PubMed Health⌕ Search

Biomedical subjects

Eric Rappaport

Publications and source records attributed to Eric Rappaport.

9 recordsLinked to original sources
PubMed2006

Integrative genomics identifies distinct molecular classes of neuroblastoma and shows that multiple genes are targeted by regional alterations in DNA copy number.

Neuroblastoma is remarkable for its clinical heterogeneity and is characterized by genomic alterations that are strongly correlated with tumor behavior. The specific genes that influence neuroblastoma biology and are targeted by genomic alterations remain largely unknown. We quantified mRNA expression in a highly annotated series of 101 prospectively collected diagnostic neuroblastoma primary tumors using an oligonucleotide-based microarray. Genomic copy number status at the prognostically relevant loci 1p36, 2p24 (MYCN), 11q23, and 17q23 was determined by PCR and was aberrant in 26, 20, 40, and 38 cases, respectively. In addition, 72 diagnostic neuroblastoma primary tumors assayed in a different laboratory were used as an independent validation set. Unsupervised hierarchical clustering showed that gene expression was highly correlated with genomic alterations and clinical markers of tumor behavior. The vast majority of samples with MYCN amplification and 1p36 loss of heterozygosity (LOH) clustered together on a terminal node of the sample dendrogram, whereas the majority of samples with 11q deletion clustered separately and both of these were largely distinct from the copy number neutral group of tumors. Genes involved in neurodevelopment were broadly overrepresented in the more benign tumors, whereas genes involved in RNA processing and cellular proliferation were highly represented in the most malignant cases. By combining transcriptomic and genomic data, we showed that LOH at 1p and 11q was associated with significantly decreased expression of 122 (61%) and 88 (27%) of the genes mapping to 1p35-36 and all of 11q, respectively, suggesting that multiple genes may be targeted by LOH events. A total of 71 of the 1p35-36 genes were also differentially expressed in the independent validation data set, providing a prioritized list of candidate neuroblastoma suppressor genes. Taken together, these data are consistent with the hypotheses that the neuroblastoma transcriptome is a sensitive marker of underlying tumor biology and that chromosomal deletion events in this cancer likely target multiple genes through alteration in mRNA dosage. Lead positional candidates for neuroblastoma suppressor genes can be inferred from these data, but the potential multiplicity of transcripts involved has significant implications for ongoing gene discovery strategies.

Chromosome Aberrations↗
PubMed2006

Rapid detection of submicroscopic chromosomal rearrangements in children with multiple congenital anomalies using high density oligonucleotide arrays.

Chromosomal rearrangements such as microdeletions and interstitial duplications are the underlying cause of many human genetic disorders. These disorders can manifest in the form of multiple congenital anomalies (MCA), which are a significant cause of morbidity and mortality in children. The major limitations of cytogenetic tests currently used for the detection of such chromosomal rearrangements are low resolution and limited coverage of the genome. Thus, it is likely that children with MCA may have submicroscopic chromosomal rearrangements that are not detectable by current techniques. We report the use of a commercially available, oligonucleotide-based microarray for genome-wide analysis of copy number alterations. First, we validated the microarray in patients with known chromosomal rearrangements. Next, we identified previously undetected, de novo chromosomal deletions in patients with MCA who have had a normal high-resolution karyotype and subtelomeric fluorescence in situ hybridization (FISH) analysis. These findings indicate that high-density, oligonucleotide-based microarrays can be successfully used as tools for the detection of chromosomal rearrangement in clinical samples. Their higher resolution and commercial availability make this type of microarray highly desirable for application in the diagnosis of patients with multiple congenital defects.

Abnormalities, Multiple↗
PubMed2005

Region-specific detection of neuroblastoma loss of heterozygosity at multiple loci simultaneously using a SNP-based tag-array platform.

Many cancers are characterized by chromosomal aberrations that may be predictive of disease outcome. Human neuroblastomas are characterized by somatically acquired copy number changes, including loss of heterozygosity (LOH) at multiple chromosomal loci, and these aberrations are strongly associated with clinical phenotype including patient outcome. We developed a method to assess region-specific LOH by genotyping multiple SNPs simultaneously in DNA from tumor tissues. We identified informative SNPs at an average 293-kb density across nine regions of recurrent LOH in human neuroblastomas. We also identified SNPs in two copy number neutral regions, as well as two regions of copy number gain. SNPs were PCR-amplified in 12-plex reactions and used in solution-phase single-nucleotide extension incorporating tagged dideoxynucleotides. Each extension primer had 5' complementarity to one of 2000 oligonucleotides on a commercially available tag-array platform allowing for solid-phase sorting and identification of individual SNPs. This approach allowed for simultaneous detection of multiple regions of LOH in six human neuroblastoma-derived cell lines, and, more importantly, 14 human neuroblastoma primary tumors. Concordance with conventional genotyping was nearly absolute. Detection of LOH in this assay may not require comparison to matched normal DNAs because of the redundancy of informative SNPs in each region. The customized tag-array system for LOH detection described here is rapid, results in parallel assessment of multiple genomic alterations, and may speed identification of and/or assaying prognostically relevant DNA copy number alterations in many human cancers.

Humans↗
PubMed2004

Cornelia de Lange syndrome is caused by mutations in NIPBL, the human homolog of Drosophila melanogaster Nipped-B.

Cornelia de Lange syndrome (CdLS; OMIM 122470) is a dominantly inherited multisystem developmental disorder characterized by growth and cognitive retardation; abnormalities of the upper limbs; gastroesophageal dysfunction; cardiac, ophthalmologic and genitourinary anomalies; hirsutism; and characteristic facial features. Genital anomalies, pyloric stenosis, congenital diaphragmatic hernias, cardiac septal defects, hearing loss and autistic and self-injurious tendencies also frequently occur. Prevalence is estimated to be as high as 1 in 10,000 (ref. 4). We carried out genome-wide linkage exclusion analysis in 12 families with CdLS and identified four candidate regions, of which chromosome 5p13.1 gave the highest multipoint lod score of 2.7. This information, together with the previous identification of a child with CdLS with a de novo t(5;13)(p13.1;q12.1) translocation, allowed delineation of a 1.1-Mb critical region on chromosome 5 for the gene mutated in CdLS. We identified mutations in one gene in this region, which we named NIPBL, in four sporadic and two familial cases of CdLS. We characterized the genomic structure of NIPBL and found that it is widely expressed in fetal and adult tissues. The fly homolog of NIPBL, Nipped-B, facilitates enhancer-promoter communication and regulates Notch signaling and other developmental pathways in Drosophila melanogaster.

Animals↗
PubMed2003

ID2 expression is not associated with MYCN amplification or expression in human neuroblastomas.

MYCN is a biologically and clinically important oncogene in human neuroblastoma as genomic amplification reliably predicts for aggressive tumor behavior and a poor prognosis. However, the mechanism by which MYCN amplification and overexpression contributes to a highly malignant phenotype remains obscure. ID2 is a dominant inhibitor of the RB1 tumor suppressor gene product and recently was suggested to be a direct transcriptional target of MYCN. Overexpression of Id2 protein has thus been postulated to result in functional inactivation of retinoblastoma in MYCN-amplified neuroblastomas, offering a potential explanation for the undifferentiated and highly proliferative nature of most MYCN-amplified neuroblastomas, as well as the paucity of retinoblastoma pathway mutations observed in clinical samples. We therefore sought to determine the likelihood that ID2 overexpression is associated with MYCN amplification and overexpression in human neuroblastoma. ID2 was not differentially expressed in 39 primary neuroblastoma specimens analyzed by oligonucleotide array-based expression analysis, and there was no correlation with MYCN expression levels. ID2 mRNA and protein expression was highly variable and independent of MYCN amplification status and mRNA expression in 10 human-derived neuroblastoma cell lines. In addition, ID2 mRNA expression was not associated with MYCN gene amplification status (P = 0.15) or MYCN expression (r = 0.22) in 131 separate diagnostic primary neuroblastoma samples analyzed by real-time quantitative RT-PCR. These data suggest that transcriptional regulation of ID2 by the MycN oncoprotein is unlikely to be a seminal molecular event resulting in a highly malignant neuroblastoma phenotype.

DNA-Binding Proteins↗
PubMed2003

CYP3A genotypes and treatment response in paediatric acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) is the most common paediatric cancer with a cure rate of approximately 80%. Relapse occurs despite treatment stratification based on clinical criteria. Relapse risk in ALL may be related to simple nucleotide polymorphisms (SNPs) of enzymes that metabolize chemotherapeutic agents. We evaluated whether SNPs in the cytochrome P450 3A family (CYP3A4*1B, CYP3A5*3 and CYP3A5*6) were associated with relapse risk on a national Children's Cancer Group (CCG) paediatric ALL trial (CCG-1891). CCG-1891 enrolled 1204 patients, and obtained both relapse and toxicity data prospectively. One hundred and twenty-four relapsed patients and 409 non-relapsed patients were assayed for each SNP. CYP3A variants were not associated with an increased risk of relapse. However, patients with the CYP3A4*1B and CYP3A5*3 genotypes had a decreased risk of peripheral neuropathy that was statistically significant on univariate analysis. After correction for multiple comparisons, the association between CYP3A*1B and CYP3A5*3 genotypes approached, but did not reach, statistical significance. CYP3 genotypes may not significantly modify the risk of relapse in childhood ALL, but may modify the risk of toxicity.

Aryl Hydrocarbon Hydroxylases↗
PubMed2002

Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss.

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .

Amino Acid Substitution↗
PubMed2002

Analysis of clinically relevant single-nucleotide polymorphisms by use of microelectronic array technology.

BACKGROUND: Microelectronic DNA chip devices represent an emerging technology for genotyping. We developed methods for detection of single-nucleotide polymorphisms (SNPs) in clinically relevant genes. METHODS: Primer pairs, with one containing a 5'-biotin group, were used to PCR-amplify the region encompassing the SNP to be interrogated. After denaturation, the biotinylated strand was electronically targeted to discrete sites on streptavidin-coated gel pads surfaces by use of a Nanogen Molecular Workstation. Allele-specific dye-labeled oligonucleotide reporters were used for detection of wild-type and variant sequences. Methods were developed for SNPs in genes, including factor VII, beta-globin, and the RET protooncogene. We genotyped 331 samples for five DNA variations in the factor VII gene, >600 samples from patients with beta-thalassemia, and 15 samples for mutations within the RET protooncogene. All samples were previously typed by various methods, including DNA sequence analysis, allele-specific PCR, and/or restriction enzyme digestion of PCR products. RESULTS: Analysis of amplified DNA required 4-6 h. After mismatched DNA was removed, signal-to-noise ratios were >5. More than 940 samples were typed with the microelectronic array platform, and results were totally concordant with results obtained previously by other genotyping methods. CONCLUSIONS: The described protocols detect SNPs of clinical interest with results comparable to those of other genotyping methods.

Factor VII↗