Molecular cytogenetic investigation of two patients with Y chromosome rearrangements and intellectual disability.

We describe two males with intellectual disability (ID) and facial dysmorphism, both of whom have non-mosaic Y chromosome rearrangements resulting in deletions of large portions of the Y chromosome. Patient A, with ID, mild dysmorphism, speech delay, Duane anomaly of the eye, hypermetropia and conductive hearing loss, had two structurally rearranged Y chromosomes resulting in both p and q arm deletions in addition to a Yp duplication. Patient B, also with speech and language delay, developmental delay and short stature, had an interstitial deletion of Yq11.21-11.23. Array-CGH excluded the presence of additional submicroscopic rearrangements at the 1 Mb resolution level. A review of males with Y chromosome rearrangements and ID was performed. Our study provides a more detailed molecular cytogenetic assessment of Y rearrangements in individuals with ID than has been previously possible, and facilitates assessment and comparison of other individuals with a Y chromosome rearrangement.

Constitutional tetrasomy 18p.

We present here the first case of constitutional tetrasomy 18p from India. A 3 year old female with developmental delay and dysmorphic features revealed 47,XX,+mar karyotype. The small meta-centric marker chromosome was identified as i(18p) with m-FISH followed by m-BAND. Parents and a normal sibling of the proband revealed normal karyotype. There was history of mental retardation and dysmorphic features in four cases on paternal side; however, their karyotype was also normal.

FISH studies on the telomeric regions of the T-cell acute lymphoblastic leukemia cell line CCRF-CEM.

So far, the problem of an influence of translocations on the telomeres of the involved chromosomes has not been addressed yet in human cells. Therefore, the telomeres of a karyotypically rather well characterized T-cell acute lymphoblastic leukemia (T-ALL) cell line (CCRF-CEM) with several marker chromosomes were examined using peptide nucleic acid (PNA) telomere FISH probes to compare the telomere length of these markers with that of the chromosome arms of their origin. In addition, chromosome libraries, centromeric probes, and subtelomeric DNA probes were used to further define the marker chromosomes. Two markers could be newly defined and a concise karyotype of the cell line could be obtained by these detailed examinations: 42-47,X,-X,del(5) (q35?),t(5;15)(q14;q13.2),t(8;9)(p11;p24),del(9)(:p13-->qter)/inv(9)(pter-->p12::q21-->p12::q21-->qter),+13,+20,+der(22)(p+ [HSR?])[cp]. The relative telomere length of all chromosomes showed considerable interchromosomal, intercellular, and inter-passage variation. However, it could be shown, that in four different passages of the examined cell line the observed differences between relative telomere lengths of the markers and the chromosomes of their origin, with two exceptions (short arms of del/inv9 and der22), were not significant. On the other hand, because of its mentioned variability, telomere length alone is not sufficient to reliably define the derivation of markers.

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.

Chromosome intrachanges and interchanges detected by multicolor banding in lymphocytes: searching for clastogen signatures in the human genome.

Genomic fingerprints of mutagenic agents would have wide applications in the field of cancer biology, epidemiology and prevention. The differential spectra of chromosomal aberrations induced by different clastogens suggest that ratios of specific aberrations can be exploited as biomarkers of carcinogen exposure. We have tested this hypothesis using the novel technique of multicolor banding in situ hybridization (mBAND) in human peripheral blood lymphocytes exposed in vitro to X rays, neutrons, heavy ions, or the restriction endonuclease AluI. In the heavy-ion-irradiated cells, we further analyzed aberrations in chromosome 5 using multicolor FISH (mFISH). Contrary to the expectations of biophysical models, our results do not support the use of the ratios of inter-/intrachromosomal exchanges or intra-/interarm intrachanges as fingerprints of exposure to densely ionizing radiation. However, our data point to measurable differences in the ratio of complex/simple interchanges after exposure to different clastogens. These data should be considered in current biophysical models of radiation action in living cells.

Identification of a dup(5)(p15.3) by multicolor banding.

A 7-year-old female was referred to the Genetics Clinic because of developmental delay and attentional difficulty. The patient was adopted and there was a nonspecific prenatal history of drug and alcohol abuse. The patient had clinical signs that were not compatible with typical fetal alcohol syndrome (FAS), although there was a history of alcohol exposure in utero, neurodevelopmental difficulties with learning and behavioral problems, and mild dysmorphisms. Cytogenetic analysis revealed an unbalanced female karyotype with a dup(5) containing additional chromosome 5 material at band 5p15.3. The dup(5) showed normal copy number of the cri-du-chat region on 5p15.2 using locus-specific probes D5S721 and D5S23. Multicolor banding of chromosome 5 (MetaSystems) using partial chromosome paint (pcp) probes showed a duplication of band 5p15.3. The karyotype of the patient was therefore interpreted as follows: 46,XX,add(5)mat.ish dup(5)(p15.3)(wcp5 +, D5S271 +, D5S23 +, C84C11/T3 + +, pcp5p15.3 + +). The patient's biological mother and maternal half-brother were found to carry the identical chromosome duplication. The clinical phenotype of the biological mother is complicated by a difficult lifestyle but there were apparent learning and behavioral difficulties at school. The half-brother is nondysmorphic and presents with learning problems and attention deficit disorder (ADD). His physical examination was normal. To the best of our knowledge, this is the first report of a limited duplication of 5p15.3. The clinical significance of the dup(5)(p15.3) is still uncertain but may be the basis for learning and attention difficulties.

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.

Comparative genomic hybridization based strategy for the analysis of different chromosome imbalances detected in conventional cytogenetic diagnostics.

Today, conventional cytogenetics (CC) is the main technique in routine genetic diagnostics for the analysis of genotype/phenotype correlations. Additionally, fluorescence in situ hybridization (FISH) has proven to be useful for the characterization of structural chromosome aberrations found in conventional cytogenetics. Comparative genomic hybridization (CGH) is a molecular cytogenetic FISH approach developed for the detection of genomic imbalances with cytogenetic resolution. CGH allows the genome-wide assessment of relative DNA copy number changes using extracted specimen DNA as a probe. We investigated the capacity of CGH in cases referred for conventional cytogenetic diagnostics for the detection of chromosome imbalances. Three different groups of conspicuous karyotypes after CC (intrachromosomal rearrangements, interchromosomal rearrangements, and additional marker chromosomes) in pre- and postnatal diagnostic cases were surveyed by CGH to characterize the underlying imbalances of chromosome segments. All together, we investigated more than 100 cases by CGH and validated the results with other molecular cytogenetic methods. Here we present eight of these cases in order to demonstrate our CGH based strategy to analyze chromosomal de novo rearrangements. CGH provided additional cytogenetic information to complement conventional cytogenetic investigations. Additionally, CGH refined the description of the aberrant chromosome segments allowing us to further characterize the underlying mechanisms involved.

Microdissection and reverse painting reveals a microdeletion 6(q26qter) in a de novo r(6) chromosome.

Ring chromosomes 6 are rare constitutional abnormalities with inconsistent phenotypic and clinical features. One of the reasons for this variability is the cytogenetically undetectable loss of chromosomal material from the telomeric segments at 6p or 6q. We have therefore used fluorescence in situ hybridization (FISH) to analyse a ring chromosome 6 that was detected in a newborn boy with dysmorphic features. Reverse painting of the microdissected ring chromosome onto normal metaphase spreads revealed a small deletion of the terminal region of the long arm, 6(q26qter). Moreover, the simple all-telomeric sequence (TTAGG)n was lost, whereas the p-specific subtelomeric sequence was still present. Our findings confirm that microdeletions occur during the formation of r(6) chromosomes and, therefore, are an important determinator of the associated phenotype.

CBFB/MYH11 fusion in a patient with AML-M4Eo and cytogenetically normal chromosomes 16.

We present a unique case of acute myeloid leukemia M4Eo (AML-M4Eo) with a CBFB/MYH11 fusion transcript and a trisomy 22, but in whom cytogenetic analyses did not disclose an inv(16). Fluorescence in situ hybridization (FISH) analysis with chromosome arm-specific painting probes as well as with the c40 and c36 cosmids also revealed no evidence for an inv(16), whereas the application of locus-specific probes confirmed the presence of a masked inv(16). The results of our comprehensive FISH investigations indicate that the events leading to this masked inv(16) were complex and concurred with deletions on both the long and short arms. The most likely explanation for the formation of the relevant CBFB/MYH11 fusion is an insertion of parts of the MYH11 into the CBFB gene, although it is also possible that it was formed by a double inversion.

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.

Mapping of the 7q31 subregion common to the small chromosome 7 derivatives from two sporadic papillary renal cell carcinomas: increased copy number and overexpression of the MET proto-oncogene.

Molecular cytogenetic analysis of several sporadic papillary renal cell carcinomas and of their xenografts in immunodeficient mice had previously allowed us to delimit a minimal overrepresented region of chromosome 7 shared by all of them to band 7q31. We have refined the location of the overlapping region to the junction of the subbands 7q31.2 and 7q31.3 by reverse painting with two differently labelled probes prepared from the small chromosome 7 derivatives microdissected from the cells of two distinct tumours. This small region was shown to contain the MET proto-oncogene, present at three to four copies per cell as determined by Southern blot analysis. The increased copy number of the MET gene was found to be associated with its overexpression at the mRNA level. However, no change in MET copy number or expression level was observed in the cells from two xenografted tumours serially transplanted into immunodeficient mice, as compared to those from the corresponding initial tumours. Our results indicate that expression of the MET proto-oncogene above a critical threshold is required for the maintenance of the tumorigenic phenotype of at least some papillary renal cell carcinomas, but does not further increase during tumour progression.

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.

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.

De novo complete trisomy 5p: clinical report and FISH studies.

We describe a de novo trisomy 5p in a 1-year-old severely retarded boy. The complete short arm of chromosome 5 segregated as an additional marker chromosome in all metaphases. The marker was identified as 5p by conventional cytogenetic techniques (GTG, GBG, CBG) and molecular cytogenetic techniques (whole chromosome-painting probe, probes for the cri-du-chat region and the centromere, and additionally high-resolution multicolor banding using a chromosome 5-specific DNA probe cocktail). The clinical findings were similar to the established trisomy 5p phenotype including macrocephaly, facial abnormalities, tracheobronchial defects with subsequent respiratory infections, hypotonia, and psychomotor retardation. To the best of our knowledge this is the first description of an isolated complete 5p trisomy without involvement of the aberrant chromosome in any structural chromosomal rearrangements.

Analysis of X-ray-induced aberrations in human chromosome 5 using high-resolution multicolour banding FISH (mBAND).

Peripheral lymphocytes were exposed to 4 Gy X-rays and aberrations were analysed in human chromosome 5 using high-resolution multicolour banding fluorescence in-situ hybridization (mBAND). This method is suited to detect simple and complex aberrations including peri- and paracentric inversions and exchanges between both chromosomes 5. Additionally, breakpoints carr be assigned to specific regions in chromosome 5. Quantitative relationships of induced aberration types are discussed.

Overrepresentation of 7q31 and 17q in renal cell carcinomas.

Xenografts from four metastatic renal cell carcinomas (RCCs) were established in immunodeficient mice. All tumors exhibited cytogenetic features specific for the papillary subtype, namely, partial or total polysomy of chromosomes 7 and 17 and integrity of 3p. Cytogenetic analysis of the initial and xenografted tumors indicated that although clonal characteristics were consistently maintained in xenografts derived from metastases, a minor clone had been selected for in the xenografts derived from the primary tumors. Reverse painting and comparative genomic hybridization (CGH) allowed us to localize minimal overrepresented genomic regions to 7q31, where the MET protooncogene is located, and to 17q. Other overrepresented regions were 8q in all xenografts and Xq22-qter in three of them. The gain of genetic material from these regions may be a key factor ensuring the papillary nature of RCCs and their survival in xenografts.

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.