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Characterisation of breakpoints in disease-associated balanced chromosome rearrangements (DBCRs), which disrupt or inactivate specific genes, has facilitated the molecular elucidation of a wide variety of genetic disorders. However, conventional methods for mapping chromosome breakpoints, such as in situ hybridisation with fluorescent dye-labelled bacterial artificial chromosome clones (BAC-FISH), are laborious, time consuming and often with insufficient resolution to unequivocally identify the disrupted gene. By combining DNA array hybridisation with chromosome sorting, the efficiency of breakpoint mapping has dramatically improved. However, this can only be applied when the physical properties of the derivative chromosomes allow them to be flow sorted. To characterise the breakpoints in all types of balanced chromosome rearrangements more efficiently and more accurately, we performed massively parallel sequencing using Illumina 1G analyser and ABI SOLiD systems to generate short sequencing reads from both ends of DNA fragments. We applied this method to four different DBCRs, including two reciprocal translocations and two inversions. By identifying read pairs spanning the breakpoints, we were able to map the breakpoints to a region of a few hundred base pairs that could be confirmed by subsequent PCR amplification and Sanger sequencing of the junction fragments. Our results show the feasibility of paired-end sequencing of systematic breakpoint mapping and gene finding in patients with disease-associated chromosome rearrangements.

Interstitial deletions of chromosome band 10q22 are rare. We report on the characterization of three overlapping de novo 10q22 deletions by high-resolution array comparative genomic hybridization in three unrelated patients. Patient 1 had a 7.9 Mb deletion in 10q21.3-q22.2 and suffered from severe feeding problems, facial dysmorphisms and profound mental retardation. Patients 2 and 3 had nearly identical deletions of 3.2 and 3.6 Mb, the proximal breakpoints of which were located at an identical low-copy repeat. Both patients were mentally retarded; patient 3 also suffered from growth retardation and hypotonia. We also report on the results of breakpoint analysis by array painting in a mentally retarded patient with a balanced chromosome translocation 46,XY,t(10;13)(q22;p13)dn. The breakpoint in 10q22 was found to disrupt C10orf11, a brain-expressed gene in the common deleted interval of patients 1-3. This finding suggests that haploinsufficiency of C10orf11 contributes to the cognitive defects in 10q22 deletion patients.

Information on chromosomal aberrations in cutaneous T cell lymphomas (CTCL), is scarce. In this study, comparative genomic hybridization (CGH) was used to analyze chromosomal imbalances (CI) in 32 patients with CTCL. CI were detected in 21 patients (66%). Euchromatic loss (dim) was localized most frequently (>16%) at the chromosomal regions 17p (28%), 13q (25%), 10q (16%), and 6q (19%), and gain of chromatin (enh) at 7 (25%), 8q (25%), and 17q (16%). The pattern dim6q-enh7-enh8-dim13 was the most frequent combination of CI. The number of aberrations per tumor sample varied between 0 and 19 and correlated with clinical tumor stages: from none in stage Ia to 8.75+/-1.8 (mean+/-SEM) in stage IVa. CI occurred more frequently in aggressive subtypes (9.33+/-2.16) than in indolent (2.88+/-0.8) subtypes. A high number of CI (>/=5) was associated with shorter survival. Gain of chromatin in 8q and loss of 6q and 13q correlated with a significantly shorter survival, whereas the most frequently observed aberrations (loss in 17p and gain in 7) did not influence the prognosis. In summary, CGH analysis revealed a characteristic pattern of recurring chromosomal gains and losses in CTCL. The association of the imbalances with the clinical course of the disease suggests that genes encoded at these loci may influence tumor development and progression.

Fanconi anemia (FA) is a condition that induces susceptibility to bone marrow failure, myelodysplastic syndrome (MDS), and leukemia. We report on a high incidence of expanding clonal aberrations with partial trisomies and tetrasomies of chromosome 3q in bone marrow cells of 18 of 53 FA patients analyzed, detected by conventional and molecular cytogenetics. To determine the clinical relevance of these findings, we compared the cytogenetic data, the morphologic features of the bone marrow, and the clinical course of these patients with those of 35 FA patients without clonal aberrations of 3q. The 2 groups did not differ significantly with respect to age, sex, or complementation group. There was a significant survival advantage of patients without abnormalities of chromosome 3q. Even more pronounced was the risk assessment of patients with gains of 3q material with respect to the development of morphologic MDS and acute myeloid leukemia (AML). Thus, our data from 18 patients with 3q aberrations reveal that gains of 3q are strongly associated with a poor prognosis and represent an adverse risk factor in FA.

Background: Chromosomal instability is observed in a wide spectrum of human cancer syndromes. However, to date, little is known of the characteristic genetic changes in sporadic neuroendocrine tumours of the gastroenteropancreatic system., Aims and Method: We have studied copy number aberrations (CNAs) in 26 sporadic neuroendocrine tumours of the enteropancreatic system (12 foregut and 14 midgut tumours) by comparative genomic hybridisation (CGH), allowing simultaneous evaluation of the entire tumour genome., Results: Nearly all tumours (25/26; that is, 96%) showed chromosomal imbalances, including full chromosomal aneuploidies, losses and gains of chromosome arms, interstitial deletions, and amplifications. Whereas gains of chromosomes 4, 5, and 19 were found in both foregut and midgut tumours, gains of chromosomes 20q (58%), 19 (50%), as well as 17p (50%), and partial losses of chromosomes 1p (42%), 2q (42%), 3p, 4q, and 6q (25% each) were frequently observed only in foregut tumours. In contrast, midgut tumours displayed less CNAs. Gains were detected for chromosomes 17q and 19p (57%). Most frequent losses affected chromosomes 18 (43%) and 9p (21%)., Conclusions: The results of our CGH analyses revealed new distinct candidate regions in the human genome associated with sporadic neuroendocrine tumours. Some of the genetic alterations were shared by foregut and midgut tumours while others discriminated between the two groups. Thus our results allude to the involvement of identical as well as discriminative genetic loci in tumorigenesis and progression of neuroendocrine neoplasms of the foregut and midgut. Based on these findings potential new candidate genes will be discussed.

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., (Copyright 2001 S. Karger AG, Basel)

Fibroblast cultures from two Werner syndrome patients were analyzed by spectral karyotyping. There were multiple, pseudodiploid clones in both cultures, mostly marked by random balanced reciprocal translocations. One of the cultures contained a clone with three-way exchanges involving chromosomes 2, 3, and 16. Duplication-deficiencies were exceptional, as were completely normal metaphases. The most frequent breakpoint occurred at 16q22 which corresponds to FRA16B, possibly reflecting difficulties of WS cells in replicating AT-rich repetitive DNA structures. Both cultures ceased proliferation after eight in vitro passages, but a single clone with exceptional growth potential emerged in one of the senescing cultures. Due to its identical translocations, the derivation of this near tetraploid clone (with tetrasomy for all autosomes except chromosomes 4 and 6) could be traced to the most prevalent pseudodiploid clone of the parental mass culture. Our study confirms the existence of variegated translocation mosaicism as the cytogenetic hallmark of WS fibroblast cultures and suggests that tetraploidization in combination with certain chromosome rearrangements and selective chromosome dosage may overcome the severely limited in vitro lifespan of WS fibroblasts., (Copyright 2001 S. Karger AG, Basel)

Permanent lymphoblastoid cell lines are of great practical value in human clinical and experimental genetics. A detailed protocol for routine use is given for the establishment of lymphoblastoid lines from peripheral blood using Epstein-Barr virus and the immunosuppressivum Cyclosporin A. In addition, the biologic basis of this transformation system is briefly summarized.