Cynthia B. Rothschild

Linkage of Genetic Markers on Human Chromosomes 20 and 12 to NIDDM in Caucasian Sib Pairs With a History of Diabetic Nephropathy

The potential contribution of maturity-onset diabetes of the young (MODY) genes to NIDDM susceptibility in African-American and Caucasian NIDDM-affected sibling pairs with a history of adult-onset diabetic nephropathy has been evaluated. Evidence for linkage to NIDDM was found with polymorphic loci that map to the long arms of human chromosomes 20 and 12 in regions containing the MODY1 and MODY3 genes. Nonparametric analysis of chromosome 20 inheritance data collected with the MODYl-linked marker D20S197 provides evidence forlinkage to NIDDM with a P value of 0.005 in Caucasian sib pairs using affected sibpair (ASP) analyses. Nonparametric analysis of chromosome 12 inheritance data collected with the MODY3-linked markers D12S349 and D12S86 provides evidence for linkage to NIDDM with P values of 0.04 and 0.006, respectively, in Caucasian sib pairs using similar analyses. No evidence for linkage of MODY1 and MODY3 markers to NIDDM in African-American sib pairs was observed. In addition, no evidence for linkage to MODY2 (glucokinase-associated MODY) was observed with either study population. Results of multipoint maximum logarithm of odds (LOD) score analysis were consistent with the ASP results. A maximum LOD score of 1.48 was calculated for linkage to MODYl-linked loci and 1.45 to MODY3-linked loci in Caucasian sib pairs. Tabulation of allele sharing in affected sib pairs with D20S197 and D12S349 suggests that affected sibling pairs may inherit susceptibility genes simultaneously from chromosome 20 and chromosome 12. The results suggest that genes contributing to NIDDM in the general Caucasian population are located in the regions containing the MODY1 and MODY3 genes.

Detection of colorectal cancer K-ras mutations using a simplified oligonucleotide ligation assay

It has been suggested that some mutations in codons 12 and 13 of the K-ras gene are associated with the progression of colorectal adenomas to carcinomas. The aim of this study was to develop a rapid, colorimetric assay for K-ras point mutations commonly associated with colorectal cancer. K-ras exon 1 was amplified from colorectal tumor DNA and K-ras activating mutations detected using an oligonucleotide ligation assay (OLA) in combination with immunological and colorimetric detection. Using the OLA with oligonucleotides specific to individual K-ras mutations, 6 (of 17 total colorectal adenomas/carcinomas) were found to have K-ras mutations. The assay could detect as little as 10% mutant allele. A simplified OLA designed to test for either the presence (+) or absence (-) of any of the K-ras activating mutations was developed. The assay was further streamlined by use of a dipstick methodology for colorimetric development. If required, assay sensitivity can be increased by the use of the recently described EDNA-ELCA detection system. The simplified (+/-) mutation OLA in combination with a dipstick or EDNA-ELCA detection system provides a rapid, sensitive assay for K-ras point mutations suitable for use as part of the clinical assessment of colorectal cancer.

Characterization of radiation/fusion hybrids containing parts of human chromosome 10 and their use in mapping chromosome 10-specific probes.

We have characterized a panel of somatic cell hybrid cell lines which contain different portions of human chromosome 10. Genomic DNA from the somatic cell hybrids was tested for hybridization with each of an ordered set of probes used previously to construct a genetic map of chromosome 10, as well as several additional probes, previously localized by in situ hybridization. Hybridization of an unmapped probe to the cell line DNAs can be used to determine its most likely position on the chromosome relative to the mapped set of probes. Genomic DNA from two of the cell lines has been used to construct region-specific cosmid and bacteriophage libraries, and clones derived from these libraries were localized by hybridization to the panel of hybrid cell lines. Several of these probes reveal restriction fragment length polymorphisms which have been genetically mapped. Three of the probes map near the locus for multiple endocrine neoplasia type 2A, and one of these probes, BG-JC353 (D10S167), maps between RBP3 and TB14.34 (D10S34). Another probe, CRI-J282 (D10S104), is close to the FNRB locus. The panel of hybrid cell lines is thus useful for rapidly localizing unmapped probes and as a source of DNA for the construction of recombinant libraries derived from specific regions of the chromosome.

D20S16 is a complex interspersed repeated sequence: genetic and physical analysis of the locus

The genomic structure of the D20S16 locus has been evaluated using genetic and physical methods. D20S16, originally detected with the probe CRI-L1214, is a highly informative, complex restriction fragment length polymorphism consisting of two separate allelic systems. The allelic systems have the characteristics of conventional VNTR polymorphisms and are separated by recombination (theta = 0.02, Zmax = 74.82), as demonstrated in family studies. Most of these recombination events are meiotic crossovers and are maternal in origin, but two, including deletion of the locus in a cell line from a CEPH family member, occur without evidence for exchange of flanking markers. DNA sequence analysis suggests that the basis of the polymorphism is variable numbers of a 98-bp sequence tandemly repeated with 87 to 90% sequence similarity between repeats. The 98-bp repeat is a dimer of 49 bp sequence with 45 to 98% identity between the elements. In addition, nonpolymorphic genomic sequences adjacent to the polymorphic 98-bp repeat tracts are also repeated but are not polymorphic, i.e., show no individual to individual variation. Restriction enzyme mapping of cosmids containing the CRI-L1214 sequence suggests that there are multiple interspersed repeats of the CRI-L1214 sequence on chromosome 20. The results of dual-color fluorescence in situ hybridization experiments with interphase nuclei are also consistent with multiple repeats of an interspersed sequence on chromosome 20.

Detection of immunoprecipitated systemic lupus erythematosus antigens by immunoblot analysis.

When antigens are isolated from staphylococcal protein A immunoprecipitation pellets for analysis by SDS polyacrylamide gel electrophoresis and immunoblotting, severe background problems, due to the presence of antisera and bacterial proteins, can result. We describe a procedure for the analysis of immunoprecipitated systemic lupus erythematosus antigens (e.g., La, Ro, and Sm) which significantly reduces this background while retaining sensitivity with respect to antigen detection. We have adapted a method previously described (MacSween, J.M. and Eastwood, S.L. Methods Enzymol. 1981; 73:459-471) in which lithium diiodosalicylate is used to separate the immunoprecipitated antigen from a covalent antibody-staphylococcal protein A complex. In addition, a modified series of immunoblot incubations was employed, in which antigenic proteins were identified by incubating blots with the antiserum used for the original immunoprecipitation (e.g., La) followed by protein A-biotin and avidin-alkaline phosphatase. Overall, the procedure is straight-forward and may be applicable to other immunoblot systems.