Am. J. Hum. Genet. 47:107-W, 1990

Lipoprotein Lipase Deficiency Resulting from a Nonsense Mutation in Exon 3 of the Lipoprotein Lipase Gene Mitsuru Emi, * Akira Hata, * Margaret Robertson, * Per-Henrik Iveriuswt Robert Hegele,*I and Jean-Marc Lalouel* *Howard Hughes Medical Institute and Department of Human Genetics, and tVeterans Administration Hospital and Department of Internal Medicine, University of Utah Health Sciences Center, Salt Lake City

Summary In DNA from a male patient of German and Polish ancestry who has lipoprotein lipase deficiency, sequencing of all nine exons and intron-exon boundaries corresponding to the coding region of the lipoprotein lipase gene detected a C-*.T transition leading to the substitution of a stop signal for the codon that normally determines a glutamine at position 106 of the mature enzyme. Hybridization with allele-specific oligonucleotides at this position established that the patient was homozygous for this mutation. This mutation must lead to the synthesis of a sharply truncated protein, accounting for the enzymatic deficiency noted in the patient.


Lipoprotein lipase (LPL) deficiency is a rare autosomal recessive condition characterized by massive fasting chylomicronemia, recurring episodes of abdominal pain or acute pancreatitis, and eruptive xanthomas, with onset in infancy or childhood. The diagnosis can be established by demonstrating profound deficiency in the enzymatic activity of LPL (triacylglyceroprotein acylhydrolase; E.C. in adipose tissue or in postheparin plasma (Nikkila 1983; Brunzell 1989). This enzyme mediates a key step in the metabolism of lipoproteins of intestinal and hepatic origin through hydrolysis of the triglyceride constituents of chylomicrons and very-low-density lipoproteins (VLDL), thereby delivering fatty acids to peripheral tissue for storage or for fuel (Garfinkel and Schotz 1987). Apolipoprotein C-II (apo C-II) serves as a cofactor; similar clinical manifestations can be observed as a result of mutations affecting the apo C-I1 gene (Nikkila 1983; Brunzell 1989). Received November 16, 1989; revision received February 26, 1990. Address for correspondence and reprints: Jean-Marc Lalouel, M.D., Howard Hughes Medical Institute, University of Utah Health Sciences Center, Wintrobe Building, 6th Floor, Salt Lake City, UT 84132. 1. Present address: Department of Medicine, Saint Michael's Hospital, Toronto. i 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4701-0015$02.00

Both the cDNA sequence (Wion et al. 1987) and the genomic structure (Deeb and Peng 1989) of human LPL have been elucidated. The gene contains 10 exons spanning 30 kb; the last exon encodes the long (1,948-bp), untranslated 3' end of two mRNA species, of about 3.35 and 3.75 kb, that result from alternative use of two polyadenylation signals (Wion et al. 1987). It encodes a protein of 475 amino acids which becomes a mature protein of 448 residues after cleavage of a signal peptide. Langlois et al. (1989) have documented the occurrence of major genomic rearrangements in five of 19 alleles examined in LPL-deficient subjects; an internal gene duplication recently has been characterized (Devlin et al. 1990). Cloning and cDNA sequencing in another LPL-deficient patient led to the identification of a single amino acid substitution (Gly--'Glu188) yielding an inactive enzyme when expressed in cultured mammalian cells (Emi et al. 1990). In the work reported here, enzymatic amplification and sequencing of all exons encoding LPL led to the identification of a nonsense mutation at amino acid residue 106 of the mature enzyme. Material and Methods Subject The male patient (K2012) investigated in the present 107

Emi et al.


followed and treated by Drs. Arthur L. Beaudet and Robert L. Nussbaum at Baylor College of Medicine in Houston. He was of German and Polish descent. The parents were kin, the father's grandfather being the mother's second cousin. At hospitalization for failure to thrive and an intercurrent febrile episode at the age of 5 mo, massive fasting hypertriglyceridemia (5,000 mg/dl) and hepatosplenomegaly suggested a diagnosis of familial LPL deficiency. A subsequent lipid profile revealed a pattern consistent with this diagnosis: total cholesterol was 299 mg/dl, total triglyceride was 2,674 mg/dl, VLDL cholesterol was 282 mg/dl, low-density-lipoprotein (LDL) cholesterol was 5 mg/dl, and high-density lipoprotein (HDL) cholesterol was 12 mg/dl. The patient's lipolytic function was investigated in Dr. Peter Herbert's laboratory at Brown University in Providence, RI. Apo C-II deficiency was excluded because the patient's plasma activated rat adipose tissue LPL in vitro. Hepatic triglyceride lipase (HTGL) and LPL activities were assayed on a sample collected 10 min after injection of heparin at a dose of 100 U/kg. These two activities were distinguished by inhibition of LPL with either protamine or sodium chloride; LPL activity was 2.0 pM free fatty acids (FFA)/ml/h in the protamine assay and 0.5 pM FFA/ml/h in the sodium chloride assay. The corresponding values for HTGL activity were 11.3 and 12.8 gM FFA/ml/h, respectively. Mixing the patient's serum with postheparin plasma from a normal subject resulted in only slight inhibition, which could be ascribed to competition between the patient's triglyceride-rich lipoproteins and the assay mixture. These experiments established LPL deficiency in the patient. Although a strict low-fat diet was prescribed, sporadic compliance led to the repeated observation of eruptive xanthomas and triglyceride concentrations in excess of 1,000 mg/dl. When the prescribed diet was strictly followed, xanthomas disappeared and triglyceride concentrations fell as low as 300 mg/dl. Subsequently, a brother of the patient presented with similar symptoms and received the same diagnosis. A fibroblast culture was established from a skin biopsy, and this specimen was contributed by Dr. Beaudet to the Camden Cell Repository under access number GM6978. report was

Isolation of LPL Cosmids Five genome equivalents (350,000 clones) of a genomic cosmid library prepared by Dr. Yusuke Nakamura were screened with the human cDNA insert of plasmid LPL35 (Wion et al. 1987), provided by Dr. R. Lawn

of Genentech in South San Francisco. Five positive clones were obtained, and DNA from these clones was prepared according to a method described elsewhere (Nakamura et al. 1988). DNA sequencing of portions of these cosmids by primers derived from the 5' and 3' ends of the LPL cDNA indicated that one of these cosmids (cRHL2A) contained all exon sequences (data not

shown). Oligonucleotide Synthesis To amplify enzymatically all exons of the LPL gene, oligonucleotide primers were synthesized on an Applied Biosystems 380A DNA synthesizer (Applied Biosystems, Foster City, CA). Each sense primer has a BamHI recognition sequence, and each antisense primer has an EcoRI recognition sequence on its 5' end. Universal and reverse primers of M13 (5'-CGCCAGGGTTTTCCCAGTCACGAC-3' and 5'AGCGGATAACAATTTCACACAGGA-3'), as well as two primers for allele-specific oligonucleotide hybridization, were also synthesized. Cloning and Sequencing of the Genomic DNA of the Patient Fibroblasts (access number GM6978) from the patient were grown and genomic DNA was extracted from the culture, both according to standard protocols. Genomic DNA was amplified enzymatically (Mullis and Faloona 1987) on a DNA Thermal Cycler (Perkin Elmer-Cetus, Norwalk, CT), using 100 pmol of each pair of primers and 5 units of Taq polymerase. The reaction mixture was denatured at 950C for 1 min., primer-annealed (at 480C for exon 1, at 43IC for exons 8 and 9, and at 540C for other exons) for 1 min, and primer-extended at 72°C for 1 min for 10 cycles of amplification. Primer-annealing was performed at 60°C for all exons in 20 subsequent cycles. Each amplified fragment was digested with BamHI and EcoRI and cloned into the M13mpl8 vector. Eight independent clones were isolated, and single-strand DNA was produced directly, by asymmetric enzymatic amplification, from plaques suspended in distilled water (Saiki et al. 1988), by using M13 universal and reverse primers. Automated analysis of DNA sequence was performed by the chain-termination method by means of an Applied Biosystems 370A DNA sequencer according to a method described elsewhere (Emi et al. 1988). Homologous sequences were aligned with the computer program

Genalign (IntelliGenetics, Palo Alto). Dot-Blot Hybridization Five microliters (1/20th) of the product of enzymati-

Nonsense Mutation in LPL Deficiency


cally amplified DNA samples of exon 3 derived from genomic DNA were spotted in duplicate on nylon membrane (BioTrace' RP, Gelman Sciences, Ann Arbor, MI), and each membrane was hybridized with 32P-endlabeled oligonudeotide probes (wild type, 5'-GTGGGACAGGATGTGGC-3'; mutant, 5'-GCCACATCCTATCCCAC-3'). The membranes were washed in 6 X SSC (1 X SSC = 0.15 M NaCI/15 mM Na citrate, pH 7.0), at 540C for wild-type probe and at 520C for mutant probe, and were autoradiographed according to a method described elsewhere (Emi et al. 1988). Results

Total genomic DNA from the patient was digested with four restriction endonucleases (PstI, StuI, XbaI, and EcoRI) and hybridized with the normal cDNA clone LPL 35; no gross alteration of IPL gene structure was apparent (data not shown). For examination of the entire coding region of the LPL gene, all nine translated exons were amplified enzymatically, and for each exon eight independent clones were sequenced. The amplification primers were designed on the basis of either the intron-exon boundaries published by Deeb and Peng (1989) or additional sequencing information derived from cosmid cRHL2A

whenever no adequately amplified product could be generated with primers sequences derived from published data (table 1). Each amplification product was cloned into M13mp18 vector as described in the Material and Methods section, and for each exon eight independent clones were isolated and sequenced. Only one patent nucleotide variant was identified: all eight clones contained a C-aT transition at nucleotide position 571. LPL is numbered as by Wion et al. (1987). This mutation generates in exon 3 a stop signal at the codon that normally specifies a glutamine at position 106 of the mature enzyme (fig. 1). The presence of this mutation in both alleles of the patient was confirmed by hybridization experiments with allele-specific oligonucleotides (fig. 2). Discussion A patient of German and Polish ancestry presented the typical clinical manifestations of familial LPL deficiency in early infancy, with minimal LPL activity in postheparin plasma. Fibroblasts stored in the Camden Cell Repository under the access number GM6978 offered an opportunity to identify the molecular defect at the origin of this deficiency. By enzymatic amplification of genomic DNA followed by cloning and sequenc-

Table I Synthetic Oligonucleotides Used for Enzymatic Amplification of Exons 1-9 from Total Genomic DNA


Primer Sequence





3............ 4............






7............ 8............





a Derived from published exon boundaries (Deeb and Peng 1989). Derived from additional sequence information obtained by partial sequencing of cosmid cRHL2A.



Emi et al. 103 104 105 106 107 108 109 Leu Val GlyGinAsp Val Al

i, SiffT- eG9aACA.' T6T-91f




3 4



8 9 10



3 4 5




7 8 9 10 Detection of the (C571-IT) transition in total 6

Figure 2 103 104 105 106 107 108 109 Ala Leu Val Asp


genomic DNA by enzymatic amplification and allele-specific oligonucleotide hybridization. Grid 1, Patient. Grids 2-8, Normal Individuals. Grid 9, Amplified DNA from M13 clone which was used for sequencing exon 3 of the patient. Grid 10, Amplified DNA from cDNA clone LPL3S.

sequence provided in the present report can be used to screen chylomicronemic or hypertriglyceridemic subjects for the presence of this mutation.

Acknowledgment Figure I Comparison of the nucleotide sequence of genomic DNA, in the vicinity of nucleotide position 571, from the patient and from a normal subject. All eight clones sequenced presented sequencing profiles similar to that for the representative done depicted. The stop codon created by the nucleotide transition is identified by an asterisk.

ing, we have established that the patient was homozygous for a mutation which should lead to the synthesis of a drastically truncated gene product, accounting for the functional deficiency noted in the patient. In consideration of the reported kinship between the parents, these two mutant alleles are likely to be identical by descent. No other nucleotide variant could be detected in any of the coding and intron-exon boundary regions subjected to sequencing. This mutation was not present in 10 chylomicronemic subjects predominantly of northern European descent who had either total or partial LPL deficiency. The mutation present in the homozygous state should lead to total absence of LPL activity. The minimal residual LPL activity detected after inhibition by either protamine or sodium chloride is in the range of assay error. Presence or absence of immunoreactive material may depend on the nature of the antibody used. The

The authors are indebted to Dr. Arthur Beaudet, who made this study possible by contributing fibroblasts from the patient to the Camden Cell Repository and who provided medical information on his patient, and to Dr. Peter Herbert, who characterized LPL deficiency in the patient. The expert technical assistance of Elaine Hillas, Rick Myers, and Zhang Shuhua and the editorial assistance by Ruth Foltz are gratefully acknowledged. This research was supported in part by the Medical Research Council of Canada. J.-M.L. is an Investigator at the Howard Hughes Medical Institute.

References Brunzell JD (1989) Familial lipoprotein lipase deficiency and other causes of the chylomicronemia syndrome. In: Scriver CR, Beaudet AL, Sly WS, Valle D, (eds) The metabolic basis of inherited disease. 6th ed. McGraw-Hill, New York, pp 1128-1180

Deeb SS, Peng R (1989) Structure of the human lipoprotein lipase gene. Biochemistry 28:4131-4135 Devlin RH, Deeb S, Brunzell J, Hayden MR (1990) Partial gene duplication involving exon-Alu interchange results in lipoprotein lipase deficiency. AmJ Hum Genet 46:112-119 Emi M, Wilson DE, Iverius PH, Wu L, Hata A, Hegele R, Williams RR, et al (1990) Missense mutation (Gly- Glu'88) of human lipoprotein lipase imparting functional deficiency. J Biol Chem 265:5910-5916 Emi M, Wu LL, Robertson MA, Myers RL, Hegele RA, Williams RR, White R, et al (1988) Genotyping and sequence

Nonsense Mutation in LPL Deficiency analysis of apolipoprotein E isoforms. Genomics 3:373379 Garfinkel AS, Schotz MC (1987) Lipoprotein lipase. In: Gotto AM (ed) Plasma lipoproteins. Elsevier, Amsterdam, pp 335-358 Langlois S, Deeb S, Brunzell JD, Kastelein JJ, Hayden MR (1989) A major insertion accounts for a significant proportion of mutations underlying human lipoprotein lipase deficiency. Proc Natl Acad Sci USA 86:948-952 Mullis KB, Faloona FA (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335-350 Nakamura Y, Lathrop M, Leppert M, Dobbs M, Wasmuth J, Wolff E, Carlson M, et al (1988) Localization of the genetic

111 defect in familial adenomatous polyposis within a small region of chromosome 5. Am J Hum Genet 43:638-644 Nikkila EA (1983) Familial lipoprotein lipase deficiency and related disorders of chylomicron metabolism. In: Stanbury JB, Wyngaarden LB, Fredrickson DF, Goldstein JL, Brown MS (eds) The metabolic basis of inherited disease, 5th ed. McGraw-Hill, New York, pp 622-642 Saiki RK, Gyllensten UB, Erlich HA (1988) The polymerase chain reaction. In: Davies KE (ed) Genome analysis: a practical approach. IRL, Oxford and Washington, DC, pp 141-152 Wion KL, Kirchgessner TG, Lusis AJ, Schotz MC, Lawn RM (1987) Human lipoprotein lipase complementary DNA sequence. Science 235:1638-1641

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

In DNA from a male patient of German and Polish ancestry who has lipoprotein lipase deficiency, sequencing of all nine exons and intron-exon boundarie...
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