Evaluation of polymorphisms in EWSR1 and risk of Ewing sarcoma: a report from the Childhood Cancer Survivor Study.

BACKGROUND: Ewing sarcoma is a malignant bone tumor characterized by a high frequency of somatic EWSR1 translocations. Ewing sarcoma is less common in people of African or African-American ancestry, suggesting a genetic etiology. PROCEDURE: Germline DNA from white patients with Ewing sarcoma (n = 135), white controls with Wilms tumor (n = 200), and African-American controls (n = 285) was genotyped at 21 SNPs in the EWSR1 gene. Intron 7 of EWSR1, the most common site of translocation, was also sequenced in all subjects. Genetic variation between groups was evaluated statistically using exact logistic regression and Fisher exact tests. RESULTS: One SNP in EWSR1 (rs2857461) showed a low level of statistical association with the diagnosis of Ewing sarcoma compared to Wilms tumor. The odds ratio for having Ewing sarcoma in people with at least one copy of the minor allele of rs2857461 was 3.57 (95% confidence interval 0.79-21.7; P = 0.07). No other SNPs or variations in intron 7 of EWSR1 were associated with Ewing sarcoma. The median relative difference in minor allele frequencies between white subjects with Ewing sarcoma and African-American controls at the evaluated EWSR1 SNPs was 45%. CONCLUSIONS: Variations in EWSR1 at known SNPs or across intron 7 are not associated with the diagnosis of Ewing sarcoma. EWSR1 does not appear to be an Ewing sarcoma susceptibility gene. The genetic basis for this disease remains unknown.

Examination of FGFRL1 as a candidate gene for diaphragmatic defects at chromosome 4p16.3 shows that Fgfrl1 null mice have reduced expression of Tpm3, sarcomere genes and Lrtm1 in the diaphragm.

Fgfrl1 (also known as Fgfr5; OMIM 605830) homozygous null mice have thin, amuscular diaphragms and die at birth because of diaphragm hypoplasia. FGFRL1 is located at 4p16.3, and this chromosome region can be deleted in patients with congenital diaphragmatic hernia (CDH). We examined FGFRL1 as a candidate gene for the diaphragmatic defects associated with 4p16.3 deletions and re-sequenced this gene in 54 patients with CDH. We confirmed six known coding single nucleotide polymorphisms (SNPs): c.209G > A (p.Pro20Pro), c.977G > A (p.Pro276Pro), c.1040T > C (p.Asp297Asp), c.1234C > A (p.Pro362Gln), c.1420G > T (p.Arg424Leu), and c.1540C > T (p.Pro464Leu), but we did not identify any gene mutations. We genotyped additional CDH patients for four of these six SNPs, including the three non-synonymous SNPs, to make a total of 200 chromosomes, and found that the allele frequency for the four SNPs, did not differ significantly between patients and normal controls (p > or = 0.05). We then used Affymetrix Genechip Mouse Gene 1.0 ST arrays and found eight genes with significantly reduced expression levels in the diaphragms of Fgfrl1 homozygous null mice when compared with wildtype mice-Tpm3, Fgfrl1 (p = 0.004), Myl2, Lrtm1, Myh4, Myl3, Myh7 and Hephl1. Lrtm1 is closely related to Slit3, a protein associated with herniation of the central tendon of the diaphragm in mice. The Slit proteins are known to regulate axon branching and cell migration, and inhibition of Slit3 reduces cell motility and decreases the expression of Rac and Cdc42, two genes that are essential for myoblast fusion. Further studies to determine if Lrtm1 has a similar function to Slit3 and if reduced Fgfrl1 expression can cause diaphragm hypoplasia through a mechanism involving decreased myoblast motility and/or myoblast fusion, seem indicated.

Characterization of microsatellite loci in the western subterranean termite, Reticulitermes hesperus, and cross-amplification in closely related cryptic species.

New, and previously reported microsatellites, were characterized for a group of four cryptic sibling species in California (USA) in the subterranean termite genus Reticulitermes with the goal of finding loci appropriate for population and species level studies. Three new microsatellites were identified originating from R. hesperus, and 19 loci previously characterized in R. flavipes and R. santonensis were examined. Of the three loci specifically developed for R. hesperus, none amplify with the other species. Variation appropriate for population level studies was found in 4-13 loci depending on the species. Fifteen loci appeared to be appropriate for use at the species level. Unique or monomorphic alleles are found among the four species, indicating these loci will be taxonomically informative for this group.

An improved CTC isolation scheme for pairing with downstream genomics: Demonstrating clinical utility in metastatic prostate, lung and pancreatic cancer.

Improvements in technologies to yield purer circulating tumor cells (CTCs) will enable a broader range of clinical applications. We have previously demonstrated the use of a commercially available cell-adhesion matrix (CAM) assay to capture invasive CTCs (iCTCs). To improve the purity of the isolated iCTCs, here we used fluorescence-activated cell sorting (FACS) in combination with the CAM assay (CAM + FACS). Our results showed an increase of median purity from the CAM assay to CAM + FACS for the spiked-in cell lines and patient samples analyzed from three different metastatic cancer types: castration resistant prostate cancer (mCRPC), non-small cell lung cancer (mNSCLC) and pancreatic ductal adenocarcinoma cancer (mPDAC). Copy number profiles for spiked-in mCRPC cell line and mCRPC patient iCTCs were similar to expected mCRPC profiles and a matched biopsy. A somatic epidermal growth factor receptor (EGFR) mutation specific to mNSCLC was observed in the iCTCs recovered from EGFR(+) mNSCLC cell lines and patient samples. Next-generation sequencing (NGS) of spiked-in pancreatic cancer cell line and mPDAC patient iCTCs showed mPDAC common mutations. CAM + FACS iCTC enrichment enables multiple downstream genomic characterizations across different tumor types.

Phylogenetic analyses of mtDNA sequences corroborate taxonomic designations based on cuticular hydrocarbons in subterranean termites.

Cuticular hydrocarbons (CHCs) are valuable characters for the analysis of cryptic insect species with few discernible morphological characters. Yet, their use in insect systematics, specifically in subterranean termites in the genus Reticulitermes (Isoptera: Rhinotermitidae), remains controversial. In this paper, we show that taxonomic designations in Reticulitermes from California (USA) suggested in light of differences among CHC phenotypes are corroborated by phylogenetic analyses using mtDNA sequences. Analyses based on CHC phenotypes and supported, in part, by behavioral and ecological differences have suggested the presence of more species than the two currently recognized: R. hesperus Banks and R. tibialis Banks. We analyze a 680 base pair fragment of the mitochondrial DNA cytochrome oxidase (COII) gene from 45 new (21 collection localities) and two previously recorded samples of Reticulitermes from California using parsimony and maximum likelihood methods. Both methods result in trees with highly similar topologies. Bootstrapping indicates support for six clades of Reticulitermes, and corroborates groupings based on cuticular hydrocarbons. One of the clades, R. hesperus, is already recognized in California, while four clades appear to be previously undescribed taxa. Although identification of the final clade is inconclusive, it includes a sample putatively identified as R. tibialis. Therefore, using phylogenetic analyses we corroborate chemical characters used to identify taxa, associate a chemical phenotype with a previously described species, and provide additional support for undescribed taxa of Reticulitermes.