Linkage of low-density lipoprotein size to the lipoprotein lipase gene in heterozygous lipoprotein lipase deficiency.

Small low-density lipoprotein (LDL) particles are a genetically influenced coronary disease risk factor. Lipoprotein lipase (LpL) is a rate-limiting enzyme in the formation of LDL particles. The current study examined genetic linkage of LDL particle size to the LpL gene in five families with structural mutations in the LpL gene. LDL particle size was smaller among the heterozygous subjects, compared with controls. Among heterozygous subjects, 44% were classified as affected by LDL subclass phenotype B, compared with 8% of normal family members. Plasma triglyceride levels were significantly higher, and high-density lipoprotein cholesterol (HDL-C) levels were lower, in heterozygous subjects, compared with normal subjects, after age and sex adjustment. A highly significant LOD score of 6.24 at straight theta=0 was obtained for linkage of LDL particle size to the LpL gene, after adjustment of LDL particle size for within-genotype variance resulting from triglyceride and HDL-C. Failure to adjust for this variance led to only a modest positive LOD score of 1.54 at straight theta=0. Classifying small LDL particles as a qualitative trait (LDL subclass phenotype B) provided only suggestive evidence for linkage to the LpL gene (LOD=1. 65 at straight theta=0). Thus, use of the quantitative trait adjusted for within-genotype variance, resulting from physiologic covariates, was crucial for detection of significant evidence of linkage in this study. These results indicate that heterozygous LpL deficiency may be one cause of small LDL particles and may provide a potential mechanism for the increase in coronary disease seen in heterozygous LpL deficiency. This study also demonstrates a successful strategy of genotypic specific adjustment of complex traits in mapping a quantitative trait locus.     

Expression of lipoprotein lipase gene in combined lipase deficiency

The expression of the gene for lipoprotein lipase (LPL) was studied in brown adipose tissue and the liver of combined lipase deficient (cld/cld) and unaffected mice. The mRNA specific for LPL was detected in both animals. Although the size of LPL mRNA in cld mice was similar to that of unaffected mice, the mRNA concentration in affected animals was higher than in unaffected animals. We also studied the LPL gene mutation in cld mice by Southern blot analysis. No restriction fragment length polymorphisms were observed after digestion with 16 endonucleases. These data indicate that there is no gene insertion or deletion, but do not exclude the possibility of point mutation in the LPL structural gene. However, the present results agree with the hypothesis that the genetic defect in cld is not due to a mutation in the LPL structural gene, but instead involves the defective post-translational processing of LPL or defective cellular function affecting transport and secretion of this enzyme group.     

Lipoprotein lipase deficiency resulting from a nonsense mutation in exon 3 of the lipoprotein lipase gene.

The gene for erythroid 5-aminolevulinate synthase has been mapped to Xpter-Xq26 by Southern blot hybridization analysis of a mouse/human hybrid cell panel. In situ hybridization maps the gene to Xp21-Xq21, with the most likely location being on band Xp11.2. The mapping of the erythroid 5-amino-levulinate synthase gene to the X chromosome suggests that a defect in this gene may be the primary cause of X-linked sideroblastic anemia.     

A missense (Asp250----Asn) mutation in the lipoprotein lipase gene in two unrelated families with familial lipoprotein lipase deficiency.

We have identified the molecular basis for familial lipoprotein lipase (LPL) deficiency in two unrelated families with the syndrome of familial hyperchylomicronemia. All 10 exons of the LPL gene were amplified from the two probands' genomic DNA by polymerase chain reaction. In family 1 of French descent, direct sequencing of the amplification products revealed that the patient was heterozygous for two missense mutations, Gly188----Glu (in exon 5) and Asp250----Asn (in exon 6). In family 2 of Italian descent, sequencing of multiple amplification products cloned in plasmids indicated that the patient was a compound heterozygote harboring two mutations, Arg243----His and Asp250----Asn, both in exon 6. Studies using polymerase chain reaction, restriction enzyme digestion (the Gly188----Glu mutation disrupts an Ava II site, the Arg243----His mutation, a Hha I site, and the Asp250----Asn mutation, a Taq I site), and allele-specific oligonucleotide hybridization confirmed that the patients were indeed compound heterozygous for the respective mutations. LPL constructs carrying the three mutations were expressed individually in Cos cells. All three mutant LPLs were synthesized and secreted efficiently; one (Asp250----Asn) had minimal (approximately 5%) catalytic activity and the other two were totally inactive. The three mutations occurred in highly conserved regions of the LPL gene. The fact that the newly identified Asp250----Asn mutation produced an almost totally inactive LPL and the location of this residue with respect to the three-dimensional structure of the highly homologous human pancreatic lipase suggest that Asp250 may be involved in a charge interaction with an alpha-helix in the amino terminal region of LPL. The occurrence of this mutation in two unrelated families of different ancestries (French and Italian) indicates either two independent mutational events affecting unrelated individuals or a common shared ancestral allele. Screening for the Asp250----Asn mutation should be included in future genetic epidemiology studies on LPL deficiency and familial combined hyperlipidemia.     

Molecular genetics of human lipoprotein lipase deficiency

Lipoprotein lipase (LPL) hydrolysis the triglyceride core of circulating chylomicrons and very-low-density lipoprotein, and modulates the levels and lipid composition of low and high density lipoproteins. Worldwide, more than 20 mutations in the LPL gene have been identified in patients with familial LPL deficiency. Most of these mutations are clustered in the region encoded by exons 4, 5 and 6 which forms the proposed catalytic domain of LPL. In French Canadians who have the highest reported frequency for LPL deficiency, three common mutations in the LPL gene have been identified which account for approximately 97% of mutant genes in this group. Simple DNA-based tests for the detection of all these mutations have been developed for the screening for carriers of LPL deficiency. This will facilitate further studies of phenotypic expression in heterozygous carriers and assessment of the risk of atherosclerosis in these individuals.     

Compound heterozygote for lipoprotein lipase deficiency: Ser----Thr244 and transition in 3'' splice site of intron 2 (AG----AA) in the lipoprotein lipase gene.

Cloning and sequencing of translated exons and intron-exon boundaries of the lipoprotein lipase gene in a patient of French descent who has the chylomicronemia syndrome revealed that he was a compound heterozygote for two nucleotide substitutions. One (TCC----ACC) leads to an amino acid substitution (Ser----Thr244), while the other alters the 3' splice site of intron 2 (AG----AA). The functional significance of the Thr244 amino acid substitution was established by in vitro expression in cultured mammalian cells.     

Trp64----nonsense mutation in the lipoprotein lipase gene.

A lipoprotein lipase (LpL) gene defect has been identified, a G----A transition at nucleotide position 446 of exon 3, resulting in a premature termination codon (Trp----stop) at amino acid residue 64. This defect was identified in a Type I hyperlipoproteinemic subject with an amino acid residue 194 defect in the other allele. Plasma lipoprotein values as well as LpL mass and activity in postheparin plasma were determined in the subjects with the residue 64 defect and in other LpL-deficient heterozygotes. LpL mass levels in both the Type I and the other subject with a 64 LpL defect were markedly reduced. This may be explained by rapid degradation of LpL protein or decreased secretion from the 64 defective allele. Alternatively, the marked reduction or absence of mass associated with the 64 defect may be due to synthesis of a severely truncated protein which escapes immunologic detection.     

脂蛋白脂酶基因的研究进展

脂蛋白脂酶(lipoprotein lipase, LPL)是脂质代谢的关键酶, 主要催化乳糜微粒和极低密度脂蛋白中的甘油三酯水解, 产生供组织利用的脂肪酸和单酰甘油。LPL基因突变影响LPL活性, 导致脂质代谢紊乱, 与2型糖尿病、高血压、动脉硬化、肥胖、冠心病的发病风险相关联。文章综述了LPL基因的结构、功能、表达调控以及与复杂疾病的关联研究进展。     

Ferrocenylnaphthalene diimide-based electrochemical hybridization assay for a heterozygous deficiency of the lipoprotein lipase gene

An electrochemical hybridization assay has been devised that enables the rapid analysis of a heterozygous deficiency of the human lipoprotein lipase (LPL) gene. PCR products of 350 base pairs (bp) containing the wild-type sequence, a mutated G(818) --> A transition or a G(916) deletion of the LPL gene were subjected to hybridization with a probe DNA of 13 or 15 bases that represented either the wild-type or the mutated sequence immobilized on a gold electrode. The differential pulse voltammetry of the electrode before and after hybridization was determined in the presence of ferrocenylnaphthalene diimide (FND) at 460 mV. The measured change in peak current, Deltai, was defined by (i - i(o))/i(o) x 100%, where i(o) and i represent the current before and after hybridization, respectively. Matched combinations of sample and probe gave Deltai values of 40-90%, whereas mismatched combinations gave values of 20-35%, enabling the discrimination of matched hybrids from mismatched ones across a slim margin. Because the heterozygote contains both the wild-type and mutated sequences, however, it alone gives large Deltai values with both the wild- and mutant-type probes. This system was validated on 10 unknown samples of each of the two types of LPL mutation, which were correctly identified in every case.     

Complete paternal isodisomy for chromosome 8 unmasked by lipoprotein lipase deficiency.

Uniparental disomy (UPD)-the inheritance of two homologous chromosomes from a single parent-may be unmasked in humans by the unexpected appearance of developmental abnormalities, genetic disorders resulting from genomic imprinting, or recessive traits. Here we report a female patient with familial chylomicronemia resulting from complete lipoprotein-lipase (LPL) deficiency due to homozygosity for a frameshift mutation in exon 2 of the LPL gene. She was the normal term product of an unremarkable pregnancy and had shown normal development until her current age of 5.5 years. The father (age 33 years) and the mother (age 24 years) were unrelated and healthy, with no family history of stillbirths or malformations. The father was a heterozygous carrier of the mutation, whereas no mutation in the LPL gene was detected in the mother. Southern blotting did not reveal any LPL gene rearrangement in the proband or her parents. The proband was homozygous for 17 informative markers spanning both arms of chromosome 8 and specifically for the haplotype containing the paternally derived LPL gene. This shows that homozygosity for the defective mutation in the LPL gene resulted from a complete paternal isodisomy for chromosome 8. This is the first report of UPD for chromosome 8 unmasked by LPL deficiency and suggests that normal development can occur with two paternally derived copies of human chromosome 8.     

Partial gene duplication and posterior pituitary peptide

We have compiled the dipeptide frequencies in 100 known protein sequences. We suggest that dipeptides which occur with low frequencies can be used to locate proteins where partial gene duplication may have taken place. The 48 residue sequence of posterior pituitary peptide contains two Cys Trp pairs. The adjacent portions of the sequence are compatible with a partial gene duplication in the evolutionary history of posterior pituitary peptide.     

Alterations in erythrocyte membrane lipid composition and fluidity in primary lipoprotein lipase deficiency.

Lipid composition of plasma lipoproteins and erythrocyte ghost membranes has been studied in 16 healthy normolipidaemic subjects and in 16 patients affected by primary lipoprotein lipase deficiency, resulting in severe chylomicronaemia and in cholesterol-depleted low-density lipoproteins and high-density lipoproteins. A significant decrease in membrane cholesterol/phospholipid ratio was observed in lipoprotein lipase deficient patients compared to controls (3.27 +/- 0.33 vs. 3.95 +/- 0.50, mean +/- S.D.; P less than 0.0001). There was also an increase in the erythrocyte membrane phosphatidylcholine/sphingomyelin ratio in lipoprotein lipase deficient patients compared to controls (1.53 +/- 0.10 vs. 1.05 +/- 0.13; P less than 0.0001) due to a concurrent increase in phosphatidylcholine and decrease in sphingomyelin relative concentrations in these patients. Erythrocyte ghost membrane fluidity was determined by fluorescence anisotropy and found to be higher in membranes from lipoprotein lipase deficient patients. This increase in membrane fluidity can be attributed in part to changes in membrane cholesterol and phospholipid concentrations in response to abnormal plasma lipoprotein composition.     

Differential effect of combined lipase deficiency (cld/cld) on human hepatic lipase and lipoprotein lipase secretion.

Combined lipase deficiency (cld) is a recessively inherited disorder in mice associated with a deficiency of LPL and hepatic lipase (HL) activity. LPL is synthesized in cld tissues but is retained in the endoplasmic reticulum (ER), whereas mouse HL (mHL) is secreted but inactive. In this study we investigated the effect of cld on the secretion of human HL (hHL) protein mass and activity. Differentiated liver cell lines were derived from cld mice and their normal heterozygous (het) littermates by transformation of hepatocytes with SV40 large T antigen. After transient transfection with lipase expression constructs, secretion of hLPL activity from cld cells was only 12% of that from het cells. In contrast, the rate of secretion of hHL activity and protein mass per unit of expressed hHL mRNA was identical for the two cell lines. An intermediate effect was observed for mHL, with a 46% reduction in secretion of activity from cld cells. The ER glucosidase inhibitor, castanospermine, decreased secretion of both hLPL and hHL from het cells by approximately 70%, but by only approximately 45% from cld cells. This is consistent with data suggesting that cld may result from a reduced concentration of the ER chaperone calnexin. In conclusion, our results demonstrate a differential effect of cld on hLPL, mHL, and hHL secretion, suggesting differential requirements for activation and exit of the enzymes from the ER.     

Functional variants in the lipoprotein lipase gene and risk cardiovascular disease.

The current report is a quantitative review of the relationship between lipoprotein lipase gene variants and cardiovascular disease based on published population-based studies. Sixteen studies, representing 17,630 individuals, report allelic distribution for lipoprotein lipase gene variants among patients and control individuals. Patient outcomes included clinical cardiovascular disease events, documented coronary disease based on angiography, or intimal media thickening by B-mode ultrasonography. Mantel-Haenszel stratified analysis was used to compute a summary odds ratio and 95% confidence intervals for the association between rare allele in the lipoprotein lipase gene and disease status. Because of potential differing effects associated with different lipoprotein lipase variants, each lipoprotein lipase mutant allele was considered separately. The lipoprotein lipase D9N/-93G to T allele has a summary odds ratio of 2.03 (95% confidence interval 1.30-3.18), indicating a twofold increase in risk of coronary disease for carriers with this allelic variant. The summary odds ratio for the relationship of the rare lipoprotein lipase G188E variant with cardiovascular disease is 5.25 (95% confidence interval 1.54-24.29). The lipoprotein lipase N291S allele is associated with a marginal increase in cardiovascular disease (summary odds ratio 1.25, 95% confidence interval 0.99-1.60, P = 0.07). However, there is stronger evidence for a positive association in certain populations. The summary odds ratio for lipoprotein lipase S447X allele is 0.81 (95% confidence interval 0.65-1.0), which indicates a cardioprotective effect of this lipoprotein lipase gene variant. Thus, lipoprotein lipase gene variants are associated with differential susceptibility to cardiovascular disease.