Identification and functional characterization of mutations in LPL gene causing severe hypertriglyceridaemia and acute pancreatitis

Abstract

Hypertriglyceridaemia is a very rare disorder caused by the mutations of LPL gene, with an autosomal recessive mode of inheritance. Here, we identified two unrelated Chinese patients manifested with severe hypertriglyceridaemia and acute pancreatitis. The clinical symptoms of proband 1 are more severe than proband 2. Whole exome sequencing and Sanger sequencing were performed. Functional analysis of the identified mutations has been done. Whole exome sequencing identified two pairs of variants in LPL gene in the proband 1 (c.162C>A and c.1322+1G>A) and proband 2 (c.835C>G and c.1322+1G>A). The substitution (c.162C>A) leads to the formation of a truncated (p.Cys54*) LPL protein. The substitution (c.835C>G) leads to the replacement of leucine to valine (p.Leu279Val). The splice donor site mutation (c.1322+1G>A) leads to the formation of alternative transcripts with the loss of 134 bp in exon 8 of the LPL gene. The proband 1 and his younger son also harbouring a heterozygous variant (c.553G>T; p.Gly185Cys) in APOA5 gene. The relative expression level of the mutated LPL mRNA (c.162C>A, c.835C>G and c.1322+1G>A) showed significant differences compared to wild-type LPL mRNA, suggesting that all these three mutations affect the transcription of LPL mRNA. These three mutations (c.162C>A, c.835C>G and c.1322+1G>A) showed noticeably decreased LPL activity in cell culture medium but not in cell lysates. Here, we identified three mutations in LPL gene which causes severe hypertriglyceridaemia with acute pancreatitis in Chinese patients. We also described the significance of whole exome sequencing for identifying the candidate gene and disease-causing mutation in patients with severe hypertriglyceridaemia and acute pancreatitis.

Keywords: LPL gene; acute pancreatitis; compound heterozygous; hypertriglyceridaemia; novel mutations.

Figure 1

Data interpretation pipeline for whole exome sequencing

Figure 2

A‐B, Family pedigree of proband 1 (A) and proband 2 (B). The filled symbol indicates the patient (proband), and the half‐filled symbol indicates the unaffected and heterozygous carrier parents. The arrow points to the proband. C‐F, Computed tomography (CT) test. (C) Arterial phase of CT enhanced scan. The white arrow indicates the disappearance of fat gap between pancreas and stomach, and local effusion. The black arrow indicates the slight oedema at both the body and tail of the pancreas. (D) Coronal reconstruction of CT enhanced scan. The white arrows indicate the peripancreatic exudation. (E) Arterial phase of CT enhanced scan. The white arrows indicate exudation and effusion beneath the body of the pancreas. (F) Sagittal reconstruction of CT enhanced scan. The white arrows indicate peripancreatic exudation

Figure 3

Sanger Sequencing. Partial DNA sequences in the LPL gene and APOA5 gene by Sanger sequencing of the proband 1 and his family members (A). Partial DNA sequences in the LPL gene by Sanger sequencing of the proband 2 (B). The reference sequence NM_000237 of LPL gene was used. The reference sequence NM_001166598 of APOA5 gene was used

Figure 4

Functional characterization of the splice donor site mutation by in vitro exon trapping assay. (A) RT‐PCR products of the c.1322+1G>A in pSPL3 minigene constructs, Lane M: the 1000 bp marker/ladder. Lanes 1 and 2: the wild‐type (WT) correctly spliced exon 7, exon 8 and exon 9 of LPL cDNA in proband 1 and proband 2, respectively. Lanes 3 and 4: the aberrantly spliced LPL cDNA with skipping of 134 bp of exon 8. Lanes 5 and 6: empty vector 265 bp. Lanes 7 and 8: the negative control. (B) Schematic representation of the splicing model. (C) Reverse transcription‐PCR and direct sequencing of LPL cDNA from cells transfected with the wild‐type construct showed normal splicing of exon 7 to exon 9. In contrast, Sanger sequencing of mutant reverse transcription‐PCR (RT‐PCR) products revealed partial loss of 134 bp from exon 8 which in turn results in removal of 45 amino acids in the LPL polypeptide due to abolition of the wild‐type donor splice site

Figure 5

Functional analysis of LPL mutants in vitro. (A) Quantitation of extracted mRNA and normalized to 18S rRNA. mRNA was extracted from transfected COS‐7 cells containing the mutant LPL genes and quantitatively determined by qPCR. The expression level of LPL mRNA was showing significant difference between the wild‐type and mutants (c.162C>A, c.835C>G and c.1322+1G>A). Values are shown as mean ± SD. (B) Lipase mass analysis of wild‐type and LPL mutants in both the cell culture medium and the cell lysates was performed. Lipase mass was measured by ELISA. There was no significant difference found in lipid mass in both cell culture medium and the cell lysate between the wild‐type and mutants. Values are shown as mean ± SD. (C) Lipase enzyme activity analysis of wild‐type and LPL mutants in both the cell culture medium and the cell lysates was performed. Lipase enzyme activity of LPL mutants was assayed as a percentage of LPL wild‐type after transfection. There was significant difference found in LPL enzyme activity between the wild‐type and mutants in cell culture medium but not in cell lysate. Values are shown as mean ± SD

Figure 6

In silico analysis of LPL mutations. (A) To understand the evolutionary conservation of the wild‐type amino acids of the missense mutation of LPL gene to perform sequence alignment between human (Homo sapiens) (GenBank Accession: NM_000237.3), bovine (Bos taurus) (GenBank Accession: NM_001075120.1), sheep (Ovis aries) (GenBank Accession: NM_001009394.1), chicken (Gallus gallus) (GenBank Accession: NM_205282.1), rat (Rattus norvegicus) (GenBank Accession: NM_012598.2) and mouse (Mus musculus) (GenBank Accession: NM_008509.2). The red coloured box showed that amino acid p.Leu279 is evolutionarily highly conserved. (B) Crystal structure of the whole LPL protein (PDB ID: http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6E7K) and predicted LPL protein structure upon these three mutations have been shown separately. (C) The p.Leu279 of the wild‐type LPL protein showed no clash. (D) The mutated residue p.Val279 causes one clash marked with “yellow” line