A clinical and genetic study of the Say/Barber/Biesecker/Young-Simpson type of Ohdo syndrome.

We report a series of eight patients with the Say/Barber/Biesecker/Young-Simpson (SBBYS) type of Ohdo syndrome, which is the largest cohort described to date. We expand on the type, frequency and severity of the clinical characteristics in this condition; comment on the natural history of Ohdo syndrome and further refine previously published diagnostic criteria. Cytogenetic investigations and microarray CGH analysis undertaken in this cohort of patients failed to identify a chromosomal aetiology. It remains possible that this rare condition is heterogeneous and therefore caution must be undertaken during counselling until the underlying genetic mechanism(s) is (are) identified.

Frequent deletion of the CDKN2A locus in chordoma: analysis of chromosomal imbalances using array comparative genomic hybridisation.

The initiating somatic genetic events in chordoma development have not yet been identified. Most cytogenetically investigated chordomas have displayed near-diploid or moderately hypodiploid karyotypes, with several numerical and structural rearrangements. However, no consistent structural chromosome aberration has been reported. This is the first array-based study characterising DNA copy number changes in chordoma. Array comparative genomic hybridisation (aCGH) identified copy number alterations in all samples and imbalances affecting 5 or more out of the 21 investigated tumours were seen on all chromosomes. In general, deletions were more common than gains and no high-level amplification was found, supporting previous findings of primarily losses of large chromosomal regions as an important mechanism in chordoma development. Although small imbalances were commonly found, the vast majority of these were detected in single cases; no small deletion affecting all tumours could be discerned. However, the CDKN2A and CDKN2B loci in 9p21 were homo- or heterozygously lost in 70% of the tumours, a finding corroborated by fluorescence in situ hybridisation, suggesting that inactivation of these genes constitute an important step in chordoma development.

Tiling resolution array CGH of dic(7;9)(p11 approximately 13;p11 approximately 13) in B-cell precursor acute lymphoblastic leukemia reveals clustered breakpoints at 7p11.2 approximately 12.1 and 9p13.1.

The dic(7;9)(p11 approximately 13;p11 approximately 13) is a recurrent chromosomal abnormality in acute lymphoblastic leukemia (ALL), mainly of B-lineage. Although more than 20 dic(7;9)-positive ALLs have been reported to date, the molecular genetic consequences of this aberration are unknown. We performed tiling resolution (32K) genome-wide array-based comparative genomic hybridization (array CGH) analysis of three cases with dic(7;9) in order to characterize the breakpoints on 7p and 9p. The analysis showed a clustering of breakpoints within 9p13.1 in all three cases and within 7p11.2 in two cases; the array CGH revealed two different breakpoints - 7p12.1 and 7p14.1 - in the remaining case. Based on these findings the abnormality should hence be designated dic(7;9)(p11.2 approximately 12.1;p13.1). Locus-specific fluorescence in situhybridization analysis of one of the cases narrowed down the 7p11.2 breakpoint to a <500-kb segment in this sub-band, a region containing three known genes. Unfortunately, lack of material precluded further molecular genetic studies, and it thus remains unknown whether the pathogenetically important outcome of the dic(7;9) is formation of a chimeric gene or loss of 7p and/or 9p material.

Combined high-resolution array-based comparative genomic hybridization and expression profiling of ETV6/RUNX1-positive acute lymphoblastic leukemias reveal a high incidence of cryptic Xq duplications and identify several putative target genes within the commonly gained region.

Seventeen ETV6/RUNX1-positive pediatric acute lymphoblastic leukemias were investigated by high-resolution array-based comparative genomic hybridization (array CGH), gene expression profiling and fluorescence in situ hybridization. Comparing the array CGH and gene expression patterns revealed that genomic imbalances conferred a great impact on the expression of genes in the affected regions. The array CGH analyses identified a high frequency of cytogenetically cryptic genetic changes, for example, del(9p) and del(12p). Interestingly, a duplication of Xq material, varying between 30 and 60 Mb in size, was found in 6 of 11 males (55%), but not in females. Genes on Xq were found to have a high expression level in cases with dup(Xq); a similar overexpression was confirmed in t(12;21)-positive cases in an external gene expression data set. By studying the expression profile and the proposed function of genes in the minimally gained region, several candidate target genes (SPANXB, HMGB3, FAM50A, HTATSF1 and RAP2C) were identified. Among them, the testis-specific SPANXB gene was the only one showing a high and uniform overexpression, irrespective of gender and presence of Xq duplication, suggesting that this gene plays an important pathogenetic role in t(12;21)-positive leukemia.

Identification of cryptic aberrations and characterization of translocation breakpoints using array CGH in high hyperdiploid childhood acute lymphoblastic leukemia.

High hyperdiploidy, characterized by non-random trisomies, is the largest cytogenetic subgroup in childhood acute lymphoblastic leukemia (ALL). It is not known whether the gained chromosomes are sufficient for leukemogenesis or if additional genetic aberrations are necessary. However, the suboptimal chromosome morphology of hyperdiploid ALLs makes detection of structural abnormalities difficult if using cytogenetic techniques; alternative methods are, therefore, needed. We performed array comparative genome hybridization (CGH) analyses, with a resolution of 100 kb, of eight cases of high hyperdiploid childhood ALL to characterize structural abnormalities found with G-banding/multicolor fluorescence in situ hybridization (FISH) and to detect novel changes. The non-centromeric breakpoints of four rearrangements, including three translocations and one 1q duplication, were narrowed down to <0.2 Mb. Furthermore, four submicroscopic imbalances involving 0.6-2.7 Mb were detected, comprising two segmental duplications involving 1q22 and 12q24.31 in one case and two hemizygous deletions in 12p13.2-31 - including ETV6 - and in 13q32.3-33.1 in another case. Notably, FISH analysis of the latter revealed an associated reciprocal t(3;13)(q?;32.2-33.1). In conclusion, the array CGH analyses revealed putative leukemia-associated submicroscopic imbalances and rearrangements in 2/8 (25%) hyperdiploid ALLs. The detection and characterization of these additional genetic aberrations will most likely increase our understanding of the pathogenesis of high hyperdiploid childhood ALL.

Genomic profiling of bone and soft tissue tumors with supernumerary ring chromosomes using tiling resolution bacterial artificial chromosome microarrays.

Ring chromosomes and/or giant marker chromosomes have been observed in a variety of human tumor types, but they are particularly common in a subgroup of mesenchymal tumors of low-grade or borderline malignancy. These rings and markers have been shown to contain amplified material predominantly from 12q13-15, but also sequences from other chromosomes. Such amplified sequences were mapped in detail by genome-wide array comparative genomic hybridization in ring-containing tumor samples from soft tissue (n = 15) and bone (n = 6), using tiling resolution microarrays, encompassing 32 433 bacterial artificial chromosome clones. The DNA copy number profiles revealed multiple amplification targets, in many cases highly discontinuous, leading to delineation of large numbers of very small amplicons. A total number of 356 (median size: 0.64 Mb) amplicons were seen in the soft tissue tumors and 90 (median size: 1.19 Mb) in the bone tumors. Notably, more than 40% of all amplicons in both soft tissue and bone tumors were mapped to chromosome 12, and at least one of the previously reported recurrent amplifications in 12q13.3-14.1 and 12q15.1, including SAS and CDK4, and MDM2, respectively, were present in 85% of the soft tissue tumors and in all of the bone tumors. Although chromosome 12 was the only chromosome displaying recurrent amplification in the bone tumors, the soft tissue tumors frequently showed recurrent amplicons mapping to other chromosomes, that is, 1p32, 1q23-24, 3p11-12, 6q24-25 and 20q11-12. Of particular interest, amplicons containing genes involved in the c-jun NH2-terminal kinase/mitogen-activated protein kinase pathway, that is, JUN in 1p32 and MAP3K7IP2 (TAB2) in 6q24-25, were found to be independently amplified in eight of 11 cases with 12q amplification, providing strong support for the notion that aberrant expression of this pathway is an important step in the dedifferentiation of liposarcomas.

High-resolution genome-wide array-based comparative genome hybridization reveals cryptic chromosome changes in AML and MDS cases with trisomy 8 as the sole cytogenetic aberration.

Although trisomy 8 as the sole chromosome aberration is the most common numerical abnormality in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), little is known about its pathogenetic effects. Considering that +8 is a frequent secondary change in AML/MDS, cryptic--possibly primary--genetic aberrations may occur in cases with trisomy 8 as the apparently single anomaly. However, no such hidden anomalies have been reported. We performed a high-resolution genome-wide array-based comparative genome hybridization (array CGH) analysis of 10 AML/MDS cases with isolated +8, utilizing a 32K bacterial artificial chromosome array set, providing >98% coverage of the genome with a resolution of 100 kb. Array CGH revealed intrachromosomal imbalances, not corresponding to known genomic copy number polymorphisms, in 4/10 cases, comprising nine duplications and hemizygous deletions ranging in size from 0.5 to 2.2 Mb. A 1.8 Mb deletion at 7p14.1, which had occurred prior to the +8, was identified in MDS transforming to AML. Furthermore, a deletion including ETV6 was present in one case. The remaining seven imbalances involved more than 40 genes. The present results show that cryptic genetic abnormalities are frequent in trisomy 8-positive AML/MDS cases and that +8 as the sole cytogenetic aberration is not always the primary genetic event.

Altered expression of TGFB receptors and mitogenic effects of TGFB in pancreatic carcinomas.

Alteration of the transforming growth factor beta (TGFB) signalling pathway is important in pancreatic carcinogenesis, as shown by the frequent inactivation of the downstream target SMAD4. We recently analysed a series of pancreatic carcinoma cell lines with respect to alterations of five SMAD genes involved in TGFB signalling, and showed that SMAD4 was structurally rearranged in 42% of these. This pathway may, however, also be affected by alterations of genes whose products regulate the activation of TGFB as well as of TGFB receptor genes. We therefore studied the expression of UPA, UPAR, IGF2R, ALK5 (TGFBR1), TGFBR2, TGFBR3, ENG, ALK1, TGFB1, TGFB2, and TGFB3 in a series of 14 pancreatic carcinoma cell lines. We also analysed ALK5 and TGFBR2 for mutations, cell surface localisation of TGFBR2 and ENG, and TGFB1 response. No mutations of ALK5 or TGFBR2 were found. However, 4 cell lines were methylated within the ALK5 promoter region. ALK5 expression was strongly reduced in 9 cases, whereas TGFBR2 expression was increased in 12 of the cell lines. The TGFB signalling associated receptors ENG and ALK1 were co-expressed in 4 of the cell lines. There was no evidence for disruption of the UPAR-IGF2R TGFB activating pathway. The response to TGFB1 was analysed in 12 cell lines, and 6 of these (50%) showed increased proliferation. The cell lines stimulated by TGFB showed frequent mutations of SMAD4, KRAS2, and TP53, as well as frequent absence of CDKN2B expression. These results suggest that the ALK5-SMAD4 part of the TGFB signalling pathway is a major target for inactivation in pancreatic carcinomas, that the expression of TGFBR2, TGFBR3, and receptors involved in TGFB activation are maintained, and that alterations of components of the TGFB signalling pathway may be accompanied by a positive effect of TGFB on cell growth.

Chromosomal breakage-fusion-bridge events cause genetic intratumor heterogeneity.

It has long been known that rearrangements of chromosomes through breakage-fusion-bridge (BFB) cycles may cause variability of phenotypic and genetic traits within a cell population. Because intercellular heterogeneity is often found in neoplastic tissues, we investigated the occurrence of BFB events in human solid tumors. Evidence of frequent BFB events was found in malignancies that showed unspecific chromosome aberrations, including ring chromosomes, dicentric chromosomes, and telomeric associations, as well as extensive intratumor heterogeneity in the pattern of structural changes but not in tumors with tumor-specific aberrations and low variability. Fluorescence in situ hybridization analysis demonstrated that chromosomes participating in anaphase bridge formation were involved in a significantly higher number of structural aberrations than other chromosomes. Tumors with BFB events showed a decreased elimination rate of unstable chromosome aberrations after irradiation compared with normal cells and other tumor cells. This result suggests that a combination of mitotically unstable chromosomes and an elevated tolerance to chromosomal damage leads to constant genomic reorganization in many malignancies, thereby providing a flexible genetic system for clonal evolution and progression.