The MLH1 D132H variant is associated with susceptibility to sporadic colorectal cancer.

Most susceptibility to colorectal cancer (CRC) is not accounted for by known risk factors. Because MLH1, MSH2 and MSH6 mutations underlie high-penetrance CRC susceptibility in hereditary nonpolyposis colon cancer (HNPCC), we hypothesized that attenuated alleles might also underlie susceptibility to sporadic CRC. We looked for gene variants associated with HNPCC in Israeli probands with familial CRC unstratified with respect to the microsatellite instability (MSI) phenotype. Association studies identified a new MLH1 variant (415G-->C, resulting in the amino acid substitution D132H) in approximately 1.3% of Israeli individuals with CRC self-described as Jewish, Christian and Muslim. MLH1 415C confers clinically significant susceptibility to CRC. In contrast to classic HNPCC, CRCs associated with MLH1 415C usually do not have the MSI defect, which is important for clinical mutation screening. Structural and functional analyses showed that the normal ATPase function of MLH1 is attenuated, but not eliminated, by the MLH1 415G-->C mutation. The new MLH1 variant confers a high risk of CRC and identifies a previously unrecognized mechanism in microsatellite-stable tumors. These studies suggest that variants of mismatch repair proteins with attenuated function may account for a higher proportion of susceptibility to sporadic microsatellite-stable CRC than previously assumed.

Characterization of herpes simplex virus type 1 thymidine kinase mutants engineered for improved ganciclovir or acyclovir activity.

Herpes Simplex Virus type 1 (HSV-1) thymidine kinase (TK) is currently the most widely used suicide agent for gene therapy of cancer. Tumor cells that express HSV-1 thymidine kinase are rendered sensitive to prodrugs due to preferential phosphorylation by this enzyme. Although ganciclovir (GCV) is the prodrug of choice for use with TK, this approach is limited in part by the toxicity of this prodrug. From a random mutagenesis library, seven thymidine kinase variants containing multiple amino acid substitutions were identified on the basis of activity towards ganciclovir and acyclovir based on negative selection in Escherichia coli. Using a novel affinity chromatography column, three mutant enzymes and the wild-type TK were purified to homogeneity and their kinetic parameters for thymidine, ganciclovir, and acyclovir determined. With ganciclovir as the substrate, one mutant (mutant SR39) demonstrated a 14-fold decrease in K(m) compared to the wild-type enzyme. The most dramatic change is displayed by mutant SR26, with a 124-fold decrease in K(m) with acyclovir as the substrate. Such new "prodrug kinases" could provide benefit to ablative gene therapy by now making it feasible to use the relatively nontoxic acyclovir at nanomolar concentrations or ganciclovir at lower, less immunosuppressive doses.

Diagnostic system for rapid and sensitive differential detection of pathogens.

Naturally emerging and deliberately released pathogens demand new detection strategies to allow early recognition and containment. We describe a diagnostic system for rapid, sensitive, multiplex discrimination of microbial gene sequences and report its application for detecting 22 respiratory pathogens in clinical samples.

Linkage disequilibrium patterns of the human genome across populations.

We studied the patterns of linkage disequilibrium (LD) in the human genome among three populations: African Americans, Caucasians and Ashkenazi Jews. These three populations represent admixed, outbred and isolated populations, respectively. The study examined defined chromosomal regions across the whole genome. We found that SNP allele frequencies are highly correlated between Ashkenazi Jews and Caucasians and somewhat distinct in African Americans. In addition, Ashkenazi Jews have a modest increase in LD compared with Caucasians, and both have greater LD than African Americans. The three populations differed more significantly with regard to haplotype heterogeneity. We found, as expected, that Ashkenazi Jews display the greatest extent of homogeneity and African Americans the greatest extent of heterogeneity. We found that most of the variance in LD can be attributed to the difference between regions and markers rather than to that between different population types. The average recombination rates estimated by low-resolution genetic maps can only explain a small fraction of the variance between regions. We found that LD (in terms of r(2)) decreases as a function of distance even within the so-called 'haplotype blocks'. This has significant consequences when using LD mapping for the genetic dissection of complex traits, as higher density SNP maps will be required to scan the genome.

Modeling bacterial evolution with comparative-genome-based marker systems: application to Mycobacterium tuberculosis evolution and pathogenesis.

The comparative-genomic sequencing of two Mycobacterium tuberculosis strains enabled us to identify single nucleotide polymorphism (SNP) markers for studies of evolution, pathogenesis, and epidemiology in clinical M. tuberculosis. Phylogenetic analysis using these "comparative-genome markers" (CGMs) produced a highly unusual phylogeny with a complete absence of secondary branches. To investigate CGM-based phylogenies, we devised computer models to simulate sequence evolution and calculate new phylogenies based on an SNP format. We found that CGMs represent a distinct class of phylogenetic markers that depend critically on the genetic distances between compared "reference strains." Properly distanced reference strains generate CGMs that accurately depict evolutionary relationships, distorted only by branch collapse. Improperly distanced reference strains generate CGMs that distort and reroot outgroups. Applying this understanding to the CGM-based phylogeny of M. tuberculosis, we found evidence to suggest that this species is highly clonal without detectable lateral gene exchange. We noted indications of evolutionary bottlenecks, including one at the level of the PHRI "C" strain previously associated with particular virulence characteristics. Our evidence also suggests that loss of IS6110 to fewer than seven elements per genome is uncommon. Finally, we present population-based evidence that KasA, an important component of mycolic acid biosynthesis, develops G312S polymorphisms under selective pressure.