Publications

Small, dense LDL particles are associated with increased risk of cardiovascular disease. To identify the genes that influence LDL size variation, we performed a genome-wide screen for cholesterol concentrations in 4 LDL size fractions. Samples from 470 members of randomly ascertained families were typed for 331 microsatellite markers spaced at approximately 15 cM intervals. Plasma LDLs were resolved by using nondenaturing gradient gel electrophoresis into 4 fraction sizes (LDL-1, 26.4 to 29.0 nm; LDL-2, 25.5 to 26.4 nm; LDL-3, 24.2 to 25.5 nm; and LDL-4, 21.0 to 24.2 nm) and cholesterol concentrations were estimated by staining with Sudan Black B. Linkage analyses used variance component methods that exploited all of the genotypic and phenotypic information in the large extended pedigrees. In multipoint linkage analyses with quantitative trait loci for the 4 fraction sizes, only LDL-3, a fraction containing small LDL particles, gave peak multipoint log10 odds in favor of linkage (LOD) scores that exceeded 3.0, a nominal criterion for evidence of significant linkage. The highest LOD scores for LDL-3 were found on chromosomes 3 (LOD=4.1), 4 (LOD=4.1), and 6 (LOD=2.9). In oligogenic analyses, the 2-locus LOD score (for chromosomes 3 and 4) increased significantly (P=0.0012) to 6.1, but including the third locus on chromosome 6 did not significantly improve the LOD score (P=0.064). Thus, we have localized 2 major quantitative trait loci that influence variation in cholesterol concentrations of small LDL particles. The 2 quantitative trait loci on chromosomes 3 and 4 are located in regions that contain the genes for apoD and the large subunit of the microsomal triglyceride transfer protein, respectively.

The beta-3 adrenergic receptor (ADRB3) has been implicated as a regulator of energy expenditure, and a polymorphism in codon 64 of this gene (Trp64Arg) has been associated in some studies with obesity and insulin resistance. However, many studies have failed to detect an effect of this variant, and the importance of the Trp64Arg variant in human obesity remains controversial. We performed a quantitative linkage analysis of the ADRB3 and obesity, using 12 markers (including the intragenic Trp64Arg polymorphism) spanning a 57-cM region of chromosome 8. The study population consisted of 470 individuals from 10 large multigenerational families of Mexican-American ancestry residing in San Antonio, TX. In two-point analysis, logarithm of odds (LOD) scores >1.0 were observed for six markers surrounding ADRB3 in a 33-cM region spanned by markers D8S1477 and D8S1136. The multipoint LOD score was 3.21, occurring between markers D8S1121 and ADRB3, approximately 2-3 cM from ADRB3. Adjusting for the presence of the Arg64 allele or excluding from the analysis the 11 individuals homozygous for the Arg64 allele did not reduce the evidence for linkage. A genome scan was conducted at 10 cM map density to detect other loci influencing variation in BMI. Multipoint LOD scores >1.0 were observed in four other regions, including two on chromosome 17, one on chromosome 6q, and one on chromosome 2p. These data suggest that the ADRB3 should continue to be regarded as a strong candidate gene for obesity even though evidence for an effect of the Trp64Arg polymorphism could not be established. It is also possible that a gene closely linked to ADRB3 may influence susceptibility to obesity.

We have identified a polymorphic insertion in the lipoprotein lipase (LPL) gene in a captive baboon colony. Mapping and nucleotide (nt) sequence analysis of the polymorphism showed that it is due to the presence or absence of an Alu repetitive element in intron 7 of the baboon LPL gene. This polymorphic Alu repeat has not been reported in humans, and we did not detect the repeat in a survey of the LPL intron 7 gene region in other non-human primates. Comparison of nt at diagnostic positions in this Alu insertion with different Alu subfamily consensus sequences showed that it most closely resembles the young AluY subfamily. These data suggest that this polymorphic Alu repeat inserted independently in the baboon lineage.

We have isolated two lipoprotein lipase (LPL)-encoding cDNA (LPL) clones from a baboon cardiac cDNA library, one of which spans a region from nucleotide (nt) 705 of the coding sequence to the poly(A) tail (2.8 kb). We used reverse transcription followed by PCR (RT-PCR), and anchor-ligated rapid amplification of cDNA ends (RACE) to amplify the remaining 5' region of the LPL transcript. Sequence comparisons reveal that the baboon nt sequence is 95% identical to the human cDNA sequence (ranging from 97.5 to 92.7% in the coding and noncoding regions, respectively). Less than 2% of nt substitutions cause changes between baboon and human amino acid (aa) sequences. The aa in the catalytic triad residues, the heparin-binding site in exon 6, as well as aa in positions where missense mutations cause LPL deficiency, are identical in baboons and humans. Characterization of the tissue-specific expression of LPL using Northern blots of total RNA showed that spinal cord expressed the most LPL transcripts of all baboon tissues examined. Like humans, baboons have two transcript sizes of approx. 3.6 and 3.4 kb in most tissues that express LPL, and sequencing of the 3' untranslated region (UTR) shows this is due to two polyadenylation sites. In contrast, only the larger 3.6-kb transcript is detected in RNA isolated from central nervous system (CNS) tissues. We used RT-PCR to show that the polyadenylation signal that produces the 3.4-kb message is present in CNS LPL transcripts, but is not utilized.

We present results from an association study between RFLPs in the lipoprotein lipase (LPL) gene and lipid and insulin levels. The study population consisted of 102 Hispanic men and 97 Hispanic women. The subjects were genotyped for two previously reported RFLPs detected with the restriction enzymes HindIII and PvuII. The frequencies of the RFLPs in the Hispanic population are similar to those seen in other Caucasian populations. Strong linkage disequilibrium was detected between the sites in Hispanics. Genotypes were used separately in analyses of variance with fasting serum triglycerides, total cholesterol, high density lipoprotein (HDL)-cholesterol, low density lipoprotein (LDL)-cholesterol, HDL2/HDL3-cholesterol, and insulin levels, as well as two measures of adiposity: waist-hip ratio and body mass index. Men and women were analyzed separately. Mean fasting insulin levels of the LPL PvuII genotypes were significantly different from each other in Hispanic men. The mean fasting insulin level of men who were homozygous for the presence of the PvuII site (+/+) was 9.20 +/- 0.24 mu units/ml, men who were heterozygous had a mean level of 10.54 +/- 0.20 mu units/ml, and men who were homozygous for the absence of the site (-/-) had a mean of 12.91 +/- 0.30 mu units/ml. This effect was not seen in Hispanic women. These results suggest that the regulation of LPL by insulin may be different in Hispanics with different LPL PvuII genotypes.

Lipoprotein lipase (LPL) plays a crucial role in plasma lipoprotein processing by catalyzing the hydrolysis of core triglycerides of chylomicrons and very low density lipoproteins. Several polymorphic restriction sites have been reported in the LPL gene, including those identified by the enzymes HindIII and PvuII. We have determined the HindIII and PvuII polymorphisms in diabetic (D) and non-diabetic (ND) Hispanics (D = 195; ND = 384) and non-Hispanic Whites (D = 76; ND = 539) from the San Luis Valley, Colorado. Both polymorphisms showed comparable gene frequencies between diabetics and non-diabetics, and between the two ethnic groups. The HindIII and PvuII polymorphisms were in strong linkage disequilibrium in both Hispanics and non-Hispanic Whites (P < 0.001). We estimated whether the two DNA polymorphisms have significant impact in determining interindividual differences in plasma levels of total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, fasting glucose, and fasting insulin. Plasma triglyceride levels varied significantly among the HindIII genotypes in the normoglycemic sample. There was a clear gene dosage effect among the three HindIII genotypes, with the (-/-) genotype having the lowest and the (+/+) genotype having the highest triglyceride levels; these levels were intermediate in the (+/-) genotype. The average effect of the (-) allele of the HindIII polymorphism was to lower triglycerides by 12.85 mg/dl in non-Hispanic White males, 8.06 mg/dl in non-Hispanic White females, 10.91 mg/dl in Hispanic males, and 12.47 mg/dl in Hispanic females. The HindIII polymorphism also showed a significant association with HDL-cholesterol levels in the normoglycemic sample.(ABSTRACT TRUNCATED AT 250 WORDS)