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Abstract Background The ultraconserved elements (UCEs) are defined as stretches of at least 200 base pairs of human DNA that match identically with corresponding regions in the mouse and rat genomes, albeit their real significance remains an intriguing issue. These elements are most often located either overlapping exons in genes involved in RNA processing or in introns or nearby genes involved in the regulation of transcription and development. Interestingly, human UCEs have been reported to be strongly depleted among segmental duplications and benign copy number variants (CNVs). However no comprehensive survey of a putative enrichment of these elements among pathogenic dose variants has yet been reported. Results A survey for UCEs was performed among the 26 cryptic genomic rearrangements detected in our series of 200 patients with idiopathic neurodevelopmental disorders associated to congenital anomalies. A total of 29 elements, out of the 481 described UCEs, were contained in 13 of the 26 pathogenic gains or losses detected in our series, what represents a highly significant enrichment of ultraconserved elements. In addition, here we show that these elements are preferentially found in pathogenic deletions (enrichment ratio 3.6 vs. 0.5 in duplications), and that this association is not related with a higher content of genes. In contrast, pathogenic CNVs lacking UCEs showed almost a threefold higher content in genes. Conclusions We propose that these elements may be interpreted as hallmarks for dose-sensitive genes, particularly for those genes whose gain or loss may be directly implied in neurodevelopmental disorders. Therefore, their presence in genomic imbalances of unknown effect might be suggestive of a clinically relevant condition.

Purpose: PCDH15, encoding protocadherin 15, is mutated in Usher syndrome type 1F (USH1F) patients. Not only point mutations, but also large deletions have been detected within this gene. However, the detection and characterization of gross deletions in the USH1F locus have been difficult. The purpose of the present work was to identify large genomic rearrangements of PCDH15 in a cohort of patients and to accurately identify the location of the junction breakpoints of the detected rearrangements., Methods: A PCDH15 MLPA (multiplex ligation-dependent probe amplification) commercial kit was used, combined with a customized oligonucleotide array-based CGH analysis (aCGH), containing almost 20,000 probes tiling the nonrepetitive sequence of the PCDH15 gene., Results: Two large intragenic rearrangements were identified-one deletion of 55 kb and one direct duplication of 82 kb-in 3 (13%) families from a cohort of 23 USH cases. The patients had been screened for mutations in the five known USH1 genes and were found to carry one or none of the pathogenic mutations in PCDH15. The exact breakpoints of both rearrangements were identified., Conclusions: This is the first time that large duplications have been associated with Usher syndrome. USH patients have not been extensively tested for large genomic rearrangements such as duplications and deletions. This type of mutation easily escapes detection by traditional PCR-based, Methods: Thus, a combination of PCR-based mutation screening, together with deletion and duplication analysis, is mandatory for the accurate screening of the PCDH15 gene in Usher patients.

Background and Objectives: Multiple myeloma (MM) is a malignant plasma cell neoplasia in which genetic studies have shown that genomic changes may affect almost all chromosomes, as shown by fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). Our objective was the genomic characterization of CD 138 positive primary MM samples by means of a high resolution array CGH platform., Design and Methods: For the first time, a high resolution array CGH with more than 40,000 probes, has been used to analyze 26 primary MM samples after the enrichment of CD138-positive plasma cells., Results: This approach identified copy number imbalances in all cases. Bioinformatics strategies were optimized to perform data analysis allowing the segregation of hyperdiploid and non-hyperdiploid cases by array CGH. Additional analysis showed that structural chromosome rearrangements were more frequently seen in hyperdiploid cases. We also identified the same Xq21 duplication in nearly 20% of the cases, which originated through unbalanced chromosome translocations. High level amplifications and homozygous deletions were recurrently observed in our series and involved genes with meaningful function in cancer biology., Interpretation and Conclusions: High resolution array CGH allowed us to identify copy number changes in 100% of the primary MM samples. We segregated different MM subgroups based on their genomic profiles which made it possible to identify homozygous deletions and amplifications of great genetic relevance in MM.

Background and Objectives: Multiple myeloma (MM) is a malignancy characterized by clonal expansion of plasma cells. In 50% of the cases, the neoplastic transformation begins with a chromosomal translocation that juxtaposes the IGH gene locus to an oncogene. Gene copy number changes are also frequent in MM but less characterized than in other neoplasias. We aimed to characterize genes that are amplified and overexpressed in human myeloma cell lines (HMCL) to provide putative molecular targets for MM therapy., Design and Methods: Nine HMCL were characterized by fluorescent in situ hybridization, comparative genomic hybridization (CGH) and cDNA microarrays for gene expression profiling and copy number changes., Results: After defining the IGH-translocations present in the cell lines, we conducted expression-profiling analysis. Supervised analysis identified 166 genes with significantly different expression among the cell lines harboring MMSET/FGFR3 (4p16), MAF (16q) and CCND1 (11q13) rearrangements. Array-CGH was then performed. Five chromosomes recurrently affected by gains/amplifications in primary samples and cell lines were analyzed in detail. Sixty amplified and overexpressed genes were found and 25 (42%) of them were only overexpressed when amplified; moreover, six showed a significant association between overexpression and gain/amplification. We also found co-amplification and overexpression for genes located within the same amplicons, such as MALT1 and BCL2., Interpretation and Conclusions: Parallel analysis of gene copy numbers and expression levels by cDNA microarray in MM allowed efficient identification of genes whose expression levels are elevated because of increased copy number. This is the first time that MALT1 and BCL2 have been shown to be overexpressed and amplified in MM.

Molecular cytogenetic techniques enabled us to clarify numerical and structural alterations previously detected by conventional cytogenetic techniques in 37 patients who had myelodysplastic syndromes with complex karyotypes. Using high-resolution comparative genomic hybridization (HR-CGH), we found the most recurrent alterations to be deletion of 5q (70%), 18q (35%), 7q (32%), 11q (30%), and 20q (24%), gain of 11q (35%) and 8q (24%), and trisomy of chromosome 8 (19%). Furthermore, in 35% of the patients, 20 amplifications were identified. These amplifications were shown by FISH to involve some genes previously described as amplified in hematological malignancies, such as ERBB2, MLL, and RUNX1. In addition, two other genes, BCL6 and BCL2, which are classically related to apoptosis and non-Hodgkin lymphoma, were shown for the first time to be involved in amplification. Genomic alterations involving different subtelomeric regions with losses in 4p16, 5p15.3, 6q27, 18p11.3, and 18q23 and gains in 1p36.3 and 19p13.3 were detected by HR-CGH. Array CGH analysis of the subtelomeric regions in some samples was able to confirm a number of these alterations and found some additional alterations not detected by conventional CGH.

The impact of the genomic imbalances on the clinical outcome of 34 patients with lymph-node positive high-risk breast cancer (HRBC) was investigated using comparative genomic hybridization. All of the patients were uniformly treated with high-dose chemotherapy and autologous stem cell transplantation. The average number of chromosomal imbalances per tumor was 11 (range, 2-24), including DNA overrepresentation on chromosomes 1q (59%), 17q (38%), 8q and 16p (35% each), 20q (32%), and 19p (26%), and genomic losses involving 9p and 18q (41%), 8p, 11q, and 18p (38%), 17p (32%), 4p and Xq (29%), and 16q (26%). The most significant association among genomic changes and clinical-pathological features was the correlation of the loss of 8p with progesterone receptor positivity (P < 0.005). With a median follow-up time of 74 months, 15 patients (44%) have relapsed. In the univariate analysis, patients with gain/amplification of 17q including the HER-2/neu gene locus had a longer disease-free survival (P = 0.02), whereas those with genomic loss of 18p had a higher probability of relapse (P = 0.003). In multivariate analysis, the loss of 18p was the only parameter correlated with shorter disease-free survival (relative risk, 4.8; 95% confidence interval, 1.57-14.8; P = 0.006). In summary, our data indicate that the tumoral genomic profile may represent a valuable marker for predicting the clinical outcome in HRBC. Furthermore, the genomic loss of 18p may identify a poor prognostic subgroup of patients with HRBC.