Association of lipoprotein lipase (LPL) gene variants with hyperlipidemic acute pancreatitis in southeastern Chinese population

ABSTRACT Objective: The study aims to explore the relationship between lipoprotein lipase (LPL) variants and hyperlipidemic acute pancreatitis (HLAP) in the southeastern Chinese population. Subjects and methods: In total, 80 participants were involved in this study (54 patients with HLAP and 26 controls). All coding regions and intron-exon boundaries of the LPL gene were sequenced. The correlations between variants and phenotypes were also analysed. Results: The rate of rare LPL variants in the HLAP group is 14.81% (8 of 54), higher than in controls. Among the detected four variants (rs3735959, rs371282890, rs761886494 and rs761265900), the most common variant was rs371282890. Further analysis demonstrated that subjects with rs371282890 "GC" genotype had a 2.843-fold higher risk for HLAP (odds ratio [OR]: 2.843, 95% confidence interval [CI]: 1.119-7.225, p = 0.028) than subjects with the "CC" genotype. After adjusting for sex, the association remained significant (adjusted OR: 3.083, 95% CI: 1.208-7.869, p = 0.018). Subjects with rs371282890 "GC" genotype also exhibited significantly elevated total cholesterol, triglyceride and non-high-density lipoprotein cholesterol levels in all the participants and the HLAP group (p < 0.05). Conclusion: Detecting rare variants in LPL might be valuable for identifying higher-risk patients with HLAP and guiding future individualised therapeutic strategies.


INTRODUCTION
H yperlipidemic acute pancreatitis (HLAP), which refers to acute pancreatitis (AP) due to hypertriglyceridemia (HTG) (1), is the second leading cause of AP in China (2).With the continuous improvement of socioeconomic levels and people's living standards, the prevalence of dyslipidemia (3) and HLAP (2,4,5) trends has increased yearly.Recent studies have shown that the risk of AP increases with the rise in serum triglyceride (TG) levels.When the TG level ≥ 5.65 mmol/L, the risk of AP increases significantly (6).The incidence of AP reaches up to 5% and 10%~20% in patients with serum TG levels >11.3 mmol/L and > 22.6 mmol/L, respectively (7).HLAP is more likely to get aggravated than other AP causes (8,9).In addition, higher TG levels independently correlate to an elevated rate of complications and a more severe course of disease (10).Timely reduction of serum TG levels should be the key to halting the disease's progression and reducing mortality.Moreover, HLAP presents a higher incidence of relapse (10).About 32% of the patients with HLAP have had a recurrence in a large cohort study of HTG and patients with pancreatitis (11).Maintaining normal blood lipid levels is a practical approach to prevent AP recurrence.
The aetiology of HLAP is complicated and could be caused by interactions between genetic and environmental factors.Primary HTG, also known as familial chylomicronemia (FCS), occurs when there are homozygous or compound heterozygous rare variants in six canonical genes involved in the TG metabolism, such as lipoprotein lipase (LPL), apolipoprotein C2 (APOC2), GPI-anchored HDLbinding protein 1 (GPIHBP1), apolipoprotein A5 (APOA5), lipase maturation factor 1 (LMF1) and glucokinase regulator protein (GCKR) (12,13).Recurrent pancreatitis is a clinical manifestation of the disease.However, multifactorial chylomicronemia syndrome (MCS) is much more clinically prevalent than FCS.People with a heterozygous rare variant or several common small-effect single-nucleotide polymorphisms (SNPs) in one of the six canonical genes involved in the TG metabolism are predisposed to MCS (12).It occurs due to genetic susceptibility and secondary factors, such as a high-fat diet, obesity, uncontrolled diabetes and alcohol intake.Patients with MCS also face a high risk of recurrent pancreatitis (12).Previous studies demonstrated genetic features of hypertriglyceridemia or chylomicronemia in China (14)(15)(16).However, the genetic factor of HLAP is not yet adequately studied.LPL is the crucial enzyme of lipid metabolism that degrades triglycerides.Recently, some rare variants in the LPL gene were reported in type 1 hyperlipoproteinemia and patients with HLAP (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27).A study in Taiwan has demonstrated that the rate of mutations in the LPL gene reached 17% among 53 cases of patients with HLAP.In contrast, 77.8% of patients with HTG and variants of the LPL gene have HLAP (28).However, Xiao-Yao Li and cols.demonstrated that not all patients had homozygote LPL variants and that LPL deficiency would develop AP (22).Studies with larger sample sizes are needed to confirm the association of LPL variations in HLAP.
This study aims to investigate the genetic characteristics of patients with HLAP, determine the correlations between HLAP and the rare variants of the LPL gene, offer fresh insights into the complex aetiology of HLAP, and provide new targets and directions for the individualised treatment of patients with HLAP.

Subjects
In total, 80 participants were recruited from the Fujian Medical University Affiliated First Quanzhou Hospital in southeastern China from 2018 to 2022.This research enrolled 34 patients with HLAP as cases and 26 patients with biliary acute pancreatitis (BAP) as controls.The diagnostic criteria for HLAP were: (a) the diagnosis of AP according to the Chinese guidelines for the management of AP (fulfilling at least two of the following three features: upper abdominal pain consistent with AP, serum lipase activity exceeds the upper limit of normal by three times, and radiographic features of AP); (b) confirmed dyslipidemia with serum TG >11.3 mmol/L or serum TG >5.65 mmol/L together with lipemia.The diagnostic criteria for the control group were: (a) the diagnosis of AP according to Chinese recommendations for the management of AP; (b) confirmed gallstones or biliary sludge on imaging methods or elevated serum levels of alanine aminotransferase (ALT) (>60 U/L).The exclusion criteria included patients with a history of alcohol consumption of more than 20 g/day, consumption of pancreatic toxic drugs, known pancreatic or periampullary tumours, pancreatic duct anomaly disorders, hereditary pancreatitis, autoimmune pancreatitis and traumatic pancreatitis.The Ethics Committee of the Fujian Medical University Affiliated First Quanzhou Hospital approved this study's protocols.

Information collection of general characteristics and biochemical measurements
The general clinical information, such as age, gender, alcohol consumption, smoking status, age of first onset, hospitalisation frequency and total hospitalisation cost, was collected via medical history examination and a questionnaire.Blood samples were collected in the morning after an overnight fast to detect biochemical parameters that include lipid and glycaemic profiles.

Genotyping analysis
The genomic DNA was extracted from peripheral blood lymphocytes.PCR reactions were performed on gDNA with each primer pair of the target gene.The PCR products of the expected size were separated by 1% agarose gel electrophoresis to confirm the presence and specificity of targeted sequences.Following further purification, the PCR products were sequenced by the dideoxy approach.DNA Baser v5.15.0 aligned the DNA fragments to target the references and detect the mutations from chromatograms.

Statistical analysis
Quantitative variables were expressed as mean ± standard deviation.Differences in the characteristics between groups were examined by the Student's t-test or Wilcoxon test.The chi-square test was applied to evaluate the categorical variables between groups.The Cox regression was used to calculate the HR value of the genotypes.Moreover, the Cox ratio risk regression model was applied to analyse the effects of genotype and gender on HLAP events.Statistical analysis was performed using SPSS 22.0 software, and two-tailed (p < 0.05) was considered statistically significant in this study.

General characteristics
Table 1 presents the general characteristics of the 54 patients with HLAP and the 26 controls with BAP.The mean age in the HLAP group was younger.The proportion of males in the HLAP group was more than that of females (p < 0.05).The mean total cholesterol (TC), TG, non-high-density lipoprotein cholesterol (non-HDL-C) and glycosylated haemoglobin (HBA) were significantly higher in the HLAP group than in the controls (p < 0.05).The proportion of smokers, drinkers and patients with fatty liver and diabetes was higher in the HLAP group (P < 0.05).More patients with HLAP had a relapse than controls (p < 0.05).

LPL gene variants
The rate of LPL gene mutation in the HLAP group is 14.81% (8 of 54).Table 2 shows the LPL variants detected in the HLAP group and controls, including four rare variants (rs3735959, rs371282890, rs761886494 and rs761265900).All the mutations were present in the heterozygous state, except one patient carried a compound heterozygous mutation of rs371282890 and rs761886494, which is reported as pathogenic or likely pathogenic in ClinVar.

DISCUSSION
HLAP is a very heterogeneous disease.Many patients with HLAP struggle to consistently achieve the threshold of TG levels required for preventing pancreatitis and are beset by the continued relapse of pancreatitis throughout their lifespan.Genetics could be a critical factor in the susceptibility to lipid metabolism abnormality and HLAP onset.However, whether and how genes involving lipid metabolism affect the occurrence of HLAP is not completely clear.This study compared the clinical characteristics and rare variants in the LPL gene of patients with HLAP and patients with BAP who have or do not have dyslipidemia.Several rare variants were observed in the HLAP group, especially rs371282890 (L279V).
Furthermore, the lipid parameters of patients with and without variant rs371282890 were compared.The findings revealed that variant rs371282890 carriers have more pronounced lipid metabolism abnormalities.LPL is the crucial enzyme of lipid metabolism, which hydrolyses circulating TGs packaged in chylomicrons or very low-density lipoproteins.The reduced LPL activity causes an increase in plasma TG levels, contributing to a higher risk of AP.A recent study revealed that patients with severe hypertriglyceridemia (STG) having a history of AP had a higher frequency of the rare variants in the LPL gene and all the LPL molecular regulating genes (14).The intensive genetic analysis for Chinese patients with TG ≥ 5.65 mmol/L exhibited 5 (27.8%) of 14 patients with AP had LPL variations (14).Khovidhunkit and cols.also reported that among 13 Thai patients with AP history, 3 (23.1%)had rare variants of LPL (29).In contrast, another study from China only found six rare variants in other LPL molecular regulating genes (APOA5, GPIHBP1 and LMF1).No LPL variations were found in 11 patients with HLAP (30).So far, there is no comprehensive investigation of the genetic characteristics of HLAP in China.Therefore, this study added more information to this field.This study identified four rare variants (rs3735959, rs371282890, rs761886494 and rs761265900) in LPL.Moreover, 8 (14.8%) of 54 patients with HLAP had LPL variations.The difference in the frequency of rare mutations between this study and the previous studies may be attributed to ethnic differences and selection bias.Moreover, the higher frequency of LPL variations in patients with HLAP was visible but not statistically provable, which was similar to those previous studies.
Previous case reports and cases series have revealed that some LPL mutations are associated with HLAP in China, such as p.W14X, p.Gln188*, p.His210Leu, p.Glu242Lys, p.Leu252VaL, p.Cys264Ter and p.L279V (19,22,27,31,32).Among them, L279V in exon 6 of LPL was first reported to be associated with HLAP from the Chinese population.Chen and cols.discovered a compound heterozygote for a missense mutation A98T and a missense mutation L279V in two probands with SHTG and AP.And beyond that, one family member and one out of 70 other HTG subjects also carried this novel LPL L279V mutation (33).Li and cols.also reported a compound heterozygous for W14X and L279V LPL gene mutations in a Chinese patient with long-term severe hypertriglyceridemia and recurrent AP (32).Khovidhunkit and cols.also observed that one in three patients with HLAP had rare variants in LPL carried L279V (29).In Jing-Lu Jin's study, the expanding genetic research included 15 TGrelated genes in 103 patients with primary SHTG, and one patient carried L279V out of 46 patients with rare variants (14).Other previous studies and reports have seldom discovered this mutation.Distinguished from these previous studies, the most frequent mutation detected in this study was rs371282890 (L279V).Five patients carried the rs371282890 variant in the HLAP group, more than those in previous studies.LPL Exon 6 (residues 232-313) encodes two structurally relevant disulphide bridges (Cys278-Cys283 and Cys264-Cys275) for the binding of heparin (34).The mutation L279V constitutes one of these disulphide bridges (Cys278-Cys283), which is crucial for the catalytic function of heparin binding.The L279V site is conserved throughout evolution from chimpanzees to zebrafish, suggesting that this residue may play a critical role in LPL function (33).This study compared the risk of AP and lipid metabolism in different rs371282890 genotypes.These five patients with the rs371282890 "GC" genotype had a higher chance of HLAP (HR: 2.843, 95% CI: 1.119-7.225,p = 0.028).Moreover, these five patients also had higher TG, TC, and non-HDL-C levels in all participants and the HLAP group.But whether rs371282890 variants can confer the risk for HLAP by simply increasing the serum TG or through other complex mechanisms need further research.
Conventional pharmacological therapies lower TG predominantly by the stimulation of LPL activity and are less effective in the case of a severely dysfunctional LPL protein (35).Due to the costs of genetic analysis and a lack of attention to molecular diagnosis, this group of patients with molecular 'defects' fail to devise in-depth diagnostic strategies.A routine sequencing of the canonical genes involved in TG metabolism (LPL, APOC2, GPIHBP1, APOA5, LMF1 and GCKR) in patients with HLAP may help identify this group and develop differential therapeutic strategies.Other than the strict low-fat diet, novel medications targeting TG-related processes, like inhibitors of APOC3, ANGPTL4, MGAT and DGAT, may reduce the risk of HLAP (35).The study has some limitations to be acknowledged.First, the study cohort was small in size.Hence, this study's subjects may not entirely represent the Chinese population.Second, LPL mass and activity measurements were not performed, thus hampering the pathogenetic interpretations of genotyping.Third, there was a difference in age between the two groups due to the features of the disease itself, so maybe some selection biases existed.Further studies are required to confirm the results.Another study limitation is that no other lipid metabolism genes were analysed.Expanded genetic testing in the future will generate more genetic information about HLAP.
In conclusion, detecting rare variants in LPL might help identify the portion of patients with HLAP at higher risk and guide future individualised therapeutic strategies.LPL variant rs371282890 is associated with HLAP and severe HTG in southeastern China.

Table 2 .
The distribution of LPL gene variants detected in patients with HLAP and controls

Table 3 .
Cox regression analysis of the impact of gender and rs371282890 on HLAP p-value <0.05 was considered statistically significant.

Table 4 .
Differences of characteristics and parameters between rs371282890 genotypes (GC, CC)Results are expressed as mean ± SD.TC: total cholesterol; TG: triglycerides; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; Non-HDL-C: non-high-density lipoprotein cholesterol; FPG: fasting plasma glucose; HBA: glycated haemoglobin; p-value < 0.05 was considered statistically significant.+p < 0.05 for statically significant difference between the GC group and the CC in the HLAP group.*p < 0.05 for statically significant difference between the GC group and the CC in all subjects.