Polymorphisms near TBX5 and GDF7 are associated with increased risk for Barrett's esophagus.

BACKGROUND & AIMS: Barrett's esophagus (BE) increases the risk of esophageal adenocarcinoma (EAC). We found the risk to be BE has been associated with single nucleotide polymorphisms (SNPs) on chromosome 6p21 (within the HLA region) and on 16q23, where the closest protein-coding gene is FOXF1. Subsequently, the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON) identified risk loci for BE and esophageal adenocarcinoma near CRTC1 and BARX1, and within 100 kb of FOXP1. We aimed to identify further SNPs that increased BE risk and to validate previously reported associations. METHODS: We performed a genome-wide association study (GWAS) to identify variants associated with BE and further analyzed promising variants identified by BEACON by genotyping 10,158 patients with BE and 21,062 controls. RESULTS: We identified 2 SNPs not previously associated with BE: rs3072 (2p24.1; odds ratio [OR] = 1.14; 95% CI: 1.09-1.18; P = 1.8 × 10(-11)) and rs2701108 (12q24.21; OR = 0.90; 95% CI: 0.86-0.93; P = 7.5 × 10(-9)). The closest protein-coding genes were respectively GDF7 (rs3072), which encodes a ligand in the bone morphogenetic protein pathway, and TBX5 (rs2701108), which encodes a transcription factor that regulates esophageal and cardiac development. Our data also supported in BE cases 3 risk SNPs identified by BEACON (rs2687201, rs11789015, and rs10423674). Meta-analysis of all data identified another SNP associated with BE and esophageal adenocarcinoma: rs3784262, within ALDH1A2 (OR = 0.90; 95% CI: 0.87-0.93; P = 3.72 × 10(-9)). CONCLUSIONS: We identified 2 loci associated with risk of BE and provided data to support a further locus. The genes we found to be associated with risk for BE encode transcription factors involved in thoracic, diaphragmatic, and esophageal development or proteins involved in the inflammatory response

Common variants at the MHC locus and at chromosome 16q24.1 predispose to Barrett's esophagus

Barrett’s Esophagus is an increasingly common disease that is strongly associated with reflux of stomach acid and usually a hiatus hernia. Barrett’s Esophagus strongly predisposes to esophageal adenocarcinoma (EAC), a tumour with a very poor prognosis. We have undertaken the first genome-wide association study on Barrett’s Esophagus, comprising 1,852 UK cases and 5,172 UK controls in discovery and 5,986 cases and 12,825 controls in the replication. Two regions were associated with disease risk: chromosome 6p21, rs9257809 (Pcombined=4.09×10−9, OR(95%CI) =1.21(1.13-1.28)) and chromosome 16q24, rs9936833 (Pcombined=2.74×10−10, OR(95%CI) =1.14(1.10-1.19)). The top SNP on chromosome 6p21 is within the major histocompatibility complex, and the closest protein-coding gene to rs9936833 on chromosome 16q24 is FOXF1, which is implicated in esophageal development and structure. We found evidence that the genetic component of Barrett’s Esophagus is mediated by many common variants of small effect and that SNP alleles predisposing to obesity also increase risk for Barrett’s Esophagus

Isolation, characterization and expression of cDNAs encoding human and marmoset cytochrome P450's

The expression of individual cytochromes P450 (P450s) has become a valuable tool for studying the structure- function relationship of these proteins and their metabolic capacities. A full-length cDNA encoding human CYP2A6 was expressed both in the baculovirus/Sf9 insect cell system and as a fusion protein with the maltose binding protein (MBP) in Escherichia coli ( E. coli) cells. The expressed proteins were detected by SDS-PAGE and western blotting. MBP-CYP2A6 fusion protein was located in the cell membrane fraction of E. coli cells. In Sf9 insect cells transfected with a recombinant baculovirus, CYP2A6 was located in the microsomal membranes. MBP-CYP2A6 fusion protein expressed in E. coli was functionally inactive towards the CYP2A6 substrate, coumarin. Although the degradation of MBP-CYP2A6 was not detected by western blot analysis, spectral analysis showed a strong P420 component suggesting misfolding of the polypeptide due to the interaction with the MBP domain. Attempts to purify MBP-CYP2A6 from E. coli were not successful. Baculovirus expressed CYP2A6 was found to be enzymatically active towards the metabolism of coumarin but not testosterone. Endogenous NADPH-cytochrome P450 reductase in Sf9 cells did interact with the expressed CYP2A6. However, the amounts of this protein were not sufficient for the amount of CYP2A6 expressed and catalytic studies required the addition of exogenous NADPH-cytochrome P450 reductase to obtain maximum CYP2A6 activity. CYP2A6 was purified from Sf9 cells by affinity chromatography. A cDNA encoding a CYP2A was isolated from marmoset liver total RNA by reverse transcription and PCR. When compared to the sequence of CYP2A6, marmoset CYP2A cDNA contained a deletion of a nucleotide C after the initiation codon which changed the reading frame. Although not useful for heterologous studies, the cDNA was used as a probe for northern blot analysis of marmoset liver total RNA isolated from the livers of untreated or phenobarbital treated animals. CYP2A mRNA was induced 20-fold on treatment of marmoset with phenobarbital

Clustering of Genetically Defined Allele Classes in the Caenorhabditis elegans DAF-2 Insulin/IGF-1 Receptor

The DAF-2 insulin/IGF-1 receptor regulates development, metabolism, and aging in the nematode Caenorhabditis elegans. However, complex differences among daf-2 alleles complicate analysis of this gene. We have employed epistasis analysis, transcript profile analysis, mutant sequence analysis, and homology modeling of mutant receptors to understand this complexity. We define an allelic series of nonconditional daf-2 mutants, including nonsense and deletion alleles, and a putative null allele, m65. The most severe daf-2 alleles show incomplete suppression by daf-18(0) and daf-16(0) and have a range of effects on early development. Among weaker daf-2 alleles there exist distinct mutant classes that differ in epistatic interactions with mutations in other genes. Mutant sequence analysis (including 11 newly sequenced alleles) reveals that class 1 mutant lesions lie only in certain extracellular regions of the receptor, while class 2 (pleiotropic) and nonconditional missense mutants have lesions only in the ligand-binding pocket of the receptor ectodomain or the tyrosine kinase domain. Effects of equivalent mutations on the human insulin receptor suggest an altered balance of intracellular signaling in class 2 alleles. These studies consolidate and extend our understanding of the complex genetics of daf-2 and its underlying molecular biology

LET-60 RAS modulates effects of insulin/IGF-1 signaling on development and aging in Caenorhabditis elegans

The DAF-2 insulin/insulin-like growth factor 1 (IGF-1) receptor signals via a phosphatidylinositol 3-kinase (PI3K) pathway to control dauer larva formation and adult longevity in Caenorhabditis elegans. Yet epistasis analysis suggests signal bifurcation downstream of DAF-2. We have used epistasis analysis to test whether the Ras pathway (which plays a role in signaling from mammalian insulin receptors) acts downstream of DAF-2. We find that an activated Ras mutation, let-60(n1046gf), weakly suppresses constitutive dauer diapause in daf-2 and age-1 (PI3K) mutants. Moreover, increased Ras pathway signaling partially suppresses the daf-2 mutant feeding defect, while reduced Ras pathway signaling enhances it. By contrast, activated Ras extends the longevity induced by mutation of daf-2, while reduced Ras pathway signaling partially suppresses it. Thus, Ras pathway signaling appears to act with insulin/IGF-1 signaling during larval development, but against it during aging