Severe hypertriglyceridemia due to two novel loss-of-function lipoprotein lipase gene mutations (C310R/E396V) in a Chinese family associated with recurrent acute pancreatitis

Abstract

Lipoprotein lipase (LPL) is widely expressed in skeletal muscles, cardiac muscles as well as adipose tissue and involved in the catabolism of triglyceride. Herein we have systematically characterized two novel loss-of-function mutations in LPL from a Chinese family in which afflicted members were manifested by severe hypertriglyceridemia and recurrent pancreatitis. DNA sequencing revealed that the proband was a heterozygote carrying a novel c.T928C (p.C310R) mutation in exon 6 of the LPL gene. Another member of the family was detected to be a compound heterozygote who along with the c.T928C mutation also carried a novel missense mutation c.A1187T (p.E396V) in exon 8 of the LPL gene. Furthermore, COS-1 cells were transfected with lentiviruses containing the mutant LPL genes. While C310R markedly reduced the overall LPL protein level, COS-1 cells carrying E396V or double mutations contained similar overall LPL protein levels to the wild-type. The specific activity of the LPL mutants remained at comparable magnitude to the wild-type. However, few LPL were detected in the culture medium for the mutants, suggesting that both mutations caused aberrant triglyceride catabolism. More specifically, E396V and double mutations dampened the transport of LPL to the cell surface, while for the C310R mutation, reducing LPL protein level might be involved. By characterizing these two novel LPL mutations, this study has expanded our understanding on the pathogenesis of familial hypertriglyceridemia (FHTG).

Keywords: acute pancreatitis; compound heterozygosity; hypertriglyceridemia; lipoprotein lipase; mutation.

Figure 1. The proband's triglyceride levels and response to treatment

Aspart 12 IU was administrated three times a day before each meal and Glargine 20 IU was administrated before sleep. Fenofibrate, 100mg three times a day.

Figure 2. Abdominal computed tomography

A typical computed tomography manifestation of enlarged pancreas with blurred outline, disappearance of peripancreatic space and thickened left renal fascia was highly suggestive of acute pancreatitis.

Figure 3. LPL gene (reference sequence NM_000237)sequencing diagram

The diagram showed the partial forward sequence of exon 6 in the proband (A), a control subject (C) and reverse sequence of exon 8 in the proband's niece (B).

Figure 4. Analysis of LPL activity and mass in plasma

The post-heparin LPL mass (A) and lipase activity (B) was measured in the proband MJH, CPH (I-1) and 10 normolipidemic subjects. Peripheral blood was collected at 10 min after heparin injection (60IU/kg) to assay lipase activity and LPL mass by enzyme-fluorescent method and ELISA, respectively. The data of controls were presented as mean.

Figure 5. A native gel western blot analysis of LPL dimerization in post-heparin plasma

Coomassie staining was used as a loading control (A). Post-heparin plasma of the proband MJH, CPH(I-1), and a normolipidemic control was analyzed by a native gel western blot (B).

Figure 6. Quantitation of extracted mRNA and normalized to 18S rRNA

mRNA was extracted from transfected COS-1 cells containing the mutant LPL genes and quantitatively determined by qPCR. The mRNA levels of LPL-310, LPL-396, and LPL-310396 were almost equal to that of LPL-wt, and there was no significant difference between the wild-type and mutants (P>0.05). Values are shown as mean±SD. Each experiment was repeated three times.

Figure 7. Functional analysis of LPL mutants in the medium

Activity of LPL mutants was assayed as a percentage of LPL wild-type after transfection. Each experiment was repeated by measuring six separate dishes. Values represent the mean ±SEM. ☆and §, significant (P<0.05) differences from LPL-wt, LPL-310, respectively.

Figure 8. Functional analysis of LPL mutants in the cell lysates

Specific activity of LPL in the cell lysates is calculated by dividing LPL activity by LPL mass. There was no significant difference between the wild-type and mutants (P>0.05). Values are shown as mean±SD.

Figure 9. Pedigree diagram of the family

Proband is shown by the arrow. hypertriglyceridemia subjects who are heterozygous for the mutant C310R LPL gene. Hypertriglyceridemia subjects who are heterozygous for the mutant E396V LPL gene. Hypertriglyceridemia subject who is compound heterozygous for the mutant C310R and E396V LPL gene combined. Normolipidemic subjects. Deceased subjects.