Anita Heller

Homologous sequences at human chromosome 9 bands p12 and q13-21.1 are involved in different patterns of pericentric rearrangements

A thorough study of the heterochromatin organisation in the pericentromeric region and the proximal long (q) and short (p) arms of human chromsome 9 (HSA 9) revealed homology between 9p12 and 9q13-21.1, two regions that are usually not distinguishable by molecular cytogenetic techniques. Furthermore, the chromosomal regions 9p12 and 9q13-21.1 showed some level of homology with the short arms of the human acrocentric chromosomes. We studied five normal controls and 51 clinical cases: 48 with chromosome 9 heteromorphisms, one with an exceptionally large inversion and two with an additional derivative chromosome 9. Using fluorescence in situ hybridisation (FISH) with three differentially labelled chromosome 9-specific probes we were able to distinguish 12 heteromorphic patterns in addition to the most frequent pattern (defined as normal). In addition, we studied one inversion 9 case with the recently described multicolour banding (MCB) technique. Our results, and previously published findings, suggest several hotspots for recombination in the pericentromeric heterochromatin of HSA 9. They also demonstrate that constitutional inversions affecting the pericentromeric region of chromosome 9 carry breakpoints located preferentially in 9p12 or 9q13-21.1 and less frequently in 9q12.

Reconstruction of the female Gorilla gorilla karyotype using 25-color FISH and multicolor banding (MCB)

The origin of the human and great ape chromosomes has been studied by comparative chromosome banding analysis and, more recently, by fluorescence in situ hybridization (FISH), using human whole-chromosome painting probes. It is not always possible, however, to determine the exact breakpoints and distribution or orientation of specific DNA regions using these techniques. To overcome this problem, the recently developed multicolor banding (MCB) probe set for all human chromosomes was applied in the present study to reanalyze the chromosomes of Gorilla gorilla (GGO). While the results agree with those of most previous banding and FISH studies, the breakpoints for the pericentric inversion on GGO 3 were defined more precisely. Moreover, no paracentric inversion was found on GGO 14, and no pericentric inversions could be demonstrated on GGO 16 or 17.

Clinical impact of nucleophosmin mutations and Flt3 internal tandem duplications in patients older than 60 yr with acute myeloid leukaemia

Background:  Nucleophosmin (NPM1) and Flt3 internal tandem duplications (Flt3‐ITD mutations) represent the most frequent molecular aberrations in patients with acute myeloid leukemia (AML). While NPM1 mutations are associated with favourable prognosis in younger AML patients, Flt3‐ITD mutations reflect an unfavourable prognostic factor in these patients. So far, especially NPM1 mutations have not yet been evaluated exclusively in older patients.

Multitude multicolor chromosome banding (mMCB): a comprehensive one-step multicolor FISH banding method

Multicolor chromosome banding (MCB) using one single chromosome-specific MCB probe set per experiment was previously reported as powerful tool in molecular cytogenetics for the characterization of all kinds of human marker chromosomes. However, a quick analysis of karyotypes with highly complex chromosomal changes was hampered by the problem that up to 24 MCB experiments were necessary for a comprehensive karyotype description. To overcome that limitation the 138 available region-specific microdissection-derived libraries for all human chromosomes were combined to one single probe set, called multitude MCB (mMCB). A typical fluorescence banding pattern along the human karyotype is produced, which can be evaluated either by transforming these profiles into chromosome region-specific pseudo-colors or more reliably by studying the fluorescence profiles. The mMCB probe set has been applied on chromosomes of normal male and female probands, two primary myelodysplastic syndromes and two solid tumor cell lines. Additionally, a cell line of Gorilla gorilla (GGO) studied previously by single chromosome-specific MCB was reevaluated by the mMCB method. All results were in concordance with those obtained in parallel or by other cytogenetic and molecular cytogenetic approaches indicating that mMCB is a powerful multicolor FISH banding tool for fast characterization of complex karyotypes.

Multicolor fluorescence in situ hybridization (FISH) applied to FISH-banding

During the last decade not only multicolor fluorescence in situ hybridization (FISH) using whole chromosome paints as probes, but also numerous chromosome banding techniques based on FISH have been developed for the human and for the murine genome. This review focuses on such FISH-banding techniques, which were recently defined as ‘any kind of FISH technique, which provide the possibility to characterize simultaneously several chromosomal subregions smaller than a chromosome arm. FISH-banding methods fitting that definition may have quite different characteristics, but share the ability to produce a DNA-specific chromosomal banding’. While the standard chromosome banding techniques like GTG lead to a protein-related black and white banding pattern, FISH-banding techniques are DNA-specific, more colorful and, thus, more informative. For some, even high-resolution FISH-banding techniques the development is complete and they can be used for whole genome hybridizations in one step. Other FISH-banding methods are only available for selected chromosomes and/or are still under development. FISH-banding methods have successfully been applied in research in evolution- and radiation-biology, as well as in studies on the nuclear architecture. Moreover, their suitability for diagnostic purposes has been proven in prenatal, postnatal and tumor cytogenetics, indicating that they are an important tool with the potential to partly replace the conventional banding techniques in the future.

FISH banding methods: applications in research and diagnostics

Recently, several chromosome banding techniques based on fluorescence in situ hybridization (FISH) have been developed for the human and the mouse genome. In contrast to the standard chromosome banding techniques presently used, giving a protein-related banding pattern, those FISH techniques are DNA-specific. Currently the FISH banding methods are still under development and no high resolution banding technique is available that can be used for a whole genome in one hybridization. Nevertheless, FISH banding methods were used successfully for research in evolution- and radiation-biology, as well as for studies on the nuclear architecture. Moreover, their suitability for diagnostic purposes has been proven in prenatal, postnatal and tumor cytogenetics, indicating that they are an important tool with the potential to partly replace the conventional banding techniques in future.

Heterogenic molecular basis for loss of ABL1‐BCR transcription: Deletions in der(9)t(9;22) and variants of standard t(9;22) in BCR‐ABL1‐positive chronic myeloid leukemia

The objective of this study was to characterize the ABL1‐BCR fusion gene in 76 BCR‐ABL1‐positive chronic myeloid leukemia (CML) patients regarding expression as well as genomic status, to assess the frequency of ABL1‐BCR gene deletion in these patients, which has been reported to be an adverse prognostic factor in Philadelphia chromosome‐positive CML. Patients were analyzed for ABL1‐BCR 1b‐b3 and/or 1b‐b4 transcription by RT‐PCR analysis. ABL1‐BCR gene status was analyzed by FISH in 16 CML patients with no ABL1‐BCR transcript. FISH revealed a partial or total deletion of the ABL1‐BCR gene in 9/16 and localized the 5′ portion of ABL1 and the 3′ portion of BCR at separated loci in 5/16 patients. The latter FISH pattern resulted from a nonreciprocal translocation in two and a complex translocation in three individuals. In 2/16 patients, FISH could not exclude an intact ABL1‐BCR fusion gene. Thus, most CML patients without ABL1‐BCR transcript could be characterized cytogenetically to belong to two major subgroups: a silent ABL1‐BCR gene was attributed to a deletion in der(9)t(9;22) in 56% of the investigated patients or to variants of a standard t(9;22) (∼ 31%). Conversely, none of the 50 patients with an ABL1‐BCR transcript exhibited a variant t(9;22) in GTG‐banding analysis. Thus, genomic aberrations such as deletions or complex genomic rearrangements are the basic and most frequent cause for ABL1‐BCR RNA negativity in CML. The heterogeneity of the underlying molecular mechanisms may explain divergent clinical implications described for patients with an ABL1‐BCR deletion and those with no ABL1‐BCR transcript.

Karyotyping of human synaptonemal complexes by cenM-FISH

The purpose of this work was to adapt the recently described centromere-specific multicolour (cenM-) FISH technique to human meiotic cells, and evaluate the usefulness of this multiplex fluorescence method for karyotyping human synaptonemal complex (SC), previously analysed by immunocytogenetic approaches. The results obtained demonstrate that cenM-FISH is a reliable one-single-step method, which allows for the identification of all SC present in pachytene spreads. Moreover, when cenM-FISH is applied after immunocytogenetic analysis, the number and distribution of MLH1 foci per chromosome can be established and recombination analysis for each chromosome can be performed easily.

Genetic imbalances in 26 cases of penile squamous cell carcinoma

To obtain more information on chromosomal changes in the up‐to‐now poorly studied tumor class of penile squamous cell carcinoma (SCC), we performed a comparative genomic hybridization study of 26 cases of this rare tumor. DNA sequence copy number alterations (CNAs) very similar to those detected in other SCC types, such as oral and esophageal SCC, were noted. The most common copy number gains were found in 8q24, 16p11–12, 20q11–13, 22q, 19q13, and 5p15, and the most common deletions were detected in 13q21–22, 4q21–32, and along the X chromosome. Classifying the patients according to the number of CNAs showed a possible correlation with clinical outcome.

Genotype/phenotype analysis in a patient with pure and complete trisomy 12p.

Reports on patients with pure and complete trisomy 12p are rare. Up to now, 12 cases have been described in the literature. Here, we report on the genotype/phenotype‐correlation of a female patient with a pure trisomy 12p. Conventional cytogenetic studies on peripheral blood chromosomes as well as molecular cytogenetic (fluorescence in situ hybridization, FISH) techniques including whole chromosome painting (WCP), comparative genomic hybridization (CGH), multicolor‐banding (MCB) detected a female karyotype with an abberant chromosome 12:46,XX,der(12).ish dup(12)(pter → q24.3::p11.2 → pter). In addition to the trisomy 12p specific clinical hallmarks, the patient showed some features of Pallister–Killian syndrome (PKS) such as sparse hair, macroglossia, and epilepsy. These findings contribute to the genotype/phenotype correlation in trisomy 12p patients.