Identification of a common variant in the lipoprotein lipase gene in a large Utah kindred ascertained for coronary heart disease: the -93G/D9N variant predisposes to low HDL-C/high triglycerides.

Defects in the lipoprotein lipase (LPL) gene are associated with dyslipidemia in the general population. Several rare mutations in the gene, as well as two common coding region polymorphisms, D9N and N291S, exhibit deleterious effects on circulating lipid levels. Using a linkage-based approach, we have identified a large Utah kindred segregating the D9N variant in the LPL gene. The kindred was ascertained for premature coronary heart disease and was expanded based on familial dyslipidemia. A genomic scan identified a region of linkage including LPL, and mutation screening identified the segregating variant. In the kindred, the variant shows high penetrance for a hypoalphalipoproteinemia phenotype, but is also associated with hypertriglyceridemia and elevated insulin levels. The strength of linkage was dependent on the combination of phenotype definition and model parameters, favoring the use of a MOD score approach. Most other studies of LPL have proceeded by mutation screening of randomly chosen individuals or selected affected probands; this is the first example identifying a segregating LPL mutation using direct linkage.

Evidence of linkage of familial hypoalphalipoproteinemia to a novel locus on chromosome 11q23.

Coronary heart disease (CHD) accounts for half of the 1 million deaths annually ascribed to cardiovascular disease and for almost all of the 1.5 million acute myocardial infarctions. Within families affected by early and apparently heritable CHD, dyslipidemias have a much higher prevalence than in the general population; 20%-30% of early familial CHD has been ascribed to primary hypoalphalipoproteinemia (low HDL-C). This study assesses the evidence for linkage of low HDL-C to chromosomal region 11q23 in 105 large Utah pedigrees ascertained with closely related clusters of early CHD and expanded on the basis of dyslipidemia. Linkage analysis was performed by use of 22 STRP markers in a 55-cM region of chromosome 11. Two-point analysis based on a general, dominant-phenotype model yielded LODs of 2.9 for full pedigrees and 3.5 for 167 four-generation split pedigrees. To define a localization region, model optimization was performed using the heterogeneity, multipoint LOD score (mpHLOD). This linkage defines a region on 11q23.3 that is approximately 10 cM distal to-and apparently distinct from-the ApoAI/CIII/AIV gene cluster and thus represents a putative novel localization for the low HDL-C phenotype.

Genetic localization to chromosome 1p32 of the third locus for familial hypercholesterolemia in a Utah kindred.

Clinical familial hypercholesterolemia has been shown to result from mutations in 2 genes, the low density lipoprotein (LDL) receptor on chromosome 19 and apolipoprotein B on chromosome 2. However, we have recently described a Utah pedigree in which linkage to both genes was clearly excluded. A multipoint linkage analysis of 583 markers genotyped on 31 (18 affected) members of this pedigree was undertaken to localize a genetic region that may harbor a third gene that could result in clinical familial hypercholesterolemia. A multipoint log of the odds score of 6.8 was obtained for markers on 1p32. Haplotype carriers and affected status are completely concordant (18/18 persons). The phenotype is also expressed in young children (ages 4 and 9). Specific recombinant individuals in the pedigree restrict the region of linkage to an approximately 17 cM interval between polymorphic markers D1S2130 and D1S1596. This region appears to overlap the region found linked to severe hypercholesterolemia in French and Spanish families. The identification of the gene in this region may provide important pathophysiological insights into new mechanisms that may lead to highly elevated LDL cholesterol and other associated dyslipidemic phenotypes.

A Drosophila photoreceptor cell-specific protein, calphotin, binds calcium and contains a leucine zipper.

The calphotin protein, encoded by the calphotin (cap) gene, is expressed in the soma and axons of all Drosophila photoreceptor cells. It is expressed early in photo-receptor cell development, at the time when cell-type decisions are being made. Expression of calphotin is not altered by the glass mutation, which blocks photoreceptor cell development. The calphotin protein binds calcium and contains a long C-terminal leucine zipper. Potential implications of these properties are discussed.

Targeted gene mutations in Drosophila.

A cloned gene can be of interest because of its expression in a particular tissue or at a certain developmental stage, or because of homology to an interesting gene from another organism. In Drosophila its location in the genome is readily determined by in situ hybridization to the banded larval salivary gland polytene chromosomes, but it is more difficult to isolate mutations that may reveal its function. This paper describes a general method for detecting transposable element insertions into the gene in question. This "reverse genetics" then offers the possibility of observing a consequent mutant phenotype, providing a key to the normal function of the gene. The sensitivity of the polymerase chain reaction makes it possible to detect the occurrence of a single appropriate P-element transposon insertion among a population of mutagenized flies. This is accomplished by the use of oligonucleotide primers--one a sequence from within the cloned gene and the other homologous to the terminal sequence of the P-element DNA--to prime synthesis into the DNA flanking an insertion site. A segment of DNA, bounded by the two primers, will be a target for amplification only in a fly in which a P-element has inserted within about 2 kilobases of the gene primer. This technique has been used to detect P-element insertions near a gene expressed in the Drosophila compound eye. Potential problems with the technique and possible refinements in the screen are discussed. In principle, it could be utilized to detect insertion of a foreign element into any gene for which at least a partial sequence is known and could be extended to other organisms.

Photophobe (Ppb), a Drosophila mutant with a reversed sign of phototaxis; the mutation shows an allele-specific interaction with sevenless.

We have isolated a dominant behavioral mutation, Photophobe (Ppb), on the second chromosome of Drosophila melanogaster. Although wild-type flies are attracted towards green light, flies homozygous for the Ppb mutation avoid it over an intensity range of six logarithms. Ppb interacts in a dominant way with mutations in the sevenless (sev) gene, an X-chromosomal gene necessary for photoreceptor cell 7 differentiation in the Drosophila retina. Specific alleles of sev alter the Ppb behavioral phenotype; of eight sev alleles tested, two alleles enhanced the negative phototaxis of Ppb, whereas six alleles had the opposite effect. In no mutant combination of Ppb and sev was photoreceptor cell 7 restored. These data show that the sev gene, in addition to its role in the differentiation of photoreceptor cell 7, plays a role along with Ppb in a common visual information-processing pathway.

Studies of the kinetics and ionic requirements for the phosphorylation of ribosomal protein S6 after fertilization of Arbacia punctulata eggs.

Fertilization of the eggs of the sea urchin Arbacia punctulata is followed by the phosphorylation of ribosomal protein S6. The increase in phosphorylation starts at the same time that protein synthesis begins to increase, and leads to the appearance of mono-, di-, and triphosphorylated S6 derivatives. Essentially all the S6 is phosphorylated by first cleavage. This phosphorylation requires the occurrence of both the normal Ca2+ transient and the consequent Na+-H+ exchange. Protein synthesis can be partially activated by an increase in intracellular pH brought about by weak bases, but this neither causes S6 phosphorylation, nor the inactivation of the specific S6 phosphatase present in unfertilized Arbacia eggs.

The control of protein synthesis during heat shock in Drosophila cells involves altered polypeptide elongation rates.

When Drosophila tissue culture cells are shifted from 25 to 36 degrees C (heat shocked) the pre-existing mRNAs (25 degrees C mRNAs) remain in the cytoplasm but their translation products are underrepresented relative to the induced heat shock proteins. Many of these undertranslated 25 degrees C mRNAs are found in association with polysomes of similar size in heat-shocked and control cells. Furthermore, the messages encoding alpha-tubulin, beta-tubulin, and actin are found associated with one-third to one-half as many total ribosomes in heat-shocked cells as in cells incubated at 25 degrees C. Increased temperature should lead to increased output of protein per ribosome. However, the 25 degrees C proteins are actually synthesized at less than 10% of 25 degrees C levels in heat-shocked cells. Thus, the rates of both elongation and initiation of translation are significantly (15- to 30-fold) slower on 25 degrees C mRNAs than they are on heat shock mRNAs in heat-shocked cells.