The test population was comprised of 28 cryopreserved, cytogenetically normal acute myeloid leukemia (AML) samples from patients registered to a randomized trial for previously untreated AML (SWOG 9031). Two normal and 12 samples with known cytogenetic aberrations were used to validate and establish the diagnostic accuracy of the spectral karyotyping assay and instrumentation in a clinical setting. Enumeration and region-specific DNA fluorescence in situ hybridization (FISH) probes verified discrepant results. In the validation data set, spectral karyotyping refined complex karyotypic rearrangements in six cases and defined the chromosomal origin of a "jumping" homogeneously staining region; however, the technology was less sensitive in the detection of subtelomeric rearrangements and double minute chromosomes. In the test population, spectral karyotyping identified previously undetected cytogenetic aberrations in two cases (7%) of karyotypically normal AML: a cryptic 11q23 translocation in 20/20 cells and a minor monosomy 7 clone in 3/21 cells (FISH, 10.5%). Both of these abnormalities are considered to confer a poor prognosis when based on classical cytogenetic prognostic criteria. As an adjunct to classical cytogenetics and standard FISH analyses, the additive resolution of spectral karyotyping, in particular, with chromosome paints spiked with subtelomeric and/or locus-specific probes, may allow significant gains to be made in diagnostic accuracy and recognition of genotype/phenotype prognostic relationships, and in defining underlying biologic mechanisms in cancer. Genes Chromosomes Cancer 28:318-328, 2000.

Cytogenetics of donkey chromosomes: nomenclature proposal based on GTG-banded chromosomes and depiction of NORs and telomeric sites.

With the expansion of comparative genome analysis across different mammals, there is an increasing need to have well-defined banded karyotypes for the species chosen for investigation. In this context, the steadily growing gene mapping data in the donkey urgently require a framework whereby alignment/comparison of genetic information can be readily made with equids and other mammalian species. Hence a GTG-banded karyotype of the donkey (Equus asinus; EAS) is presented, along with schematic drawings and nomenclature of the banded chromosomes. In addition, the most characteristic features of individual chromosomes are described and their relative size estimated. Using the FISH approach, the location of nucleolous organizer regions (NORs) and telomeric repeat sequences (TTAGGG) were detected. Where possible, information on asine chromosomes is supplemented with known/likely equine and human homologues. The study thus primarily aims to provide an appropriate cytogenetic basis for the donkey chromosomes, so that research focused on gene mapping and comparative genomics in this species can be reported under a common format.

The long-term effect of external quality assessment on performance in service cytogenetics.

In 1993 a nationwide cytogenetic external quality assessment program (EQA) was initiated in the Federal Republic of Germany. Presently, some 70 laboratories, representing approximately 90% of all cytogenetic diagnostic tests, are participating in the study. Based on the quality assessment scheme of the Association of Clinical Cytogeneticists (1988) in the United Kingdom, quality of chromosome preparation and speed of routine diagnostic services are being evaluated. As a result of continuous external quality assessment, the mean banding level of participating laboratories rose from below 400 bands per haploid set (bphs) in 1994 to approximately 450 bphs in 1999 in prenatal tests and from about 400 to approximately 500 bphs in postnatal tests over the same 5-yr period. The percentage of participants achieving a banding level of 400 bphs or higher rose from approximately 50% to 93% in prenatal tests and from 60% to 96% in postnatal tests. No significant differences were observed in banding scores achieved by private laboratories compared to university institutes. The impact of the assessment of interpretation, reporting, and documentation remains difficult to evaluate. Preliminary data point to a more stringent adherence to ISCN nomenclature in karyotype designation by participants.

Standard G-, Q-, and R-banded ideograms of the domestic sheep (Ovis aries): homology with cattle (Bos taurus). Report of the committee for the standardization of the sheep karyotype.

Revised G-, Q- and R-banded karyotypes and ideograms for sheep chromosomes at the 420-band level of resolution are presented. The positions of landmark bands on the sheep chromosomes are defined by their distance relative to the centromere to facilitate comparison with equivalent cattle chromosomes. Chromosome-specific (reference) molecular markers that have been mapped to sheep chromosomes and their equivalent cattle chromosomes are proposed. Reference markers will facilitate genome comparisons between sheep and cattle and minimise confusion due to chromosome nomenclature. Numbering of the Robertsonian translocation chromosomes remains as previously reported.

Standard G-, Q-, and R-banded ideograms of the domestic sheep (Ovis aries): homology with cattle (Bos taurus). Report of the committee for the standardization of the sheep karyotype.

Revised G-, Q- and R-banded karyotypes and ideograms for sheep chromosomes at the 420-band level of resolution are presented. The positions of landmark bands on the sheep chromosomes are defined by their distance relative to the centromere to facilitate comparison with equivalent cattle chromosomes. Chromosome-specific (reference) molecular markers that have been mapped to sheep chromosomes and their equivalent cattle chromosomes are proposed. Reference markers will facilitate genome comparisons between sheep and cattle and minimise confusion due to chromosome nomenclature. Numbering of the Robertsonian translocation chromosomes remains as previously reported.

A confirmation analysis method for identification of chromosomal fragile sites.

Fragile sites are chromosome bands that do not manifest a presumed breakage pattern. Identification of fragile sites is a way to investigate the mechanism of carcinogenesis because the fragility at a specific chromosome position may be the causation of an associated cancer. A problem in the identification of fragile sites is the high false positive rate arising from simultaneously carrying out a large number of significance tests. To control it, we propose to find a reference study to confirm the identification result of an objective study. We utilize the Bayesian concept for linking two studies. Basically, our method demonstrates a conservative way to take account of the prior information of a binomial parameter. The derived estimate of breakage probability can be interpreted as a resampling weighted sample-pooling method. It is applied to confirm the identification of fragile sites for a data set of neuroblastoma patients.

A proposed nomenclature of the domestic cat karyotype.

High-resolution G- and Q-band patterns of cat (Felis catus) prometaphase chromosomes with more than 450 numbered bands are presented. This number represents approximately twice the number of bands per haploid set exhibited by feline chromosomes at mid-metaphase. A diagrammatic representation of G-banded cat chromosomes has already been described (O'Brien and Nash, 1982); however, precise numbering of bands and landmarks, as in the human karyotype and in the karyotypes of other domestic and laboratory animals, has not yet been available for the domestic cat karyotype, except for the RBG-banded ideograms constructed by Shibasaki et al. (1987) and Rønne and Storm (1995). In this report, we propose a numbering system for the G-banded ideogram reported previously (O'Brien and Nash, 1982) and an extended ideogram at the high-resolution level (473 numbered bands) as a contribution to the future standardization of the feline karyotype.

Report on the progress of standardization of the G-banded canine (Canis familiaris) karyotype. Committee for the Standardized Karyotype of the Dog (Canis familiaris).

Karyotyping of dog chromosomes is a difficult task owing to the high diploid number of chromosomes (2n = 78) and the similar morphology of autosomes, all of which are acrocentrics. In this report 22 of the 39 G-banded chromosome pairs and their corresponding ideograms have been standardized. The ideogram comprises altogether 235 bands. The need for the introduction of molecular techniques such as chromosome painting and physical mapping of genetic markers for the identification of small acrocentrics is discussed.

Combined Q-banding and fluorescence in situ hybridization for the identification of bovine chromosomes 1 to 7.

Eleven probes were assigned to bovine chromosomes 1 to 7 by fluorescence in situ hybridization (FISH). The identification of chromosomes was based on QFQ-banding prior to in situ hybridization and comparison with the Reading Conference (1976) and ISCNDA (1989) standards. The probes used for FISH can now be utilized as identification and discrimination features for bovine chromosomes 1 to 7 and particularly for chromosomes 4 and 6, which are difficult to distinguish. Comparison of our mapping data with previous assignments and of the standard chromosome banding patterns prompt us to propose a change in the ISCNDA nomenclature: ISCNDA chromosome 4 should be named chromosome 6 and vice versa. Chromosome 4 is marked by the ribosomal RNA cluster RNR3, and chromosome 6 is characterized by the casein gene cluster and an anonymous satellite (D6Z1).

Metaphase quality can be monitored by automatic counting of bands.

A high quality of cytogenetic preparations is important for maintaining a high standard in the cytogenetic laboratory. In the past, quality has been assessed by visual counting or estimation of the number of bands on chromosomes. In this paper we have counted bands automatically during karyotyping by using the Magiscan chromosome analysis system. A high correlation between automatic and visual counting was found, the automatic counting was reproducible and count figures were comparable when the karyotyping machines were operated by different technicians. The automatic counting procedure has now been included in our routine for 1 year. The highest numbers of bands were counted on slides from peripheral blood, with fewer bands for amniotic fluid and the lowest number for cells from chorionic villi. All three cell types showed significant variations in band number over time, which may be due to changes in weather conditions and/or the reagents used for cell culture, harvest and staining. It is concluded that the automatic counting procedure may be used to monitor the quality of metaphases over time and to set limits for band number on routine preparations acceptable for analysis.

Quality control in routine chromosome analysis: prediction of total number of bands for the individual case analyzed.

In routine diagnosis it is of importance for each single analysis to provide information about the level of quality at which the chromosomal analysis has been performed. The number of bands counted can be used as a direct criterion for the level of quality. Statistical prediction methods via linear regression were employed in order to predict the total number of bands of a haploid set of the metaphase (TNB) from those of single chromosomes or pairs of chromosomes. It was demonstrated that TNB can be predicted successfully by counting bands on just one chromosome, where chromosomes C2, C4, C5 or C7 were found suitable for prediction. If pairs of chromosomes are used to predict TNB, we would recommend C4/C7, C5/C6, and C2/C9.

Sex determination and species exclusion in forensic samples with probe cY97.

A total of 120 human samples of blood, saliva and semen stains, hair roots, bone and skin fragments, obtained from 30 males and 16 females were analyzed in Southern blots with probe cY97. Only the male samples gave a specific band of 5.7 kb. In dot blot, under high stringency conditions, male DNA gave signals equivalent to a quantity of female DNA eight times higher. Probe cY97 did not react with 9 different vertebrate species but gave a signal for monkey DNA when used at low stringency. The advantage of using a probe specific for the centromeric region for sex determination and species exclusion is discussed.