Structure and polymorphic map of human lipoprotein lipase gene

Lipoprotein lipase (LPL) catalyzes the key step for the removal of triacylglycerol-rich lipoproteins from the circulation. In this paper, we report the cloning and structure of the normal human LPL gene, which was isolated in three overlapping lambda phage clones that span about 35 kilo bases (kb) of the genetic locus. The peptide coding region of the gene is approx. 23 kb in length and contains nine exons with intron sizes ranging from 0.7 to 8.7 kb. The entire 3' untranslated region is in the tenth exon. Specific sequences in this region support the hypothesis that two mRNA species found for human LPL are generated by differential utilization of polyadenylation signals. The first exon occurs in the 5' untranslated region and the region coding for the signal peptide. The second exon includes the protein domain coding for the N-linked glycosylation site that is required for the expression of enzyme activity. The fourth exon contains the region that was proposed as a lipid binding domain, the sixth for one putative heparin binding domain, and the eighth codes for a domain containing another N-linked glycosylation site. These results suggest that the unique structural and functional domains are confined to specific exons. The PvuII polymorphic site was located within the intron between exon 6 and 7 and the HindIII polymorphic site to the 3' flanking region. The location of these polymorphic sites suggests that the PvuII restriction fragment length polymorphism (RFLP) associated with lipase deficiency in a few Japanese kindred may be a linkage marker for a functional defect of LPL, while the HindIII RFLP associated with hypertriglyceridemia may be important for gene regulation of LPL.     

Lipoprotein lipase activity and common gene variants in severely hypertriglyceridemic patients with and without diabetes

OBJECTIVES: In severe type IV hypertriglyceridemia (triglyceride levels >10 g/l), it is yet unknown whether lipoprotein lipase (LPL) differs according to the presence or not of diabetes. METHODS: We compared LPL activity and the presence of four common variants in the LPL gene (Asp 9 Asn (exon 2), Gly 188 Glu (exon 5), Asn 291 Ser (exon 6) and Ser 447 Ter (exon 9)) in a group of 34 patients of whom 17 presented diabetes mellitus. RESULTS: Maximum triglyceride, cholesterol levels and distribution of apolipoprotein E phenotypes did not differ between the two subgroups. Mean post-heparin LPL activity was lower in non-diabetic compared to diabetic patients (9.74 vs. 12.98 micromol FFA/ml/h, p=0.033). Four patients were carrying a mutation in exon 9 (1 non-diabetic), 6 patients in exon 2 (4 non-diabetic) and 1 patient in the non-diabetic subgroup in exon 5. All mutations were at the heterozygous state. CONCLUSION: We found that LPL activity was lower in type IV hyperlipidemia in the absence of diabetes. Genetic defects in the LPL gene that could lead to this lower LPL tended to be more frequently observed in patients without diabetes. These data suggest that the pathomechanisms which contribute to severe type IV hyperlipidemia are different according to the presence or not of diabetes.     

Guinea pigs possess a highly mutated gene for L-gulono-gamma-lactone oxidase, the key enzyme for L-ascorbic acid biosynthesis missing in this species.

Guinea pigs cannot synthesize L-ascorbic acid because of their deficiency in L-gulono-gamma-lactone oxidase, a key enzyme for the biosynthesis of this vitamin in higher animals. In this study we isolated the L-gulono-gamma-lactone oxidase gene of the rat and the homologue of this gene of the guinea pig by screening rat and guinea pig genomic DNA libraries in lambda phage vectors, respectively, using a rat L-gulono-gamma-lactone oxidase cDNA as a probe. Sequencing analysis showed that the amino acid sequence of the rat enzyme is encoded by 12 exons and that all the intron/exon boundaries follow the GT/AG rule. On the other hand, regions corresponding to exons I and V were not identified in the guinea pig L-gulono-gamma-lactone oxidase gene homologue. Other defects found in this gene homologue are a deletion of the nucleotide sequence corresponding to a 3' 84-base pair part of rat exon VI, a 2-base pair deletion in the remaining exon VI-related region, and nonconformance to the GT/AG rule at one of the putative intron/exon boundaries. Furthermore, a large number of mutations were found in the amino acid-coding regions of the guinea pig sequence; more than half of them lead to nonconservative amino acid changes, and there are three stop codons as well. Thus it is clear that the guinea pig homologue of the L-gulono-gamma-lactone oxidase gene exists as a pseudogene that randomly accumulated a large number of mutations without functional constraint since the gene ceased to be active during evolution. On the basis of the neutral theory of evolution, the date of the loss of L-gulono-gamma-lactone oxidase in the ancestors of the guinea pig was roughly calculated to be less than 20 million years ago.     

Genetic screening of the lipoprotein lipase gene for mutations associated with high triglyceride/low HDL-cholesterol levels

The lipoprotein lipase (LPL) enzyme plays a major role in lipid metabolism, primarily by regulating the catabolism of triglyceride (TG)-rich lipoprotein particles. The gene for LPL is an important candidate for affecting the risk of atherlosclerosis in the general population. Previously, we have shown that the HindIII polymorphism in intron 8 of the LPL gene is associated with plasma TG and HDL-cholesterol variation in Hispanics and non-Hispanic whites (NHWs). However, this polymorphism is located in an intron and hence may be in linkage disequilibrium with a functional mutation in the coding region or intron-exon junctions of the LPL gene. The aim of this study was to initially screen the LPL coding region and the intron-exon junctions by single-strand conformation polymorphism (SSCP) analysis for mutation detection in a group of 86 individuals expressing the phenotype of high TG/low HDL, followed by association studies in a population-based sample of 1,014 Hispanics and NHWs. Four sequence variations were identified by SSCP and DNA sequencing in the coding region of the gene, including two missense mutations (D9N in exon 2 and N291S in exon 6), one samesense mutation (V108V in exon 3), and one nonsense mutation (S447X in exon 9). Multiple regression analyses, including these four mutations and the HindIII polymorphic site, indicate that the association of the HindIII site with plasma TG (P=0.001 in NHWs and P=0.002 in Hispanics) and HDL-cholesterol (P=0.007 in NHWs and P=0.127 in Hispanics) is independent of all other LPL variable sites examined. These observations reinforce the concept that the intronic 8 HindIII site is functional by itself and provide a strong rationale for future comprehensive functional studies to delineate its biological significance.     

Organization of the mouse microfibril-associated glycoprotein-2 (MAGP-2) gene

A 1.4-kb EST clone encoding mouse microfibril-associated glycoprotein-2 (MAGP-2), identified by its similarity with the reported human cDNA, was used to screen a mouse 129 genomic bacterial artificial chromosome (BAC) library. The mouse gene contains 10 exons spanning 16 kb, located on the distal region of Chromosome (Chr) 6. The exons range in size from 24 to 963 bp, with the ATG located in exon 2. The tenth and largest exon contains 817 bp of 3′ untranslated sequence, including a B2 repetitive element. Northern analysis demonstrates abundant expression of MAGP-2 mRNA in skeletal muscle, lung, and heart. Sequence analysis of additional cDNA clones suggests that the two mRNA forms of MAGP-2 in the mouse arise from alternative polyadenylation site usage. The promoter does not contain an obvious TATA box, and the sequence surrounding the start site does not conform to the consensus for an initiator promoter element. Additionally, the mouse promoter contains 22 copies of a CT dinucleotide repeat sequence located ∼155 bp 5′ to exon 1. Received: 27 August 1999 / Accepted: 2 November 1999     

Branch point selection in alternative splicing of tropomyosin pre-mRNAs.

The rat tropomyosin 1 gene gives rise to two mRNAs encoding rat fibroblast TM-1 and skeletal muscle beta-tropomyosin via an alternative splicing mechanism. The gene is comprised of 11 exons. Exons 1 through 5 and exons 8 and 9 are common to all mRNAs expressed from this gene. Exons 6 and 11 are used in fibroblasts as well as smooth muscle whereas exons 7 and 10 are used exclusively in skeletal muscle. In the present studies we have focused on the mutually exclusive internal alternative splice choice involving exon 6 (fibroblast-type splice) and exon 7 (skeletal muscle-type splice). To study the mechanism and regulation of alternative splice site selection we have characterized the branch points used in processing of the tropomyosin pre-mRNAs in vitro using nuclear extracts obtained from HeLa cells. Splicing of exon 5 to exon 6 (fibroblast-type splice) involves the use of three branch points located 25, 29, and 36 nucleotides upstream of the 3' splice site of exon 6. Splicing of exon 6 (fibroblast-type splice) or exon 7 (skeletal muscle type-splice) to exon 8 involves the use of the same branch point located 24 nucleotides upstream of this shared 3' splice site. In contrast, the splicing of exon 5 to exon 7 (skeletal muscle-type splice) involves the use of three branch sites located 144, 147 and 153 nucleotides, upstream of the 3' splice site of exon 7. In addition, the pyrimidine content of the region between these unusual branch points and the 3' splice site of exon 7 was found to be greater than 80%. These studies raise the possibility that the use of branch points located a long distance from a 3' splice site may be an essential feature of some alternatively spliced exons. The possible significance of these unusual branch points as well as a role for the polypyrimidine stretch in intron 6 in splice site selection are discussed.     

Novel compound heterozygous mutations for lipoprotein lipase deficiency. A G-to-T transversion at the first position of exon 5 causing G154V missense mutation and a 5' splice site mutation of intron 8.

We systematically investigated the molecular defects causing a primary LPL deficiency in a Japanese male infant (patient DI) with fasting hyperchylomicronemia (type I hyperlipoproteinemia) and in his parents. Patient DI had neither LPL activity nor immunoreactive LPL mass in the pre- and post-heparin plasma. The patient was a compound heterozygote for novel mutations consisting of a G-to-T transversion at the first nucleotide of exon 5 [+1 position of 3' acceptor splice site (3'-ass) of intron 4] and a T-to-C transition in the invariant GT at position +2 of the 5' donor splice site (5'-dss) of intron 8 (Int8/5'-dss/t(+2)c). The G-to-T transversion, although affecting the 11 nucleotide of the 3'-consensus acceptor splice site, resulted in a substitution of Gly(154) to Val (G154V; GG(716)C(-->)GTC). The mutant G154V LPL expressed in COS-1 cells was catalytically inactive and hardly released from the cells by heparin. The Int8/5'-dss/t(+2)c mutation inactivated the authentic 5' splice site of intron 8 and led to the utilization of a cryptic 5'-dss in exon 8 as an alternative splice site 133 basepairs upstream from the authentic splice site, thereby causing joining of a part of exon 8 to exon 9 with skipping of a 134-bp fragment of exon 8 and intron 8. These additional mutations in the consensus sequences of the 3' and 5' splice sites might be useful for better understanding the factors that are involved in splice site selection in vivo.     

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.