CFTR mutation analysis and haplotype associations in CF patients.

Most newborn screening (NBS) laboratories use second-tier molecular tests for cystic fibrosis (CF) using dried blood spots (DBS). The Centers for Disease Control and Prevention's NBS Quality Assurance Program offers proficiency testing (PT) in DBS for CF transmembrane conductance regulator (CFTR) gene mutation detection. Extensive molecular characterization on 76 CF patients, family members or screen positive newborns was performed for quality assurance. The coding, regulatory regions and portions of all introns were sequenced and large insertions/deletions were characterized as well as two intronic di-nucleotide microsatellites. For CF patient samples, at least two mutations were identified/verified and four specimens contained three likely CF-associated mutations. Thirty-four sequence variations in 152 chromosomes were identified, five of which were not previously reported. Twenty-seven of these variants were used to predict haplotypes from the major haplotype block defined by HapMap data that spans the promoter through intron 19. Chromosomes containing the F508del (p.Phe508del), G542X (p.Gly542X) and N1303K (p.Asn1303Lys) mutations shared a common haplotype subgroup, consistent with a common ancient European founder. Understanding the haplotype background of CF-associated mutations in the U.S. population provides a framework for future phenotype/genotype studies and will assist in determining a likely cis/trans phase of the mutations without need for parent studies.

Emerging areas of research reported during the CDC National Conference on Pfiesteria: from biology to public health.

Since its identification in 1996, the marine dinoflagellate Pfiesteria piscicida Steidinger & Burkholder has been the focus of intense scientific inquiry in disciplines ranging from estuarine ecology to epidemiology and from molecular biology to public health. Despite these research efforts, the extent of human exposure and the degree of human illness directly associated with Pfiesteria is still in the process of being defined. Unfortunately, during this same time Pfiesteria has also stimulated media coverage that in some instances jumped ahead of the science to conclude that Pfiesteria presents a widespread threat to human health. Political and economic forces also came into play when the tourism and seafood industries were adversely impacted by rumors of toxin-laden water in estuaries along the east coast of the United States. Amid this climate of evolving science and public concern, Pfiesteria has emerged as a highly controversial public health issue. In October 2000 Centers for Disease Control and Prevention sponsored the National Conference on Pfiesteria: From Biology to Public Health to bring together Pfiesteria researchers from many disparate disciplines. The goal of this meeting was to describe the state of the science and identify directions for future research. In preparation for the conference an expert peer-review panel was commissioned to review the existing literature and identify research gaps; the summary of their review is published in this monograph. During the meeting primary Pfiesteria researchers presented previously unpublished results. The majority of those presentations are included as peer-reviewed articles in this monograph. The discussion portion of the conference focused upon researcher-identified research gaps. This article details the discussion segments of the conference and makes reference to the presentations as it describes emerging areas of Pfiesteria research.

The role of mismatch repair in the prevention of base pair mutations in Saccharomyces cerevisiae.

In most organisms, the mismatch repair (MMR) system plays an important role in substantially lowering mutation rates and blocking recombination between nonidentical sequences. In Saccharomyces cerevisiae, the products of three genes homologous to Escherichia coli mutS-MSH2, MSH3, and MSH6-function in MMR by recognizing mispaired bases. To determine the effect of MMR on single-base pair mismatches, we have measured reversion rates of specific point mutations in the CYC1 gene in both wild-type and MMR-deficient strains. The reversion rates of all of the point mutations are similar in wild-type cells. However, we find that in the absence of MSH2 or MSH6, but not MSH3, reversion rates of some mutations are increased by up to 60,000-fold, whereas reversion rates of other mutations are essentially unchanged. When cells are grown anaerobically, the reversion rates in MMR-deficient strains are decreased by as much as a factor of 60. We suggest that the high reversion rates observed in these MMR-deficient strains are caused by misincorporations opposite oxidatively damaged bases and that MMR normally prevents these mutations. We further suggest that recognition of mispairs opposite damaged bases may be a more important role for MMR in yeast than correction of errors opposite normal bases.

Selectable cassettes for simplified construction of yeast gene disruption vectors.

Cassettes based on a hisG-URA3-hisG insert have been modified by the addition of an KmR-encoding gene and flanking polylinker sites, greatly simplifying construction of gene disruption vectors in Escherichia coli. After gene disruption in yeast, URA3 can then be excised by recombination between the hisG repeats flanking the gene, permitting reuse of the URA3 marker.

Mutations in the MSH3 gene preferentially lead to deletions within tracts of simple repetitive DNA in Saccharomyces cerevisiae.

Eukaryotic genomes contain tracts of DNA in which a single base or a small number of bases are repeated (microsatellites). Mutations in the yeast DNA mismatch repair genes MSH2, PMS1, and MLH1 increase the frequency of mutations for normal DNA sequences and destabilize microsatellites. Mutations of human homologs of MSH2, PMS1, and MLH1 also cause microsatellite instability and result in certain types of cancer. We find that a mutation in the yeast gene MSH3 that does not substantially affect the rate of spontaneous mutations at several loci increases microsatellite instability about 40-fold, preferentially causing deletions. We suggest that MSH3 has different substrate specificities than the other mismatch repair proteins and that the human MSH3 homolog (MRP1) may be mutated in some tumors with microsatellite instability.