Publications of the Genome Center

1. P. O'Connell, R. J. Leach, D. Rains, T. Taylor, D. Garcia, L. Ballard, P. Holik, J. Weissenbach, S. Sherman, P. Wilkie, J. L. Weber, S. L. Naylor. A PCR-Based Genetic Map for Human Chromosome 3. Genomics, 24 , 557-567, 1994.

   Oligonucleotide primers for 125 simple sequence repeat microsatellite-based genetic markers have been assayed by polymerase chain reaction (PCR) in the CEPH reference family panel. These microsatellites include 101 di-nucleotide repeats as well as 24 new tetra-nucleotide repeats. The average heterozygosity of this marker set was 72. 4%. Genetic data were analyzed with the genetic mapping package LINKAGE. A subset of these microsatellite markers define a set of 56 uniquely ordered loci (>1000:1 against local inversion) that span 271 cM. Sixty-seven additional loci were tightly linked to markers on the uniquely ordered map, but could not be ordered with such high precision. These markers were positioned by CMAP into confidence intervals. One hundred-thirteen of the microsatellite markers were also tested on a chromosome 3 framework somatic cell hybrid panel that divides this chromosome into 23 cytogentically defined regions, integrating the genetic and physical maps of this chromosome. The high density, high heterozygosity and PCR format of this genetically and physically mapped set of markers will accelerate the mapping and positional cloning of new chromosome 3 genes.

2. V. Pekkel, M.C. Schmitz, Y.-Y. Yu, C. K Osborne, D.C. Allred, P. O'Connell. DNA isolation and pre-amplification from microdissected paraffin-embedded tissues for loss of heterozygosity studies. Submited to Biotechnique.

   We are studying loss of heterozygosity precursor lesions of invasive breast cancer (i.e. typical hyperplasia, atypical hyperplasia, and in situ cancer). Samples for DNA extraction are microdissected from formalin-fixed, paraffin-embedded archival tissue samples. These preparations permit efficient PCR amplification of highly polymorphic microsatellite (simple sequence repeat) polymorphisms for genetic analysis. However, the variable yields of DNA from particularly small lesions has made extensive testing of these samples difficult. In order to equalize the recovery of DNA from the various morphologically defined lesions and normal tissue, we have implemented the method of primer-extension pre-amplification (PEP). To demonstrate the fidelity of the PEP technique in this application, we selected a series of 23 normal/lesion DNA sets from formalin-fixed, paraffin-embedded tissues. These samples were analyzed for loss of heterozygosity by direct PCR amplification with highly polymorphic microsatellite markers before and after PEP to determine whether the effects of pre- amplification of the genetic characteristics of the samples. For the eight microsatellite loci tested, nearly all of the samples amplified and showed the same patterns of LOH before and after PEP.

3. Peter O'Connell, Vladimir Pekkel, Suzanne A. W. Fuqua, C. Kent Osborne, and D. Craig Allred. Molecular genetic studies of early breast cancer evolution. Breast Cancer Research and Treatment, 32: 5-12, 1994.

   In the past few years there has been an explosion in the number of patients diagnosed with hyperplastic breast disease and in situ breast cancer. Based on epidemiological data, these morphologically defined lesions may be categorized as those with little malignant potential (e.g. typical hyperplasia or proliferative disease without atypia [PDWA]), those with significant malignant potential which may already be "initiated" (e.g. atypical ductal hyperplasia [ADH]), and early "transformed" lesions which are malignant but not yet invasive (e.g. ductal carcinoma in situ [DCIS]). They may represent sequential evolutionary stages in the ontogeny of invasive breast cancer, with each morphologically defined stage resulting from accumulating genetic changes culminating in a transformed clonal lineage capable of invasion and metastasis. Using loss-of- heterozygosity (LOH) analysis, we are studying the genetic changes associated with these lesions in archival tissue samples.

4. Braxton D. Mitchell⁽¹⁾, Candace M. Kammerer⁽¹⁾, Peter O'Connell⁽³⁾, Chantal R. Harrison⁽³⁾, Madelene Manire⁽⁴⁾, Patricia Shipman⁽³⁾, Mary Pat Moyer⁽⁵⁾, Michael P. Stern⁽²⁾, and Marsha L. Frazier⁽⁴⁾ Evidence for Linkage of Post-Challenge Insulin Levels with Intestinal Fatty Acid Binding Protein (FABP2) in Mexican Americans DIABETES, vol.44, pp.1046-1053, 1995.

   From the:
   Department of Genetics(1) Southwest Foundation for Biomedical Research P.O. Box 28147 San Antonio, TX 78228-0147	Departments of Medicine/Epidemiology(2), Pathology(3), and Surgery(5) University of Texas Health Science Center 7703 Floyd Curl Drive San Antonio, TX 78284	Department of Gastrointestinal Medical Oncology and Digestive Diseases(4) University of Texas MD Anderson Hospital 1515 Holcomb Blvd. Houston, TX 77030
   Address correspondence to:
   Braxton D. Mitchell, Ph.D. Department of Genetics Southwest Foundation for Biomedical Research P.O. Box 28147, San Antonio, TX 78228-0147 phone: (210) 674-1410 ext. 471 FAX: (210) 670-3316

   Key words: insulin, linkage analysis, segregation analysis, FABP2, Mexican Americans

   Running title: Linkage of FABP2 with 2-hr insulin

   ABSTRACT

   Single genes with large effects may contribute to insulin resistance or influence susceptibility to noninsulin-dependent diabetes mellitus (NIDDM). In the Pima Indians, results from sib-pair analysis have suggested that a gene on chromosome 4q influences both fasting insulin levels and maximal insulin action. We conducted sib-pair and lod-score linkage analysis to seek evidence for linkage between genes influencing insulin levels and chromosome 4q loci. Analyses were conducted on nondiabetic individuals from 28 different families participating in the San Antonio Family Diabetes Study. All subjects received a 2-hr oral glucose tolerance test. Fasting insulin levels were measured on 382 nondiabetic individuals and 2-hr insulin levels on 366 individuals. Initial sib-pair linkage analysis revealed a possible association between 2-hr post-glucose challenge insulin levels and the intestinal fatty acid binding protein (FABP2) locus located in the region of chromosome 4q28-31 (p=0.006). Subsequent sib-pair linkage analysis of eleven additional chromosome 4q markers supported this hypothesis. We next conducted segregation analyses to estimate allele frequencies and other model parameters for the putative locus influencing 2-hr insulin levels. Results of lod-score linkage analysis indicated possible linkage between the major gene described by the segregation model and FABP2. Using combined segregation and linkage analysis, we obtained a lod-score of 2.80 at recombination frequency of 0.0 between FABP2 and the putative locus influencing 2-hr insulin levels. The maximum likelihood estimate of the allele associated with low insulin levels was 0.21. Individuals having one or two copies of this allele had a mean ln(2-hr insulin level) equal to 3.484 (back-transformed mean = 898.1 pmol/L), compared to 4.480 (back-transformed mean = 331.7 pmol/L) for individuals in whom this allele was absent. Approximately 32% of the total phenotyic variance in ln(2-hr insulin levels) could be attributed to this locus. These results are consistent with the hypothesis that FABP2, or a tightly linked gene, influences 2-hr insulin levels. This gene may be associated with insulin resistance.

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