Identification and Characterization of Two Novel Compounds: Heterozygous Variants of Lipoprotein Lipase in Two Pedigrees With Type I Hyperlipoproteinemia

Abstract

Background: Type I hyperlipoproteinemia, characterized by severe hypertriglyceridemia, is caused mainly by loss-of-function mutation of the lipoprotein lipase (LPL) gene. To date, more than 200 mutations in the LPL gene have been reported, while only a limited number of mutations have been evaluated for pathogenesis.

Objective: This study aims to explore the molecular mechanisms underlying lipoprotein lipase deficiency in two pedigrees with type 1 hyperlipoproteinemia.

Methods: We conducted a systematic clinical and genetic analysis of two pedigrees with type 1 hyperlipoproteinemia. Postheparin plasma of all the members was used for the LPL activity analysis. In vitro studies were performed in HEK-293T cells that were transiently transfected with wild-type or variant LPL plasmids. Furthermore, the production and activity of LPL were analyzed in cell lysates or culture medium.

Results: Proband 1 developed acute pancreatitis in youth, and her serum triglycerides (TGs) continued to be at an ultrahigh level, despite the application of various lipid-lowering drugs. Proband 2 was diagnosed with type 1 hyperlipoproteinemia at 9 months of age, and his serum TG levels were mildly elevated with treatment. Two novel compound heterozygous variants of LPL (c.3G>C, p. M1? and c.835_836delCT, p. L279Vfs*3, c.188C>T, p. Ser63Phe and c.662T>C, p. Ile221Thr) were identified in the two probands. The postheparin LPL activity of probands 1 and 2 showed decreases of 72.22 ± 9.46% (p<0.01) and 54.60 ± 9.03% (p<0.01), respectively, compared with the control. In vitro studies showed a substantial reduction in the expression or enzyme activity of LPL in the LPL variants.

Conclusions: Two novel compound heterozygous variants of LPL induced defects in the expression and function of LPL and caused type I hyperlipoproteinemia. The functional characterization of these variants was in keeping with the postulated LPL mutant activity.

Keywords: hypertriglyceridemia; lipoprotein lipase (LPL); pedigree; type 1; variants.

Figure 1

The clinical characteristics of proband 1. (A) The proband 1 triglyceride and total cholesterol levels. The dashed line indicates the normal reference range. Abbreviations: TG, triglycerides; TC, total cholesterol. Normal range: TG: 0.4–1.8 mmol/L; TC: 3.6–6.2 mmol/L. (B) Abdomen and lower limbs of patient 1. (C) Abdominal CT scan of patient 1. The pancreas was irregular in shape; the liver was normal in shape and size; no abnormal density shadow was observed in the parenchyma; no expansion of the bile ducts in and outside the liver was observed; the shape and size of the gallbladder were fine; no abnormal density shadows were found in the gallbladder; and the spleen was normal in shape and size.

Figure 2

The pedigree including the patients. The arrow refers to the proband. Black indicates that the person has type I hyperlipoproteinemia. The shading indicates that the person carries gene mutations but has a healthy phenotype. Circles indicate female individuals reared, and squares indicate male individuals reared. The question mark means that it cannot be determined whether the individual carries a genetic mutation. (A) The pedigree of patient 1. (B) The pedigree of patient 2.

Figure 3

LPL gene sequencing diagram. (A) LPL gene (reference sequence NM_000237) sequencing diagram of patient 1 and her children. (B) LPL gene (reference sequence NM_000237) sequencing diagram of patient 2 and his parents.

Figure 4

Analysis of LPL activity in plasma. Peripheral blood was collected at 10 min after heparin injection (60 IU/kg) to assay lipase activity by the enzyme-fluorescent method.

Figure 5

HEK 293T/17 cells transiently transfected showed a reduction in the production of LPL variants. HEK293T/17 cells transfected with the p. M1? mutation, p. L279Vfs*3 mutation, or p. M1? and p. L279Vfs*3 mutation showed virtually no protein in the cell lysate (p<0.01). Compared with cells transfected with the plasmids containing the pcDNA3.1-LPL-WT, there was no significant difference in the amount of protein reduction produced by the cells transfected with the p. S63F mutation or the two variants (p. S63F and p. I221T), but protein synthesis was reduced by 48.33 ± 1.78% in cells transfected with the p. I221T variant (p<0.01). Abbreviations: EV, empty vector; WT, wild type; LPL, lipoprotein lipase.

Figure 6

Functional analysis of LPL mutants in the medium. Except for the p. L279Vfs*3 mutation, LPL activity in the medium transfected with other plasmids containing pcDNA3.1-LPL-MU was significantly reduced compared with the wild type. Specifically, cells transfected with the p. M1? or the two variants (p. M1? and p. L279Vfs*3) showed reductions in protein activity of approximately 55.93 ± 3.28% (p<0.01) or 59.84 ± 4.47% (p<0.01), while LPL activity was reduced by 62.76 ± 9.90% (p<0.01) or 63.25 ± 10.55% (p<0.01) in the media of HEK 293T/17 cells transfected with p. Ser63Phe or the two variants (p. Ser63Phe and Ser63Phe+Ile221Thr). The media of cells transfected with p. Ile221Thr demonstrated a maximum reduction in LPL activity of approximately 81.56 ± 4.35% (p<0.01).