[Show abstract][Hide abstract] ABSTRACT: The mechanism for generating double minutes chro-mosomes (dmin) and homogeneously staining re-gions (hsr) in cancer is still poorly understood. Through an integrated approach combining next-generation sequencing, single nucleotide polymor-phism array, fluorescent in situ hybridization and polymerase chain reaction-based techniques, we in-ferred the fine structure of MYC-containing dmin/hsr amplicons harboring sequences from several differ-ent chromosomes in seven tumor cell lines, and char-acterized an unprecedented number of hsr inser-tion sites. Local chromosome shattering involving a single-step catastrophic event (chromothripsis) was recently proposed to explain clustered chromosomal rearrangements and genomic amplifications in can-cer. Our bioinformatics analyses based on the listed criteria to define chromothripsis led us to exclude it as the driving force underlying amplicon genesis in our samples. Instead, the finding of coexisting het-erogeneous amplicons, differing in their complexity and chromosome content, in cell lines derived from the same tumor indicated the occurrence of a multi-step evolutionary process in the genesis of dmin/hsr. Our integrated approach allowed us to gather a com-plete view of the complex chromosome rearrange-ments occurring within MYC amplicons, suggesting that more than one model may be invoked to explain the origin of dmin/hsr in cancer. Finally, we identified PVT1 as a target of fusion events, confirming its role as breakpoint hotspot in MYC amplification.
Nucleic Acids Research 07/2014; · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Clear cell renal cell carcinomas are characterized by 3p loss, and by inactivation of Von Hippel Lindau (VHL), a tumorsuppressor gene located at 3p25. Recently, SETD2, located at 3p21, was identified as a new candidate ccRCC tumor-suppressor gene. The combined mutational frequency in ccRCC tumors of VHL and SETD2 suggests that there are still undiscovered tumor-suppressor genes on 3p. We screened all genes on 3p for mutations in 10 primary ccRCC tumors using exome-sequencing. We identified inactivating mutations in VHL, PBRM1, and BAP1. Sequencing of PBRM1 in ccRCC-derived cell lines confirmed its frequent inactivation in ccRCC. PBRM1 encodes for BAF180, the chromatin targeting subunit of the SWI/SNF complex. BAP1 encodes for BRCA1 associated protein-1, involved in histone deubiquitination. Taken together, the accumulating data suggest an important role for aberrant chromatin regulation in ccRCC development.
Human Mutation 03/2012; 33(7):1059-62. · 5.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Whole-genome analysis using genome-wide arrays, also called "genomic arrays," "microarrays," or "arrays," has become the first-tier diagnostic test for patients with developmental abnormalities and/or intellectual disabilities. In addition to constitutional anomalies, genomic arrays are also used to diagnose acquired disorders. Despite the rapid implementation of these technologies in diagnostic laboratories, external quality control schemes (such as CEQA, EMQN, UK NEQAS, and the USA QA scheme CAP) and interlaboratory comparisons show that there are huge differences in quality, interpretation, and reporting among laboratories. We offer guidance to laboratories to help assure the quality of array experiments and to standardize minimum detection resolution, and we also provide guidelines to standardize interpretation and reporting.
Human Mutation 03/2012; 33(6):906-15. · 5.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Formalin-fixed, paraffin-embedded (FFPE) archival tissue is an important source of DNA material. The most commonly used technique to identify copy number aberrations from chromosomal DNA in tumorigenesis is array comparative genomic hybridization (aCGH). Although copy number analysis using DNA from FFPE archival tissue is challenging, several research groups have reported high quality and reproducible DNA copy number results using aCGH. Aim of this study is to compare the commercially available aCGH platforms suitable for high-resolution copy number analysis using FFPE-derived DNA. Two dual channel aCGH platforms (Agilent and NimbleGen) and a single channel SNP-based platform (Affymetrix) were evaluated using seven FFPE colon cancer samples, and median absolute deviation (MAD), deflection, signal-to-noise ratio, and DNA input requirements were used as quality criteria. Large differences were observed between platforms; Agilent and NimbleGen showed better MAD values (0.13 for both) compared with Affymetrix (0.22). On the contrary, Affymetrix showed a better deflection of 0.94, followed by 0.71 for Agilent and 0.51 for NimbleGen. This resulted in signal-to-nose ratios that were comparable between the three commercially available platforms. Interestingly, DNA input amounts from FFPE material lower than recommended still yielded high quality profiles on all platforms. Copy number analysis using DNA derived from FFPE archival material is feasible using all three high-resolution copy number platforms and shows reproducible results, also with DNA input amounts lower than recommended.
Genes Chromosomes and Cancer 12/2011; 51(4):344-52. · 3.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The correct interpretation of copy number gains in patients with developmental delay and multiple congenital anomalies is hampered by the large number of copy number variations (CNVs) encountered in healthy individuals. The variable phenotype associated with copy number gains makes interpretation even more difficult. Literature shows that inheritence, size and presence in healthy individuals are commonly used to decide whether a certain copy number gain is pathogenic, but no general consensus has been established. We aimed to develop guidelines for interpreting gains detected by array analysis using array CGH data of 300 patients analysed with the 105K Agilent oligo array in a diagnostic setting. We evaluated the guidelines in a second, independent, cohort of 300 patients. In the first 300 patients 797 gains of four or more adjacent oligonucleotides were observed. Of these, 45.4% were de novo and 54.6% were familial. In total, 94.8% of all de novo gains and 87.1% of all familial gains were concluded to be benign CNVs. Clinically relevant gains ranged from 288 to 7912 kb in size, and were significantly larger than benign gains and gains of unknown clinical relevance (P < 0.001). Our study showed that a threshold of 200 kb is acceptable in a clinical setting, whereas heritability does not exclude a pathogenic nature of a gain. Evaluation of the guidelines in the second cohort of 300 patients revealed that the interpretation guidelines were clear, easy to follow and efficient.
European journal of human genetics: EJHG 09/2011; 20(2):161-5. · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The epidemiological relationship between squamous cell lung cancer (SCC) and chronic obstructive pulmonary disease (COPD), both smoking-related diseases, suggests that they have also a genetic basis. We compared 35 SCC patients with and without COPD with whole-genome gene expression profiles of laser microdissected tissue. Validation of differential expression was performed for 25 genes using quantitative (q)RT-PCR. Subsequently, we performed array-based CGH on the same tumor samples. We found that 374 probes were differentially expressed in SCC from patients with and without COPD. Forty-four probes were derived from genes with mitochondrial functions and 34 probes were located on 5q. All these probes showed a lower expression level in SCC from non-COPD patients. For a random selection of 25 mitochondrial and 5q genes, we confirmed the differential expression by qRT-PCR. Loss of 3p, 5q and 9p was observed, via array-CGH, to be more frequent in SCC from non-COPD patients as compared to SCC from COPD patients. Combination of chromosomal aberrations and the location of the differentially expressed genes showed significant association for loss of the 5q31.2-31.3 region and reduced expression of the 5q genes. In conclusion, a more frequent loss of 5q and a low expression of genes located on 5q in SCC tumors of non-COPD patients compared to tumors from COPD patients was identified suggesting that different oncogenetic pathways are operational in patients with and without COPD.
Lung cancer (Amsterdam, Netherlands) 05/2011; 72(2):177-83. · 3.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Double minutes (dmin) and homogeneously staining regions (hsr) are the cytogenetic hallmarks of genomic amplification in cancer. Different mechanisms have been proposed to explain their genesis. Recently, our group showed that the MYC-containing dmin in leukemia cases arise by excision and amplification (episome model). In the present paper we investigated 10 cell lines from solid tumors showing MYCN amplification as dmin or hsr. Particularly revealing results were provided by the two subclones of the neuroblastoma cell line STA-NB-10, one showing dmin-only and the second hsr-only amplification. Both subclones showed a deletion, at 2p24.3, whose extension matched the amplicon extension. Additionally, the amplicon structure of the dmin and hsr forms was identical. This strongly argues that the episome model, already demonstrated in leukemias, applies to solid tumors as well, and that dmin and hsr are two faces of the same coin. The organization of the duplicated segments varied from very simple (no apparent changes from the normal sequence) to very complex. MYCN was always overexpressed (significantly overexpressed in three cases). The fusion junctions, always mediated by nonhomologous end joining, occasionally juxtaposed truncated genes in the same transcriptional orientation. Fusion transcripts involving NBAS (also known as NAG), FAM49A, BC035112 (also known as NCRNA00276), and SMC6 genes were indeed detected, although their role in the context of the tumor is not clear.
Genome Research 09/2010; 20(9):1198-206. · 14.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sporadic clear cell renal cell carcinoma (cRCC) is genetically characterized by the recurrent loss of the short arm of chromosome 3, with a hotspot for copy number loss in the 3p21 region. We applied a method called "gene identification by nonsense-mediated mRNA decay inhibition" to a panel of 10 cRCC cell lines with 3p21 copy number loss to identify biallelic inactivated genes located at 3p21. This revealed inactivation of the histone methyltransferase gene SETD2, located on 3p21.31, as a common event in cRCC cells. SETD2 is nonredundantly responsible for trimethylation of the histone mark H3K36. Consistent with this function, we observed loss or a decrease of H3K36me3 in 7 out of the 10 cRCC cell lines. Identification of missense mutations in 2 out of 10 primary cRCC tumor samples added support to the involvement of loss of SETD2 function in the development of cRCC tumors.
Cancer Research 06/2010; 70(11):4287-91. · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chromosomal microarray (CMA) is increasingly utilized for genetic testing of individuals with unexplained developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), or multiple congenital anomalies (MCA). Performing CMA and G-banded karyotyping on every patient substantially increases the total cost of genetic testing. The International Standard Cytogenomic Array (ISCA) Consortium held two international workshops and conducted a literature review of 33 studies, including 21,698 patients tested by CMA. We provide an evidence-based summary of clinical cytogenetic testing comparing CMA to G-banded karyotyping with respect to technical advantages and limitations, diagnostic yield for various types of chromosomal aberrations, and issues that affect test interpretation. CMA offers a much higher diagnostic yield (15%-20%) for genetic testing of individuals with unexplained DD/ID, ASD, or MCA than a G-banded karyotype ( approximately 3%, excluding Down syndrome and other recognizable chromosomal syndromes), primarily because of its higher sensitivity for submicroscopic deletions and duplications. Truly balanced rearrangements and low-level mosaicism are generally not detectable by arrays, but these are relatively infrequent causes of abnormal phenotypes in this population (<1%). Available evidence strongly supports the use of CMA in place of G-banded karyotyping as the first-tier cytogenetic diagnostic test for patients with DD/ID, ASD, or MCA. G-banded karyotype analysis should be reserved for patients with obvious chromosomal syndromes (e.g., Down syndrome), a family history of chromosomal rearrangement, or a history of multiple miscarriages.
The American Journal of Human Genetics 05/2010; 86(5):749-64. · 11.20 Impact Factor