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.


| INTRODUC TI ON
Hypertriglyceridaemia (HTG) is a very rare autosomal recessive disorder which unlikely causes acute pancreatitis (AP). 1,2 It has been reported that HTG leads to AP only in 10% of all the cases. Patients with triglyceride (TG) level >2000 mg/dL (22.6 mmol/L) are clinically diagnosed as 'very severe HTG', because this level of TG inevitably leads to pancreatitis. 1,3,4 In addition, patients with fasting TG level in between 1000 (11.3 mmol/L) and 2000 mg/dL (22.6 mmol/L) are diagnosed as 'severe HTG', carrying increased risk of developing pancreatitis as TG level may rise above 2000 mg/dL (22.6 mmol/L) after eating. Severe HTG patients with serum TG level >11.3 mmol/L (1000 mg/dL) gradually and progressively developed hepatosplenomegaly, stomach ache, lipaemia retinalis and eruptive xanthomas with an increased risk of AP. [5][6][7] However, the specific mechanism by which HTG patients gradually develop AP is still unknown. One obvious explanation is that both HTG and increased level of chylomicrons (CM) lead to increase the plasma viscosity, which leads to ischaemia in pancreatic tissue and organ inflammation. 7,8 Germline mutations in LPL gene cause an extremely rare autosomal recessive familial lipoprotein lipase deficiency (LPLD) manifested with severe HTG and chylomicronaemia with recurrent AP. 8,9 According to the aetiology, HTG is classified into two types, primary and secondary. 9 In addition, primary HTG is usually caused by germline mutations of lipoprotein lipase (LPL) gene. In non-hepatic tissues, LPL catalyses TG. 3 The LPL gene is located on chromosome 8 and comprises of 10 exons. LPL gene encodes an enzyme called lipoprotein lipase, consisting of 475 amino acids. 10,11 Wild-type lipoprotein lipase hydrolyses TG in TG-rich lipoproteins. Homozygous or compound heterozygous mutations in LPL gene lead to the complete or partial loss of function of both copies of the LPL gene which finally results into severe HTG. 12 Till date, hundred mutations of LPL gene have been reported. 13 In this study, we identified two unrelated Chinese probands, presented with HTG and AP. Clinical diagnosis found phenotypic heterogeneity between these two probands. The proband 1 was presented with very severe HTG and AP, while the proband 2 was manifested with severe HTG and AP. The clinical phenotype of proband 1 is comparatively more severe than patient 2. Whole exome sequencing identified a novel splice donor site mutation as well as two previously reported mutations in LPL gene in these two probands. In vitro functional analysis of these mutations was performed to understand their effect underlying the disease phenotype as well as the phenotypic heterogeneity between these two unrelated Chinese probands.
Our present study also strongly described the significance of whole exome sequencing for identifying the candidate gene and diseasecausing mutations in patients with heterogeneous inherited disorders.

| Patient and clinical samples
In our present study, we investigated two Chinese probands manifested with HTG and AP, from two unrelated Chinese families. The proband 1 and proband 2 belong to the families 1 and 2, respectively. Moreover, the proband 1 and proband 2 are the only affected individuals in their family. In family 1, we screened the proband 1 (II-2), proband's mother (I-2), elder sister (II-1), wife (II-2) and his two sons (III-1 and III-2). But in family 2, we only screen the proband due to unavailability of blood samples of the other family members. All of the members of these two families have provided informed consent for their participation in this study. The study was approved by the institutional review board at The Ethics Committee of the First Affiliated Hospital, Sun Yat-sen University.

| Whole exome sequencing
Blood samples were collected, and genomic DNA was extracted from these two probands by using a QIAamp DNA Blood Mini Kit (Qiagen) based on the manufacturer's instructions. Both the proband 1 and proband 2 were subjected to whole exome sequencing. Agilent SureSelect version 6 (Agilent Technologies) was used for capturing sequences. Then, the enriched library was sequenced on an Illumina HiSeq 4000. Next, whole exome sequencing reads were aligned to the GRCh37.p.10 by using Burrows-Wheeler Aligner software (version 0.59). After that, GATK IndelRealigner was used for local realignment of the Burrows-Wheeler aligned reads. Then, the base quality recalibration of the Burrows-Wheeler aligned reads was performed by using GATK Base Recalibrator. Next, identification of single-nucleotide variants (SNV) and insertions or deletions (indel) has been done by GATK The function of the variant and their correlation with the disease phenotype was done by OMIM database and previously published literature. Schematic presentation of the detailed and comprehensive data interpretation process is described in Figure 1.

| Sanger sequencing
Sanger sequencing was performed to validate the variants identified by whole exome sequencing. Sanger sequencing was performed with these primers: The reference sequence NM_000237 of LPL was used.

| Minigene construction
In vitro exon trapping study was performed to understand the effect of the splice donor site mutation (c.1322+1G>A) of the LPL gene at the transcriptional level. Minigene construction has been done according to the previously reported protocol. 15 The genomic DNA from proband 1 and proband 2 was used as template for constructing minigene. Minigenes were consisting of one wild-type (WT) and one mutant allele, including exon 7, intron 7, exon 8, intron 8 and exon 9 with 5′ and 3′ intronic flanking sequences. Minigenes were amplified by PCR and cloned into pSPL3 (exon trapping vector) vector through double digestion by two restriction endonucleases: BamHI and XhoI. 15 After constructing both the wild-type and mutant minigenes, they were transiently transfected into COS-7 cell line.

| Transfection and Sanger sequencing
Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum, 1% penicillin-streptomycin and 1% glutamine was used to culture the COS-7 cells within a humidified incubator with 5% CO 2 and 37°C temperature. Total RNA was extracted

| Real-time PCR (RT-PCR)
Transfected COS-7 cells were collected, and total RNA was extracted RT-PCR was performed by ABI 7500 real-time PCR system (Applied Biosystems). The relative expression level was calculated using 2 −ΔΔCt , and the software SPSS20 was used for the analysis of the data.

| Analysis of LPL activity and mass
Post-heparin transfected COS-7 cell culture medium was harvested and centrifuged at 4°C for 20 minutes at 12 000 g. The remaining precipitated cells were washed with PBS and then solubilized in 80 μL cell lysis buffer (Tiangen Biotechnology Co., Ltd.). The cell lysates were centrifuged at 4°C for 20 minutes at 12 000 g, and the supernatant was stored at −80°C. LPL mass and activity were determined by Human LPL ELISA kit (Shanghai Xinfan Biotechnology Co., Ltd.) and LPL activity assay kit (Cell Biolabs, INC.), respectively, by using M5 full-band multifunctional enzyme label instrument (Molecular Devices). In addition, for LPL activity assay, the parameters were set to 485/520 nm and filter set with cut-off of 495 nm.

| Pedigree and family
In this present study, we identified two Chinese patients from two unrelated Chinese families, manifested with severe HTG and AP ( Figure 2A,B). In both the families, only the proband is the affected The lipid profiles of all the subjects

Lipids
Reference range

Wife of proband 1 (II-3)
Elder son of proband 1 (III-1) individual. We clinically diagnosed the proband 1 with very severe HTG and AP, while the proband 2 was identified with severe but comparatively milder phonotype than proband 1. Here, we aimed to identify the candidate gene and disease-causing mutation underlying the disease phenotype in both of these two probands and functionally characterize the variants by performing in vitro experiments. In vitro functional analysis allowed us to understand the basis of phenotypic heterogeneity between these two patients. In addition, proband 1 and all the living members of his family have been studied well, whereas proband 2 is the only member of her family has been studied due to the early death of her parents. The lipid profiles of all the subjects are described in Table 1. Routine blood test result of all the subjects is given in Table 2.

| Patient 1
The proband 1 is a 32-year-old Chinese man belongs to nonconsanguineous Chinese parents (Figure 2A) The proband had never been identified with hypertension, diabetes, coronary heart disease or any other diseases. His parents were born and brought-up at the same town but not consanguineous. His father had been suffering from hepatitis and accidentally died at age of 57. The blood lipids' levels for his mother, sister and two sons were all normal ( Table 1). The full blood test result is given in Table 2.

| Patient 2
The proband 2 is a 42-year-old Chinese woman presented with HTG and AP ( Figure 2B). The proband 2 was first admitted at our hospital at the age of 36 years with AP. She was also identified with HTG (TG: 17.1 mmol/L).
We recommend her with the treatment of fenofibrate 200 mg QD. After

| Proband 2
In the proband 2, whole exome sequencing identified two het- we were unable to test them for these two heterozygous mutations.
These two mutations were not detected in 100 ethnically matched normal healthy control individuals. These two mutations were also not present in the ExAC, dbSNP, gnomAD, 1000 Genome Database as well as in BGI's database which is consisting of ~50 000 Chinese Han samples. Hence, both of these two mutations could be regarded as potential pathogenic mutations, causes disease in proband 2 with compound heterozygosity.

| Functional analysis of the novel splice donor site mutation
In vitro exon trapping assay found that the novel heterozygous   Figure 5A). Each experiment was repeated three times. 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

| Evolutionary conservation test
In the proband 2, whole exome sequencing identified a heterozygous transversion, c.835C>G in exon 6, leads to the replacement of a leucine by valine at the position of 279 amino acid (p.Leu279Val) of the wild-type LPL protein. Multiple sequence alignment showed that p.Leu279 is evolutionarily highly conserved among different species, indicating its importance in both the structure and the functions of the wild-type LPL protein ( Figure 6A).

| In silico protein structural analysis
In order to understand the effect of the missense mutation into the occurrence one clash. The p.Leu279 is shown in Figure 6C.
The mutated residue p.Val279 causes one clash marked with "yellow" line ( Figure 6D).

| D ISCUSS I ON
In the present study, we identified two Chinese probands with severe HTG and AP. Proband 1 was presented with very severe HTG and AP, while proband 2 was manifested with similar but relatively less serious HTG phenotype than proband 1. In this study, our result showed that these three mutations  Figure 5B). However, significant decrease in LPL enzyme activities was found in all of these three mutant-construct transfected COS-7 cells in cell culture medium ( Figure 5C), but no change in cell lysate ( Figure 5C). Hence, these three mutations exhibited both decreased catalytic activity and secretion ability.
HTG with AP is an extremely rare disorder of lipoprotein metabolism caused by germline mutations in LPL gene with an autosomal recessive mode of inheritance. In this study, we identified a novel and two previously reported mutations in the LPL gene causing HTG and AP. HTG with AP is an extremely severe and potentially fatal. 1,3 However, very severe and severe HTG generally develop with genetic mutations in LPL gene which impair the catabolism of TG in CM and very low-density lipoproteins (VLDL). 10 In adipose tissue, muscle, islets and macrophages, lipoprotein lipase is playing the key role in hydrolysis of the TG in CM and VLDL. 29 Hence, mutations in LPL gene cause the formation of partially or completely non-functional lipoprotein lipase which cannot catabolize the TG in CM leads to the gradual and progressive increase in CM levels and increased the risk for AP, HTG, diabetes mellitus and other metabolic disorders. 30 In order to provide specific and effective treatment to the patients with type I hypertriglyceridaemia, analysis of LPL mass and LPL enzyme activity is very crucial and significant. 31 Patients with no LPL mass could not be successfully treated with alipogene tiparvovec due to immune response to the injected functional LPL protein. 32 Hence, analysis of both lipid mass and lipase enzyme activity assay is very significant for the patients with HTG and AP. In our present study, we identified two unrelated Chinese patients from two Chinese families. Both probands were identified and clinically diagnosed with HTG and AP. Proband 1 has been suffering from very severe HTG with AP, while the proband 2 was identified with severe HTG. Whole exome sequencing identified two pairs (c.162C>A and c.1322+1G>A in proband 1 and c.835C>G and c.1322+1G>A in proband 2) of variants in LPL gene in both the probands. Both the probands was found to harbour a common splice donor site mutation (c.1322+1G>A). Proband 1 was identified with a nonsense mutation (c.162C>A; p.Cys54*), while proband 2 was carrying a missense mutation (c.835C>G, p.Leu-279Val). The effect of a nonsense truncated protein is more than that of a protein with a missense change. Hence, proband 1 was identified with more severe phenotype than proband 2.
In conclusion, in this study, we identified two unrelated Chinese patients with extremely rare and severe HTG with AP. Both the patients sharing a common splice donor site mutation in LPL gene.
Phenotypic heterogeneity was also identified between these two patients. Functional characterization of identified mutations has been done. Additionally, we also described the importance of whole exome sequencing for identifying candidate gene with disease-causing mutations in rare disease of metabolism.

ACK N OWLED G EM ENTS
We are thankful to the proband and all the family members for participating in our study. We are thankful to the China National GeneBank and Shenzhen Peacock Plan (No. KQTD20150330171505310).

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
Santasree Banerjee and Zhihong Liao designed the study and su-