Missense Mutation (Gly + Gldss) of Human Lipoprotein Lipase Imparting Functional Deficiency*

Cloning and sequencing of lipoprotein lipase (LPL) cDNA prepared from the adipose tissue of a patient with classical LPL deficiency revealed a G to A transition at nucleotide 818 in all sequenced clones, leading to the substitution of glutamic acid for glycine at residue 188 of the mature protein. Hybridization of genomic DNA with allele-specific oligonucleotides confirmed that the patient was homozygous for this mutation and revealed that carrier status for this mutation among relatives of the patient was significantly associated with hypertriglyceridemia. Assay of the patient's plasma for immunoreactive enzyme and activity demonstrated the presence of a circulating inactive enzyme protein, the concentration of which was further increased by injection of heparin. The mutant sequence was produced by oligonucleotide-directed mutagenesis, and both normal and mutant sequences were cloned into the expression vector pSVL and transfected into COS-1 cells. The normal sequence led to the in vitro expression of an enzyme that bound to heparin-Sepharose and had a specific catalytic activity similar to that of normal postheparin plasma enzyme. By contrast, the mutant enzyme expressed in vitro was catalytically inactive and displayed a lower affinity for heparin than the normal enzyme. We conclude that this single amino acid substitution leads to the in vivo expression of an inactive enzyme accounting for the manifestations of LPL deficiency noted in the patient.

bution of energy from fatty acids among various tissues (1). In familial LPL deficiency, a rare autosomal recessive disorder, chylomicrons accumulate in plasma as a consequence of impaired lipolysis. Subjects usually present with episodes of abdominal pain, recurrent attacks of acute pancreatitis, and eruptive skin xanthomas in early infancy or childhood; occasionally they remain asymptomatic (2, 3). Analysis of fasting plasma typically reveals type I hyperlipoproteinemia with triglyceride concentrations usually above 1500 mg/dl, normal or moderately elevated cholesterol in total plasma and in VLDL, and markedly reduced cholesterol concentrations in the low density and high density lipoprotein fractions. Absence of significant LPL activity in postheparin plasma as well as in adipose tissue establishes the diagnosis; deficiency in the cofactor apolipoprotein CII is ruled out by specific activation assays (2). The phenotype of heterozygotes for LPL deficiency remains poorly characterized (2-5). Human LPL cDNA has been cloned and its sequence reported (6). It includes a coding region of 1425 nucleotides encoding 475 amino acids. The genomic structure of the LPL gene was elucidated while the present work was in progress (7). Although abundant in adipose, muscle, and adrenal tissues, LPL mRNA has not been detected in white blood cells (6). A recent report suggests that major genomic rearrangements of the LPL gene may account for a substantial proportion of mutations causing LPL deficiency (8). We previously reported a patient with lipoprotein lipase deficiency and documented the occurrence of hyperlipidemia among her relatives (4). By cloning and sequencing LPL cDNA isolated from her adipose tissue, we have now determined that she is homozygous for an amino acid substitution at position 188 (Gly + Glu). Furthermore, in vitro expression of an LPL sequence including this mutation led to the production of an inactive enzyme. The identification of this point mutation has also allowed us to investigate the relationship between carrier status and hyperlipidemia in relatives of the patient. spotted on nylon membranes in duplicate and hybridized with '*P end-labeled oligonucleotide probes (818G, 5'-CCAGGGGACCCT-CTGGTGA-3' or 818A, 5'-TCACCAGAGAGTCCCCTGG-3') corresponding, respectively, to normal or mutant cDNA. The membranes were washed at 62 "C and autoradiographed as previously described (21).

RESULTS
Assay of the proband's grossly lipemic plasma for lipase activity and LPL mass was carried out after removal of the Total RNA from the proband and a normal control were electrophoresed, blotted, and hybridized with pLPL35. Lane I, 3 Kg of RNA from the proband; lanes 2, 3, and 4, 1,3, and 6 fig, respectively,  show the extent and the direction of each sequencing experiment. large lipid particles by centrifugation. Treatment of the plasma with deoxycholate before centrifugation prevented the simultaneous removal of adsorbed lipases, thus facilitating the demonstration of immunoreactive LPL-like material in pre-and postheparin plasma as well as hepatic triglyceride lipase in postheparin plasma (Table I). The LPL activity in the postheparin plasma of this patient was extremely low compared with the established range of 227 f 58 nmol/min/ ml (mean f S.D.) for normal females (XI), confirming our previous measurements which relied on a different assay (4). The low measured LPL activity could be due either to analytical error or to a minor cross-reactivity of the inhibiting antibody with hepatic triglyceride lipase, resulting in a slight overestimation of LPL activity. The virtual absence of LPL activity in pre-as well as postheparin plasma, in spite of the presence of LPL-like immunoreactive material, suggested that LPL deficiency in this patient resulted from the synthesis of a dysfunctional protein.
In the absence of documented inbreeding, the proband could be either a homozygote for a single mutation or a compound heterozygote for two different mutations in the LPL gene. Southern blot analysis of the proband's genomic DNA, with pLPL35 as a probe, showed no gross alteration of LPL gene structure (data not shown). Hybridization experiments with RNA from adipose tissue indicated an apparently normal amount of full-length LPL mRNAs, 3.75 and 3.35 kilobases long (Fig. 1). Because both messages have been identified in normal subjects and the difference in size appears to result from the use of different polyadenylation signals (6), we planned to clone LPL cDNA from the proband's biopsy specimen. We amplified a 1567-bp region of LPL cDNA containing the entire coding region by means of the polymerase chain reaction (20), after synthesizing cDNA from adipose tissue total RNA (Fig. 2). Agarose gel electrophoresis of the amplified DNA showed a single DNA band of the size anticipated (data not shown).
After the amplified cDNA was cloned in Ml3 vector, eight independent clones were isolated and sequenced. Two additional clones amplified from pLPL35 were also sequenced as normal controls. The sequences obtained for each of the eight clones from the patient's cDNA were aligned and compared with the normal sequence. Thirteen apparent mutations were unique to individual clones; we ascribe these occasional mismatches to either sequencing artifacts or misincorporations during the amplification (24). By contrast, a single point mutation, a G + A transition at position 818 of the LPL cDNA, hereafter referred to as the 818A mutation, was observed in all eight clones from the patient but not in the two normal clones, in agreement with the published sequence (6). The consequence is a glycine (GGG)-to-glutamic acid (GAG) substitution at the 188th amino acid residue of the mature enzyme (Fig. 3). This result implied that either the proband was homozygous for this mutation, or she was a compound heterozygote for two distinct LPL mutant alleles and we were able to amplify and sequence mRNA species from only one of them.
Two methods were employed to examine these possibilities and to identify heterozygous carriers of the 818A mutation. As the 818A mutation abolishes a Sau961 recognition site located at position 818-822 of the normal cDNA sequence (from GGNCC to AGNCC), Southern blot analysis of Sau961digested genomic DNA was performed with an amplified 1567bp LPL cDNA as a probe. As shown in Fig. 4, the patient and both parents presented a restriction fragment consistent with the loss of a Sau961 site. Densitometric analysis suggested that the patient was homozygous for the 818A mutation, while her parents were heterozygous; however, the presence of another DNA fragment comigrating with the 810-bp fragment prevented a definitive inference. In a further experiment, the 818A mutation was detected directly by amplification with specific primers of a 58-bp region of genomic DNA encompassing nucleotide 818, followed by hybridization with two allele-specific oligonucleotide probes for the normal (818G) and mutant (818A) sequences. The fact that our experiments yielded the expected 58-bp fragment indicated that no intron interrupts this region of the LPL gene. Indeed, the mutation and both primer sequences lie within exon 5 of the LPL gene (7). Dot-blot hybridization of amplified genomic DNAs confirmed that the proband is homozygous for mutation 818A and that both parents are heterozygous (Fig. 5). The 818A mutation was likely to be responsible for the deficient activity of LPL in the patient because 1) she is homozygous for the 818A mutation; 2) no other mutation was detected in the entire coding region of the transcribed LPL sequence; 3) the mutation was not detected in 60 unrelated random subjects (data not shown); 4) almost no LPL enzymatic activity was detected in her adipose tissue; and 5) a significant amount of immunoreactive LPL mass was detected in postheparin plasma (Table I).
Definitive confirmation of the functional significance of the 818A mutation was established by in vitro expression of the normal and mutant LPL sequences. LPL mRNA was present at similar levels in cells transfected with vectors containing either normal or mutant LPL sequences, while such a message was undetectable after transfection with pSVL alone (Fig. 6). Cells transfected with the normal sequence (pSVL-LPL) expressed LPL activity as well as immunoreactive LPL, whereas cells transfected with the mutant sequence (pSVL-LPL818A) expressed immunoreactive material lacking significant LPL activity (Table II) The amplified genomic DNA was hybridized to two allelespecific probes for the normal and mutant sequences surrounding position 818. Representative results from the core family members of the proband are shown. Shading for symbols and identification designations are as in Fig. 8. Cloned Ml3 DNAs containing mutant (MU) and normal ( WT) LPL sequences were amplified and hybridized as controls.
expressed neither activity nor immunoreactive material (data not shown). When the respective culture media were analyzed by affinity chromatography on heparin-Sepharose, the medium from cultures expressing the normal gene resulted in two peaks of immunoreactive LPL (Fig. 7A). The most retarded peak, having the highest affinity for heparin, was enzymatically active and had a specific activity (Table II) similar to that of postheparin plasma enzyme (25). The nature of the inactive material has not been investigated at this time; it could be either inactivated previously active enzyme or enzyme synthesized in an inactive form, possibly due to defective post-translational processing in this particular Total RNA isolated from COS-1 cells were electrophoresed, blotted, and hybridized with pLPL35. Lane I, cells transfected with normal LPL sequence (pSVL-LPL); lane 2, cells transfected with mutant LPL sequence (pSVL-LPL818A); Lane 3, cells transfected with pSVL alone; lane 4, untransfected cells. The origins of the two major transcripts detected, of estimated sizes equal to 3900 and 3000 nucleotides, respectively, were investigated by enzymatic amplification with primers spanning either the polyadenylation signal or the VP1 splice junction of pSVL after first strand cDNA synthesis of oligo(dT)-primed total RNA (data not shown). These experiments showed that a single polyadenylation signal was used and that the two species reflected the utilization of alternative acceptor sites located at nucleotides 558 and 1463 of the VP1 splice junction in a manner analogous to the generation of 19 S and 16 S mRNA in SV40. Media from three dishes were pooled, treated with deoxycholate, and 10 ml were loaded on the column using the IO-ml Superloop, which was cooled by submersion in ice. The column fractions were analyzed for total lipase activity and LPL concentration by immunoassay. The NaCl gradient profile was calculated by assuming an elution volume of 2.2 ml for low molecular weight buffer components. The first immunoreactive peak in panel A and the only peak in panel B had the same elution volume (36 ml) which corresponded to a concentration of 0.9 M NaCl. The second immunoreactive peak in panel A eluted at 38 ml corresponding to 1.2 M NaCl. Further experimental detail is provided under "Experimental Procedures." 1 Fractions 70-74 (Fig. 7A).
-, not calculated. c The peak measures of total LPL protein and activity were calculated by adding up the contents of each of the contributing fractions.
Lipoprotein determinations in the patient and 27 of her relatives are reported in Fig. 8. Nine relatives of the proband presented plasma triglyceride concentrations above the 95th percentile for sex and age (26). In none of these subjects was the LDL-cholesterol concentration notably elevated. Dot-blot hybridization detected 11 heterozygous carriers of the 818A mutation (Fig. 5)

DISCUSSION
The investigation of an LPL-deficient proband and her close relatives, some of whom exhibited less severe hypertriglyceridemia, has led to the identification of a missense mutation of LPL with a single amino acid substitution. This mutation, present in a double dose in the proband and resulting in the elaboration of a dysfunctional enzyme protein, was also segregating in several relatives. These findings provide new opportunities for clinical as well as biochemical investigations, whether exploring the contribution of heterozygous LPL deficiency to genetic hyperlipidemia or elucidating structure-function relationships within the LPL molecule. The detection of carriers of this mutation with allelespecific oligonucleotide probes in the present pedigree, or in other pedigrees in which the same mutation may be segregating, may be a first step toward the identification of other factors, whether genetic or environmental, which lead to the expression of hypertriglyceridemia. That as yet unidentified 4. Wilson, D. E., Edwards, C. Q., and Chan, I.-F. (1983) Metabolism 32,1107-1114 factors modulate the expression of the heterozygous state for LPL deficiency is supported by the observation of a normal lipid profile in some carriers, including the father of the proband (subject 3 in Fig. 8), or of hypertriglyceridemia in noncarriers (e.g. subject 13).
Lipoprotein lipase is a unique enzyme which in addition to its catalytic site (27) also has been postulated to possess domains for interfacial lipid binding (28), heparin binding (29,30), apolipoprotein C-II binding (31), and self-association (13). The structure and precise localization of these domains have not yet been elucidated. Therefore, any naturally occurring mutation in LPL leading to a single amino acid substitution which imparts defective function may provide helpful clues for an improved understanding of structure-function relationships within this protein.
The substitution of glutamic acid for glycine at position 188, as described here, resulted in the elaboration of an apparently inactive enzyme protein which also displayed lower affinity for heparin than normal active LPL (Fig. 7). This mutation has affected the central region of homology which exhibits strong sequence conservation among lipases (32). This region harbors the domain for interfacial lipid binding and probably also the catalytic site (28,33). Since the putative domain for heparin binding resides farther away in the carboxyl-terminal direction of the enzyme, the decreased affinity for heparin is probably due to a conformational change extending outside the region of central homology. This notion is supported by the application of the Chou-Fasman algorithm (34) which predicts that the 818A mutation should disrupt a P-turn in the secondary structure of the protein. It remains to be established whether the predicted change in conformation is present in the nascent enzyme protein or happens after the enzyme has been secreted and which other functional domains are affected, There is some evidence that the affinity of normal bovine LPL for heparin decreases when the enzyme becomes inactive (35). By analogy, the nascent mutant enzyme might be fully active and then inactivated at a much faster rate than the normal enzyme, thus leading to functional deficiency, decreased heparin binding, and circulating inactive enzyme.