Allelic Loss in a Minimal Region on Chromosome 16Q24 Is Associated with Vitreous Seeding of Retinoblastoma
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Research Article Allelic Loss in a Minimal Region on Chromosome 16q24 Is Associated with Vitreous Seeding of Retinoblastoma Sandrine Gratias,1 Harald Rieder,4 Reinhard Ullmann,5 Ludger Klein-Hitpass,2 Stephanie Schneider,6 Re´ka Bo¨lo¨ni,3 Martin Kappler,7 and Dietmar R. Lohmann1 1Institut fu¨r Humangenetik, 2Institut fu¨r Zellbiologie, and 3Augenklinik, Universita¨tsklinikum Essen, Essen, Germany; 4Institut fu¨r Humangenetik und Anthropologie, Universita¨tsstraße 1, Universita¨tDu¨sseldorf, Du¨sseldorf, Germany; 5Max-Planck Institute for Molecular Genetics, Berlin, Germany; 6Institut fu¨r Klinische Genetik, Universita¨tsklinikum Marburg, Marburg, Germany; and 7Berufsgenossenschaftliches Forschungsinstitut fu¨r Arbeitsmedizin, Ruhr-Universita¨tBochum, Bochum, Germany Abstract Loss of all or parts of chromosome 16 is observed in 31% of In addition to RB1 gene mutations, retinoblastomas frequently retinoblastomas (51 of 162; summarized in ref. 6). In most of these show gains of 1q and 6p and losses of 16q. To identify tumors, the whole long arm of one homologue is lost (40 of suppressor genes on 16q, we analyzed 22 short tandem repeat 51, 78%). A survey of comparative genomic hybridization (CGH) loci in 58 patients with known RB1 mutations. A subset of analyses has indicated that most partial deletions on chromosome tumors was also investigated by conventional and matrix 16q include chromosome band 16q22 (7 of 11, 64%; ref. 3). To study comparative genomic hybridization. In 40 of 58 (69%) tumors, alterations of this region in further detail, Marchong et al. (6) did LOH analysis of seven microsatellite markers located on 16q21-23.3 we found no loss of heterozygosity (LOH) at any 16q marker. 8 LOH was detected in 18 of 58 (31%) tumors, including five with (Mb 60.9–81.5, Ensembl v36 ) and quantitative multiplex PCR of allelic imbalance at some markers. In one tumor LOH was only five sequence-tagged sites in Mb 61.5 to 75.1 (Ensembl v36) and of observed at 16q24. As the parental origin of allele loss was six exons of the cadherin 11 (CDH11) gene. They found frequent unbiased, an imprinted locus is unlikely to be involved. allelic loss at D16S398 (located at Mb 64.7, observed in 11 of 28 Analysis of gene expression by microarray hybridization and tumors, 39%) and at D16S422 (Mb 81.5, observed in 9 of 23 tumors, quantitative RT real-time PCR did not identify a candidate 39%). Quantitative multiplex PCR showed that sequences located suppressor in 16q24. Cadherin 13 (CDH13), CBFA2T3, and within the CDH11 gene (Mb 63.5–63.7) are most frequently lost (41 WFDC1, which are candidate suppressors in other tumor of 71 tumors, 58%). The long arm of chromosome 16 is a frequent entities with 16q24 loss, did not show loss of expression. In target of deletions in various cancers. Three candidate regions, one in 16q22.1 and two in 16q24.3, have been identified. In some tumors addition, mutation and methylation analysis showed no somatic alteration of CDH13. Results in all tumors with with 16q22.1 loss, E-cadherin (CDH1) is inactivated by mutations or chromosome 16 alterations define a single minimal deleted silenced by epigenetic mechanisms (11, 12). This provides good region of 5.7 Mb in the telomeric part of 16q24 with the evidence for a tumor-suppressor role of CDH1 in these cancers. centromeric boundary defined by retention of heterozygosity The tumor suppressors underlying loss in the telomeric candidate for a single nucleotide variant in exon 10 of CDH13 (Mb 82.7). regions are not defined yet. Here, we used LOH analysis and Interestingly, clinical presentation of tumors with and without microarray expression analysis to identify candidate tumor 16q alterations was distinct. Specifically, almost all retino- suppressors on 16q in retinoblastoma. Moreover, we investigated blastomas with 16q24 loss showed diffuse intraocular seeding. if clinical manifestation is distinct depending on the presence of This suggests that genetic alterations in the minimal deleted alterations on 16q. region are associated with impaired cell-to-cell adhesion. [Cancer Res 2007;67(1):408–16] Materials and Methods Samples and extraction of DNA and RNA. Tumor and blood samples Introduction were obtained from retinoblastoma patients at the time of operative treatment. In addition, blood samples were received from parents of most Retinoblastoma, a rare childhood eye tumor, has served as a patients. Tissues were snap frozen in liquid nitrogen. Informed consent was model to elucidate the genetic events underlying the development obtained from all patients or their parents. Storage and processing of of sporadic and hereditary cancer (reviewed in refs. 1, 2). For samples as well as extraction of nucleic acids was done as previously initiation of this tumor, mutations in both alleles of the RB1 gene, described (10). Material from 58 patients (54unilateral and 4bilateral cases) a tumor suppressor on chromosome 13q14, are required. Retino- with known mutations in the RB1 gene was used for the present study. blastomas often show gains on chromosomes 1q and 6p and losses Microsatellite analysis of markers on chromosome 16. A total of 22 of chromosome 16q (summarized in ref. 3). Therefore, it is reason- short tandem repeat loci with high polymorphic information content were able to assume that mutations in additional genes contribute to analyzed (Fig. 1A; primer sequences are available on request). Intermarker promotion and progression of this tumor (4). The minimum regions distances were even (1–2 Mb along 16q) except a gap of 15 Mb between markers D16S3080 and D16S3050 (at 16q12.1 and 16q21, respectively). PCR of genomic gains on 1q and 6p have been defined and this has led with labeled forward primers (FAM, PET, or NED fluorescent dyes at the to the identification of putative target oncogenes (5–10). 5¶-end, Applied Biosystems, Weiterstadt, Germany) was done in multiplexed assays and analyzed as described (10). If loss of one allele in the tumor was incomplete, the allele ratio was determined as follows: (PI allele tumor / PI Requests for reprints: Dietmar R. Lohmann, Institut fu¨r Humangenetik, 1 Universita¨tsklinikum Essen, Hufelandstrasse 55, D-45122 Essen, Germany. Phone: 49- 201-7234562; Fax: 49-201-7235900; E-mail: [email protected]. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-1317 8 http://www.ensembl.org. Cancer Res 2007; 67: (1). January 1, 2007 408 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. 16q Loss in Retinoblastoma and Vitreous Seeding Figure 1. Pattern of chromosome 16 alterations in retinoblastoma samples. A, results of microsatellite analysis (MSA) in 40 tumors with no LOH (black squares) at any informative marker. Allelic imbalance (blue squares) is scored for makers with allele ratios between 1.3 and 2.5. Gray squares, noninformative markers. Headers of columns with microsatellite analysis results give a summary of the results of conventional CGH and matrix CGH (matrix CGH-a, reported in ref. 17; matrix CGH-b, original): n, no copy number changes on chromosome 16; L, losses; gL, gains and losses; —, not done. B, results of microsatellite analysis in 18 tumors with LOH at some or all informative markers. Parental origin of allele losses for markers with LOH on chromosome 16: m, maternal allele retained; p, paternal allele retained. C, synopsis of the results of microsatellite analysis and matrix CGH in five tumors with complex copy number changes. Green bars, gains; orange bars, losses. Scale (left), map position of chromosome 16 markers according to Ensembl v36. allele2 tumor) / (PI allele1 blood / PI allele2 blood), where PI is the peak as previously described (14, 15). The cDNAs were purified by phenol/ integral. To obtain allele ratio values >1, the allele with the larger peak area chloroform/isoamyl alcohol/phase lock gel (Eppendorf, Hamburg, Germany) in the tumor was defined as allele1. We used the criteria established in extraction, precipitated, and used to generate biotinylated cRNA by in vitro a previous study (13) to categorize the results as follows: LOH for values transcription for 16 h at 37jC (Bioarray High Yield RNA Transcript Labeling of allele ratio >2.5; allelic imbalance for 1.3 V allele ratio V 2.5; normal for kit, Enzo Life Science, Farmingdale, NY). Purification of cRNA was done values of allele ratio < 1.3. using RNeasy mini columns (Qiagen, Hilden, Germany). Fragmentation of Microarray expression analysis. Synthesis of double-stranded cDNA cRNA, hybridization to HG-U133A oligonucleotide arrays (Affymetrix, Inc., was done with f2.5 Ag of total RNA and anchored T7-oligo-d(T)21-V Santa Barbara, CA), washing, staining, and scanning (GeneArray scanner primer [5¶-GCATTA-GCGGCCGCGAAATTAATACGACTCACTATAGGGA- 2500, Agilent, Palo Alto, CA) were done following standard Affymetrix GA(T)21V-3¶, MWG Biotech, Ebersberg, Germany] for first-strand synthesis protocols (Technical Manual). Signal intensities and detection calls for www.aacrjournals.org 409 Cancer Res 2007; 67: (1). January 1, 2007 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Cancer Research Table 1. List of genes in 16q24 differentially expressed between tumors with and without LOH at chromosome 16q c b Gene symbol Position* (bp) Affymetrix accession no Median expression P < Expression in retina No LOH LOH MBTPS1 82644869 201620_at 3,383.4 1,727.3 0.003 2,901.9 KIAA1609 83068608 65438_at