Integrated detection and population-genetic analysis of SNPs and copy number variation

Dissecting the genetic basis of disease risk requires measuring all forms of genetic variation, including SNPs and copy number variants (CNVs), and is enabled by accurate maps of their locations, frequencies and population-genetic properties. We designed a hybrid genotyping array (Affymetrix SNP 6.0) to simultaneously measure 906,600 SNPs and copy number at 1.8 million genomic locations. By characterizing 270 HapMap samples, we developed a map of human CNV (at 2-kb breakpoint resolution) informed by integer genotypes for 1,320 copy number polymorphisms (CNPs) that segregate at an allele frequency >1%. More than 80% of the sequence in previously reported CNV regions fell outside our estimated CNV boundaries, indicating that large (>100 kb) CNVs affect much less of the genome than initially reported. Approximately 80% of observed copy number differences between pairs of individuals were due to common CNPs with an allele frequency >5%, and more than 99% derived from inheritance rather than new mutation. Most common, diallelic CNPs were in strong linkage disequilibrium with SNPs, and most low-frequency CNVs segregated on specific SNP haplotypes.     

A genome-wide association study identifies colorectal cancer susceptibility loci on chromosomes 10p14 and 8q23.3

To identify colorectal cancer (CRC) susceptibility alleles, we conducted a genome-wide association study. In phase 1, we genotyped 550,163 tagSNPs in 940 familial colorectal tumor cases (627 CRC, 313 high-risk adenoma) and 965 controls. In phase 2, we genotyped 42,708 selected SNPs in 2,873 CRC cases and 2,871 controls. In phase 3, we evaluated 11 SNPs showing association at P < 10(-4) in a joint analysis of phases 1 and 2 in 4,287 CRC cases and 3,743 controls. Two SNPs were taken forward to phase 4 genotyping (10,731 CRC cases and 10,961 controls from eight centers). In addition to the previously reported 8q24, 15q13 and 18q21 CRC risk loci, we identified two previously unreported associations: rs10795668, located at 10p14 (P = 2.5 x 10(-13) overall; P = 6.9 x 10(-12) replication), and rs16892766, at 8q23.3 (P = 3.3 x 10(-18) overall; P = 9.6 x 10(-17) replication), which tags a plausible causative gene, EIF3H. These data provide further evidence for the 'common-disease common-variant' model of CRC predisposition.     

Chymotrypsin C (CTRC) variants that diminish activity or secretion are associated with chronic pancreatitis

Chronic pancreatitis is a persistent inflammatory disease of the pancreas, in which the digestive protease trypsin has a fundamental pathogenetic role. Here we have analyzed the gene encoding the trypsin-degrading enzyme chymotrypsin C (CTRC) in German subjects with idiopathic or hereditary chronic pancreatitis. Two alterations in this gene, p.R254W and p.K247_R254del, were significantly overrepresented in the pancreatitis group, being present in 30 of 901 (3.3%) affected individuals but only 21 of 2,804 (0.7%) controls (odds ratio (OR) = 4.6; confidence interval (CI) = 2.6-8.0; P = 1.3 x 10(-7)). A replication study identified these two variants in 10 of 348 (2.9%) individuals with alcoholic chronic pancreatitis but only 3 of 432 (0.7%) subjects with alcoholic liver disease (OR = 4.2; CI = 1.2-15.5; P = 0.02). CTRC variants were also found in 10 of 71 (14.1%) Indian subjects with tropical pancreatitis but only 1 of 84 (1.2%) healthy controls (OR = 13.6; CI = 1.7-109.2; P = 0.0028). Functional analysis of the CTRC variants showed impaired activity and/or reduced secretion. The results indicate that loss-of-function alterations in CTRC predispose to pancreatitis by diminishing its protective trypsin-degrading activity.     

Serine peptidases: Classification, structure and function

Serine peptidases play key roles in human health and disease and their biochemical properties shaped the molecular evolution of these processes. Of known proteolytic enzymes, the serine peptidase family is the major cornerstone of the vertebrate degradome. We describe the known diversity of serine peptidases with respect to structure and function. Particular emphasis is placed on the S1 peptidase family, the trypsins, which underwent the most predominant genetic expansion yielding the enzymes responsible for vital processes in man such as digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis and immunity. Received 13 December 2007; received after revision 8 January 2008; accepted 22 January 2008     

Recombinant scorpine: a multifunctional antimicrobial peptide with activity against different pathogens

Scorpine is an antimicrobial peptide whose structure resembles a hybrid between a defensin and a cecropin. It exhibits antibacterial activity and inhibits the sporogonic development of parasites responsible for murine malaria. In this communication we report the production of scorpine in a heterelogous system, using a specific vector containing its cloned gene. The recombinantly expressed scorpine (RScp) in ^{Anopheles gambie} cells showed antibacterial activity against ^{Bacillus subtilis} and ^{Klebsiella pneumoniae}, at 5 and 10 μM, respectively. It also produced 98% mortality in sexual stages of ^{Plasmodium berghei} at 15 μM and 100% reduction in ^{Plasmodium falciparum} parasitemia at 5 μM. RScp also inhibited virus dengue-2 replication in C6/36 mosquito cells. In addition, we generated viable and fertile transgenic ^Drosophila that overexpresses and correctly secretes RScp into the insect hemolymph, suggesting that the generation of transgenic mosquitoes resistant to different pathogens may be viable. Received 6 May 2008; received after revision 24 July 2008; accepted 29 July 2008     

TGFβ-induced protein mediates lymphatic endothelial cell adhesion to the extracellular matrix under low oxygen conditions

TGFbeta-induced protein (TGFBI) is an extracellular protein that mediates cell adhesion to collagen, laminin and fibronectin through its interaction with different beta integrins. We had previously reported that hypoxia-induced TGFBI mRNA expression in lymphatic endothelial cells (LEC). Here, we demonstrate that TGFBI can contribute to hypoxia-induced increases in LEC adhesion to the ECM. We show that while there are no changes in alpha1, alpha4, alphav, beta1, beta2, beta3, alpha5beta1, alphavbeta3, alphavbeta5 integrin expression on the LEC surface after hypoxia exposure, there exists an accumulation of TGFBI adaptor protein in LEC supernatants. We also demonstrate that hypoxia driven TGBFI expression is dependent on TGFbeta production by LEC. Furthermore, we show that TGFBI mediated LEC adhesion and migration through the ECM by its binding to the beta3 integrin. The identification of the specific mechanisms regulating LEC-ECM interactions may help us design new therapeutic applications for diseases in which lymphatic vessel function is compromised.     

Mutations in the CEL VNTR cause a syndrome of diabetes and pancreatic exocrine dysfunction

Dysfunction of the exocrine pancreas is observed in diabetes, but links between concurrent exocrine and endocrine pancreatic disease and contributing genetic factors are poorly characterized. We studied two families with diabetes and exocrine pancreatic dysfunction by genetic, physiological and in vitro functional studies. A genome-wide screen in Family 1 linked diabetes to chromosome 9q34 (maximal lod score 5.07). Using fecal elastase deficiency as a marker of exocrine pancreatic dysfunction refined the critical chromosomal region to 1.16 Mb (maximal lod score 11.6). Here, we identified a single-base deletion in the variable number of tandem repeats (VNTR)-containing exon 11 of the carboxyl ester lipase (CEL) gene, a major component of pancreatic juice and responsible for the duodenal hydrolysis of cholesterol esters. Screening subjects with maturity-onset diabetes of the young identified Family 2, with another single-base deletion in CEL and a similar phenotype with beta-cell failure and pancreatic exocrine disease. The in vitro catalytic activities of wild-type and mutant CEL protein were comparable. The mutant enzyme was, however, less stable and secreted at a lower rate. Furthermore, we found some evidence for an association between common insertions in the CEL VNTR and exocrine dysfunction in a group of 182 unrelated subjects with diabetes (odds ratio 4.2 (1.6, 11.5)). Our findings link diabetes to the disrupted function of a lipase in the pancreatic acinar cells.     

Identification of the gene responsible for methylmalonic aciduria and homocystinuria, cblC type

Methylmalonic aciduria and homocystinuria, cblC type (OMIM 277400), is the most common inborn error of vitamin B(12) (cobalamin) metabolism, with about 250 known cases. Affected individuals have developmental, hematological, neurological, metabolic, ophthalmologic and dermatologic clinical findings. Although considered a disease of infancy or childhood, some individuals develop symptoms in adulthood. The cblC locus was mapped to chromosome region 1p by linkage analysis. We refined the chromosomal interval using homozygosity mapping and haplotype analyses and identified the MMACHC gene. In 204 individuals, 42 different mutations were identified, many consistent with a loss of function of the protein product. One mutation, 271dupA, accounted for 40% of all disease alleles. Transduction of wild-type MMACHC into immortalized cblC fibroblast cell lines corrected the cellular phenotype. Molecular modeling predicts that the C-terminal region of the gene product folds similarly to TonB, a bacterial protein involved in energy transduction for cobalamin uptake.