mBAND: a high resolution multicolor banding technique for the detection of complex intrachromosomal aberrations.

Precise breakpoint definition of chromosomal rearrangements using conventional banding techniques often fails, especially when more than two breakpoints are involved. The classic banding procedure results in a pattern of alternating light and dark bands. Hence, in banded chromosomes a specific chromosomal band is rather identified by the surrounding banding pattern than by its own specific morphology. In chromosomal rearrangements the original pattern is altered and therefore the unequivocal determination of breakpoints is not obvious. The multicolor banding technique (mBAND, see Chudoba et al., 1999) is able to identify breakpoints unambiguously, even in highly complex chromosomal aberrations. The mBAND technique is presented and illustrated in a case of intrachromosomal rearrangement with seven breakpoints all having occurred on one chromosome 16, emphasizing the unique analyzing power of mBAND as compared to conventional banding techniques.

Evidence for a new microdeletion syndrome in 15q21.

We report on the fourth known case with an interstitial deletion in 15q21. In the present case the breakpoints have been determined by GTG-banding, microdissection and the recently developed multicolor banding (MCB) technique as 15q21.1-q21.3. Common features in all four cases are mental retardation, growth retardation, a beak-like nose with hypoplastic alae nasi and a thin upper lip. Additional frequent features are small hands and feet, hypotonia, low hair implantation, low set ears, clinodactyly and obesity. The possibility that a critical region for a new microdeletion-syndrome is situated in 15q21 is discussed.

Maternal insertion of 18q11.2-q12.2 in 18p11.3 of the same chromosome analysed by microdissection and multicolour banding (MCB).

OBJECTIVES: Different aberrations in one chromosome 18 were prenatally detected during each of three different pregnancies of a healthy woman. Routine cytogenetic analysis revealed a morphologically altered maternal chromosome 18 as well. The purpose of the current study was to characterize these cytogenetic changes in detail and thus to clarify the reason for the recurrent appearance of morphologically altered chromosomes 18 in this family. METHODS: As GTG banding did not allow resolution of the kind of aberrations present in these four cases, the following molecular cytogenetic approaches were used: microdissection combined with reverse painting and multicolour banding (MCB) analysis using a chromosome 18 specific probe set. RESULTS: Molecular cytogenetic approaches revealed that fetus 1 had a derivative chromosome del(18)(q11.2q12.2), fetus 2 and the mother had the identical derivative chromosomes ins(18)(pterp11.32::q12.2q11.2::p11.32q11.2::q12.3qter) and fetus 3 had a dup(11.2q12.2). CONCLUSION: Partial monosomy in fetus 1 and partial trisomy in fetus 3 can be explained by crossing over events during maternal meiosis.

Improved definition of chromosomal breakpoints using high-resolution multicolour banding.

Characterisation of chromosome rearrangements using conventional banding techniques often fails to determine the localisation of breakpoints precisely. In order to improve the definition of chromosomal breakpoints, the high-resolution multicolour banding (MCB) technique was applied to identify human chromosome 5 breakpoints from 40 clinical cases previously assessed by conventional banding techniques. In 30 cases (75%), at least one breakpoint was redefined, indicating that MCB markedly improves chromosomal breakpoint localisation. The MCB pattern is highly reproducible and, in contrast to conventional banding pattern, is consistent in both short and elongated chromosomes. This might be of fundamental interest for the detection of chromosomal abnormalities, especially in tumour cells. Moreover, MCB even allows the detection of abnormalities that remain cryptic in GTG-banding analysis.

The homeobox gene MEIS1 is amplified in IMR-32 and highly expressed in other neuroblastoma cell lines.

Neuroblastoma is a childhood tumour of the sympathetic nervous system that demonstrates striking clinical heterogeneity. In order to determine which genes are abnormally expressed in neuroblastoma, we screened regions of amplification from the short arm of chromosome 2 in the neuroblastoma cell line IMR-32 and found that the homeobox gene, myeloid ecotropic integration site 1 (MEIS1), is highly amplified. MEIS1 normally maps to chromosome band 2p14. High expression of MEIS1 without amplification was also found in other neuroblastoma cell lines, with and without MYCN amplification, and in medulloblastoma and crythroleukaemia cell lines. MEIS1 is highly expressed in cerebellum and ubiquitously expressed in normal immunohaematopoietic tissues and is thought to be important in cell proliferation and differentiation. While several lines of evidence point towards a role for homeobox genes in the development of other malignancies, this is the first report showing the amplification of a homeobox gene in neuroblastoma.

Molecular cytogenetic characterisation of partial trisomy 9q in a case with pyloric stenosis and a review.

Partial trisomy 9q represents a rare and heterogeneous group of chromosomal aberrations characterised by various clinical features including pyloric stenosis. Here, we describe the case of a 1 year old female patient with different dysmorphic features including pyloric stenosis and prenatally detected partial trisomy 9q. This partial trisomy 9q has been analysed in detail to determine the size of the duplication and to characterise the chromosomal breakpoints. According to the data gained by different molecular cytogenetic techniques, such as fluorescence in situ hybridisation (FISH) with whole and partial chromosome painting probes, yeast artificial chromosome (YAC) probes, and comparative genomic hybridisation (CGH), the derivative chromosome 9 can be described as dup(9)(pter-->q22. 1::q31.1-->q22.1::q31.1--> q22.1::q31.1-->qter). Four breakpoint spanning YACs have been identified (y806f02, y906g6, y945f5, and y747b3) for the proximal breakpoint. According to this new case and previously published data, the recently postulated putative critical region for pyloric stenosis can be narrowed down to the subbands 9q22.1-q31.1 and is the result of either partial trisomy of gene(s) located in this region or a gene disrupted in 9q31.

Microdissection based comparative genomic hybridization analysis (micro-CGH) of secondary acute myelogenous leukemias.

Comparative genomic hybridization (CGH) is a well established technique in molecular cytogenetics. However, leukemias, and especially secondary acute myelogenous leukemias (sAML) are not very well analyzed by this technique, even though such diseases are often characterized by complex karyotypic changes, not resolvable by conventional cytogenetic banding analysis. This lack of CGH-studies might be due to the fact, that in most cases bone marrow aspirate is too limited to do DNA-extraction additionally to the cytogenetic analysis. To circumvent this problem a new CGH technique has been applied to analyze 10 AML cases with complex karyotypic changes. In each case 15 interphase nuclei of the harvested and fixed bone marrow cell-suspension have been microdissected from the coverslip surface and collected in a tube. Subsequently, DNA was amplified by DOP-PCR. With this micro-CGH technique additional cytogenetic information from 10 highly aberrant AML cases was obtained and confirmed by FISH on metaphase of the corresponding AML case.

An SRY-negative 47,XXY mother and daughter.

Females with XY gonadal dysgenesis are sterile, due to degeneration of the initially present ovaries into nonfunctional streak gonads. Some of these sex-reversal cases can be attributed to mutation or deletion of the SRY gene. We now describe an SRY-deleted 47,XXY female who has one son and two daughters, and one of her daughters has the same 47,XXY karyotype. PCR and FISH analysis revealed that the mother carries a structurally altered Y chromosome that most likely resulted from an aberrant X-Y interchange between the closely related genomic regions surrounding the gene pair PRKX and PRKY on Xp22.3 and Yp11.2, respectively. As a consequence, Yp material, including SRY, has been replaced by terminal Xp sequences up to the PRKX gene. The fertility of the XXY mother can be attributed to the presence of the additional X chromosome that is missing in XY gonadal dysgenesis females. To our knowledge, this is the first human XXY female described who is fertile.

An easy and reliable procedure of microdissection technique for the analysis of chromosomal breakpoints and marker chromosomes.

Microdissection in combination with reverse painting fluorescence in-situ hybridization (FISH) is a very effective method to identify breakpoints and rearrangements of derived chromosomes and reveal the chromosomal origin of marker chromosomes. We describe an innovation that allows a convenient, fast and safe isolation of microdissected fragments as currently available protocols. The microdissected chromosomes are harvested in a collection drop located in a movable micropipette adjusted to a second micromanipulator under microscopic observation. We used this technique to analyze several cytogenetic aberrations. In order to evaluate the efficiency of our microdissection procedure, we compared the results obtained with microdissection probes made from only one fragment with those obtained with more than six microdissected fragments. In all cases, the single-fragment microdissections were sufficient to provide probes.

Maternal UPD 20 in a hyperactive child with severe growth retardation.

Maternal uniparental disomy was observed in a 4-year-old boy with severe pre- and postnatal growth retardation (body height: 85 cm = 12 cm < third percentile, head circumference: 48 cm = 10 cm < third percentile), a few minor facial findings, and with apparent hyperactivity. His intelligence is within the normal range for his age. Karyotype analysis revealed two cell lines, one apparently normal with 46,XY, the other with a tiny marker (47,XY, + mar). Microdissection and reverse chromosome painting using the marker DNA library as a probe, as well as PCR analysis revealed that the marker is from chromosome 20 and contains only the centromere and pericentromeric segments, but none of the pericentromeric loci for microsatellites. Microsatellite analysis of 25 chromosome 20 loci disclosed maternal uniparental disomy for all 16 informative markers. Maternal heterodisomy was evident for seven loci of the short arm segment 20p11.2-pter. Maternal isodisomy was found at five loci, three of them map to the proximal 20p11.2 segment and two to 20q. To our knowledge, this is the first case of maternal disomy 20 in humans.

High resolution multicolor-banding: a new technique for refined FISH analysis of human chromosomes.

A new multicolor-banding technique has been developed which allows the differentiation of chromosome region specific areas at the band level. This technique is based on the use of differently labeled overlapping microdissection libraries. The changing fluorescence intensity ratios along the chromosomes are used to assign different pseudo-colors to specific chromosome regions. The multicolor banding of human chromosome 5 is presented as an example.

Expressed copies of the MN7 (D15F37) gene family map close to the common deletion breakpoints in the Prader-Willi/Angelman syndromes.

Approximately 70% of patients with Prader-Willi syndrome or Angelman syndrome have a similar sized de novo deletion of 3-4 Mb in the proximal region of 15q. The distal breakpoints appear to cluster between the P gene (OCA2) and D15S24, whereas two deletion breakpoint clusters have been identified on the proximal side (one centromeric to D15S541 and one between D15S541 and D15S9). Based on the identification of a gene family in 15q11-->q13 (MN7, D15F37), we have previously proposed that the presence of multiple copies of this sequence may be related to the instability of this region. Using fluorescence in situ hybridization and YAC mapping, we have found that at least one D15F37 locus is centromeric to D15S9 and at least two are between OCA2 and D15S24. As determined by cDNA cloning and sequence analysis, each of the individual loci is expressed. The close proximity of the D15F37 loci and the deletion breakpoints suggests that the common deletions arise by unequal crossover events at or near these loci.

Complex chromosomal rearrangements associated with congenital erythrophagocytotic histiocytosis.

We describe a patient with a congenital malignant blood disorder and a constitutional de novo chromosomal rearrangement that includes four breakpoints. By conventional cytogenetic analysis an obviously reciprocal balanced translocation with the breakpoints 1p36 and 5q11.2 was diagnosed. Due to a suspicious dark band in the breakpoint area of 1p a more detailed analysis of the breakpoints was performed using microdissection and reverse chromosome painting. This revealed a small inversion at 1p36 that must have occurred prior to the reciprocal translocation. The three breakpoints in chromosome 1 (1p36.11, 1p36.21 and 1p36.31) are within or close by regions known to contain tumor suppressor genes. The chromosomal rearrangement might have resulted either in a submicroscopic deletion, in loss of heterozygosity of one or more imprinted genes, or in gene position effects as possible explanations for the clinical course of our patient.

Physical map of human 6p21.2-6p21.3: region flanking the centromeric end of the major histocompatibility complex.

We have physically mapped and cloned a 2.5-Mb chromosomal segment flanking the centromeric end of the major histocompatibility complex (MHC). We characterized in detail 27 YACs, 144 cosmids, 51 PACs, and 5 BACs, which will facilitate the complete genomic sequencing of this region of chromosome 6. The contig contains the genes encoding CSBP, p21, HSU09564 serine kinase, ZNF76, TCP-11, RPS10, HMGI(Y), BAK, and the human homolog of Tctex-7 (HSET). The GLO1 gene was mapped further centromeric in the 6p21.2-6p21.1 region toward TCTE-1. The gene order of the GLO1-HMGI(Y) segment in respect to the centromere is similar to the gene order in the mouse t-chromosome distal inversion, indicating that there is conservation in gene content but not gene order between humans and mice in this region. The close linkage of the BAK and CSBP genes to the MHC is of interest because of their possible involvement in autoimmune disease.

Prenatal diagnosis of a half-cryptic translocation using chromosome microdissection.

This report describes a case of a paternal balanced, but apparently non-reciprocal, insertion of chromosome 15 material into the short arm of chromosome 17 with difficulties in distinguishing between the normal and the deleted chromosome 15 in prenatal karyotype analysis. Microdissection and degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) of the paternal 17p+ chromosome was performed to generate a painting probe specific for the small region inserted from chromosome 15 into chromosome 17. Fluorescence in situ hybridization (FISH) of this probe simultaneously with a differentially labelled 15q microdissection probe enabled the identification of a balanced karyotype in the fetus. In this case, microdissection combined with FISH was the only method for obtaining a reliable result within the short time available for prenatal diagnosis. In addition, it was possible to identify with certainty the originally suspected reciprocal translocation as an insertion of the region 15q22.3-->q23 or 24 into the sub-telomeric region of 17p [ins(17;15)(p13;q22.3q23 or 24)]. Thus, the chromosomal defect of two family members with a partial trisomy of chromosome 15 having severe mental retardation and dysmorphic features was identified precisely.

Molecular cytogenetic characterization of del(7q) in two uterine leiomyoma-derived cell lines.

Uterine leiomyoma cytogenetically exhibits at least six chromosomally abnormal subgroups. The largest subgroup is characterized by deletions of the long arm of chromosome 7. Few molecular and fluorescence in situ hybridization data are available that have aimed at a better definition of the lesion. Here, we report the results of a partial molecular cytogenetic characterization of two del(7q) chromosomes that were derived from cell lines established from two uterine leiomyomas with del(7)(q22q32). By using a large series of ordered 7q markers, we were able to identify the most proximal and the most distal conserved markers, which delineate the size of the deletion and which allow for a more targeted approach to the nature and function of genes that are possibly relevant for the pathogenesis of the disorder.

Nephropathic cystinosis (CTNS-LSB): construction of a YAC contig comprising the refined critical region on chromosome 17p13.

A yeast artificial chromosome (YAC) contig was constructed encompassing the entire region on chromosome 17p13 where the autosomal recessive disorder infantile nephropathic cystinosis (MIM 21980, CTNS-LSB) has been genetically mapped. It comprises seven clones ordered by their content of a series of six sequence-tagged sites (STSs). Fluorescence in situ hybridisation (FISH) revealed two chimaeric clones. The order of four polymorphic STSs mapped with the contig was consistent with that of the known genetic map with the exception of markers D17S1583 (AFMb307zg5) and D17S1798 (AFMa202xf5) where a telomeric location of D17S1583 was inferred from the contig; two non-polymorphic STSs were localised within the marker frame-work. From the analysis of recombination events in an unaffected individual as defined by leucocyte cystine levels we support the high-resolution mapping of this region to a small genetic interval and show that it is entirely represented on a single, non-chimaeric YAC clone in the contig.

Interstitial deletion of chromosome 6q: precise definition of the breakpoints by microdissection, DNA amplification, and reverse painting.

Routine chromosomal analysis using GTG-banding alone showed a mosaic terminal deletion of 6q in a 14-week-old boy with developmental retardation, facial anomalies, agenesis of corpus callosum, cleft palate, hypotonia, short neck and pterygium colli, and minor anomalies of hands and feet. Discrepancies between the clinical findings on our patient and those described in the literature on patients having terminal deletions led to a more precise analysis of the karyotype. Reverse painting was performed on normal G-banded metaphases for exact determination of the breakpoints and on metaphases of the patient for evaluation of mosaicism. A DNA library that was obtained by microdissection of three deleted chromosomes 6 was used as a painting probe. Subsequent DNA amplification was performed with the help of topoisomerase-pretreated degenerate oligonucleotide primers. Unexpectedly, the hybridization pattern on normal metaphase chromosomes revealed an interstitial deletion with breakpoints at 6q25.1 and 6q27 instead of a terminal deletion. Hybridization on metaphases of the patient showed one deleted chromosome 6 in all metaphases analyzed at a higher resolution rather than mosaicism as previously assumed [karyotype, 46,XY,del(6)(q25.1 --> q27)]. We assume that in the single cases of 6q- described in the literature the deletions are misclassified. This might be due to difficulties in distinguishing between interstitial and terminal deletions at 6q and in precisely defining chromosomal breakpoints after GTG-banding alone.