Molecular pathobiology of the human lipoprotein lipase gene

ISSN 0163-7258/96 $32.00 PI1 SOl63-7258(96)00005-S zyxwvutsrqp Pharmacol. Ther. Vol. 70, No. 2, pp. 101-135, 1996 Copynght 0 1996 Elsev~er Science Inc. ELSEVIEK Editor: D. Shugur Associate Molecular Pathobiology of the Human Lipoprotein Lipase Gene Ven Murthy,*t Pierre zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Julien,+ and Claude Gag& *MOLECULAR BIOLOGY LABORATORY ON HUMAN DISEASES. DEPARTMENT OF BIOCHEMISTRY, FACULTY OF MEDICINE, LAVAL UNIVERSITY, STE-FOY, QUEBEC GIK 7P4, CANADA + DEPARTMENT OF MEDICINE AND CENTRE DE RECHERCHE SUR LES Lll'lDlQUES.LAVALUNIVERSITY MEDICAL CENTRE,STE-FOY,QUEBEC GlV 4G2,CANADA MALADIES ABSTRACT. diseases, appear Lipoprotein including to be directly of a superfamily human LPL mature protein. of Quebec, these and directed tional coronary or indirectly homology, evolutionary gene, which There to abnormalities hepatic in LPL indicate function. and its function, of LPL LPL in the normal of LPL Familial lipoprotein lipase deficiency, syndrome, gene mutations. LPL mutations (207 and suggesting we discuss of abnormal hepatic 70(2): LPL of the in the Province A study of structural and func- the interrelationships in humans, 101-135. lipase, pancreatic 188) in the created zyxwvutsrqponmlkjihgfedcbaZYXWV i n vi t r o by site- into multiple THER. that they functions population. as well as those is organized etiology is a member lipases are charac- have been discovered In this review, PHARMACOL. W ORDS. protein, to the French-Canadian molecules, enzyme. the molecular deficiency. Human lipase. These mutations of lipids. M any and atherosclerosis, sites in the gene and affect different exclusive mutant that the sequence aspects of natural of two of these is almost (207) that act in concert at different prevalence occurring of LPL structure micronemia in the metabolism (pancreatitis), lipase and pancreatic A large number are located and therapeutic KEY is a key enzyme disease, chylomicronemia both at the level of the gene and the mature is a high naturally mutagenesis, units related origin. and one of them other E.C. 3.1.1.34) heart of lipases that includes terized by extensive have a common lipase (LPL; obesity, zyxwvutsrqponmlkjihgfed and the clinical 1996. lipase, lipase gene family, chylo- CONTENTS 1. INTRODUCTION . . . . . .. . . . . . . . . .. . . THE LIPASE SUPERFAMILY . . . . . . . . .. . 2.1. HUMAN LIPASES AND THEIRFUNCTION . . . . . . . .. . . . . . 2.2. GENEORGANIZATIONOFLIPASES .. 2.3. CONSERVATION ANDEVOLUTION OF LIPASES ANDTHEIR STRUCTURALDOMAINS ......... 3. LIPOPROTEIN LIPASE . . . . . . . . . . . . . . . 3.1. FUNCTIONALANATOMYOFTHE LIPOPROTEIN LIPASE MOLECULE ... 3.2. MOLECULARPHYLOGENYOF LIPOPROTEIN LIPASE . . . . . . . . . . . 3.3. MUTATIONSINVOLVINGTHE NONCODINGSEQUENCESOFTHE HUMAN LIPOPROTEIN LIPASEGENE . . . . . . . . . . .. . . . . . 3.4. MUTATIONS INVOLVING THE CODING SEQUENCES OF THE HUMAN LIPOPROTEIN LIPASE GENE 4. THE CHYLOMICRONEMIA SYNDROME .. 4.1. DESCRIPTION . . . . . . .. . . . . . . . . 4.2. DIAGNOSIS . . . . . . . . . . . . . . . . . . . 4.3. CLINICAL MANIFESTATIONS ...... 4.3.1. SYMPTOMS OF CHYLOMICRONEMIA . . .. .. 2. 102 102 102 103 105 107 107 109 113 114 118 118 118 119 119 4.3.2. SIGNSOFCHYLOMICRONEMIA 4.3.3. ANOMALOUS LABORATORY FINDINGSIN CHYLOMICRONEMIA . . .. . . 4.4. CAUSESOFCHYLOMICRONEMIA SYNDROME . . . . . .. . . . . . . . .. . . . 5. PRIMARY LIPOPROTEIN LIPASE DEFICIENCY . .. . . . . . . . . . .. . . . . . . . . 5.1. HOMOZYGOTESTATEOFLIPOPROTEIN LIPASE DEFICIENCY . . . . . . . . . . . . . 5.2. HETEROZYGOTESTATEOF LIPOPROTEIN LIPASE DEFICIENCY . . 5.3. ORIGIN ANDDISSEMINATION OF LIPOPROTEIN LIPASEGENEDEFECTS INQUeBEC . . . . .. . . . . . . .. . . . . . 6. TREATMENT OF LIPOPROTEIN LIPASE DEFICIENCY . . . . . . . . . . . . . . . . . . . . . . 6.1. IDENTIFICATION AND CORRECTION OFSECONDARYFACTORS . . . . . .. . . 6.2. FAMILY SCREENING AND COUNSELING . . . . . . . . .. . . . . . . . 6.3. DIETARYREGIMEN . . . . . . . . .. . . . 6.4. DRUGTHERAPY . . . . . . . . . . . .. . 6.5. PROSPECTS• FGENETHERAPY . . . . ACKNOWLEDGEMENTS . . . . . . .. . . . . . . . . REFERENCES . . . . . . . . . .. . . . . .. . . . . . . . 120 121 121 122 122 122 124 125 125 125 125 126 127 127 127 ABBREVIATIONS, apo, apolipoprotein; FCH, familial combined hyperlipidemia; HDL, high-density lipoprotein; HL, hepatic lipase; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; LPL, lipoprotein lipase; LRP, LDL receptor-related protein; MCT, medium-chain TG; PL, pancreatic lipase; TG, triglyceride; VLDL, very low-density lipoprotein. ~Corresponding author. V. zyxwvutsrqponmlkjihgf Murthy et al 102 1, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA INTRODUCTION (PL; EC. 3.1.1.3). All three enzymes hydrolyze lipid emulLipoprotein lipase (LPL; E.C. 3.1.1.34) is the enzyme respon- sible for the influx of free fatty acids into peripheral tissues for storage in the form of triglycerides (TGs) or as a source sions and have similar aqueous-lipid interfacial catalytic activity. The three lipases show several individual characteristics, as well as differences in their properties of energy. It is known that the action of LPL on TG-rich functions lipoproteins cells of several tissues, including proteins promotes the exchanges of lipids between lipo- and that LPL is, thus, indirectly maturation involved in the of the majority of plasma lipoproteins (Brunzell, 1989). There is evidence to suggest that LPL has a contributory role in the manifestation conditions of a number of pathologic related to the metabolism ofTG-rich (Bergeron et uI., 1991). Dysfunction activity thus has been associated various dyslipoproteinemias atherogenic lipoproteins and deficiency of LPL with the pathogenesis and with the production particles, such as intermediate-density teins (IDL) and low-density lipoproteins (LDL) 1989; Eckel, 1989). Because of its physiological and its possible role in lipid-related and physiological in the parenchymal skeletal muscle, heart, adi- pose tissue, lung, lactating mammary gland, etc. (Borensztajn, 1987). It is expressed transiently in the embryonic liver, but et al., 1988; Gimenez Llort et al., not in the adult (Vilaro 1991). It is transported from the site of synthesis to the luminal surface of vascular endothelia, interaction with heparin where it is anchored sulfate proteoglycans by ion and/ or by of glycosyl phosphatidylinositol of It can be released from the bound form into the circulation lipopro(Brunzell, importance pathologies, (Table 1). LPL is synthesized LPL has by the administration (Braun and Severson, of heparin. This property 1992). has been widely used to release LPL from cell membranes and to purify LPL by heparin-Sepharose son and Olivecrona, affinity chromatography (Bengts- 1977). In its active form, LPL is a gly- cosylated noncovalent homodimer (Osborne et al., 1985) been intensively examined in many in s~ivoand zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA in vitro studies chat reversibly dissociates to form inactive monomers under in both human and animal models. The functions of LPL and its regulation have been high- physiological conditions, as well as under the effects of pH, lighted in a number of excellent and detailed recent articles ionic strength, and temperature (Olivecrona (Borensztajn, Olivecrona, 1987; Brunzell, and Bengtsson- 1987). For enzyme activity, it has an obligatory Severson, 1992; requirement 1992; Lalouel et al., 1992; Santamarina-Fojo, 1992; of 79 amino acid residues. Each LPL subunit contains a site 1991; Auwerx Dolphin, 1989; Eckel, 1989; Bensadoun, et al., 1992; Braun Wang et (II., 1992; Olivecrona 1993; Santamarina-Fojo and for heparin and Bengtsson-Olivecrona, and Dugi, 1994). The objective of ous LPL deficiencies known to occur in humans and to provide an assessment of the possible therapeutic Olivecrona approaches as well as a site for interaction reviewed in detail by Olivecrona (1987). The enzymatic and activity Bengtsson- is optimal at a pH between 8.0 and 8.5 and it is stabilized by the presence of lipids or by binding logic conditions. with The physical and kinetic properties of LPL enzyme have been available for the treatment of these patho- binding, (apo) CII, a small protein ape CII. the present review is to discuss the molecular basis of vari- that are currently for apolipoprotein to lipid-water interfaces gents, such as deoxycholate. Although and deter- LPL is activated by its cofactor, apo C-II, it is inhibited by various factors, includ2. THE LIPASE SUPERFAMILY ing fatty acids, apo CIII and, possibly, apo E. It is inhibited 2.1. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Human Lipases and Their Function also by high salt concentrations (1 M NaCI), an observation Apart from LPL, there are two other lipases with important that is used to differentiate lipolytic activities: hepatic lipase (HL) and pancreatic activities. The later remains unaffected TABLE lipase plasma LPL and HL under these condi- 1. Properties of the Enzymes of the Human Lipase Family HL LPL Site of synthesis Site of action Subunits Glycosylation Binding to cell surface Released into circulation Activators Inhibitors Aqueous-lipid interfacial activity Lipoprotein-ligand activity Substrates for lypolysis Parenchymal cells Capillary endothelia of several Homodimcr + Glycosaminoglycan links By heparin Apo CII Apo CIII and possibly apo E f + Chylomicrons, VLDL Enzyme TG Specificity between activity lipasc, Sri-1 and minor phospholipase Sri-3 ester bonds tissues Hepatocytes Liver sinusoids M onomer + Glycosaminoglycan By heparin Possible stimulation PL links Exocrine pancreas Duodenum Monomer + Free by apoE Colipase + + Chylomicron remnants, IDL, HDLL TG lipasc, phospholipase Sri-1 and Sri-3 ester bonds + Alimentary TGs TG lipase, variable phospholipase Sn-1 and Sn-3 ester bonds Human Lipoprotein Lipase Gene 103 tions. In vitro, LPL can hydrolyze a number of substrates, transports PL and procolipase, along with other pancreatic such as long- and short-chain glycerides, phospholipids, and hydrolases, into the duodenum, where the enzyme completes various synthetic but at much lower rates than the hydrolysis of alimentary TGs begun anteriorly by lingual substrates, TG (see Olivecrona and Bengtsson-Olivecrona, a review). Chylomicrons (VLDL) constitute 1987, for and very-low-density lipoproteins the major substrates for lipolysis by LPL in viva (Wang et al., 1992). In the resulting shows a high degree of substrate reaction, sn-3 bonds of lipoprotein TGs are preferentially generating sn-2 monoglycerides 1992). The sn-2 bond enzymatic conversion LPL specificity. The sn-1 and hydrolyzed, and free fatty acids (Dolphin, is attacked subsequently after non- to the sn-1 isomeric form. In addition lipase (Verger, 1984). The colipase, which helps to anchor the PL to the lipid-water interface, lytic rate (van Tilbeurgh does not affect its cata- et al., 1992), in contrast to the role of apo CII in LPL activity. Like LPL and HL, PL acts preferentially on the sn-1 and sn-3 bonds of TGs (Dolphin, It shows variable phospholipase 1992). activity, depending on the animal species. The active enzyme is glycosylated like the other two lipases. LPL and HL share two conserved sylation sites, whereas the glycosylation to its lipolytic activity, LPL also fulfills other important func- different tions in lipid metabolism. 1990; Ben Zeev et al., 1994). Like HL, PL is active in the It recently has emerged that lipo- proteins may bind to cell surfaces through binding, a ligand function independent lipase-mediated of the enzymatic position monomeric anchored (Olivecrona glyco- site of PL is in a and Bengtsson-Olivecrona, form, but unlike the other two lipases, it is not surfaces, but acts as free molecules. zyxwvutsrq to membrane activities of the lipases (Beisiegel et al., 1994). The heparinbinding capacity of LPL (shared by HL, but not by PL) may facilitate the uptake and internalization of plasma lipopro- teins through specific receptors (Beisiegel, scriptional and posttranscriptional 1995). Both tran- steps of LPL gene factors and by hormones as insulin, thyroid hormone, et al., 1976; Nilsson-Ehle and glucocorticoids et al., 1980; Cryer, Gene Organization of Lipases The exon-intron organization and nucleotide sequences of human LPL and HL genes have been studied by several investigators (Martin et al., 1988; Cai et al., 1989; Deeb and Peng, expression appear to be regulated by various environmental, dietary, and developmental 2.2. such (Pykalisto 1981). 1989; Kirchgessner et al., 1989; Ameis et al., 1990). Some of the salient features of their gene structure are shown in Table 2. The two lipases are situated on two different chromo- In contrast to LPL, the synthesis of HL is confined mainly somes (8~22 for LPL and 15q21 for HL). The size of the HL to liver where it is localized on the surface of liver sinusoids gene is twice that of LPL, due mainly to the longer introns through glycosaminoglycan Bengtsson-Olivecrona, links (Table 1) (Olivecrona and 1993). Treatment with heparin, there- in HL. The LPL gene contains 10 exons separated fore, leads to the release of HL, as well as LPL, into circu- Intron 9 is present only in LPL and interrupts lation. nation codon TG/ A. The LPL and HL mRNAs HL is a glycosylated monomer. Although protein glycosylation cally affect the secretion and is active as a has been shown to criti- and the activity of LPL, it does by 9 introns, and the HL gene has 9 exons separated by 8 introns. the termicode for signal peptides of 27 and 22 amino acid residues and mature proteins of 448 and 477 amino acid residues, respectively. et ul., The greater length of HL is due mainly to the larger number 1991; Ben Zeev et u1., 1992). HL possesses both TG lipase of amino acid residues coded by its exons 2 and 8. The LPL and phospholipase mRNA not seem essential for HL catalytic activities acts on TGs of chylomicron activity (Stahnke (Deckelbaum et al., 1992). It remnants to further reduce their size, on the TGs of IDL to produce LDL, and on the TGs and phospholipids of HDL: of the high-density lipoproteins to form HDLI. The conversion is important mechanism extrahepatic in the reverse cholesterol believed to be necessary transport of excess cholesterol. for an obligatory LPL, which needs apo CII for its function, of HL may be stimulated process, a for the protection tissues from accumulating HL has no requirement (HDLs) of HDLL to HDLi occurs in two isoforms in the human (3.75 kb and 3.35 kb), but only a single isoform of HL mRNA is observed. This is due to the existence polyadenylation mRNA. sites in LPL mRNA Although and only one in HL the LPL gene is only half the size of the HL gene, each of the two LPL mRNAs the HL mRNA. (1.7 kb) of two alternate This difference due to the very large noncoding is twice as long as in mRNA size is primarily exon 10 that is present in activator, unlike LPL, but absent in HL. Apart from these size differences, but the activity the other 9 exons of LPL and the 9 exons of HL are very by apo E. In contrast, as noted similar in size and are organized in a very similar fashion. earlier, apo CIII and, possibly, apo E may act as LPL inhib- Except for introns itors. It has been suggested that the opposite effects of apo at sites identical to HL (Cai zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPO et al., 1989). There are multiple E on LPL and HL could serve to direct the action of these TATA Box and CAAT two plasma lipases toward specific lipoproteins gene, but there (Thuren et ul., 1992). Thus, the preferred substrates of LPL are the apo CII-rich lipoproteins, chylomicrons, and VLDL, those of HL are the TGs of apo E-containing i.e., chylomicron remnants, PL is synthesized IDL, and HDL2. activator, the precursor colipase. The pancreatic mole- for LPL, as well as for HL. Both duct initiation site genes possess potential 5’-elements responsive to glucocorticoids, cium ions, and adipocyte cyclic AMP, cal- specific enhancer motifs. of human PL is not known. How- ever, such data are available for canine scriptional are located Box consensus sequences in the HL is one major transcription The gene organization by the acinar cells of the exocrine pan- creas (Table I), which also synthesize cule of its protein whereas lipoproteins, 1, 4, and 9, all LPL introns PL, whose tran- unit is 10 times larger than the mature mRNA and is organized into 13 exons (Mickle et al., 1989). Exon 104 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA TABLE 2. Organization of Human LPL and HL Genesa LPL zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF HL 8p22 15q21 (kb) 30 60 Number of exons 10 9 Number of introns Chromosome Gene length Intron/ exon Major ratio mRNA Exon size (kb) 3.75; 1 9 8 9 37.5 3.35 1.7 275 nt Untranslated Signal 188 nt peptideh Uncleaved 130 nt 43 nt 81 nt: 27 aa Met-!’ - Ala-’ portion 66 nt: 22 aa Met-22 - Ala-I 6 nt: 2 aa Ala’ - Asp? 21 nt: 7 aa Leu’ - Glu’ Exon 2’ 162 nt: 54 aa Gln’(c/ !A) - Thri” 186 nt: 62 aa G~u” (G/ AG)- Se@ Exon 3 180 nt: 60 aa Va15’ - GIu” ~ 183 nt: 61 aa Val’(’ - Glu” @ Exon 4 117 nt: 39 aa 111 nt: 37 aa zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIH Thrli' - Thr’h” Glu” ’ Glulli Exon 5 234 nt: 78 aa Asn?” 234 nt:78 aa Glyli’ _ Gly” l G~Y” “ (G/ GG) Exon 6 243 nt: 81 aa As~?‘?(c/ AT) - Lvs” : 243 nt: 81 aa A~~?‘~(G/ cc) - Lys jjh Exon 7 120 nt: 40 aa V~~“ )(G/ TC) - Thr’sl Vd'*"(G/TT) - Exon 183 nt: 61 aa 8 LeUii3(CT/G) Exon 219 nt: 73 aa Gln’-‘l’ Lcu'~"(cT/G) - LyS’” I05 nt: 35 aa L~s~‘~(AA/ G)- Gly++” 9 Termination codon Noncoding sequence Exon - 3 nt (TG/A) sequence Noncoding Number sequence 3 nt (TGA) None 49 nt None None 1948 nt None 4’75 499 of aa coded by mRNA Total Signal peptide Mature protein 5’-flanking Start 27 23 448 476 elementsc -188 nt of transcription -43 TATA Box 215 to -210 nt CAAT Box 253 to -249 nt Nuclear factor-A Glucocorticoid Cal+ 111 nt: 37 aa Arg4+‘(AG/ A) - Arg” ;’ IO” Coding Cyclic 117 nt: 39 aa Thr” ‘; AMP binding responsive responsive Adipocyte specific i’-flanking elements? Poly-A Polyadenylation sites -424 to -419 nt -512 to -508 nt - 1332 to ~ 1327 nt to -227 to -761 nt nt -832 to -827 nt ~ 1022 to 1008 nt -560 to -554 nt -577 t 2952 to + 2957 signals nt to -66 nt to -111 nt -234 -768 -242 to -235 nt _ 550 to 543 nt responsive -70 -116 +3348 to +3353 nt nt t2981 nt; +3376 nt to -571 113 to -91 +1525 nt nt to t1531 +1545 a Compiled from Deeh and Peng (1989) and Kirchgessner et ui. (1989) for LPL, and Marnn et ui. (I%?), Cai et al. (1989) and Am& et ul. (1990) for HL. ’ The ammo acids are numbered starting with the first residue (+ 1) nf the mature protein. ’ Codons that are split between two succcss~vc exons and the correspondmg armn~ acids arc shown in parentheses. d The number of nucleotides in exon 10 of LPL ~ncludcs the third nuclrotidc of the i’-terminal sr<q~ codon (TG/ A) and up to nr 3376 (the second polyadenylatlon site). ’ The position of the 5’- and 3’-flank’mg eIements areidentltied m the s<ienrlfic ltteraturcusinga wmetv of startmg sites. ~g., the first nucieotide of a given cDNA clone (Wion et ul., 1987) or the transcnpnon initiation site (Deb and Peng, 1989; A meis et al., 1990). Because these reference points are not unique and may be subject to change, we have numbered the nuclcotidea in this xwew using the first nucleotlde of the Initiator codon (ATG) as + I, becaux there occurs only one such codon 111both LPL and HL. nt, nucleotides; aa, amino acids. Human Lipoprotein Lipase Gene 105 1 codes for all of the untranslated Exon 2 starts with the initiator mRNA codon sequence, and ATG and encodes the major part of the signal peptide. The stop codon, polyadenylation are contained signal, and the 3’ untranslated the sequence in exon 13. The presence of an intron imme- diately upstream of the initiator codon may afford a mechanism for the use of alternate promoters splicing to achieve tissue-specific From a comparison and differential expression of the intron-exon 1990; Winkler et al., 1990). The sequences of these three lipases are aligned in Fig. 1 so as to obtain maximum homology with the minimum number of gaps. The putative structural and functional domains common to these lipases are also indicated. The sequence similarity between LPL and HL (53%) is much greater than that between either LPL and PL (35%) or HL and PL (36%) (Table 3). It is reasonable of PL. organization of that amino acid sequences that are associated to assume with critical PL, HL, and LPL, it has been proposed that these three major functions lipolytic enzymes may belong to a superfamily of lipase genes served than others. This, indeed, appears to be the case for in an enzyme protein may be much better con- and may have descended from a single ancestral gene (Kirch- certain gessner et al., 1989). On the basis of amino acid sequence family (Table 4). For example, the catalytic triad consisting homology, of serine, aspartate, and histidine the membership of this superfamily is occasion- functional and ally extended to include certain conserved regions of Drosoph- regions surrounding ila yolk proteins between YPl, YP2, and YP3 (Kirchgessner 1987; Komaromy and Schotz, et al., 1987; Datta et al., 1988; Kirch- structural domains of the lipase is identical and even the the triad residues are well conserved the members of the lipase family (Fig. I) (Kirch- gessner et al., 1989). A second series of conserved regions are those identified as the potential lipid binding sites. Two gessner et al., 1989). The zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Drosophila yolk proteins, however, show no lipolytic or other activities in common with the putative N-linked glycosylation sites are present in HL and three lipases in the group. Two other lipase-like enzymes, LPL, which are conserved lecithin-cholesterol and A5 in Fig. I). However, HL appears to have two addi- acyltransferase found to have no significant PL and, presumably, (Komaromy and lingual homology with LPL, HL, and do not belong and Schotz, lipase are to the same family 1987). tional potential and in the same positions sites where N-glycosylation (A2 can occur (Al and A4), and only one such consensus sequence is identified in PL (A3) (Hide et al., 1992). The sequence of the lid covering the active site is not well conserved, formation although the con- of the lid and its structural relation to other parts 2.3. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Conservation and Evolution of of the enzyme may be stabilized by the disulfide bridge that Lipases and their Structural Domains links the two conserved Of the three human lipases, only the three-dimensional ture of PL has been established far (Winkler struc- by X-ray crystallography so et al., 1990). The results show the presence of cysteines servation rounded on protein structure. Asp” ‘, triad: Ser153, HisZh4(in the porcine enzyme, the catalytic numbered Ser152, Asp *7h, HisZh4, because residue Serj’ C-terminal of human domain (residues 337-449) acid has a &sandwich fold site. The active site domain is shielded by a flap or a lid con- sisting of an amphipathic helix. On binding to the substrate, the helical lid is supposed the molecule, the amino PL is deleted in this species). The and contains the main colipase binding in the N-terminal triad is to roll back upon the body of thus enhancing the hydrophobicity around the active site. This movement of the lid is related to interfacial activation, a phenomenon by which the lipase activ- flanking wings) hole, presumed to be involved in control- ling access of the substrate the three amino acid residues of the catalytic the lid. Other are the p-5 loop (or hydrophobic and the oxyanion two distinct domains in the PL molecule. The N-terminal domain (residues l-336) consists of a central P-sheet surby ol-helices (the cr/ P-hydrolase fold) and encloses flanking putative functional structures that do not show marked con- to the active site. Proline is an amino acid that can have profound The &-tram isomerism acid generally influences the conformation effects of this amino of proteins. LPL and HL have a striking resemblance quency of proline residues, but are differ- and distribution in regard to the fre- ent from PL in this regard (Table 5). The four proline residues conserved in all three lipases (Pro’60, I’ro1T3, ProZ07, and Pro214of LPL) are all clustered within a region that contains the lipid-binding domains B3 and B4 and the lid (Fig. 1). There are also other proline residues that are not conserved, but that are present within various lipid-binding domains of LPL: e.g., Pro95, Pro157, and Prolgo. Cysteine residues contribute to protein conformation and stability by their capacity to form covalent disulfide linkages ity is increased in the presence of lipid-water interface (Ollis that bring distant regions of the protein molecule into closer et al., 1992; van Tilbeurgh proximity. Not all cysteines, however, have such a role (Fig. 1, protein interface activator et al., 1992, 1993). Colipase, the of PL, helps to bind the enzyme to the in the presence of bile salts in the intestine that Table 5). The eight cysteines conserved in all the three lipases participate in the formation of disulfide bridges. One of these, might otherwise interfere with this binding. Similar lids are involving also presumably flap or lid region of the lipases (Fig. 1). The two additional present in all three enzymes of the lipase family. cysteines that LPL shares with HL, but not with PL (Cys*7 The amino acid sequences have all been established of human either LPL, HL, and PL by direct sequencing of selected polypeptide regions or by deciphering from the corresponding CY~*‘~ and Cyszj9 of LPL, encloses the cr-helical cDNA and Cys40 of LPL), are not linked by an S-S bond, but are highly conserved in LPL and HL molecules of different species. Of the six cysteines unique to PL, four are linked sequences (Wion et al., 1987; Ameis et ui., zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB by S-S bridges not found in LPL or HL. Of the other two, 106 ++ hLPL:----ADQRRD hHL: -------LGQ hPL: KEVCYERLGC Al + + ++ ---------RTPEDT---AQAVETmL HEMK----TR TE--RPLHIL PWSPKDVNTR ++ FIDIESKFAL SLKPEPFGRR FSDDSPWSGI + +++ ++ ___------ A FLLGETNQ-FLLY-TNENP 33 EDTCHLIPGV --GCQIRINH NNFQEVAA-D 45 56 O------o A2 + ++ ++** + + *+ hLPL:AESVATCHFNSKTFMVI H SSLPLVMII Jd hHL: PDTLQECGFN TNRKTRFIIH hPL: SSSISGSNFK D *+ + * *+ GWTVTGMYES GWSVDGVLEN GFIDKGE-EN A Bl +*+ + +++ +** +** hLPL:mVSAGYTK LVGQDVARFI hHL: =IAVRNTR LVGKEVAALL hPL: UQASQNIR IVGAEVAYFV B2 A3 ++**++*+* ****++* ** t ++++ + ++ + + NWMEEEFNYP LD -GYS LGAHAAGIAG S----LTNKK 148 RWLEESVQLS RS SAHVSGFAG SSIGG--THK 163 EFLQSAFGYS PS HVHVIGHS J&AHAAGEAG RRTNG----T 169 AA B3 ++******+* +* *++ hLPL:VNRITGLDJ?A GPNFEYAEAP hHL: IGRITGLDAALFEGSAPS hPL: IGRITGLDPA EPCFQGTPEL A *** hLPL:NGGTFQPGm hHL: NGGSFQPGhPL: NGGVEMPGLDF 0 + * ++*+ **+*+** * **++**+++ SRLSPDDADF VDVLHTFTBS; NRLSPDDANF VDAIHTFTPF, VRLDPSDAKF VDVIHTDGAP C ++*** ++ * ++ RrJaGDVD-OL HGFMIT-OT + ++ ICEAIRVIAF. +* +++*+ * +++* FIVPKLVAALY WIWQMVAALK WLANVCKNLF 0 + *+ + + *++ KREPDS-NV1 SQPAQPVNVG KVE--SVNCI B5 +*+*+** + +++**+++ VKCSHERSIH LFIDSUNEE ESHERSVH LF-HAG SNHLRSYKNPD .A +++* * ++ ++t SCRKNRCNNL SCKKGRCNTL PCPSGGCPQM o--a A4 + ++++ hLPL:GTESETHTNQ hHL: ImETPIQT hPL: GKKVTGHIL- A5 ** *++ +++* +*+++ +** +++++ + + + AFEISLYGTV AESENIPFTL P--EVSTm YSFLIYTEVD TFTMSLLGTK EKMQKIPITL GKGIA-Sm YSFLITLDVD ---VSLFGNK GNSKQYEIFK GTLKPDS--T HSNEFDSDVD * + + +++* hLPL:APAVFVKCHD hHL: QEKIFVKCEI hPL: VLLTLTPC-0 ----DSYFSW WDTVQTIIPW --INPTL--- ++ + + + KSLNKKSG-KSKTSKRKIR -----_____ ++ SDWWSSPGFA STGPRHSGLV ------PRVG 448 476 449 R---SSKMYL K---SKRLFL TNDVGQKFYL +*+*++++ IQKIRVKAGE LKTIRVKAGE ASKIIVET-N *** VVDWLSRim LVDWITLAm CVDWKGGSm 0 92 105 113 B4 +++**+*++* +++ *+ SP--Q KPVGHVDIYP 207 HM-GJSVGLK QPIGHYDFYP 222 IVPNJaaGMS zyxwvutsrqponmlkjihgfedcbaZY QWGHLDFFP 229 hLPL:KEAFEKGLCL hHL: MNSFSQGLCL hPL: YNVFTANKCF d hLPL:WKS------hHL: WENSAVWANV hPL: WYNNV----- GYEINKVRAK GYHVRQEPRS GHYADRYPGK + + KTRSQMPYKV VTRAQSPFKV DTGDASNFAR +++ +** TQKKVIFCSR TQQRMTPCSE VGKQFNFCSP + +++* + NPSKAYRCSS TQSMAYPCGD GF-AGFPCAS 266 281 28 8 l+*++++ ++ FHYQVKIHFS YHYQLKIQFWRYKVSVTLS +*+* * *++ IGELLMLKLK IGELIMIKFK VGDLQMVKFI +++ 323 337 348 381 396 402 + + EKVSHLQKGK NTDDLLLRPT ETV----REE 430 456 441 Human Lipoprotein Lipase Gene zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA TABLE 3. Amino Acid Sequence Homology in Human Lipases (“4 LPL and HL LPL and PL HL and PL ,’ From Hdc tions of the lipases. On the basis of these data, a number of structural Dayhoff Homology Lipases and Divergence distancea 53 35 36 domains with possible specific functions to be organized large and distinct amino terminal 72 132 130 structural domains. The N-domain is the seat of many important LPL, including catalysis, and the C-domain implicated in such functions lipoproteins into two (N: residues 1-312) and carboxy terminal (C: residues 313-448) er ui. (1992). have been identified in the LPL molecule (Table 4). Thus, by analogy with PL, LPL is considered (W 107 activities of appears to be as the initial interaction and the LPL-mediated with uptake of lipoproteins by cell surface receptors. one (Cys’02 of LPL) is situated within the sequence bordered by one of the unique S-S bridges of PL, and the other The active site of LPL can be considered of (a) the catalytic triad, (b) the oxyanion (Cys’HJ of LPL) is part of one of the presumed lipid binding binding domains catalytic (B3, Fig. 1). LPL and HL are much closer to each other are to PL in both their amino acid sequence their evolutionary homology and divergence (Table 3). In the course of evo- lution of the lipase gene family, it is probable gene branched than they that the PL off earlier than the other two. Although PL gene has the highest number of introns, the it has approx- imately the same number of codons as the other two lipases site, (d) the lid, and (e) the p-5 loop. Except for the triad, most residues in the catalytic and PL are hydrophobic for hydrogen diminishing the affinity of the catalytic bonding. This has the effect of groups of the phospholipids 1994), thus explaining sites of LPL and their main chains are not readi- ly accessible phoryl to be made up hole, (c) the lipid site for the phos- the lower phospholipase these enzymes, as compared et al., (van Tilbeurgh activities of with their TG lipase activities. The catalytic triad of LPL is made up of the three amino (e.g., 448, 477, and 449 amino acid residues in mature pro- acid residues Ser’jz, Asp’j6, and Hi@‘, teins of hLPL, hHL, and hPL). As seen in Fig. 1, the func- in all the three members of the human lipase family (LPL, tional HL, and PL) (Fig. 1) and in all other species so far studied domains of the lipases often coincide axons. Based on these observations, that the molecular curred introns, through evolution of the lipases could have oc- gene duplication events, and fusion of exons (Kirchgessner et ul., 1992), a mechanism explain the evolution with specific it has been suggested selective loss of et al., 1989; Hide that has also been applied to of other multidomain proteins, such (Hide et al., 1992). The oxyanion formed by the main chain site constitutes phobic binds the aliphatic groove that probably the acyl-enzyme intermediate. may also show substrates. to the catalytic interactions to permit the substrate to have access domain. The lid sequence tween two conserved Lipase Molecule The structural and functional mostly been extrapolated from the available physical infor- existing by (a) nucleotide have been further and amino acid homologies among the three lipases, (b) analysis of degrees of conservation in different regions of the enzyme molecules, (c) identification of naturally and characterization and (d) production examination occurring of the biochemical cysteines is contained (Cys*‘h to Cysz” ), be- which form one of the four disulfide bridges in the LPL molecule domains of LPL and HL have mation on PL. Some of these inferences corroborated with the lipid a mobile surface loop in the putative 3.1. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Functional Anatomy of the Lipoprotein chain of structure of LPL that covers the catalytic site and is rearranged LII’ASE a very hydro- Tyry4, ProiS’, AlalSH, and Ile’“ ’ hydrophobic The lid represents three-dimensional LIPOPROTEIN of Trpis and Leu” ‘, et al., 1994). The lipid-binding as the LDL receptor (Sudof et al., 1985) and serine proteases 3. hole in LPL is probably nitrogens which are next to Ser” : of the catalytic triad (van Tilbeurgh (Rogers, 1985). which are identical human mutations lesions involved, and could have the effect of stabilizing the lid within the protein during its action. Dugi et al. (1992) have identified two highly amphiphilic a-helical structures (residues Asn?ii to Arg2ls and Asp?‘? to Lys?‘” connected by a p-turn) in the lid with opposite polar and hydrophobic faces. In addition to its role in the hydrolysis of TGs and phospholipids, the lid may be essential for specifying For the lipase substrate. example, there are found to be differences in the open (active) of in zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA vitro site-directed mutations and conformation of LPL and PL, which may account for their of their effect on the catalytic or other func- different substrate specificities (van Tilbeurgh et al., 1994). FIGURE 1. Amino acid sequences of human LI’L (hLPL), human HL (hHL), and human PL (hPL). The amino acid sequences are shown in single letter notation and are aligned according to Hide et al. (1992). Amino acid identity between all three sequences is indicated by an asterisk (*) and identity between any two of the three sequences is indicated by a plus sign (+). The following putative functional sites are underlined: (Al-AS) N-linked glycosylation, (Bl-BS) lipid-binding domain, (C) o-helical lid, and (d) p-5 loop. The three amino acids representing the catalytic triad (serine, aspartate, and histidine) are indicated by solid triangles (A). Amino acids of the oxyanion hole are indicated by open triangles (A). The four cysteine pairs involved in disulfide linkages in all three lipases (0) and the two pairs unique to PL zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJ (0) are each connected by continuous lines. 108 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA TABLE 4. Functional and Structural Anatomy Functional Catalytic domain Amino triad Oxyanion site Trp5j, Leu” ’ Glu9’ Pro’57 ArglR; Vallzb up to up to up to up to et al. LPL Gene acid residues implicated Reference Ser’32, AsP’~~, His*4’ hole Lipid-binding of the Human V. Murthy Kirchgessner Emmerich van ProYi Prolh” Ile” ” Gly” q et nl., 1989; Faustinella et al., 1992; Faustinella Tilbeurgh et al., 1994 1990; Hide et et al., 1994 Winkler et al., van Tilbeurgh et al., 1991a,b; et al., 1992 al., 1992; Ser*“ ” up to Leulj’ Lid Cys?‘h p-loop Apo Glyj’ CII binding site,’ Heparin-binding LYS clusters van Tilbeurgh up to Trp”4 et al., 1992; et al., 1994 14'_Lys14" Yang et al., 1989; Davis zyxwvutsrqponmlkjihgfedcbaZYXWVU et al., 1992; Dichek et ul., 1993 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA &I"', Arg""_L 2110, A$82 YS Lys’WArg?W, Arg:“ ‘, LYS Dugi et al., 1992; Tashiro Henderson et al., 1993 up to cysl’q 148 "i-Lys Davis et al., 1992; Berryman and Bensadoun, 1993; Dichek et al., 1993; Hata et al., 1993; Ma et al., 1994a; van Tilbeurgh et al., 1994 LYs’O” Arg151 Lys’“ ‘, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Arg4”;, Lys4”‘, Lys”l) Lys+“-Lys4’+ Glycosylation Initial Asn-“ -His44-Ser-‘5 Asn~jY_Lys’h@_Thr’hl sitesh interaction Dimerization C-terminal residues with lipoproteins Semenkovich et al., Busca et ul., 1995 56 amino acid 1990; Ben Zecv et al., 1994; Wong et ul., 1991; Dichek et al., Bengtsson Olivecrona, 1993 site van Tilbeurgh et ul., 1993; Lookene and 1994 Intact carboxy terminal folding domain Williams et al., 1992; Al-Haideri et nl., 1993; Nykjaer et ul., 1993; Zhang et al., 1994a Prolines Residues: 19, 31, 66, 77, 95, 122, 157, 160, 168, 173, 190, 199, 207, 214, 258, Bruin Cysteines Residues: 27, 40, 216, 239, 264, 275, 278, 283, 418, 438 LPL-mediated lipoproteins uptake of by cell surface receptors 310, 350, 354, et al., 1994a 397, 432 :’ LyslqY of LPL in the human is changed to Thr m the gutnea pig. h Ser” 1s changed to Thr m chicken, and Lys “ ” is changed to Asn m the guinea pig. Asn” ‘-Pro” ‘~Srr” ‘, (Won et al., 1987), may not be eflic~ent because of the presence of proline (Marshall, 1974). Using deletion Henderson of charge mutants affecting et al. (1993) and have periodicity different shown in the of the lid is crucial for normal apical residues contribute tion. Ile225Thr tion that The is located p-5 loop structure that ogy with the &loop rendering the occupies hole necessary LYS ‘t7-Lys’qH, its protein LPL muta- LPL protein to Trph” . In anal- on opening of the LPL lid, the core of the protein, more accessible thus and bringing competent position 1994). The sequence functional One to be responsible CII. The domains of them the N-terminal sites neutral other The in the endothelial These heparin-binding presence consensus proteins, in LPL may heparin binding. confer Additional heparin-binding (in the N-terminal region) Lysj’j, His” ” is the site, tetrapeptide of region). Lys” ‘q, sulfated and A noncharged the consensus including sequence proteoglycans, and up and X is a small are found in apo E and apo B. and CysL;“ regions charged Lys’q’, clusters Lys” “ , upon with Arg’j’ LYS~“ ‘, Arg” Oi, LyP, GIYJ” ~ (in the tetrapeptide, C-terminal Trp’“ P up to Trp’“ j Trp-Set--Asp-Trp, is also and X-B-B-B-X-X- on these Lys” “ , to two heparin- (Cy~?~‘-Cys?‘~ are: for the wall. The sequences activity in addition and residue positively in medi- LPL. hypothetical stability site for the interaction X-B-B-X-B-X charged of two S-S bridges Cys?“ ‘) and vessel to the sequences, B is a positively residue. implicated CII wall glycosaminoglycans correspond consensus where apo sites are essential cell of LPL as a pClsn1ble glycosylatlorl has been between of activities for the binding C-terminal with localization B-X, Glu-Glu) interaction heparin-binding of LPL possible catalysis. apo the binding in the Hisi in a complex for the to this func- human loop a catalytically multiple believed activator, upon into is a participant those that seg- that of apo CII (Lys-Gly ating region. region site even et al., and has, therefore, to this distal and minimally mobile the may fold back the active Tilbeurgh LPL within is another of the lid, maintenance and catalysis occurring PL, it is supposed oxyanion (van is a naturally the proximal ments of the loop parts that proposed present capable in the with of binding C-terminal Human region. Lipoprotein Lipase Gene The relative importance arin binding 109 of these various sites in hep- is not clear, although it may be presumed that they may have differential activities, depending ent reaction conditions. has no significant As compared interaction symmetrical charge distribution Two putative glycosylation shows a more (van Tilbeurgh et al., 1994). sites have been identified in human LPL. In the absence of glycosylation at the N-terminal domain (Asn” up to Serq5), LPL is completely the enzyme is not secreted trast, glycosylation inactive and (Ben Zeev et al., 1992). In con- at the C-terminal domain (Asn3jy up to Thr36’) does not appear to affect either enzyme activity or secretion (Semenkovich to be mediated 56 amino acids (Lookene by the carboxy terminal change the last Olivecrona, of LPL with the lipoproteins 1993). is supposed in the enzyme mole- cule, leading to the opening of the lid that normally triad. This opening masks exposes the hydrophobic residues of the lid in a process called interfacial and Pro- Prolines 10 (4 SS) 10 (4 SS) 20 21 14 (6 SS) 26 0 0 6 (2 SS) 11 10 16 10 (4 SS) 8 (4 SS) 8 (4 SS) 7 6 8 8 (4 SS) 4 to: Common to: hydro- the region containing and Bengtsson to result in a conformational the catalytic Unique LPL HL PL LPL, HL and PL domain of LPL, particularly interaction Total number LPL HL PL of lipoprotein substrates with LPL, The initial interaction of Cysteine Cysteines LPL and HL LPL and PL HL and PL which is a necessary prerequisite for their subsequent The and Distribution in Human Lipasesa Residues et al., 1990; Ben Zeev et al., 1994; Busca et al., 1995). lysis, is believed line upon differ- with LPL, PL, which with heparin, TABLE 5. Frequency activation ;’ Derlvcd from Fig. 1. SS, disulfide linkages. 3.2. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJI Molecular Phylogeny of Lipoprotein Lipase The amino acid sequences of chicken, guinea-pig, mouse, rat, bovine, and human LPL are shown in Fig. 2. There are two extra amino acid residues at the N-terminus of the mature LPL of bovine, sheep, and chicken. also 15 additional In chicken, there are amino acids following the stop codon of (Tashiro et aI., 1992) and forms a cleft lined by the hydro- the human LPL. In rat and mouse, there is deletion of the phobic amino acid side chains of the LPL backbone residue corresponding amphiphilic or the helices of the lid (Dugi et al., 1992). The fatty to Asn444 in the human. The sheep, rat, mouse, guinea-pig, and chicken LPL mRNAs code for acid chains of TGs are thought to bind to this hydrophobic 28, 27, 27, 17, and 23 amino acids of the signal peptide as cleft, with the glycerol moiety occupying the oxyanion hole. compared The hydrolysis of the TG is then brought peptide has not yet been determined. participation of the catalytic 1985). Both head-to-head (Osborne et al., and head-to-tail dimeric forms have Based on structural pancreatic lipase, van Tilbeurgh analysis of LPL and et al. (1994) have proposed a in exon 1 (Glys in the ox replacing Arg5 in the sheep). The 447 residues. In spite of these differences, there exists a very high proportion degree of sequence domain acid residues in the rodents, rat and mouse, differ from each other in 11 of their each monomer with the C-terminal signal The bovine and sheep mature protein, differ from each other by only one residue head-to-tail dimeric form in which the N-terminal domain of is in contact LPL. The bovine LPL, both of which have 450 amino The active form of LPL is a homodimer been considered. about with the triad. with 27 in the human of identical amino acids (92-94%) similarity (96-97%) and high in the LPLs of the of the other in such a manner that the heparin binding sites rodents and the ruminants are available for reaction and both lids are free to open upon the human interfacial in the guinea-pig (87% and 92%) and even less in the chicken activation. the two monomers The specific amino acid residues in that may participate in dimer formation In addition lipoproteins identity (74% and 82%). These differences consistent with their evolutionary have not been identified. to its action role as an intermediary (sheep and ox) as compared with (Table 6). The on TGs, LPL may also play a in the uptake and degradation by cells. Although of LPL, by itself, may mediate by the Dayhoff The distance proportions and similarity between are less the species are divergence, as determined (Hide et al., 1992). of amino acid residues in the various LPL species, identical or similar to the human enzyme, are the binding of lipoproteins to cell surfaces by the interaction the highest in the middle exons and they tend to be lower of the free heparin binding sites of the LPL-lipoprotein towards the N-terminal plexes with the membrane proteoglycans, com- it appears that or C-terminal exons. The similarity is even greater if only those regions of the LPL molecule cell surface receptors, such as the LDL receptor or the LDL are considered receptor-related tions of the enzyme. There are also specific amino acids, such as proline and phenomenon protein (LRP), may also have a role in this (Williams et al., 1992; Nykjaer et al., 1993). Using various deletions, spanning positively charged amino acids, in the C-terminal region of LPL, Zhang et al. (1994a) found that the binding of lipoproteins dent of catalytic function of LPL, but was dependent and heparin to LRP was indepenbinding properties on the region Ile404 up to Lys4’4, which includes the very highly conserved residue Glyq” “ . that are presumed to subserve specific func- cysteine, that may have a disproportionate tein conformation and remain unchanged influence on proeven within cer- tain regions of the LPL molecule that are not generally well conserved. For example, Proi60, Proiij, ProZoi, and Pro214 of LPL are not only conserved in all species of LPL, but also in HL and PL (Figs. 1 and 2). Prolines in positions 95, 157, Exon 1 hLPL: - ------ N- _--__-_fJ-- "--__--___ _-sy_----- TlhLPL: ADQRRDFIDI I bLPL:DRITGGK--R-sLPL:DRITRGK--R---GG---S-rLPL: --AG-m-S-mLPL: gLPL: -NCQK-YT-cLPL:SDPEAEMN-EG- ESKFALRTPE DTAEDTCHLI ----------------se- ------------------- ____----___-------- PGVAESVATC HFNXSKTFM _--T----N---T-_--N- __-----___-----_--- --L-j-j--SN- --L-j-,--SN- -----_---v ----- ------------------ V -----R-m-----S----A N-"------EPD--V-Y-V --QMD-L-Q- N---T----V QEHYPVSAGY TKLVGQDVAR _--T----N- +--- -----i---____-,--------A---- hLPL: hLPL: YEVPKLVA ALYKREPDSN VIVVDWLSRA bLPL: sLPL: rLPL: mLPL: gLPL: cLPL: _--------________-_ _____----------------------- --------__ _____----__------__ --------__ _-------__ -------_-- zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFE -Q-----------m----K -M---AD_-----------Q----___---------K +,--_f(D-----____y--Q____--------N__-_ ----L_,____ _------y__ -a----____ -----N_------_+__K -------R__ -H---E--D_ -----E___- _--------D -----___-- __A_-e-V-- -Q------A- -----K---M Exon hLPL: hLPL: bLPL: sLPL: rLPL: mLPL: gLPL: cLPL: ---------- -----R--S- 82 YPLDNVHLLG YSLGAHAAGI A A --- G -------------_--- G --------------- 4 -E-------_ AGSLTNKKVN RI ------------------- ------------------- --------._v ---------- ---------v ---------- -sv-----_- -------m-V ---N-_-__- ---------- __f_______ --D------- -C---T---G NFEYAEAPSR LSPDDADFVD ------------------- ----_____----___--- ---------- ---------- ---------- ---------- ---R--T--S -------T-s ------Q--- -----K-_-- T----D--I- ----______ Exon hLPL: R _-Q-_--E_- __-TE---_- -a-ES-L-@- -----S---- 0---T----- 5 - -_--__ a- EM zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA C I hLPL: VLHTFTRGSP GRSIGIQKPV GHVDIYPNGG bLPL: sLPL: rLPL: mLPL: gLPL: cLPL: ---------_ -_--__---_ ---------_ ------------------- ____-----____--------------------------------- ___----__----------L-__-----+-------__-----_-----------L___-----I-------------------------------K ------------------------------t------------------------ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA S-------QD -L---SQK-F _ M------- ----y____- D--------- --I-----__ TFQPGCNIGE G_____-L__ AIRVIAERGL -L-L---K-F GDVDQLVKCS S _------f i3 hLPL: hLPL: bLPL: sLPL: rLPL: mLPL: gLPL: cLPL: --HT-----N B5 HERSIHLFID A CA------R_ TH---P---- ______---- ----- SLLNEENPSK AYRCSSKEAF 0 EKGLCLSCRK 0.4 NRCNNLGYEI -_--V----_ ---------- ----N---_- ------____ ----- -_--V----_ _-------__ ----N-m--- ----__---- -----M---- ---------- ---------- ----N--___ ------____ ----- ---------------------------- --------__ ------------Y--K--M ----N-m-----_N-------_NT_--- -_____--------_--------_---_ P ---- M ---- V -----___---------V__---------KV S__------NKVRAKRSSK ---------___---------------- -__----_----_------R--T--NT- Human Lipase Gene zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Lipoprotein Exon 6 --------em hLPL: ----__---- ---T-____- ____------ 1 _I@------ YKVFHYQVKI HFSGTESETH TNQAFEISLY GTVAESENIP FTLPEVSTNK ------- _______N-y -____----- ----_----- -- ----_-- -------N-y _____----_ ____------ -- ------- ------- ------NDKQ N------m-m ____------ -- ------- I _____-- -- I hLPL : bLPL : ---______- -- SLPL : ---------- -- rLPL : -________- -- mLPL gLPL : : CLPL : MYLKTRSQMP Exon 111 7 I i A2 -----__ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA ------NGKQ H------s-m ____------------- -----____- -- ------- ---------- -- ----e-s y----_TT-y ---------- ---------- -- --____A__- -- ----_-- --F-KTNV-K VD-p-L---- --LD------ --+___s-- ------__-- l-3 -fi-----___ ---------_ Exon --------- ----A-N t a --- hLPL: V----- 8 V --- -1 ------ hLPL : TYSFLIYTEV DIGELLMLKL KWKSDSYFSW SDWWSSPGFA IQKIRVKAGE TQKKVIFCSR bLPL : ___--L---_ ---------- --I---__-- _N--_----D _G__------ --- -----L---_ ---------- -- -N-------D -G-- sLPL: I ------- : ---------- ------- M -- ---------- -----_-s-v _E__------ mLPL : ---------- ------- M -- --M_------ p----_-s-v _ER_------ gLPL : ---------- ---------- --ITE-____ -S--GR-T-T -E-_------ CLPL : _F-------_ --- Q-EK-TF--- -N--TPFA-T --RV---S-- hLPL : ---------- ---------- hLPL : EKVSHLQKGK bLPL : --M-Y----- SLPL : : : _--------_ DR-------- ---X-__ _____----_ DS-------- ---X--- ----K--__- EA P--___-- DGS-R-G--E EA-I---- D ------ Exon mLPL ------ 9 ------ R --M-Y--__- gLPL: CLPL ------ ______ rLPL rLPL : FIGURE 2. Amino sLPL, rLPL, mLPL, acid replacements in LPL sequences of various gLPL, and cLPL refer to mature LPL sequences species and in human mutant LPL. Note that hLPL, bLPL, from human (Wion et al., 1987), bovine (Senda et al., 1987), sheep (Edwards et al., chicken (Cooper etal., are aligned according acids that differ from 1993), rat (Bra& et al., 1992), mouse (Kirchgessner et al., 1987), guinea-pig (Enerback et al., 1987), and 1989), respectively. The human mutant LPL molecule is indicated by hLPL (in italics). The LPL sequences to Hide et al. (1992). The amino acid sequence of the hLPL is shown in full; for others, only those amino the human sequence are indicated at the appropriate sites. The nine coding exons of human LPL are identified by the corresponding numbers at their C-termini and a vertical line following each number. When a vertical line passes through the letter symbol of an amino acid, it indicates that the codon for that amino acid is split between two successive exons by an intronic sequence within the gene. Mutations in the human LPL gene involving amino acid substitutions are indicated by amino acid letter symbols, stop codons by p, frame shift mutations by 0, and silent mutations by a. Specific amino acid or polypeptide regions of the human LPL protein suspected of being involved in the spatial structure and functions of the active enzyme (Table 10) are overlined with the following identifications: (4) N-linked glycosylation; (g) lipid-binding domain; (c) lid; (B) the p-5 loop. The th ree amino acids of the catalytic triad (Ser13Z, As~‘~~, and HisZG1) are denoted by solid triangles (A). Amino acids of the oxyanion hole are indicated by open triangles (A). The four cysteine pairs involved in disulfide linkages are denoted by solid circles (o), and the corresponding pairs are connected by continuous lines. and 190 of LPL are conserved necessarily lines in other in positions LPL by alanine plete loss prolines ment no 168, effect by another tion) or replacement of another but not and of pro- 310 of human to result in almost similar substitutions but 199, 350, of LPL, substitution 214, 258, reported activity, 173, significant of proline in all species Site-directed 157, 190, 207, has been of catalytic 160, to have lipases. 397, and 432 et al., 1994a). amino acid (Type com- may both LPL Two naturally activity. mutations are Prol57Arg activate enzyme activity present in human replaced in the Replace- animals of A substitu- acid by proline substitution) found were (Bruin amino zyxwvutsr (Type B with B mutations are Ser266Pro undesirable occurring evident in humans and consequences on Type A human LPL and Pro207Leu, (Table LPL (Pro’??, natural no have LPL Pro’@, that Proj9i molecules adverse Leu286Pro. both 7). Certain effect. of which other and Pro432) are of other Examples lead to the inactivation This in- prolines loss of enzyme species of of Type of LPL activity 112 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA TABLE 6. Amino Acid Replacements in the LPL of Different Species as Compared V. Murthv et al. zyxwvutsrq with the Human Enzymea Exon 1 LPL (species) Uncleaved 27 2 UK? 4-o-4 0 0 54-2-5 60-3-l 87 91 93 37-1-1 95 98 97 4-o-4 0 0 54-2-5 60-3-l 79 82 87 91 93 98 37-1-1 95 27-3-2 81 93 2-o-o 100 100 54-3-5 85 91 60-3- 1 37-1-o 93 98 97 100 27-3-2 2-o-o 54-3-5 60-2-I 37-1-l 81 93 100 100 85 91 95 98 95 100 97 100 17-l-16 0 6 2-1-o 50 100 54-4-6 81 89 60-l-4 37-4-4 78 89 78-4-6 87 92 23-2-21 4-I-3 0 25 54-6- 16 59 60- 3-4 88 78-8-5 83 70 93 37-1-2 92 95 Human Number of amino acids Bovine T-C-NJ % identity % homologyi Sheep T-C-N % identity % homology 28-l-5 Rat T-C-N % identity % homology Mouse T-C-N % identity % homology Guinea-pig T-C-N % identity B homology Chicken T-C-N % identity o/ ohomology Exon Signal 0 9 2 3 4 5 6 7 8 Mature protein 9 Functional regionsb 60 81 61 35 54 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 37 78 40 448 Y7 92 93 78-1-O 99 100 81-3-O 96 40-O-2 95 61-2-3 92 100 95 95 78-1-O 99 100 81-3-O 96 100 40-O-2 95 95 61-2-3 78-1-O 99 81-2-O 98 100 40-3-2 88 95 61-3-l 78-0-O 81-I-O 40-I-4 88 90 61-4-3 81-2-I 96 09 40-O-4 90 90 61-2-Y 8 l-4-8 85 YO 100 Y9 100 94 35-2-4 83 89 450-14-20 92 115-3-O 96 100 35-2-4 83 89 450-14-20 34-o-4 88 88 447-16-13 34-o-4 88 88 447-12-18 93 96 34-3-3 82 91 448-21-37 82 85 87 92 115-4-10 88 Yl 40-4-8 61-7-10 50-7-26 465-4 l-82 I 15-5-h 70 80 72 34 48 74 82 YO 95 92 95 93 98 89 95 84 96 115-3-O 92 96 97 100 115-2-l 94 97 97 99 115-2-O 98 100 ,’ Derived from Fig. 2. ” Onlv those regions of LPL presumed to participate in specific funcm)ns (If rhc CIIZWWare taken into ionslderation (Table 4. Fig. 2). LWhen an amino aud m human LPL 1s coded bv a codon split between two successive axons, due to an ~nrervcn~ng sequrnce (intron), it IS nrbitrarlly assigned to the following cxon in calculating the number of amino acids III each coon. The same rule is applied to other LPL sequences, in the absence of specific mformation on their gene organization. J T 3C I and N refer to the total number of eron ammo acids and the number of ronscrvat1ve or nonconscr~x~v~ replacements in the var~~b LPL m&c&s. cThe bovine signal peptide has nor been idenuficd (Senda er ui., 1957). f Homology is calculated by adding the number of ammo acids that are &ntlcnl and those that represent conservative suhstlturlons. The amino xld changes are classified as conscrx~ative or nonconservative from the Venn diagram of ammo acid subsets &rived by Taylor (lY86), hxxd on Dayhoff’s amino acid changes are arhltrarily defined as those involving not more than a single property. mutation odds matrix (Davhoff et al., 1978). C onservative Substitutions of one amino acld by another differing m more than one propertv are rlarsed as nonconscrwtlw. Extra amino RCI~\ or deletions, compared with the normal human LPL, are also considered nonconservative seems line to be due to the appearance and not because due substitutions amino acids other species of LPL On the other line with at certain present in Ala” ’ to inactivation same sites species the effect but of the and LPL (Ser’“ ‘, apparent human of the importance (Table acid effect 7). by pro- appears to are naturally Ser’“ “ , Alaq” , may and not always also on the nature For example, its replacement by in other molecule substitutions proacids, Leu?“ ” activity of an amino of substitution involved. amino naturally but may depend has no LPL, S@h occur in the such additional original loss of catalytic LPL residue by proline in the chicken In view no due to proline, of the other the proline because However, be entirely of the substitution other some of a new loss of than hand, be well tolerated Se+?). to the replacement because of it occurs by threonine leads and proteins, residues in the for- stability of the three-dimensional it is to be expected by amino acid Replacement occurring lytic activity LPL inactivation (Table highly in conserved bridge and is close ing that 8). There place Arg243Cys, S-S A $” in human resulting residues SerliZCys, would by serine of Cys” ” Pro’“ . to His?“ ’ by cysteine (Table the of other amino with Ser’;’ encloses amino triad. acids, in enzyme cases of human introduction is in close of new acids, e.g., is next to the proximity the to an lid sequence, Substitution such and/ or of as histidine inactivation, the S-S bridge as as LPL. to loss of cata- are also known that structure, of a naturally leading from of of cystcines such is the cause of the catalytic 8), results the enzyme LPL, Arg” ’ or other interference affect and SerZ5ICys. (Cy~~‘~-Cys!~‘) structures loss or gain of a multidomain mutation cysteine that substitutions well as the function, or leucine LPL. of cysteine mation indicatthe His” ’ Human Lipoprotein TABLE 7. Various LPLs A mino A cid Substitutions as Compared Proline 113 Lipase Gene with A mino to other amino Involving the Human in TABLE 8. Various LPLs amino A mino amino acids A cid Substitutions as Comoared Cysteine acid to proline or other acids Proline Species with Involving the Human A mino to other acid other acids Replac- ing ing ing amino amino amino amino LPL species Site Ala’ Ala’ Ser guinea-pig Ax human Thr guinea-pig Thr12 LW human Phe chicken Arg Ser rat mutant mutant acid LPL k-0 chicken bovine, Se+ Ile Pro Pro Se+” Thr Site species acid Cys216 or acids Ser LPL soecies Site acid human mutant Glu3 Ser’7L Cys guinea-pig CYS human human human human sheep LPL ArgZ4j Cys Arg2q3 His Arg2q7 Leu Serz5’ CYS chicken human mutant chicken soecies mutant mutant mutant mutant human mutant human mutant Pro bovine, sheep Met LeuL8” All armn~ acid residues are numbered as in human LPL. Conserved cyst&e rat, guinea-pig Val Led”6 guinea-pig, sites in LPL are 27, 40, 239, 264, 275, 278, 283, 418, and 438. chicken Pro chicken zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Ala”+ Thr Aias3’ human mutant Pro Serj”’ mouse Pro chicken Ser”h new splice sites and leading to the formation of aberrantly Ser396 A rg guinea-pig Ala”’ Pro guinea-pig spliced mRNAs. In a recent report, Yang et al. (1995) have Ala”’ V al bovine, sheep described a naturally occurring mutation in the LPL gene, Pro Serqq2 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA chicken LeuLsh mouse A la which occurs in the binding All amino acid residues are numbered as in hLPL. Conserved proline sites in LPL are 19, 31, 66, 77, 95, 160, 173, 190, 199, 214, 258, 310, 350, and 354. Ott-I of the catalytic triad may be involved in this action. forms a part of one of the lipid-binding SerZ5’ domains (B5 in Fig. 2). Among the LPL species so far investigated, the guineapig LPL is the only natural molecule that contains 11 cys- teines instead of 10. This extra cysteine, which replaces Glu’ LPL, occurs very near the extreme N-terminus of exon 1 and, therefore, may be expected to have little effect on enzyme activity. site of the transcription and results in a highly reduced Although mutations of the coding factor promoter activity. sequences are often revealed by their effects on enzyme mass and/ or function, mutations of the noncoding of human in Replac- Replac- ing acid to cysteine amino Replac- Site Cysteine Soecies sequences may have more subtle effects and may require indirect and investigation. The approaches occurrence for detection of alternate types of nucleotides in the same position in the nucleic acid sequence, with no concomitant apparent phenotypic generally referred to as polymorphism. differences, is Such polymorphisms in nucleic acids can be detected easily if they lead to alterations in restriction in the noncoding sites. In fact, many of the polymorphisms sequences of the human LPL gene, which have now been characterized in terms of the actual base change, were originally detected by restriction fragment analysis and, even now, continue to be identified in the same 3.3. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Mutations Involving the Noncoding manner. A number of studies have been published supportSequences of the Human Lipoprotein Lipase Gene ing or refuting claims that certain restriction polymorphisms Although intron nucleotides do not code for amino acids, at least some parts of their sequence, particularly the intron-exon junctions, ing of the mRNA play a critical role in the process- precursor the coding exons. Mutations affect the maturation products sequences also contain ing the transcription human pathologies in humans. been reported on the possible association polymorphism therefore, of the mRNA, and the nature may as well and number 5’ and 3’ noncoding various regulatory elements, initiation site, polyadenylation includsite, etc. of the elements present in the LPL gene), whose activity may be influenced tively or negatively by a given polymorphism. posi- Several muta- ciated with hypertriglyceridemia introns or in the flanking regions (Table 9). Some of these occur in, or near, the splice acceptor or splice donor sites and are found to interfere with gene expression by creating (Chamberlain et al., 1989; Ahn et aI., 1993b), levels of total and HDL cholesterol mann et at., 1991; Mitchell et al., 1994), coronary (Heiz- heart dis- ease (Chamberlain et al., 1989; Thorn et al., 1990; Mattu et al., 1994), and insulin resistance (Ahn et al., 1993a; Cole et al., 1993). Even though the association remains to be clarified of et al., ences in test subjects. The Hind111 site is reported to be asso- transitions sequence between the PvuII 1989; Ahn zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK et al., 1993a), which may be due to ethnic differ- lar LPL polymorphic in the nucleotide investigated and plasma TG levels (Chamberlain tions have been discovered in the human LPL gene involving or transversions The most extensively of these are the PvuII and Hind111 sites. Variable results have in the introns, formed. The (see Table 2 for a description of the LPL gene may be associated with various lipid-related and in the correct splicing of and turnover as its size, its translatability, of the protein those near between a particu- site and a pathology whether is confirmed, this association it is due to the LPL gene itself or due to its metabolic or genetic linkage to a separate unidentified sible for the observed gene that may be primarily responabnormality. 114 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA TABLE 9. M utations in the Noncoding Sequences of the Human V. Murthy et al. Gene zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK LPL Restriction Location A lternate Type Intron 1 (SD Exon 2-Intron 1 bp SB ACCgta 2 (SD site)” 1 bp SB GACGgt 3 (SA 1 bp site) Intron 2-Exon Intron 3 (20 bp from SD site) site)h SB 3 (SA 3 (6 bp from SA site) site) aaGTAAC Hata et al., 1990a GAGCCT Gotoda 1 bp SB G - 6 (SA 1 bp SB C-A Intron 6 (1.57 kb from SA site)” 9’ - tttcagGT A et al., 1992 Holzl et ul., 1994 (TTTA)n, AAGCTT - TAGCTG Fisher rt al., 1987; Li er al., 1988a; Gotoda et ul., 1989; Oka et al., 1989; Gotoda er al., 199213 PVUII n = 9- 13 - Zuliani and Hobbs, Ahn et al., 1992 AAGCGT 101: 1990; Hind111 Heinzmann et al., 1987; Oka et al., 1990, 1991; Gotoda et al., 1992b XbaI Heizmann BstNI Funke BamHI Fisher et al., 1987; Chamberlain et u1., 1989 Li et al., 1988b Hegele et al., 1989a Hegele et al., 198913 BgIII PstI TaqI element” 1992 et ul., 1994 Wiebusch BstI Promoter et al., Nakamura Mb011 Inn-on 9 3’ flank of exon rt ul., 1992b Wiebusch CAGCTG Repeats Intron Gotoda zyxwvutsrqponmlkjihgfedcbaZY et cd., 1990, 1991a,b GACGat - 4 (SD site) 8 (495 bp from SD site) - - Intron Intron Chimienti et al., 1992; Pepe and Chimienti, 1993 ACCcta agGTAAC Intron Intron 6, at the 3’-end of an alu sequence’ Reference GAGACT cttcagGT site)’ site C -T 1 bp SB Intron Intron - forms et al., 1991 et cd., 1988 Hegele et al., Repeats (CA)n Narcisi 1 bp SB T-C Yang 198913 er al., 1993 et ul., 1995 L’This mutatwn, involwng the first nucleotide of intron 2 at the junction of exon 2 and intron 2, creates multiple cryptic splice sites, givq rise to several aberrantly sphced mRNAs. h This represents a transition mutation of the last nucieotlde of mtron 2 in rhe splice acceptor we at the lunction of intron 2 and exon 3. LThis mutation causes aberrant splicing, resulting in the deletion of c‘xons 6 through 9 in the mRNA. d The region containmg the PvuII site resembles the splicing sire in its homology towards the consensus sequence required for 3’-splicing and formation of lariat structure, suggesting that the C - T change may interfere with the correct splicing of mRNA. v A total of five different alleles were found containing this tetranucleotide repeat. ‘Occurs only among Blacks, not in Caucasians, in the populations surveyed. c Two IOCI of CA repeats were found with a total of 10 d&rent alleles. ‘>This mutation occurs at the blnding site of rhe transcriptlon factor Ocr-I (nt-39 starting from the major transcriptional start site or nt-227 from the ATG initiator codon). It is reported to inhibit promotor activity by 85%. SA, splice acceptor; SB, substitution; SD, sphce donor. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 3.4. Mutations Involving the Coding the Sequences of the Human Lipoprotein Mutations merous Table in the coding thev continue and 10 lists 71 different that affect LPL protein a majority amino the acid of 46, to be detected mutations structure gene at a rapid in the human and function. pace. substitutions, 9 missense mutations involving of an amino of various of various mutations in which the amino codon acid codons stops the 1 bp 1 bp to 2 kb) are altered Although polypeptide the with creation growth to 6 kb), and no 4 5 silent change in of a termiimmediately, lead of interesting presented and even- with is higher be made from in exon number many of and 5 (Fig. sites as exons, exons with those that the LPL are also functional The exons C-terminal 3). The of mutations the distributed. in the middle the N-terminal it is highest highest can 10. are not uniformly of mutations contain to frame-shifts deductions in Table LPL mutations and the insertions of translation. compared by a termination sizes (from sizes (from acid. codon number data density include involving A the and termination The iPL^gene These deletions tual are nu- mutations insertions nation LPL are missense 7 deletions coded of the which substitution codon, regions Lipase Gene of molecule. Some regions, to be more example, and even susceptible 3 of the some specific to mutations 4 amino acid codons, than residues appear others; for situated be- Human Lipase Gene zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Lipoprotein TABLE 10. Mutations Tvue Mutation D9N’ in Coding Sequences of the Human Location Exon MS 2 LPL Gene Base or codon change GAC - AAC Amino acid change - Asp - Asn Reference Rouis et al., 1996; Elbein 1994; Mailly et al., 11 bp DL-FS-Ter Exon 2 Multiple D2lV’ MS Exon 2 GAC - GTC N43S” H44Yi MS Exon 2 AAT - ACT MS Exon 2 - TAC Exon 2 His _ Tyr 1 bp IN CA C _ Kobayashi et al., 1994 Gag& et al., 1994 Y61Ter Ter 3 3 TAT - TAA Tyr Ter Foubert Gotoda TGG - TGA Trp - Ter Sprecher 3 GTG - CTG Val - Leu Bruin W64Ter Ter V69Lh MS Exon Reina Multiple Asp Asn - Val Ser - et ul., 1995 T18” Exon Exon 115 et al., 1992 Gag& et al., 1994 et al., 1994 et al., 1991a et al., et al., 1992 199413 Y73Ter Ter Exon 3 TAC - TAG Tyr - Ter Wilson et al., 1993 R75S W86R MS Exon 3 AGA - ACT Arg - Ser Wilson et al., MS Exon 3 TCG - CGG Trp - Arg Ishimura K102’ 5 bp IN-FS-Ter Exon 3 Multiple Q 106Te@ Ter Exon 3 CAG - TAG Gln - Ter Emi et al., 1990a; Ishimura Oka et al., 1992a Vlcw Silent Exon 3 GTG - GTA Val - Val Gagne Multiple 1993 Oka et al., Henderson et al., et al., 1992a 1990 1994; Nevin et al., 1994 _ 2 bp IN Exon 3 E118E Silent Exon 4 GAG N120-Y1219 Exon 4 Exon 4 A A CTA C - AC CAT - CGT Multiple H136R 4 bp DL-FS-Ter MS His - Arg Deeb et al., G139S’@ MS GGC CGA - AGC GAA Ser MS 4 4 Gly G142E” Exon Exon Gly - Glu Bijvoet zyxwvutsrqponmlkjihgfedcbaZYX et al., 1994 Ameis et al., 1991 v149v Silent Exon 4 GTG - GTC Val - Val Elbein G154P MS GGC CAT - AGC AAT - Ser Bruin MS 5 5 Gly D156N” Exon Exon Asp - Asn Ma et al., D156G” MS Exon 5 CAT - GGT MS Exon 5 CAT - CAT - Gly His Faustinella D156H” Asp Asp PI 57R” MS Exon 5 CCA - CGA Pro - Arg Bruin E163D s172clj MS Exon 5 GAG - GA(T/ C) Glu Asp Gehrisch et al., MS Exon 5 TCT Hayden and Ma, - - GAA TGT Foubert Glu - Glu - - Ser - et al., 1994 Gagne et al., Emi et al., Cys 1994 1990a 1991 et al., 1994 et al., Foubert 1993 1992a et al., et al., et al., et al., 1992 MS Exon 5 5 A CA GAG Beg et al., Exon - Thr Asp Glu Haubenwallner H183QlS MS MS GCA GAC Ala - D180G” Exon 5 CA C - CAG His - Gln G188E’” MS Exon 5 GCG - GAG Gly Glu - G188R’4 MS Exon 5 MS Exon 5 GGG ATT G195E MS Exon 5 CGA - GAA Gly - Glu H202H Silent Exon 5 CAT - CA C His - His - (A/ C)GG ACT Gly - Ile - D204E2’ MS Exon 5 GAC GAG Asp 1205Sz2 MS Exon 5 ATT - ACT Ile - P207L:’ MS Exon 5 CCC - CTG Pro - - Arg Thr - Glu Ser Leu 1990 et al., et al., Emi et al., 1990b; G2092” 1 bp DL-FS-Ter Exon 5 CGA MS Exon 5 TGT - GG ACT Multiple Cys - et et al., 1992; Henderson et al., 1992 Foubert et al., 1994 Dichek er al., 1991; Henderson et al., 1991; Ma et al., 199413 Hata et nl., 1992 Gehrisch et al., 1994 Gotoda Reina et al., et al., Ma et al., Wiebusch Ser Hayden et al., A 221L” 1 bp DL-FS-Ter Exon 5 GCT - CT Multiple MS Exon 5 ATT - ACT Ile - Deeb Thr 1991a 1991; Normand et al., and Ma, Major C239Ter2” Ter Exon 6 TGC - TGA Multiple R243HL4 MS Exon 6 CCC - CA C Arg - His R243P MS Exon 6 CCC - TGC Arg - Cys DL Exons 3-5 Multiple Multiple et al., et al., 1994 1992 1992; Ma 1992 ec al., Henderson et al., 6 kb DL Monsalve 1992 1991; Goroda 1992a; Takagi 1225T2’ 1993 1994 1990; Bergeron 1992; Levesque C216P 1992; Ma Tenkanen al., 1194T’@ 1994 1993 A176T’” - 1991a 1994 et al., et al., et al., 1992 1993; Ma 1993 Langlois et al., 1989 1994 Dichek et al., 1991; Gotoda et al., 1991a; Ma et al., 1994b Ma et al., 1994b Takagi et al., V. M urthy 116 TABLE et al. 10. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA (continued) Base or M utation Type 6 Exon 6 Exon 6 D250N” MS MS MS S251C L252A’: S259R MS MS MS A26lT’: Y262H” Y262Ter’ ’ S266P’” L286P N291S” MS MS -I-U MS MS MS 2 kb IN’j Duplication A3 34Tlh T3521 L353’7 T36lT MS MS 2 bp DL-FS-ter Silent L365V’” MS W382Trr’” W382Ter’z,“’ Ter Ter E410Vf” MS S447Ter-” Ter 3 kb DL’? Majpr DL R243LL” S244T’O The amino acid residue (ATG), when mentioned Location change CCC - CTC TCC - ACC GAC - AAC A mino acid change Reference Arg - Leu Ser - Thr Asp - Asn Appelman et al., 1994 Hata et al., 1990a Ishimura Oka et cd., lYY2b; Ma et ul., 199213 Wiebusch zyxwvutsrqponmlkjihgfedcbaZYXWVU et cd., 1992 Exon 6 TCT - TGT Ser - Cys Leu - Arg Exon 6 CTG - CGG Ma et al., 1994~ Exon 6 ACT - CGT Ser - Arg Wiebusch et al., 1992; Foubert or AG(A/G) et al., 1994 Ala - Thr Ma et al., 1994~ Exon 6 CCC - ACC Tyr - His Rouis et ul., 1996 Exon 6 TAC - CAC Tyr - Ter Funke et al., 1990 Exon 6 TAC - TA(A/G) Wiebusch et ul., 1992 Exon 6 TCC - CCC Ser - Pro Foubert et ul., 1994 Exon 6 CTG - CCG Leu - Pro AAT - ACT Asn - Ser Wiebusch et al., 1992; Ma et al., Exon 6 1994~; Reymer et al., 1995 Langlois et ul., 1989; Devlin Exon 6Multiple Multlple er al., 1990 Inn-on 6 Ala - Thr Kobayashi et a!., 1993 Exon 7 GCC - ACC Thr - Ile Wiebusch et al., 1992 Exon 7 ACT - ATT Wiebusch et ul., 1992 Exon 7 CTG - G MultIpIe Thr - Thr Exon 8 ACC - ACA Reina et al., 1992; Gagne et al., 1994; Gehrisch et al., 1994 Exon 8 CTA - GTA Leu - Val Wlebusch et al., 1992; Pepe et UI., 1994 Exon 8 TGG - TAG Trp - Ter Ma rr al., 1994~ Trp - Ter Gotoda et cd., 1991a; Kozaki Exon 8 TGG - TGA er al., 1993 Wiebusch ec al., 1992; Previato GAG - GTG Glu - Val Exon 8 et ul., 1994 TCA - TGA Ser - Ter Hara et ul., 1990b; Kobayashi et Exon 9 al., 1992; Kozaki er ucI., 1993 Multiple Multiple Be&an et al., 1995 Exon 9 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Exon numbers codon refer to the mature LPL protein as in Wion et al. (1987). &dons are numbered starting from the imtiator DL, deletion; FS, frameshlft; IN, insertion; MS, missense; Ter, translation terminarion. codon in the following text. ’ Alters : Leads ’ Asp” TaqI sate. Appears to contribute to hypertriglyceridemia in some families. In e‘~tr” expression shows the presence of both LPL mass and activity. to the formation of a truncated protein of only 19 residues. is highly conserved in all species examined, except guinea-pig and chlcken, where it is replaced by Asn or Glu, both conservative subsritutlons. The rrpiacemenr of Asp hv Val residue leads tu a change in charge and generates a new HaeIII site m the exon. in wtro studies show no effect on catalytic activity. i Asn” is thought to be an N-hnked glycosylation site of the mature LPL proteln. Expression studies show that this mutation affects both enzyme actlvxy and secretion. i HIS+~ is a highly conserved residue. His4iTyr represents a nonconser\‘anve wbstltutlon. Found in two Individuals wth famihal combined hypcrlipidemia, but m rlitro studies show no change m catalytic activity. ’ Located in a conserved hydrophobic region of LPL. Expresa~on of the mutant cDNA prepared by site-directed mutagenesis m COS cells shows 80% reducrion in catalytic activity. (ACC ; Found in an LPL-deficient patient of Malaysian descent, it consists of a 6 hp msertior (TGGGCT) at th e site of n single base d&non AC) at the residue Thr”“ in exon 3, causing a frameshlft. This results in a markedly truncated LPL protein rhat rrrminates prematurely in exon 4 with a random sequence of 44 ammo acid residues in the carhoxy terminal portlo”. of the first base of ’ Found in suhlects of Polish, German, and English descent, this mutation leads to a truncated LPL molecule due to substitution with resultmg suhstirution of the Gln codon (CAG) by T. Th c s a me base is replaced by G and by A m normal guinea-pig and chicken LPL mRNA, Gln by Glu and Lys. In rat and mouse, both rhe first and the third bases of this codon are replaced to give Asn in place of Gin. ’ This deletion causes a frameshIft mutation by removing the last two nucleorides of Asn”” (AAC - A) and the hrst Two nucleotides of Tyr”’ (TAC C), producing a truncated proteln of 142 residues, h>Ith substitution of 23 C-terminal ammo acids. This mutation alters the MSEI restriction site. “I Found in an LPL-d&cent indrvidual of Spanish descent. In tiitro mutagenesis shows that this mutation completely abolishes LPL functmn. normal amounrs ” The region surrounding Gly ‘I’ is highly conserved among lipases from dlffcrent specws. Expression m COS-i cells shows that, although of the proteln are produced, It is deticienr m both catalytic acriviry and secretion. ” This mutation affects the last base of exon 4 and is sxuated within the 5’-consensus sequence for splicing. It cgeates a new BfaI restrlction site. It inactive enzyme when expressed occurs withln a conserved P-sheet region close to Asp”‘, which forms a part of the catalytic site and produces catalytlcally in z’ltr” changes of the first base and the third of ” Three d&rent mutations affect Asp”+ of the catalytic triad, SIX”‘, Asp”“, HIS!“, two of them involving the second base of wdon 181 and resultmg in substxution of aspartare by asparagine, histldme, or glyc~ne. All three mutations alter the TaQl restriction site and give rise ta inactlve LPL when expressed m vitro. proline. It alters a PvuII site. ” This muratmn 1s situated next to Asp”” and results in the loss of a conserved Ii Leads ro only partial loss of LPL catalytic activity, compared with several other m~ssensc mutations. ” Also termed LPL-Bethesda, this mutant LPL shows loss of catalytic activity and alrered affinity for heparln. Also alters a SfaNl site. (contmued) Human Lipase Gene zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Lipoprotein 117 TABLE 10. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA (cont i nued) ‘7 Although this amino acid substitution is highly conservative, is no effect on secretion or heparm binding. Ii: His’“ ’ hes close to a putanve lipld-blnding production of lnacuve ” Glyl88Glu, LPL. The mutant highly prevalent domain of the LPL molecule gene is thought in the Province and outside the proposed (B4, Fig. 2), and KS replacement to be of Russian of Quebec, is believed active site of LPL, the catalytic by glutamine triad and the lid that covers the hydrolytic site for phosphorylation heparin or glycosylation affinity. The occurrence to be a ubiquitous and probably ” Alters a Hincl site in cxon 5. 1LIk? 1s a highly conserved ammo Substitutions in two different ancient acid and is situated DNA in strand of Quebec. It alters the BslI restriction The mutation haplotypes is suggested 10 of the proposed in the Northwest leads to charge there alteration region of France, The frequency with pancreatic and to indicate between a multicentric structure strands of occurrence lipase, it is in the proximity group of threonine provides a potential results in loss of enzyme activity, three-dimensional interactions mutation. of the hydroxyl changes. of these residues with other amino acids could disrupt hydrophobic of the central catalytic domain. li This mutation, which appears to have originated lation pocket of the enzyme. Introduction that could induce conformational of this mutanon is abolished; or Swiss origm. and distribuuon of Glyl8HArg is not known. Both mutations alter Sau961 and AvaII sites. x Ilc” ’ is located wthm a highly conserved, putative lipid-binding domain of LPL (84, Fig. 2). By analogy of both the catalync activity but retains normal origin. of LPL (as do Pro’@’ and Asp” +). 10 and 11, thus changing is now found almost exclusively the conformation in the French-Canadian popu- site. after 223 residues. ” This involves deletion of the thnd nucleotide of the codon for Gly” “ , resulting in termination :’ This mutatlon destroys a conserved dlsulfide bridge that may be crmcal for LPL structure and function. “ ~The frameshift abolishes an AluI caused we I; Ile’!i is located the rnportance in the proximal of maintaining !’ This mutation, :” Three by the single bp d&non leads to termination xctwn charge of the amphipathic and periodicity three residues downstream. The deletion of G (GCT - CT) surface loop shielding have been reported the active site of LPL. L oss of LPL activity by this mutation suggests in this region of the molecule. which results in total loss of LPL activity mutations of translation (AGCT). that change Arg” ’ or mass, abolishes an HgiAl site and creates (which is close to the catalytic triad: Set” ‘, an Mbol site. Asp’jh , Hi@‘) to histidine, cyst&e, ot leucine. One of them affects the first base of codon 270 and the other two the second base of the same codon. Al1 three mutations alter HhaI and Eco47111 restriction sites. Arg243His was found m Caucasian, Chinese, and Japanese subjects, and Arg243Cy s was found in those of French and German descent. Haplotype analysis indicates two separate origins for the ArgZ43Cys mutation. This, together with ArgZ43His, suggests high mutability of this CpG dinucleotide in the LPL gene. ‘j’ Also occurs near H&l’ ” The location III the armno of the catalytic of Asp” ” terminal in the proposed region of LPL. The mutation ” L~u’~‘, like the neighboring XI other wld-type AsnZUlSer, rosine by hlsndine that result in parnal and the other effect on dimcr formation. the suhstltutlon serine ” This mutatwn of different - threonine 1s produced ancestries to Ala334Thr, ‘- The codon involved charged involving by alteranon human represents to be associated The Tyr262His formanon 1s conserved ‘! This represents ” This deletion The the longest (2.116 lntron prematurely activities 9, the mutant duplicated has no effect on LPL activny, regions exons by residues wth similar size and hydrophobicity and results in the observed or one of the other three mutatmns of the amino LPL has reduced partial (Ala261Thr, acid Ty?, one leading to replacement heparin-binding activity of ty- and a destabihzmg 6. m the serine codon. T - A change exon 6 and involves exon-Alu because this substitution 7 and 8, but both the deleted normal the end of intron LPL contains of pancreatic in the codon terminated than 8 and an Alu sequence in the same codon interchange. is found bases of the codon results in It is found in subjects in normal are in exon natural member a close undergo of the catalytic neighbor chicken LPL. 7. The frameshift leads m intron mutations. Alu sequence Gly188Glu, Trp3sz. One is very prevalent tion (Bergeron 3. The presence in the Qukbec may affect The the normal mutation of mtron protein generates 9. Of the three suggesting at the C-terminus. a MnII restriction site. AIu sequences present that a stem-loop structure in this mutation. properties at a given position in the protein sequence may be critical for enzyme activity, and replacement by any other residue may not be tolerated. Such appears which is to be the case for most of the human LPL mutations. However, in some cases, LPL from other species are resulting found to contain Two mis- residue whose replacement of normal variants of the amino amino human these, AspZlVal, Leu286Pro, popula- acid residue with unique leads to mutation LPL. Thus, for example, although substitutions and Ala334Thr and Ala334Pro acid in the Asp9Asn, are mutational in the human LPL, Asp9Gly, (Giu), Leu286Met(Val), et al., 1992). of an amino termination. As~‘~~, a affect each of the following Tyr262, and protein. plus the right arm of the second, 9 may have been involved of the triad, in three different amino acid substitutions. Gly’ss, with only two residues less than for the truncated translation of this residue by mutatcon site. 8, the whole of exon 9, and about two-thirds each suffer three missense mutations sense mutations species. Replacement a Mae111 restnction LPL molecule, only the third member hpase in different have been reported triad, and Arg2” , of another and assembly of active LPL homodimers. the second or the thrd base. Both lead to immediate generates and Gly?@“ , and all the 3 successive residues in Asp” 5” -Serzi’-Leu?iZ acids: an cY-helix LPL. and Pro ‘57, 5 of the 8 situated between tween Gly’j’ His!“ , wth mutation. is split between A to T base change kb) includes the end of lntron HiP2 mutant C change of intron 6 to a partially an ancient in LPL and in analogous of homodlmers. in the normal interaction after 355 residues. Less, equal, or higher catalytic hetwccn in a charge of spatial structure with this mutation site in exon ii It is suggested that this mutation interferes with the correct folding “ I Two different mutatwns of this codon have been reported, involvmg ” Glu” “ replacedonly the third base of the codon in an Awl chicken apparently m this mutation is may lead to disruption LPL, is caused by T - in the normal Ala334Pro is reported acid and amino arginine of translation. by the juxtaposition and possibly “ ’ In contrast to termination to termination that inactivates of LPL suggests that it may be involved LPL deficiency. are reported, It can be detected a mutation is a highly conserved chylomicronemia types of substitutions structure alters a TaqI site. with the positively Pregnancy-induced Trp382Ter) ” Two dlffcrent ” Ser266Pr0, AspziO and SC?‘, lipases. Substwutmn loss of LPL actwty. wad. three-dimenslonal AspZlAsn substitutions are normal variants in other LPL species (Fig. 2). 118 ct ul. zyxwvutsrqpo V. Murthv mucosa and response to the consumption cleared appear from (Cohen, the son types lipoproteins, and I is defined the lipoprotein in which lipoprotein the presence I-V), profiles clinical into based manifestations. resulting in LPL chylomicrons The Type activity are the predomi- Type V pattern chylomicrons on of plasma in Types I and V disorders. fasting of elevated Fredrick- disorders (Types disorder, species. hr from com- in the plasma. associated as a genetic deficiency nant classified is present 8-10 results large macromolecular presence in are, then, within and the electrophoretic Chylomicronemia stream syndrome of dyslipoproteinemias the concentrations 0 of these have blood fat. They compartment continued et al. (1967) several the chylomicronemia in the clearance plexes and their in of dietary vascular 1989). The defects transiently is marked and VLDL, by resulting EXON zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA from genetic causes, as well as various secondary causes FIGURE 3. the human Distribution of mutations LPL gene. The number of mutations number of amino mutation in a specific acid residues in different density exon exons is defined divided of as the by the total (Brunzell All LPL mutations the same manner. arin binding 0: these tural domains are also found degrees dimensional A search involving the acid alternate acid and, the therefore, that depending the codon and disorders mature atic studies effects at least protein (Table the same result protein to be affected, the to the rules change and, possible degree the consequence have been conducted rate of the rate of synthesis of such mutations However, to examine polymorphisms func- preference rate of mRNA action. in the but it is con- may influence the turnover 10). of acceptability of codon hence, to amino The on five in regard not of endonuclease of codon three- does the nucleotide sites acid resi- overall are silent specify mutation of mRNA the variable acid by another. in the human, ing codon in lipid syndrome Brunzell, by alter- no system- the quantitative on LPL mRNA CHYLOMICRONEMIA 4.1. Description The chylomicronemia or protein. presence (>15 Diagnosis is characterized hypertriglyceridemia Chylomicrons are formed from (Brunzell the and of chylomicronemia ing the appearance (lactescent, more clinical recently, it is consequences Bierman, white). in the fasting in the the state intestinal After 1982; of Chait for 24 hr, chylomicrons plasma clear (Type in large amounts, V). With an and chylomicronemia, and only in specialized laboratory of plasma of heparin into the plasma assay is also ity can (Peeva U/ kg into body circulation used to determine is released 1992). diagnosis of apo et al., (Hixson gene mutations 1991, 1992b). In the newborn the which releases 1989). LPL LPL 1992). of adipose with primary tissue useful syndrome activation 1990), and detection 10) (Monsalve enzyme LPL activ- techniques of chylomicronemia and Vernier, LPL Postheparin mass by immuno- et ul., of LPL using injection 1987; Huff et ul., 1990), analysis (Table cause procedure, i.v. holus in S.C. biopsies assay such In this defective laboratory CII, available TG substrate et cd., (Brunzell Other of evalua- by measurement after if a catalytically the plasma are is one weight), to measure causes As will be discussed activity. (Brun also be determined et al., usually with a synthetic 10 min to formulate by clinical deficiency lipolytic obtained (50-100 blood LPL (Type frequently exact and can be confirmed is measured samples that layer present plasma may The laboratories. 4.4, surface accuracy can be pinpointed postheparin LPL activity levels sufficient or plasma left in the re- are also lactescent TG techniques in Section VLDL approach. lipid of chylomicronemia plasma with then, tion in detail plasma therapeutic by observ- form a creamy or frankly practice, visually, immediate has been I) or, when a turbid some performed (“ cream of tomato” ) the plasma on CII (Connelly by is easily of the blood frigerator genotypes of marked mmol/ L). SYNDROME syndrome but has been in the plasma Diagnosis 4.2. measurements THE classification 1992). the differential 4. past, of chylomicrons metabolism, arises chylomicronemia be estimated of the of translation in changing to shows that according Another the exons of the the mutations of the amino and struc- the site of a given literature is not expected ceivable may consist functions and of LPL. LPL of one amino Different upon local published therefore, of protein. these probably, sequence forms replacement new affect may be of LPL to different the molecule. structure mutations tion, to of the amino functions and the contribution affected The to depending, mutation due within In the in certain 10). This nature in and hep- and to Table of the multifunctional assignment function secretion, differentially (see footnotes a reflection the activity, are affected combinations LPL do not influence Catalytic 1973). by that exon. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA based on the detection of molecular defects. The chylomicro- coded nemia 4. et ul., made based on the appearance et al., in are by apo of apo E of LPL 1990; Ma et ul., LPL deficiency, chylomicro- Human Lipoprotein 119 zyxwvutsrq Lipase Gene TABLE 11. Symptoms, Signs, and Laboratory Abdominal pains with or without memory Peripheral nemia is present infants very often after early milk, plications of the disease children, diffuse reported (Black patients have and the observation of LPL a less severe first year of life and high (Fig. been early childhood make The nal pain, vious are more TGs patients do not common with comparatively show when clinical plasma signs, TGs and are very 4). major plasma the 1995). However, of plasma complication with levels eating be elicited. of TGs habits, The of chylomicronemia or without pancreatitis, (Gagne et al., unusually precise rich reasons and 1989). in fatty is abdomi- is related to the History of pre- foods, for repetitive can often colicky abdomi- the evaluation have rarely of tomato” tooth patients. Two grandmothers they reported In another that their case, hyperbilirubinemia conditions leading was unusually because than g-o- - 330- and often : n 2O4 L;: lo- of the aspect 0 - et al., 1989). are diverse purposes had led to suspicions (Gagne suspected for other in our investiga- newborn which to diagnosis to LPL deficiency, drawn icterus, in and that have the clinical of LPL deficiency * of LPL deficiency grandchild 60- in blood asthma, a chylomicronemic without and, later, confirmation of the conditions initiated E 5 Physical led to diagnosis LPL ages and, pains. mononucleosis, of primary the 1 ao- 2 was made screening appearance abscess, diagnosis between of abdominal 100 disease. of patients diagnosis or family 1983). to avoid of the in 25% in 50% adults et al., learned form are some of the coincidental blood to com- in young have 70%, findings the of the signs experience, (Hoeg was made In almost “ Cream trauma, unrelated such have and levels from our deficiency fortuitously diagnosis allowed Thus, In 1994a) have clinic, signs of chylomicronemia tion when 1989; Brunzell, lower 1993). In young since et al., and/ or following “ pale? 1993). individuals coincidental pregnancy related hemorrhages such through epistaxis, may refrain problems that 20 years. deficiency. pains, and Sprecher, (I’erron is made of 1 and others, growth diagnosis In our referral during colicky may These progression. patients foods 1977). Sprecher, medical It is presumed fatty diagnosis et al., gastrointestinal school older and (Sadan (Black growth Sometimes, and have and under normal expected feeding, have repetitive drinking loss first findings Pseudohyponatremia Pseudohypocalcemia Pseudo-increased hemoglobinemia Normal amylasemia with pancreatitis Pseudohyperbilirubinemia Splenomegaly birth Syndrome TG > 15 mmol/ L Fasting chylomicronemia neuropathy the after Laboratory Lipemia retinalis Eruptive xanthomas Hepatomegaly pancreatitis Dyspnea Paresthesias Flushing with alcohol be made of Chylomicronemia Signs Symptoms Recent Findings 5. I I I I measurement of lipids. Clinical 4.3. Manifestations 4.3.1. Symptoms plasma triglyceridemia the chylomicronemia presence of one or more in Table 11. However, tions is unpredictable equal lence 1995). of each Thus, of these to or greater syndrome the occurrence for each than patient, and/ or of clinical and by there is no consensus manifestations the signs listed manifesta- some may have no symptoms clinical with 15 mmol/ L, is characterized of the symptoms with severe chylomicronemia (Brunzell, In patients of chylomicronemia. patients or signs FIGURE 4. Plasma TG concentrations ical signs of chylomicronemia mary LPL deficiency. of the first clinical the Quebec asterisk The frequency evaluation Lipid Research (*) indicates Clinic a significant plasma TG concentration compared no clinical * (M ean SD). of physwith pri- was evaluated on the basis (Gagne difference on the preva- signs. in patients of 56 patients, (Gagne et al., and frequency syndrome et carried out at al., 1989). (I’< 0.05) The for the with that of patients with V. Murthy FIGURE 5. Retinal photographs of normolipidemic subject (A) and chylomicronemic ciency (B) showing lipaemia retinalis. Eruptive xanrhomas in female (C) and male deficiency. nal pains and pancreatitis presumably et al., related 1967). Pregnancy ciency (Ma et al., the ceptives, production and of 1992). Rarely, diabetes mellitus pancreatitis to and Ooi, triglyceridemia (Howard tion (Kraus and LPL defi- contra- (Chait alcohol well as patients The is often It is difficult relation overlooked to determine is the cause or the consequence mono- if hyper- of pancreatitis plain and eridemia. plain Patients 1992). only are memory loss disappear subjects, of dyslipidemia, often However, on treatment with chylomicronemia are unex- of hypertriglycoccasionally consumption of alcohol, present, as com- mechanisms manifestations are rare and, when elicited syndrome Chylomicronemic forms of the extremities. upon 1995). et al., and recent psychoneuropathic of flushing symptoms (Cantin of chylomicronemia with other as yet and of abnormally lysophosphatidylcholine or polyparesthesias, Brunzell, in plained and patients of numbness involved by the presence of plasma likely symptoms and with fat malabsorption is supported in chylomicronemic dyspnea, even sub- patient homozygote for familial LPL defi(D) homozygote patients with familial LPL concentrations trimesters, et al., 1986; Chait 1977). possibility elevated Other pancreatitis, Levy, This Oral types, (Stuyt repetitive are at increased third is increased. estrogenic pancreatitis 1992). incipient and chylomicronemia com- but are most often such subtle by a questionnaire. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA er ul., 1964; Cameron et al., 1973), because evalua- of hyperlipidemia indeed, with creatitis, fasting, plasma zell, 1992). such causes mechanism The is often which done pathogenesis of irritation is generally Inflammation in large quantities microns in the blood rapidly by the acute event; (Chait and PL from capillaries (Chait and Brun- resulting from but the leads to pancreatitis of the pancreas acids of pan- understood, chylomicronemia by fatty the of pancreatitis as cholelithiasis by which after is part of the treatment TG levels decrease is still unknown. result more they (Fredrickson patients the second of VLDL the leads to chronic (Searles put these the risk of pancreatitis and Brunzell, clinical, and during especially may increase although levels may also unmask 1993) risk of pancreatitis when are unknown, to TG-chylomicron et al. could lysolecithins the circulating and Brunzell, be the released 4.3.2. Signs of chylomicronemia. tive xanthomas, nemia that are observed Lipemia retinalis nal vessels, due to thomas, are the circulating which chylo- macrophages 1992). They have intermittently (Table chylomicrons a yellowish of the reti- examination, (Fig. in groups accumulation in the skin (Parker erup- 11 and Fig. 4). appearance by funduscopic may cluster of the retinalis, are signs of chylomicro- refers to the whitish as revealed result Lipcmia and hepatomegaly 5B). Eruptive and become appearance 1970; Brunzell, with is xan- confluent, of chylomicrons et al., and a reddish by the 1995). ring at Human Lipoprotein Lipase the base, and may become tive xanthomas 121 zyxwvutsrqp Gene pruritic (Fig. 5C and D). Erup- are found mainly on the extensor of the arms, the back, the buttocks, Lipemia retinalis, eruptive xanthomas, should be considered surfaces and the thighs. and hepatomegaly signs of very elevated plasma TG levels, as well as high risk factors for pancreatitis. These signs may also be used to evaluate the efficacy of therapeutic ment. In contrast LPL-deficient patients, TG levels (Gagne of interest to these, splenomegaly treat- often persists in despite adequate control of plasma et al., 1990*). It is et al., 1989; Bertrand to note that 28% of the patients with spleno- megaly had no other clinical sign and their plasma TGs were only 16.7 f 3.7 mmol/ L, splenomegaly combined whereas other patients TABLE 12. Causes of the Chylomicronemia Syndrome Primary LPL deficiency Primary apo CII deficiency Familial LPL inhibitor Autoimmune chylomicronemia Combination of primary and/ or heterozygous Primq hypertriglyceridemia, Common secondan h~pertriglyceridemia Familial hypertriglyceridemia Dysbetalipoproteinemia Familial combined factors factors Secondary hypertriglyceridemia Diabetes mellitus Hvuothvroidism I. Nephrotic syndrome Chronic renal failure hyperlipidemia with secondary state for LPL deficiency with one or more extra clinical signs Alcohol had plasma TG levels of 50.7 f 16.4 mmol/ L (mean ? zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB SEM ). Obesity Drugs raising 4.3.3. nemia. Anomalous laboratory In chylomicronemic are encountered patients, laboratory in the analytical membrane clinical consequences, There et al., 1995). This of lipoprotein and the ambient et ul., 1992). This hemolysis inter- P-Blockers Pregnancy zyxwvutsrqponmlkjihgfedc fragility of the erythrocyte resulting from exchange the membrane of diagnosis. anomalies due to chylomicron patients for analysis (Cantin is possibly due to an increased between the level of plasma TGs Estrogens Oral contraceptives Corticosteroids Tamoxifen procedures (Table 11). In zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA vitro hemoIsotretinoids when blood is collected from chyloDiuretics or indirectly lysis is often observed micronemic in chylomicro- that are caused either directly by high levels of blood chylomicrons ference findings material plasma (Cantin does not appear to have any but can become misleading are other instances in terms where chylomicro- 4.4. Causes of the Chylomicronemia The most common genetic and nongenetic to chylomicronemia causes leading are listed in Table 12. The catabolism nemia gives rise to false blood analysis results. Pseudohypo- of chylomicrons natremia is a well-known anomaly in chylomicronemia. activity of LPL and the availability Thus, Syndrome and VLDL is dependent on the catalytic of its protein activator, a plasma level of 15 mmol/ L of TGs may decrease natremia the apo CII. Defects in either LPL or apo CII may lead to by about 1 mmol/ L in the presence of normal plasma osmal- chylomicronemia. ity (Steffes and Frier, 1976). Similarly, ciency are autosomal recessive disorders, each of which can 12 mmol/ L of chylomicron TGs increases hemoglobin surement by 10 g/ L, as determined method globin (Gagne a concentration by the Coulter of mea- counter et al., 1977a). This false increase in hemo- concentration may be corrected plasma with an isotonic solution. by replacing Ordinarily, the pancreatitis Familial LPL deficiency cause massive chylomicronemia due to complete or partial loss of LPL activity. Primary LPL deficiency is discussed in Section recent reviews 5. Readers are referred 1987; Connelly is associated with increases in plasma amylase concentrations. 1992; Tuzgol et al., 1994; Brunzell, inhibitor in pancreatitis due to chylomicronemia. finding has been attributed This paradoxical to an interference factor (Fallat et ul., 1973) or to the presence of an enzyme inhibitor (Lesser to detailed and articles on familial apo CII deficiency al., 1978; Breckenridge, However, amylasemia has often been found to be normal and apo CII defi- 1995). An inherited leading to chylomicronemia heparin LPL activity (autoantibodies et LPL and very low post- has also been reported (Brunzell et al., 1983). Chylomicronemia disorders (Breckenridge et al., 1987; Dolphin, in a family due to autoimmune against LPL) has been reported and Warshaw, 1975; Warshaw et al., 1975). Real values are in a patient with idiopathic thrombocytopenic obtained Grave’s disease (Kihara et al., 1989), and heparin resistance if amylase activity is determined using diluted plasma (Fallat er al., 1973). Pseudohyperbilurinemia been observed laboratory in chylomicronemia. in analyses may make the diagnosis and the follow- up of chylomicronemic oratory has also These uncertainties patients somewhat difficult. All lab- results, therefore, tion in the presence should be interpreted of a lactescent with cau- plasma. was noted lupus erythematosus (Glueck et al., 1969a,b). Abnormalities in the LPL and apo CII genes do not represent the most frequent causes of the chylomicronemia syndrome in the general population. Familial forms of hypertriglyceridemia, such as familial hypertriglyceridemia, dysbetalipoproteinemia, and familial combined hyperlipidemia (FCH), *Bertrand. M., Gagne, C.. Bun, L. D., J&en, P., Pineault, S., White, J., hlurthy, M. R. V. and Lupien, P. J. (1990) Familial hyperchylomicronemia and splenomegaly. In: International Symposium on Triglycerides: The Role in Diabetes and Atherosclerosis, May 23-26, 1990, p. 130, Vienna, Austna. in a case of disseminated purpura and are often associated with one or more other genetic and/ or nongenetic factors that tend to increase the plasma TG levels (Table 12). The genetic predisposition triglyceridemia is exacerbated by secondary to hyper- factor(s), lead- 122 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA ing to the chylomicronemia syndrome 1992). The same individual may possess ent genetic hypertriglyceridcmic triglyceridemia, type, heterozygocity Gaudet rt al., Uncontrolled nemia 1973; the d., Gleeson (especially such 1987; Iverius et al., (Molitch and and Connolly, P-blockers to moderate uals, may familial forms and (Stuyt et u1., 1988), 1992), which 1.31 t 0.55 4.92 + 0.92 Chylomicrons TGS Cholesterol 22.35 t 5.44 t VLDL TG:: Cholesterol Cholesterol/ npo Cholestert-rl/ apo HDL T(;s Cholcstcrol Cholcs~crol/ apo B ns ns ns 0.36 k 0.25 0.49 k 0.26 1.19 + 0.35 0.021 0.0001 B O.lY k 0.11 3.14 t 0.78 3.M k 0.71 AI 0.27 t 0.04 1.25 ? 0.33 0.78 * 0.10 0.36 * 0.10 0.33 * 0.08 0.45 + 0.06 0.0042 0.0001 0.0001 in normal Correction to rapidly in one tissue concentrations individ- mmol/ L, carrying 36 mmol/ L The of these eliminate the and normal have been with a mean lipoprotein in plasma sionally due total to severe which statistically VLDL is impaired (Brunzell, of these particles. of chylomicrons is unclear, in the but it could of HL or the direct endothelial The of ingested variations between different viduals, over no that not but there continue profiles significant differences between long-chain patients, long periods (Table of plasma postheparin TGs. The lesterol activity activity is related as well as within Patients LPL However, et ul., the secretion produce have been activity, and/ or reported but show con- indi- Variations the trans- heterogeneity in the who explain the found B and that in et al., 1989). apo AI), as in both these particles reduced atherosclerotic homozygotes of pancreatitis; observed level, which relative in the attacks and the presence such as chylomicrons, ably been but also drastically are rarely cholesterol ratio, HDL cardio- (Nikkila, 1983). the low LDL-to-HDL cho- of large plasma lipoproteins, are nonatherogenic, freedom complications could of homozygote (Gagne prob- patients et ul., 1989). to the observed the same 1989). indicating in recurrent of LPL low for with females, were reduced of chylomicronemia mainly LDL by the reticulo- acids. morbidity The of (apo 13. occa- 13). for the turnover TG levels have been (Gagne 10) may plasma fatty fractions, (Table is expressed from in plasma and HDL have ratios, were not only poor in cholesterol, in number Except the sexes (GagnC apos and in Table level are observed chylomicronemia. profiles complications a process of the catalytic of chylomicrons to an even- the LDL for females are shown cholesterol TG 6 and 234 1989). is lower in males as compared vascular exists absence be the result occurs reach mechanism complete that may influence of LPL levels do plasma, of hypcrchylomicronemia amount in factors and system. severity siderable port also The uptake lesions chylomicron hypertriglyceridt-mia in the indicating of genetic of both Chylomicrons indefinitely equilibrium, turnover as a result catabolism and massive 1995). accumulate tual is defective the to vary between et ul., (Brun- the plasma of 26 mmol/ L PRIMARY LIPOPROTEIN LII’ASE DEFICIENCY The levels of cholesterol and 5.1. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Homozygote State of Lipoprotein Lipase Deficiency well as the cholesterol-to-apo gene, 0.0001 in another patients, (Gagne 5. activity activity found value for males plasma Increases no LPL 12.84 3.91 4.33 2 7.41 1.64 rt 3.00 17.80 t 25.12 zell, 1989). In the French-Canadian the lipoprotein LPL ns TGs :lllii cholesterol values xc expressed as mmol/ L. Mean * SD (n = 16). I,‘, mu slgnificnnt. Datn from CantIn et 01. ClYO?). cholesterol, When 0.0001 0.72 + 0.48 0.38 + 0.19 6.79 -’ 8.33 LDL I-G Cholesterol enzyme condition. in the 27.39 k 18.23 7.91 k 6.42 oral induce in patients found Plasma TGs Cholesterol P (Bag- Flynn of hypertriglyceridemia. chylomicronemic (Ma Homozygotes Controls et ul., 199513). Brunzell, Brunzell, has been trimester) rt cd, 1986), isotretinoids et al., 1987), diuretics, (Brun and et condition corticosteroids chylomicronemia factors with (Pykalisto (julien hypertriglyceridemia produce secondary combined the third 1974), 1980; (Chait mild failure, as estrogens et ul., as hypo- renal physiological during drugs, TV (IL., 1992). Chylomicronemia by obesity dade rt u1., 1970), tamoxifen (Dicken such when by a transient lipid-raising 1986; diseases, and chronic 1983, particles (Brunzcll to hypertriglyceridemia et al., 1993), and exacerbated contraceptives of LPL V arious and Brunzell, produc- with chylomicron syndrome, may also be induced Several may induce the hepatic chylomicronemia such as pregnancy and Brunzell, and alcohol activity 1994). predisposition 1976; Chait et ul., use are the 1966; Chait Diabetes catalytic induce E: geno- (Julien in the chylomicro- by increasing nephrotic also familial to apo alcohol involved that compete et al., Wilson thyroidism, and et ul., 1993). TGs saturate may factors chylomicronemia tion of VLDL and mellitus (Bierman et al., 1983; Wilson fasting due et al. 13. Plasma Lipoprotein Concentrations in Control and Homozygotes for Primary LPL Deficiency TABLE Subjects hyper- 1995). secondary syndrome of differ- such as familial for LPL deficiency diabetes most frequent and Brunzell, an amalgam traits, hypertriglyceridemia and/ or 1994; (Chait V. Murthy have 5.2. Heterozygote State of Lipoprotein Lipase Deficiency The heterozygote ically zygotes and are state for LPL biochemically considered chylomicronemia to and other deficiency is still not well characterized. The be asymptomatic in regard usual manifestations clin- heteroto of the dis- ease, and they escape unambiguous clinical identification. abnormal zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Human Lipase Gene zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Lipoprotein TABLE 14. Phenotypic Expression of the Heterozygote M innicha Subjects LPL gene mutation Number of subjects Male/ Female Reduction in LPL activity Het 291 19 nd slight - or Total TG Total cholesterol VLDL cholesterol LDL cholesterol HDL cholesterol State for LPL Deficiency M aillyb Babirakc W ilsond Emi’ Het (Obligate Het 9 nd Het 188 Het 188 25 25/ o 2Om30% 14 8/ 6 52% 29 II/ 18 Het 188 11 5/ 6 8 4/4 23/25 50% - 50% 62% 40-50% 1 - or I t nd t I t nd nd nd nd M iesenback’ Julieng Het 207 48 t - or 1 t nd nd Apo R Ape AI Denser LDL 1 nd 1 HDLL 1 HDLL I - zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA or t nd 1 nd nd nd nd nd nd presence Het, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA he te ro zyg o te ; nd, not determined; -, unchanged; 1, increased; I, decreased. ‘Minmch et al. (1995): hMa~llv et al. (1995): <Babirak et al. (1989): dWilson et ai. (19901; ‘E mi et ui. (1990b); ‘Mxsenbock (1994). (1995~4 (unpubil;hed results) and Sniderman et al. (1995). presence et ui. (1993); EJulien et al. Babirak et al. (1989) have reported that carriers of LPL defi- dense in subjects carrying LPL gene mutations ciency could be identified on the basis of reduced postheparin (Miesenbbck plasma LPL activity and mass. However, in our own study for LPL gene deficiency show impaired TG tolerance and postprandial lipemia (Miesenbbck ity in heterozygotes Postprandial was half the normal level, we could not individual carriers from individual non- carriers based on postheparin of significant overlapping groups of subjects. plasma LPL activity because in LPL activity between these two Wilson et al. (1990) also reported that condition hyperlipidemia French-Canadian tainties in biochemical In view of these uncer- and clinical data, the only reliable way to identify the carriers of LPL deficiency is by analysis of the gene defects. Phenotypic is found to result in a 20-62% in heterozygotes decrease in postheparin LPL activity (Table 14). Plasma lipoprotein of these individuals than normal found are abnormal, not only in individuals LPL activity (40-60% profiles in most characterized by higher with a marked is decrease in of normal), but also in those with only in LPL activity (<30%). is significant heterogeneity However, there in hypertriglyceridemia (normal is consistent the presence hyperapo particles) (Julien in Table 14, an investigation particles (TG- and et al., 1995a). As shown of a group of Quebec clearance hetero- LDL of triglyceridemia than LPL deficiency may also play a role in the phenotypic is supported by the observation French-Canadian are carriers of an LPL gene defect (Julien et al., et al., 1995). Age, obesity, hyperinsulinemia, and lipid-raising expression drugs have been shown to contribute of hypertriglyceridemia apo AI, indicating as in the homozygotcs. lipoprotein observed in some heterozygotes metabolism (Chait and Brunzell, for LPL deficiency protects patients against increased fraction the importance no significant inherited could defect in 1983; Wilson et al., 1983). Similarly, it has been shown that homozygosity In two other studies, only the particles in the HDLI subwere found to be altered. Generally, to in heterozygotes (Wilson et al., 1990, 1993; Julien et al., 1995b). Thus, severe as well as reduced reduction, was not as pronounced of patients with Type IV and V hyperlipo- hypertriglyceridemia But this This possibility that as high as 13-20% be the result of a second independently that these HDL par- in the heterozygote expression of this familial dyslipoproteinemia. subjects had reduced and poor in cholesterol. and without indicates that factors other these heterozygote ticles are less numerous particles B. zygotes, the largest of any group studied so far, shows that HDL cholesterol, state, thus, of plasma TGs, due hypoalphalipoproteinemia, carriers of LPL gene mutations the et al., 1995). defective LPL enzyme. This probably leads The heterogeneity 1994; Minnich of larger than normal VLDL of apo B. However, of the heterozygote of cholesterol-poor proteinemia cholesterol-rich expression with impaired tion. The major feature of this hypertriglyceridemia is the in these do not exhibit increases in either total apo B or LDL apo B (Sniderman The phenotypic to severe), even in subjects carrying the same LPL gene mutapresence heterozygotes to mild hypcrtriglyceridemia, plasma levels of plasma TG. Hypertriglyceridemia moderate reduction by an overproduction to a catalytically expression of LPL mutations 1979). However, deficiency could represent a subgroup of FCH, a dyslipidemia characterized the carriers from the noncarriers. (Zilversmith, has not been investigated carriers. Babirak et al. (1989, 1992) have suggested that LPL but this reduction of et al., 1993). is known to be a predisposing for atherosclerosis precocious atherogenesis adipose tissue LPL activity was reduced by 50% in carriers, did not allow reliable identification 188 and 207 et al., 1993; Julien et al., 1994, 1995a). Heterozygotes (Gagne et al., 1977b), even though adipose tissue LPL activclearly distinguish 123 familial hypercholesterolemic LDL cholesterol. of gene-gene interactions changes were found in total plasma apo B and LDL choles- expression of dyslipoproteinemia terol. However, LDL particles have been reported to be more thermore, This indicates in the phenotypic (Zambon et u1., 1993). Fur- a variable lipid phenotype has also been dem- V. zyxwvutsrqponmlkjihgfe Murthy et ul. 124 TABLE 15. Activity Among Frequency of LPL 82 Homozygous Gene M utations and French-Canadian Exon M utation W) tury from the Northwestern part of France, especially from Perche (Fig. 7A) (Dionne zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONML et al., 1993). DNA haplotype anal- Plasma ysis has revealed that LPL alleles containing Number of alleles LPL Probands= postheparin are homozygous LPL H2 (Hind111 and PvuII: + / +), indicating activity mutation for a specific LPL gene haplotype 207 named a common origin and a founder effect for this mutation (Ma et al., 1991). After 5 pro’“; - Le” j Gly’s” - Glu 37 (22) 0 arrival in Charlevoix, 6 A ,$” - A sn 4 (2) 0 the Northeast along the St. Lawrence River and then along - Ser 1 (1) 0 1 (1) 8 (5) 0 0 the Saguenay River towards Lac St-Jean (Fig. 7B) (Dionne 6 ASTP 9 Se+ 113 (69) Ter Unidentified - (’Data from J&n (1992) and unpublished 0 et al., 1992). These descendants some of the settlers migrated towards are the regions now populated of the original immigrants by the who carried muta- tion 207 in their LPL gene (Fig. 6). data. The second most common mutation, 188, is present mainly onstrated in a family affected by combined and LPL activities (Auwerx deficiency of HL et al., 1990). The in Western Quebec (Fig. 6) (Bergeron logical reconstruction interaction et al., 1992). Genea- of 14 families carrying mutation of genetic, dietary, and other factors in the phenotypic expres- has led to identification sion of dyslipidemia (in LPL heterozygote ent regions of France, one of them possibly of Scottish subjects) has not et al., 1992; Dionne gin (Fig. 7A) (Bergeron yet been investigated. Mutation 188 of 4 founders originating from differori- et al., 1993). 188 has been shown to be specifically associated with haplotype Hl (Hind111 and PvuII: +/ -), suggesting 5.3. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Origin and Dissemination of a common origin for this mutation (Ma et al., 1991). The Lipoprotein Lipase Gene Defects in Qukbec occurrence of this mutation in different parts of the world As mentioned in Sections 3.3 and 3.4, gene analysis of indi- viduals with documented identification enzyme deficiency has led to the of a large number of LPL gene variants. These variants have been found in subjects from different ethnic origins, including Black Americans Caucasians, Japanese, Malaysian, been carried out in two large populations to Canada by the city of Quebec. Their descendants migrated towards the Mauricie region, where they settled. Some of them, however, also moved along the fertile St. Lawrence Valley towards (Fig. 7B). We find most of the current carriers of 188 in these areas (Fig. 6). The relative isolation Northeastern and the other of affected families have been population of homozygosity of Quebec in the has been to date in the Province of Quebec, and they all lead to com- plete loss of plasma postheparin LPL activity (Table 15). Of the 82 French-Canadian homozygotes during the past centuries have a long period of history, to the regions where these immiHaplotype settled (Dionne analysis of other clinically homozygous unidentified et al., 1993). French-Canadian patients for LPL deficiency, and carrying still mutation(s) of the LPL gene, suggests that there may exist at least 3 additional mutations, besides 188, 207, a majority are and 250, underlying LPL deficiency in the French-Canadian heterozygotes for mutations 207 population patients identified, or compound movements served to confine the carriers of mutations 207 and 188, over grants originally is as low as 1 in 1 million (Julien rt ul., 1994). Five LPL gene defects have been identified of different regions of the Province of Quebec and the limited demographic of Quebec. for LPL deficiency region of the Province at- to be at least one in 10,000, and the incidence in the general population (Julien et al., 1995a). Recently, 2 new mutations at positions 291 and 447 of the LPL protein were identified and 188. Most of the French-Canadians Quebec are descendants from France neau and Robert, living in the Province of of approximately between 8500 settlers who in this was the result of a founder effect (De Braekeleer et al., 1991). Genealogical reconstructions show two different sets of founders tion 207, which is almost exclusively more prevalent in the Northeastern (Fig. 6) (Normand of affected families for mutation 207. Analyses of geographic distributions is region of the Province et al., 1992). Genealogical of families carrying mutation 188 and indicate that muta- French-Canadian, reconstruction 207 has enabled us to identify 16 founders who migrated to Quebec homozygote in the early 17th cen- patients (Table 15) (Min- nich et ul., 1995). gene mutations have hypothesized of familial LPL deficiency in 2 French-Canadian The total heterozygote 1608 and 1759 (Charbon- 1987). We previously that the high frequency population immigrants Montrkal European in the French-Canadian migrated The European 188 arrived at the site presently occupied mutation The incidence calculated Indian populations. carrying mutation (Wilson et al., 1990; Julien et al., 1994). Genea- one North logical reconstructions tempted affected by LPL groups suggests that it may be an French- deficiency, Canadian and (Lalouel et al., 1992). Detailed studies have and in various ethnic ancient mutation predating the spread of European and East Weinberg carrier rate for all of these LPL has been estimated, equilibrium Saguenay-Lac-St-Jean based on the Hardy- (Table 16) (Julien et al., 1994). The (Northeastern Qukbec) and Mauricie (Western Quebec) regions are found to have the highest carrier rate, with frequencies of l/ 48 and l/ 107, respectively. However, within the Charlevoix Quebec region, the incidence area, a subdivision is estimated the basis of the number of homozygote the Lipid Clinics, of the to be l/ 33. On patients referred to the total number of carriers for these two major mutations is believed to be at least 45,000 in the Province of Quebec (J&en et ul., 1994). 125 zyxwvutsr Human Lipoprotein Lipase Gene Lac St-Jean 08 Administrative Regions 01: Bas-St-Laurent 02: Saguenay - Lac St-Jean 03: Quebec 04: Mauricie - Trois-Rivikres 05: Estrie 06: Montreal 07: Outaouais 08: Abitibi - Tkmiscaminque 09: C&e-Nord 10: Nouveau-QuGbec - 207-m 188-m FIGURE 6. of Quebec. of patients Canadian 6. Geographic The distribution distribution identified TREATM ENT of LPL-deficient patients is based on the place of birth in each administrative Cardiology Publications, Inc., region. Reprinted M ississauga, LIPASE homozygous of each Ontario, patient. from Julien DEFICIENCY for different LPL Heterozygote gene mutations - H zyxwvutsrqponm in the Province The size of each box is proportional to the number et al. (1994), with permission of the copyright holder, Canada. 6.2. OF LIPOPROTEIN Compound Unknown - 0 250-m Family Screening and Counseling Statistically, the siblings of a patient with primary LPL defi- 6.1. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Identification and Correction of Secondary Factors ciency have a relative risk of 25% of having the disease and As mentioned in Section 50% of being heterozygote. Diagnosis of chylomicronemia 4.4, primary LPL deficiency is not Understanding the multiple etiology underlying chronic and transient ilies where primary LPL deficiency is detected, the only way chylomicronemia to predict the risk of having a child with the disease is to the most common of this syndrome, plications. directed The is necessary for the proper management as well as to avoid recurrent clinical comlong-term goal of the treatment towards the prevention be achieved mmol/ L. cause of chylomicronemia. by maintaining of recurrences. plasma TG must be This can levels below 10 Factors that may aggravate hypertriglyceridemia, such as alcohol consumption, drugs, oral contraceptive avoided. Careful attention use of hypertriglyceridemic agents, and estrogens, should be should be given to detection If a woman desires pregnancy, up should be planned in advance, as pregnancy plicated and has the potential 1992; Ma et al., 1993). identify the mutation at the molecular level in both prospective parents. As discussed in Section of postheparin of heterozygotes, 5.2, the measurement LPL activity is not useful for identification because enzyme levels change and overlap with those of normal individuals, mean is lower in heterozygotes (Gag& over time although the (about 50% of normal levels) et al., 197713). and treatment of secondary factors, such as diabetes mellitus and hypothyroidism. in one individual, therefore, implies family screening. In fam- the followmay be com- for high risk (Watts et al., 6.3. Dietary Regimen Fat restriction applies to patients with familial LPL defi- ciency, as well as to patients with secondary eridemia, when secondary hypertriglyc- causes cannot be treated or treat- 126 et al. V. Murthy familial LPL deficiency. limiting the intake it by complex should caloric and minerals. cess depends, The low-fat increased by using fatty acids are directly administration a milk of MCT formula amounts PROVINCE Formulation in the daily diet. be limited in iron. B: Founding regions of mutations France for mutations ince of Quebec and movement 188 (solid line) and 207 to Quebec in the 17th 188 and 207 century. in the Prov- of settlers along the St. Lawrence and milk The pregnant of these reduction reduce causes of triglyccridemia. triglyceridemia signs of zell, 1992). are can be achieved at lo-20 The aim must for adequate phase, TG (Watts et al., pancreatitis by other nutrition the therapy is to is progressively symptoms and eliminated. Prevention by maintaining fasting of plasma are identified also be desirable to consider drugs, necessary. is the only dietary treatment available for 16. Gene Estimation M utations A dministrative 01 Rx of the Heterozygote in the Province region St Laurent 02 Saquenay 03 Quebec if judged et al., as their in the same (Chait and Brun- be given, but lipidic lipids are metabolized After while possible the secondary For long-term acute alimentation factors therapy, it may the use of hypotriglyceridemic 6.4. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJ Drug Therapy Drug TABLE and corrected. Ma et u1., 1985). be monitored introduced, and 1992; as chylomicrons. should should Sprecher, not nurse, causes as these may adapted and is treated should be avoided, mechanisms diet attention should in fats (Steiner to iron, the Special produced levels be given (Black of are present conditions. children by a mmol/ L. Fat restriction LPL minerals be periodically poor should same of the where et quantities diet should women Parenteral by the is not sufficient to a level chylomicronemia pancreatitis TGs secondary available Perron be made for whom with LPL deficiency as that should and children growing is extremely emulsions ment the adequate 1990; adequate attention Chylomicron-induced manner diet and the Saguenay. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA River and er al., that vitamins, and to infants, 1993). Women from (Schluter Special in women 1993), A: Spread MCT with For infants, is commercially to ensure nutrients, especially be given 7. vein. Chylo- et u1., 1992). acids of an adequate dietitian tc age and physiological line) and MCT 199413). all necessary FIGURE chylomicrons relieved mostly fatty may be oils for cook- via the portal oil (Shirai Mead-Johnson) professional (dashed into significantly containing of essential (Portagen@, ill., be with the of the diet (MCT) to Suc- for their catabolism. transported can compliance TG on LPL activity thus, of fat is limited palatability fats are not incorporated micronemia, the diet with the usual 40%. medium-chain are not dependent by replacing However, consumption The and as well as essential on the patient’s diet. accomplished acids intake, compared however, extremely fatty and proteins. adequate 10-l 5% of total calories, ing. MCT is efficiently carbohydrates provide vitamins This of long-chain Lac-St-Jean” (Charlcvolx)” 04 Maurlcie’ 06 Montr~ul 00 core Nod Eastern Quebec” Western Quebec’ Whole Quebec Carrier Rate for of Quebeca Estimated and carrier l/ l/ l/ l/ 245 48 l16 107 l/24’) l/ 166 l/M l/ 220 l/ l43 rate therapy is used to lower plasma triglyceridemia, dysbetalipoproteinemia, LPL deficiency glyceridemia. effective action who Fibric drugs and lipolysis develop moderate acid derivatives is on plasma plasma in adipocytes, activation LDL in HDL of fibrates and high-carbohydrate when heterozygotes. are known levels. VLDL However, hyperFCH, inhibition cholesterol through produc- Lowering with are elevated, use of fibrates of in denser (Shepherd, acid) diet is useful in lowering the principal of VLDL with a reduction (or nicotinic levels as the most The 1994). for hypertri- metabolism of LPL (Davignon, and an increase especially in primary to severe TG TG plasma TG levels is often associated A combination TGs hypertriglyceridemia, as well as in heterozygotes for lowering of fibrates reduced tion, such as familial 1993). a low-fat plasma TGs, as in the case of to treat homo- Human Lipoprotein zygote patients is not indicated 127 Lipase Gene for primary because LPL- they and apo respond Two approaches (X-deficiencies only to low-fat oped. diet. In the to somatic gene therapy ex viva strategy, the target are being cells devel- are harvested from the patient, transfected by the normal functioning 6.5. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Prospects of Gene Therapy and, then, transplanted back into the patient. This As described in Sections of LPL deficiency restrictions or lipidemia. methods the part of medical sive and vances genes in mammalian possibilities normal cells of direct could means a cure of continuous Several that are directly mouse in animals (Field, 1993; Liu et al., et al., 1994), its activator and HL (Busch tions to expect in humans. also that could The and deficiency LPL LPL et al., gene, er al., studies lipid-related 1994). would permit no recognize the target can significant be properly the possible sophisticated to migrate and of attention, more work is needed can be tested technical the gene expressed. to develop loss, to in a form Although is receiving in clinical direct to the target or qualitative and to deliver where (Wilson method, of the a highly techniques treat- cells in viuo. This, the vector integrated use of such they consists to the target quantitative cells, disease An alternative less invasive, require that would in the hypercholesterol- a great deal them to a point situations. and LPL itself et al., Animal et al., success familial that 199413; are useful Ackno~,lrdgements-The Shugar for his interest authors are deeply indebted co Professor David and careful correction of the drafts of this article. We wish to express our thanks to Professor Alan Sniderman for reading the final manuscript and for useful suggestions. We thank Gervais Lapointe, Merck Frosst Canada, and Car& Bra&, CHUL Research Centre, for assistance in complling the references. Research from the authors’ laboratories discussed in this review was supported by grants from the Heart and Stroke Foundation of Canada, Parke-Davis Canada, the Canadian Diabetes Association, the Natural Scirnces and Engineering Research Council of Canada and the Medical Research Council of Canada. Dr. I? Julien was a Career Scwnt~st of the Fends de la Recherche en Sante: du Quebec. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON of the LPL transgene 1994). The the causes a deficiency is transcribed et ul., develops but within (il., 1990; Masuno the et al., glycosylation processing pathway, such cytes (Davis above two The because models could help by lesions in other an secretory genes. if (Davis for the to the lipase glyco- in cld adipo- characteristics the LPL deficiency of the efficacy of is caused itself or indirectly of the insulin-resistance M. I., Hamman, M. I. (1993a) with the physsyndrome Diabetes in the Care 16: R. F., Cole, S. A. and Ferrell, R. E. (1993b) Two DNA polymorphisms gene and their associations in the lipoprotein lipase with factors related to cardiovascu- lar disease. J. Lipid Res. 34: 421-428. Al-Haideri, M., Granot, E., Schwiegelshoh, I. J. and Deckelbaum, B., Vogel, T., Gorecki, R. J. (1993) Apoprotein E simulates non receptor triglyceride-rich Circulation terization particle cellular uptake. 88: I-321. M., Schotz, et R. E and Kamboh, lipase gene variation 1502-1506. Ameis, D., Stahnke, protein at the lipoprotein 90: 184. of the San Luis Valley, Colorado. M., Goldberg, abnormally N-linked normally in the LPL gene related for LPL gene inactive in evaluating when and appears gene responsible are processed in situations The components Ahn, Y. I., Kamboh, of chylomicronemia days. to be specific other and iological of lipoprotein population a single, LPL reticulum I erent 1990). Th e d’ff et al., directly endoplasmic for repeat polymorphism Ahn, Y. I., Ferrell, R. E., Hamman, Association com- homozygote it produces 1990). has of both catalytically appears as adipsin LPL gene therapy but mannose), is (Gin- is the in utero l-3 lipase (LPL) locus. Hum. Genet. T/ t complex The massive dies within normally, remains normally exhibits and (high 1983). LPL of Arg model M. I. and Ferrell, R. E. (1992) Tm,o new alleles in the tetranucleotide base change substitution animal Ahn, Y. I., Kamboh, xan- deficiency by a single within cld mutation glycosylated LPL References show to human mutation the that subcutaneous which (Paterniti to suckle, LPL transgene 412 of the LPL protein second of for the devel- of cats mouse, 17 that at birth, models animal feline in the (cld/ cld) activities normal attempted two similar The residue chromosome allowed is eventually for human activity resulting autosomal by defects Zhang retinalis, 1983). acid HL either genes et al., to be caused lipase-deficient proteins con- apo CII (Shachter is a colony lipemia and is found defective a prevalent be much site with that treatthan deficiency serve as test systems model reduced at the amino that 1994). protocol first (Jones inherited recessive 1994; are available chylomicrons, bined this of human the expression of an optimum zinger up have been in LPL when such a procedure There expression. Gly et al., how LPL deficiency in the opened highly partial from of the gene of interest however, design some in zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA viuo and to evaluate the nature of complica- is regulated thomas, ad- found suffering Grossman would transfer of foreign 1983), including et al., fasting Recent rather recently a number Zsigmond opment which inva- genes by their disease, implicated lipid diseases in understanding on the part accomplished, for the (Shimada 1994), 1992; have of defective models overexpress or indirectly the attendant another et al., hyper- management. transgenic structed emia, on control side effects. If properly represent of a patient and expression replacement counterparts. ment and has already ment is physiologically lead to unpredictable of transfer nique of dietary supervision and compliance use of drugs in the techniques to long-term professionals treatment of a regimen of drugs require Chronic may consists administration Both of the patient. 6.3 and 6.4, the current in humans gene tech- G., Kobayashi, J., McLean, J., Lee, G., Buscher, M. C. and Will, H. (1990) Isolation and charac- of the human hepatic lipase gene. J. Biol. Chem. 265: 6552-6555. Ameis, D., Kobayashi, Kane, J. I?, Lee, (1991) Familial due to a single J. Clin. Appelman, LPL 87: (type mutation R. J. and M. J., Schotz, M. C. I hyperlipoproteinemia) in the lipoprotein lipase gene. 1165-1170. E. E. G., Bijvoet, T, Hayden, mutation in the deficiency. H., Havel, chylomicronemia missense Invest. P W., Bruin, de novo J., Davis, R. C., Ben Zeev, O., Malloy, G., Wong, S. M., Wiebusch, M. R. and lipoprotein Atherosclerosis H., Ma, Y., Reymer, Castelein, lipase 109: 63. (LPL) J. J. (1994) gene A causing V. Murthv et al. zyxwvutsrqp 128 Auwerx, J. H., Babirak, S. P., Hokanson, H., Deeb, S. S. and Brunzell, J. E., Stahnke, malities of hepatic lipase and lipoprotein Am. J. Hum. Genet. of abnor- lipase in a large family. contributions K. (1992) Lipoprotein from molecular biology. Crit. lipase: Rev. Clin. S. P., Iverius, (1989) Detection I? H., Fujimoro, and state for lipoprotein W. Y. and Brunzell, characterization of the lipase deficiency. J. D. heterozygotr Arteriosclerosis 9: 326- S. I’., Brown, B. G. and Brunzell, J. D. (1992) Familial com- bined hyperlipidemia scler. Thromb. and abnormal lipoprotein Arch. A complication Intern. Med. U., Meng, corticosteroid therapy. Bethesda: M. S., Skarlatos, S. I., Previato, a single amino acid substitution heparin binding their U. (1995) Receptors L., Brunzell, lipase (Ala176-Thr) leads and loss of enzymic activity. Proc. for triglyceride-rich role in Ilpoprotein metahollsm. lipoproteins Curr. Opin. and Lipidol. 6: U., Krapp, A., Weber, W. and Olivecrona, role of alpha 2M receptor/ LRP olism. Ann. Bengtsson, lipase affinity T. Biochem. P., itienne, metab- A., Arnault, recombination. Evaluation J., de Gennes, exon 9 causes hpoprotein of lipoprotein of conditions for F., Hamelin, L., Brault, J., Fouhert, E (1995) Homozygous lipase deficiency; possible D.. L., Chuat, dcletlon of intron-Alu in dietary lipase. Annu. Rec. Nutr. 11: M. H., Davis, R. C., Elovson, J. and Schotz, M. C. (1992) Maturation activity of lipoprotein requires tion to rhe cis-Golgi Ilpase. Expression glucose trimmlng compartment. of full hut not transloca- J. Viol. Chem. Xi: asparagine-linked G., Liu, G., Da\%, R. C. and Doolittle, lipase and hepatic glycosylation lipase: the role of in the expression of a functional J., Julien, la lipoprotcln I? and Murthy, lipase humaine: M. R. V. 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