Yuanhong Ma

A Mutation in the Human Lipoprotein Lipase Gene as the Most Common Cause of Familial Chylomicronemia in French Canadians

BACKGROUND Lipoprotein lipase hydrolyzes the triglyceride core of chylomicrons and very-low-density lipoproteins and has a crucial role in regulating plasma lipoprotein levels. Deficiencies of lipoprotein lipase activity lead to aberrations in lipoprotein levels. Worldwide, the frequency of lipoprotein lipase deficiency is highest among French Canadians. We sought to determine the molecular basis of the disorder in this population. METHODS The entire coding sequence of the lipoprotein lipase gene from one French Canadian patient was amplified by the polymerase chain reaction and sequenced. Exon 5 from 36 other French Canadian patients was amplified and analyzed by dot blot hybridization with allele-specific oligonucleotides. RESULTS Sequence analysis revealed a missense substitution of leucine (CTG) for proline (CCG) at residue 207 in exon 5. This mutation was found on 54 of the 74 mutant alleles (73 percent) in the patients. Studies of site-directed in vitro mutagenesis have confirmed that this mutation generates inactive lipoprotein lipase and is the cause of lipoprotein lipase deficiency. CONCLUSIONS We have identified a missense mutation at residue 207 of the lipoprotein lipase gene that is the most common cause of lipoprotein lipase deficiency in French Canadians. This mutation can be easily detected by dot blot analysis, providing opportunity for definitive DNA diagnosis of the disorder and identification of heterozygous carriers.

Patients With ApoE3 Deficiency (E2/2, E3/2, and E4/2) Who Manifest With Hyperlipidemia Have Increased Frequency of an Asn 291→Ser Mutation in the Human LPL Gene

Approximately 1% to 2% of persons in the general population are homozygous for a lipoprotein receptor-binding defective form of apoE (apoE2/2). However, only a small percentage (2% to 5%) of all apoE2/2 homozygotes develop type III hyperlipoproteinemia. Interaction with other genetic and environmental factors are required for the expression of this lipid abnormality. We sought to investigate the possible role of LPL gene mutations in the development of hyperlipoproteinemia in apoE2/2 homozygotes and in apoE2 heterozygotes. As a first step, we performed DNA sequence analysis of all 10 LPL coding exons in 2 patients with the apoE2/2 genotype who had type III hyperlipoproteinemia and identified a single missense mutation (Asn 291-->Ser) in exon 6 of the LPL gene. The mutation was then found in 5 of 18 patients with type III hyperlipoproteinemia who had the apoE2/2 genotype (allele frequency = 13.9%; P < or = 7.4 x 10(-5)) and 6 of 22 hyperlipidemic E2 heterozygous patients with the apoE3/2 and E4/2 genotype (allele frequency = 13.6%; P = 2.2 x 10(-5)). In contrast, this mutation was found in only 3 of 230 normolipidemic controls (allele frequency = 0.7%). In vitro mutagenesis studies revealed that the Asn 291-->Ser mutant LPL had approximately 60% of LPL catalytic activity and approximately 70% of specific activity compared with wild-type LPL. The heparin-binding affinity of the mutant LPL was not impaired. Our data suggest that the Asn 291-->Ser substitution is likely to be a significant predisposing factor contributing to the expression of different forms of hyperlipidemia when associated with other genetic factors such as the presence of apoE2.

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.

Genetic variants affecting human lipoprotein and hepatic lipases

The genes for human lipoprotein and hepatic lipases have been cloned facilitating genetic analysis. Numerous naturally occurring mutations in these two enzymes are described which give preliminary insights into regions of functional importance in these proteins.

Mapping of the epitope on lipoprotein lipase recognized by a monoclonal antibody (5D2) which inhibits lipase activity

A monoclonal antibody, 5D2, which inhibits human lipoprotein lipase (hLPL) activity has been widely used for assessment of LPL immunoreactive mass in the clinical evaluation of patients [1] and for analysis of structure-function relationships of LPL [2,3]. We have mapped the epitope on LPL, recognized by the 5D2 antibody, within residues 396-405. Ala400 is the critical amino acid residue conferring epitope specificity. This knowledge confirms that the C-terminal domain of LPL plays a critical role in LPL activity and also provides important information for studies exploring the structure-function relationship of LPL using this antibody.

A missense mutation (Asp250→Asn) in exon 6 of the human lipoprotein lipase gene causes chylomicronemia in patients of different ancestries

We have previously reported two common lipoprotein lipase (LPL) gene mutations underlying LPL deficiency in the majority of 37 French Canadians (Monsalve et al., 1990. J. Clin. Invest. 86: 728-734; Ma et al., 1991. N. Engl. J. Med. 324: 1761-1766). By examining the 10 coding exons of the LPL gene in another French Canadian patient, we have identified a third missense mutation that is found in two of the three remaining patients for whom mutations are undefined. This is a G to A transition in exon 6 that results in a substitution of asparagine for aspartic acid at residue 250. Using in vitro site-directed mutagenesis, we have confirmed that this mutation causes a catalytically defective LPL protein. In addition, the Asp250----Asn mutation was also found on the same haplotype in an LPL-deficient patient of Dutch ancestry, suggesting a common origin. This mutation alters a TaqI restriction site in exon 6 and will allow for rapid screening in patients with LPL deficiency.

Gene environment interaction and plasma triglyceride levels: the crucial role of lipoprotein lipase

In most people, plasma triglycerides are in dynamic equilibrium, mediated by a balance between very low density lipoprotein (VLDL) and chylomicron (CM) synthesis, lipolysis of triglyceride-rich lipoproteins (Brunzell 1989) and by uptake of remnant particles through appropriate receptors (Fig. 1) (Beisiegel et al. 1991). However, with abnormalities involving synthesis, lipolysis or remnant uptake, hypertriglyceridemia may ensue (Fig. 2). For example, in diabetes, pregnancy or after alcohol intake VLDL synthesis is significantly increased, which in some persons manifests as hypertriglyceridemia. In most individuals, however, even after significant alcohol intake or in pregnancy, with an intact lipolytic system, hypertriglyceridemia may not ensue or is only mild to moderate. A rise in serum levels of triglyceride-rich lipoproteins facilitates the transfer of cholesterol esters from HDL to VLDL and CM in exchange for triglycerides. As a consequence, the HDL particle becomes triglyceride enriched and measured HDL cholesterol decreases (Patsch et al. 1992). We have recently demonstrated that environmental factors such as normal pregnancy, which results in a twoto threefold increase in triglyceride levels, may trigger chylomicronemia and associated

Compound heterozygosity for a known and a novel defect in the lipoprotein lipase gene (Asp250 → Asn; Ser251 → Cys) resulting in lipoprotein lipase (LPL) deficiency

Two missense mutations in exon 6 of the LPL gene were identified on separate alleles in a Dutch patient with lipoprotein lipase (LPL) deficiency. The first mutation is a G1003-->A transition resulting in a D250N mutation, which has been shown previously to result in a catalytically defective protein in patients of French-Canadian ancestry. The second mutation, a C to G transition at nucleotide 1007, predicts a S251C residue change in the highly conserved region of LPL surrounding the loop structure the covers the catalytic triad. This mutation constitutes a novel defect among LPL gene mutations reported so far. Site-directed mutagenesis experiments provide in-vitro evidence for the complete loss of LPL activity resulting from this latter missense mutation. The G1003-->A nucleotide substitution underlying the Asp250 mutation deletes a TaqI endonuclease recognition site and the C1007-->G change that leads to the S251C alteration abolishes a HinfI recognition site. This will facilitate rapid screening for these mutations in LPL-deficient patients.

Support for founder effect for two lipoprotein lipase (LPL) gene mutations in French Canadians by analysis of GT microsatellites flanking the LPL gene.

Mutations in the human lipoprotein lipase (LPL) gene are one of the major causes of familial chylomicronemia. We have characterized two polymorphic GT microsatellites flanking this gene. Two LPL mutations that are extremely frequent in French Canadians appear to be in complete linkage disequilibrium with specific LPL microsatellite haplotypes indicating a founder effect within this population.

Evidence for a cholesterol-lowering gene in a French-Canadian kindred with familial hypercholesterolemia

We describe a four-generation kindred with familial hypercholesterolemia (FH) in which two of the eight heterozygotes for a 5-kb deletion (exons 2 and 3) in the low density lipoprotein (LDL) receptor gene were found to have normal LDL-cholesterol levels. In our search for a gene responsible for the cholesterol-lowering effect in this family, we have studied variation in the genes encoding the LDL receptor, apolipoprotein (apo) B, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, apoAI-CIII-AIV, and lipoprotein lipase. The analysis showed that it was unlikely that variation in any of these genes was responsible for the cholesterol-lowering effect. Expression of the LDL receptor, as assessed in vitro with measurements of activity and mRNA levels, was similar in normo and hyperlipidemic subjects carrying the deletion. Analysis of the apo E isoforms revealed that most of the e2 allele carriers in this family, including the two normolipidemic 5-kb deletion carriers, were found to have LDL-cholesterol levels substantially lower than subjects with the other apo E isoforms. Thus, this kindred provides evidence for the existence of a gene or genes, including the apo e2 allele, with profound effects on LDL-cholesterol levels.