SRA1, APOA5, and AIFM2 identified as novel genetic risk factors in recurrent hypertriglyceridemia-associated acute pancreatitis by whole-exome sequencing

To the Editor: Hypertriglyceridemia-associated acute pancreatitis (HTG-AP) is the second most common cause of acute pancreatitis (AP) in China, characterized by more complications, higher intensive care unit (ICU) admissions, prolonged hospital stays, and increased recurrence rates (16.0–49.2%) compared with other etiologies.^[1] Recurrent HTG-AP (HTG-RAP) imposes a substantial disease burden, including multiple hospitalizations, diminished quality of life, and an elevated risk of chronic pancreatitis (CP) and pancreatic cancer. Therefore, identifying the key risk factors for HTG-RAP is clinically essential. Hypertriglyceridemia (HTG) is a major risk factor for HTG-RAP recurrence and is caused by genetic and environmental factors. Genetic mutations in genes such as lipoprotein lipase (LPL) and apolipoprotein C-II (APOC2) have been shown to correlate with higher recurrence rates of HTG-RAP in Caucasian populations. However, the genetic predisposition contributing to the risk and recurrence of AP in patients with HTG, particularly in Asian populations, remains poorly understood. This study aimed to identify genetic risk factors for HTG-RAP and investigate their association with recurrence and severity using whole-exome sequencing.

This study included the discovery and validation cohorts [Supplementary Figure 1, http://links.lww.com/CM9/C484]. HTG-RAP was defined as fulfilling both HTG-AP and recurrent acute pancreatitis (RAP) criteria: HTG-AP was diagnosed in patients with triglyceride (TG) >1000 mg/dL or >500 mg/dL with chylaemia, excluding other causes, and RAP by ≥2 AP episodes with symptom resolution for >3 months and no CP. In the discovery phase, 10 HTG-RAP patients and 10 matched HTG patients from Shanghai General Hospital were enrolled. In the validation phase, 62 HTG-RAP patients from Shanghai General Hospital and Shanghai Songjiang District Central Hospital (August 2022–2023) and 53 HTG patients with TG >1000 mg/dL from the Health Screening Center were recruited. This study was approved by the Ethics Committees of both hospitals (Nos. 2022KY071 and 2023SQ060). Written informed consent was obtained from all the patients. The study was registered in the Chinese Clinical Trial Registry (ChiCTR2300071094).

Whole-exome sequencing was performed on genomic DNA extracted from patients in the discovery cohort. Variants were identified using the Genome Analysis Toolkit (GATK) and annotated with Annotate Variation (ANNOVAR). Non-synonymous variants with significant differences in allele frequencies between HTG-RAP and HTG patients (P <0.05) were selected and filtered using Combined Annotation Dependent Depletion (CADD), Polymorphism Phenotyping version 2 (PolyPhen-2), and Sorting Intolerant From Tolerant (SIFT). Rare variants (global allele frequency <0.01) were prioritized based on the 1000 Genomes 2014 October_East Asian (1000g2014oct_eas) and Exome Aggregation Consortium_East Asian (ExAC_EAS) databases [Supplementary Figure 2, http://links.lww.com/CM9/C484]. Polymerase chain reaction (PCR) and Sanger sequencing were used for variant genotyping [Supplementary Figure 3, http://links.lww.com/CM9/C484]. All the primers used are listed in Supplementary Table 1, http://links.lww.com/CM9/C484. Data were expressed as frequencies, mean (standard deviation [SD]), or medians (interquartile range [IQR]), and statistical analyses were performed using IBM SPSS Statistics 25 (IBM SPSS Statistics, IBM Corporation, Armonk, New York) and GraphPad Prism 9 (GraphPad Software Inc, California, USA). P values less than 0.05 were considered statistically significant.

The discovery cohort comprised 10 patients with HTG-RAP and 10 with HTG, with no significant differences between the groups in terms of sex, serum TG levels, and body mass index (BMI), or prevalence of diabetes, hypertension, and fatty liver (Supplementary Table 2, http://links.lww.com/CM9/C484, P >0.05). Raw reads and related quality control information are listed in Supplementary Table 3, http://links.lww.com/CM9/C484. Whole-exome sequencing identified 3,393,798 genetic variants, of which 41,808 non-synonymous variants were prioritized for downstream analysis. A comparison of allele frequencies between the groups revealed 141 protein-affecting variants that were significantly enriched in patients with HTG-RAP (P <0.05). A comparison of allele frequencies revealed 141 protein-altering variants that were enriched in patients with HTG-RAP (P <0.05). After excluding 20 non-pathogenic variants, the remaining 121 variants were filtered against the 1000g2014oct_eas and ExAC_EAS frequency databases, resulting in a final candidate list of 39 rare variants (allele frequency <0.01), ranked by P-value [Supplementary Table 4, http://links.lww.com/CM9/C484].

To validate the candidate list of the 39 variants, an independent cohort comprising 62 patients with HTG-RAP and 53 patients with HTG from two hospitals was analyzed. The demographic and clinical characteristics showed no significant differences between the groups [Supplementary Table 5, http://links.lww.com/CM9/C484]. Sanger sequencing confirmed significant enrichment of steroid receptor RNA activator 1 (SRA1), apolipoprotein A5 (APOA5), and apoptosis-inducing factor, mitochondrion-associated, 2 (AIFM2) variants in HTG-RAP patients (P <0.05, Table 1 and Supplementary Table 6, http://links.lww.com/CM9/C484). The SRA1 variant (C/G) at chr5:140552044 showed an odds ratio (OR) of 5.5 (95% confidence interval [CI]: 1.8–16.5, P = 0.001), APOA5 at chr11:116790676 exhibited an OR of 3.5 (95% CI: 1.7–7.4, P = 0.001), and AIFM2 at chr10:70115028 demonstrated an OR of 3.4 (95% CI: 1.2–9.6, P = 0.015) for HTG-RAP vs. HTG patients [Table 1]. These findings suggest that the three variants are significantly associated with HTG-RAP and may represent novel genetic risk factors.

Table 1.

Significant variants identified in the validation cohort of hypertriglyceridemia-associated acute pancreatitis.

Gene	Position (GRCh38.p13)	Variant	Var/Ref alleles HTG-RAP	Var/Ref alleles HTG	OR (95% CI)	P-value
SRA1	chr5:140552044	C/G	22/102	4/102	5.5 (1.8–16.5)	0.001
APOA5	chr11:116790676	C/A	36/88	11/95	3.5 (1.7–7.4)	0.001
AIFM2	chr10:70115028	C/T	18/106	5/101	3.4 (1.2–9.6)	0.015

AIFM2: Apoptosis-inducing factor, mitochondrion-associated, 2; APOA5: Apolipoprotein A5; HTG: Hypertriglyceridemia; HTG-RAP: Recurrent hypertriglyceridemia-associated acute pancreatitis; OR: Odds ratio; SRA1: Steroid receptor RNA activator 1; Ref: Reference; Var: Variant.

To further identify the relationship between variants in SRA1, APOA5, and AIFM2 and the recurrence rate of HTG-RAP, a joined cohort of 10 patients with HTG-RAP from the discovery cohort and 62 from the validation cohort were included in the subsequent analysis. Recurrence rates were significantly higher in patients with SRA1, APOA5, or AIFM2 variants than with negative patients (P <0.0001 for SRA1 and APOA5, P = 0.002 for AIFM2; Supplementary Table 7, http://links.lww.com/CM9/C484). Given that clinical factors beyond genetic predisposition could influence AP recurrence, univariate and multivariate linear regression analyses were performed to validate the association between these variants and recurrence rates. Univariate linear regression analysis showed that SRA1, APOA5, and AIFM2 variants, Ranson score, white blood cell (WBC) count, local complications, systemic inflammatory response syndrome (SIRS), ICU admission, and prolonged hospitalization days were positively associated with the HTG-RAP recurrence rate (P <0.05, Supplementary Table 8, http://links.lww.com/CM9/C484). These risk factors were further analyzed using multivariate linear regression to control for potential confounders. The results showed that the recurrence rate of HTG-RAP displayed a positive association with SRA1 (B = 0.578, P <0.001), APOA5 (B = 0.558, P <0.001), and AIFM2 (B = 0.364, P = 0.004) variants using a stepwise entry method [Supplementary Table 9, http://links.lww.com/CM9/C484]. These findings imply that SRA1, APOA5, and AIFM2 variants contribute to increased HTG-RAP recurrence as independent risk factors.

The severity of AP is a critical determinant of disease prognosis. It is commonly assessed using classical scoring systems such as the Acute Physiology and Chronic Health Evaluation II (APACHE II), Ranson Bedside Index for Severity in Acute Pancreatitis (BISAP), and Pancreatitis Activity Scoring System (PASS), along with local and systemic complications, as defined by the Atlanta classification. Laboratory indicators, including WBC count, serum albumin level, and clinical factors, such as ICU admission and extended hospital stay, are also associated with AP severity. We analyzed these factors in patients with HTG-RAP with or without SRA1, APOA5, and AIFM2 variants. SRA1-positive patients exhibited higher Ranson and PASS scores, local complications, SIRS, WBC count, neutrophil percentage, and platelet-to-lymphocyte ratio than those negative patients (P <0.05, Supplementary Table 10, http://links.lww.com/CM9/C484). APOA5-positive patients exhibited elevated APACHE II and Ranson scores, increased local complications, and lower lymphocyte percentage than APOA5-negative patients (P <0.05; Supplementary Table 11, http://links.lww.com/CM9/C484). AIFM2-positive patients showed significantly higher APACHE II and Ranson scores and SIRS incidence than those negative patients (P <0.05, Supplementary Table 12, http://links.lww.com/CM9/C484).

Univariate linear and logistic regression analyses were performed to determine whether the three genetic variants were associated with AP severity. Four severity scores were analyzed [Supplementary Table 13, http://links.lww.com/CM9/C484]. Regarding the APACHE II score, the AIFM2 variant exhibited a significant positive correlation with disease severity (P <0.05), whereas the SRA1 and APOA5 variants demonstrated no significant differences. All three variants showed a statistically significant positive association with the Ranson score (P <0.05). Regarding the BISAP score, APOA5 and AIFM2 variants positively correlated with disease severity (P <0.05). None of the variants were significant determinants of the PASS score. Complications were included in the analysis [Supplementary Table 14, http://links.lww.com/CM9/C484]. SRA1 and APOA5 variants were associated with a higher incidence of local complications (P <0.05). All three variants showed a strong positive relationship with SIRS (P <0.05). Additionally, none of the variants were significant determinants of other systemic complications. These results revealed that these genetic variants might serve as potential predictors of AP severity.

In this pioneering study of an Asian HTG-RAP cohort, we identified three novel genetic variants (SRA1, APOA5, and AIFM2) that were significantly associated with HTG-RAP. These findings were confirmed in an independent validation cohort, and further analysis demonstrated a positive association between these variants and HTG-RAP recurrence and severity. Previous studies established HTG as a major RAP risk factor and found genetic mutations such as LPL in severe HTG and recurrent AP patients. However, these findings do not fully explain HTG-RAP susceptibility in Asian populations. Chang et al^[2] reported associations between cystic fibrosis transmembrane conductance regulator (CFTR) and tumor necrosis factor (TNF) gene polymorphisms and HTG-RAP, supporting the role of genetic factors in lipid dysregulation. This study expands on this by identifying novel variants, suggesting that novel genetic mechanisms are involved in HTG-RAP pathogenesis.

The SRA1 variant, essential for lipid metabolism and inflammation, was positively correlated with Ranson and PASS scores, local complications, and systemic inflammation indicators.^[3] These findings suggest that the SRA1 variant may impair its regulatory functions in lipid and inflammatory pathways, leading to more severe HTG-RAP. The APOA5 variant, a regulator of plasma triglycerides, was associated with an increased risk of HTG-RAP recurrence and elevated APACHE II and Ranson scores, as well as local complications.^[4] These findings indicate that APOA5 variants may influence HTG-RAP by exacerbating HTG and the related inflammatory responses. The AIFM2 variant, which regulates ferroptosis and modulates inflammation through interaction with coenzyme Q10 (CoQ10), was linked to higher APACHE II and Ranson scores and SIRS incidence.^[5] These correlations suggest that dysregulation of ferroptosis and glycolysis may intensify inflammatory responses, contributing to the severity of HTG-RAP.

In conclusion, this study identified SRA1, APOA5, and AIFM2 variants as genetic risk factors for HTG-RAP, suggesting that the dysregulation of lipid metabolism, inflammatory responses, and ferroptosis contributes to disease recurrence and severity. These findings highlight the potential of these variants as biomarkers, advancing our understanding of HTG-RAP, and suggesting pathways for targeted interventions. Further research on these genetic mechanisms may lead to the development of novel strategies for HTG-RAP management.

Funding

This work was financially supported by grants from the National Natural Science Foundation of China (Nos. 82270671, 82200714, and 82300731), the Program for Science and Technology Development from Songjiang Science and Technology Committee (No. 22SJKGGG28), the USST Medical-Engineering Cross-project (No. 10-21-308-420), the Clinical Project from Shanghai General Hospital (No. CCTR2022-B02), the National High Level Hospital Clinical Research Funding (No. 2022-PUMCH-E-003), and the CAMS Innovation Fund for Medical Science (No. 2023-I2M-3-001).

Conflicts of Interest

None.

Supplementary Material

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