Objective SLE is associated with increased cardiovascular risk (CVR). High serum concentrations of triglyceride-rich lipoproteins and apolipoprotein B-rich particles constitute the characteristic dyslipidaemia of SLE.
Methods A cross-sectional study was conducted to study the relationship between genetic variants involved in polygenic hypertriglyceridaemia, subclinical atherosclerosis and lipoprotein abnormalities. 73 women with SLE and 73 control women age-matched with the case group were recruited (age range 30–75 years). Serum analysis, subclinical atherosclerosis screening studies for the detection of plaque, and genetic analysis of the APOE, ZPR1, APOA5 and GCKR genes were performed.
Results Triglyceride concentrations and the prevalence of hypertension, dyslipidaemia and carotid atherosclerosis were higher in women with SLE than in the control group. Multivariate logistic regression showed that CC homozygosity for the GCKR rs1260326 gene (OR=0.111, 95% CI 0.015 to 0.804, p=0.030) and an increase of 1 mmol/L in triglyceride concentrations were associated with a greater risk of carotid plaque in women with SLE (OR=7.576, 95% CI 2.415 to 23.767, p=0.001).
Conclusions GCKR CC homozygosity (rs1260326) and serum triglyceride concentrations are independently associated with subclinical carotid atherosclerosis in women with SLE. Subclinical carotid atherosclerosis is also more prevalent in these women compared with the control group. The study of GCKR rs1260326 gene variants may contribute to more precise assessment of CVR and modulation of the intensity of lipid-lowering treatment in patients with SLE.
- cardiovascular diseases
- lupus erythematosus, systemic
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
SLE is associated with polygenic hypertriglyceridaemia, but the effect that hypertriglyceridaemia polymorphisms may have on cardiovascular risk (CVR) in these patients is unknown.
WHAT THIS STUDY ADDS
CC homozygosity for GCKR rs1260326 was shown to have a protective effect against carotid atherosclerosis compared with TT homozygosity in patients with SLE.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
The study of GCKR polymorphisms can improve CVR stratification in patients with SLE who could benefit from more intensive lipid-lowering pharmacological strategies.
SLE is a systemic autoimmune disease that predominantly affects adult women and is associated with increased cardiovascular risk (CVR).1 2 The interaction of classical cardiovascular risk factors (CVRFs) with the chronic inflammatory state of SLE has been linked to acceleration of the atherosclerotic process and premature cardiovascular events (CVEs) in these patients. The most characteristic dyslipidaemia of SLE is atherogenic dyslipidaemia, which is characterised by an increase in plasma triglyceride concentrations, a decrease in the concentration of high-density lipoprotein cholesterol (HDL-C), and a generalised disorder of structure and function of all lipoproteins that worsens within disease flares.3 Triglycerides are transported in the plasma by very low-density lipoproteins (LDLs), chylomicrons and their remnants. Similar to LDLs, all these lipoprotein particles have one apolipoprotein B molecule per particle and may enter the arterial wall and cause atherosclerotic cardiovascular disease (ASCVD). Under these circumstances, low-density lipoprotein cholesterol (LDL-C) concentrations may be virtually normal but with an increase in small dense LDL particle concentrations.4
According to the Spanish Society of Arteriosclerosis (SEA) and the European Atherosclerosis Society, hypertriglyceridaemia is defined as a plasma triglyceride concentration greater than 1.7 mmol/L or 150 mg/dL.4 5 Most cases of hypertriglyceridaemia have a polygenic nature. The so-called polygenic hypertriglyceridaemia can be caused by the sum of rare genetic variants inherited heterozygously that encode essential proteins in the metabolism of triglycerides, including the LPL, APOAV, APOCII, LMF1 and GPIHBP1 genes.6 However, the Global Lipids Genetics Consortium genome-wide association studies have demonstrated that polygenic hypertriglyceridaemia is more frequently the result of an excessive aggregation of single nucleotide polymorphisms.7 More specifically, a study carried out by our group concluded that the pathogenic variants of the ZPR1, APOA5 and GCKR genes most frequently conditioned the development of hypertriglyceridaemia in patients in our area. To date, the effect that these polymorphisms may have on the lipoprotein metabolism of patients with SLE is unknown. Likewise, it is not known whether their interaction with the remaining CVRFs influences the development of cardiovascular disease (CVD) in patients with SLE.
The objective of this study was to analyse the relationship between the genetic variants of the ZPR1, APOA5 and GCKR genes, subclinical atherosclerosis and lipoprotein abnormalities in a population of patients with and without SLE.
Materials and methods
Population and study design
This was a cross-sectional, observational study that included 146 women in two groups. A total of 73 female subjects with SLE were consecutively recruited from the systemic autoimmune diseases unit of our hospital. The diagnosis of SLE was based on the revised criteria of the American College of Rheumatology. The control group consisted of 73 women without SLE, matched by age (±2 years) with the patient group (age range 30–75 years) and were selected from the outpatient clinics (mainly dermatology or occupational medicine follow-up) of the hospital, where they were visited for illnesses unrelated to autoimmune diseases, atherosclerosis, or any systemic or serious illness. Women with previous CVE, autoimmune diseases other than SLE, non-cardiovascular lower limb amputation and inability to obtain ultrasound (US) images of the carotid arteries were excluded.
Clinical and anthropometric information was collected. CVRF was defined according to the SEA5; the dietary pattern was assessed using the Mediterranean Diet questionnaire8; and SLE disease activity was measured with index scales (Systemic Lupus Erythematosus Disease Activity Index9 and Systemic Lupus International Collaborating Clinics/ACR Damage Index (SLICC/SDI).10
Blood and urine samples were collected after 8 hours of fasting. All the biochemical analyses were performed in plasma using a COBAS 711 (Roche Diagnostics, Basel, Switzerland), while homocysteine levels were determined using the Immulite 2000 XPi analyser (Siemens Healthcare GmbH, Erlangen, Germany). Haematology values were measured (Sysmex S.L XN 900), and coagulation parameters were determined (ACTLOP analyser).
Genetic analysis of polymorphisms involved in triglyceride metabolism
Variants of the APOE gene were genotyped using validated TaqMan MGB probes. Genetic analysis of the allelic variants of the ZPR1, APOA5 and GCKR genes, which mainly determine the presence of polygenic hypertriglyceridaemia, was performed. The three possible genotypes were dichotomised in homozygotes so as not to present a risk allele (0) or risk allele carriers (either heterozygous or homozygous) c*724C>G (ZPR1); (0=CC vs 1=G), c.56C>G (APOA5); (0=CC vs 1=G), c.1337T>C (GCKR); (0=CC vs 1=T).
Evaluation of insulin resistance
The triglyceride/glucose (T&G) index described by Simental-Mendía et al was chosen for the evaluation of insulin resistance. It was calculated as the natural logarithm (Ln) of the product of glucose and plasma triglycerides according to the following formula: Ln [fasting triglycerides (mg/dL)×fasting glucose (mg/dL)]/2. As a cut-off point for the diagnosis of insulin resistance, a T&G index greater than 4.65 proposed by the same authors was used.11 12
Carotid US and ankle–brachial index (ABI)
Certified vascular technologists measured the carotid US and the ABI using standardised protocols. Carotid US was performed with the commercially available scanner (ACUSON Antares, Siemens Medical Solutions USA) using a 6 MHz linear array transducer. According to the Mannheim consensus, plaque criteria were defined as a focal protrusion in the lumen with a carotid intima–media thickness (cIMT) of >1.5 mm, a protrusion at least 50% greater than the surrounding cIMT or an arterial lumen of >0.5 mm.13
The ABI was performed using an automatic waveform analyser (Vascular Handheld Doppler Bidop V.7; Hadeko, Kawasaki, Japan) and was calculated as the ratio of ankle to brachial pressure. The ABI was evaluated according to standardised criteria.14
Qualitative variables were analysed by χ2 or Fisher’s exact test. Analysis of variance and Mann-Whitney U test were used for normally and not-normally distributed quantitative variables, respectively. To analyse the relationship between genetic variants and the presence of plaque, multivariate logistic regression analysis was performed. The presence of plaque was considered as a dependent variable and genetic variables as independent variables adjusted for different covariates that were related to subclinical atherosclerosis in a population with SLE in a previous study by our group15: the group (control and SLE), arterial hypertension, triglycerides (both presented statistical significance in the bivariate analysis) and age. Carotid US could not be performed in 15 women of the control group due to organisational difficulties caused by the COVID-19 pandemic. To determine whether the sample of controls with carotid US was representative of all the women in the control group, an analysis of the differences between the two subgroups, with and without US, was performed. The results of this analysis are shown in online supplemental table 1. For all analyses, a p value less than 0.05 was considered significant.
Comparison between women with SLE and the control group
The most relevant data of this analysis are shown in table 1. When comparing both groups, it was observed that women with SLE had a higher prevalence of hypertension and dyslipidaemia (45.2% vs 5.5%, p<0.001, and 52.1% vs 34.2%, p=0.030, respectively), performed less physical activity (1 point vs 2 points, p=0.001), had a worse dietary pattern (11 points vs 12 points, p=0.003) and had a higher prevalence of premature menopause (12.3% vs 1.4%, p=0.009) than the control group. None of the subjects in either group exceeded the limit of low-risk alcohol consumption for women (10 g of alcohol per day).
Statin treatment was also more frequent among the group of women with SLE than those in the control group (45.2% vs 11%, p<0.001) and, consequently, the women in the SLE group presented lower total cholesterol concentrations (4.9 mmol/L vs 5.3 mmol/L, p=0.007), LDL-C (2.7 mmol/L vs 3.0 mmol/L, p=0.002), apolipoprotein B (0.9 mmol/L vs 1.0 mmol/L, p=0.014) and non-HDL-C (3.2 mmol/L vs 3.5 mmol/L, p=0.026) than the women in the control group. However, women with SLE had higher triglyceride concentrations than the control group (1.0 mmol/L vs 0.8 mmol/L, p=0.008). Despite the absence of differences in creatinine values, parathyroid hormone (PTH) concentrations (5.6 pmol/L vs 4.4 pmol/L, p=0.024) and proteinuria detected by the albumin:creatinine ratio (0.8 g/mol vs 0.0 g/ mol, p<0.001), all these values were higher in the patients with SLE. C reactive protein (CRP) (2.7 mg/L vs 1.0 mg/L, p<0.001) and homocysteine (11 µmol/L vs 9 µmol/L, p<0.001) concentrations were higher, while albumin (44.0 g/L vs 46 g/L, p<0.001) and vitamin B12 (324 pmol/L vs 384 pmol/L, p=0.006) concentrations were lower in the women in the SLE group compared with the women in the control group. The remaining variables studied are shown in online supplemental tables S1 and S2.
As shown in table 2, there were no significant differences between patients with SLE and control women in either the analysis of the APOE gene polymorphisms or in that of the genetic variants of the ZPR1, APOA5 and GCKR genes. The Hardy-Weinberg equilibrium remained constant in both groups.
The study of subclinical atherosclerosis revealed that there were more women with carotid plaque in the SLE group than in the control group (20.5% vs 6.9%, p=0.028, respectively) with a statistically significant difference (table 3). There were no significant differences in ABI values. The maximum and minimum ABI data are shown in online supplemental table S2. The statistical analysis of the genetic variants of the ZPR1, APOA5 and GCKR genes with carotid plaque is shown in online supplemental table S3.
A multivariate logistic regression model was made to evaluate the contribution of the genetic variants of the GCKR gene, SLE, plasma triglyceride concentrations, hypertension and age to the risk of having carotid plaque (table 4). The whole model explained the 40.3% (Nagelkerke R2) risk of having carotid plaque. CC homozygosis for the GCKR rs1260326 gene (OR=0.111, 95% CI 0.015 to 0.804, p=0.030) showed that it has a protective effect against carotid atherosclerosis compared with TT homozygosis. In addition, a 1 mmol/L increase in plasma triglyceride concentrations was associated with a 7.6-fold increase in the risk of presenting carotid plaque (OR=7.576, 95% CI 2.415 to 23.767, p=0.001). Hypertension was associated with a trend towards increased risk of carotid plaque (OR=3.577, 95% CI 0.872 to 14.676, p=0.077). In addition, SLE was associated with a trend towards a higher risk of having carotid plaque (OR=1.588, 95% CI 0.352 to 7.168, p=0.548). The same model was analysed, adding the interaction between the group (SLE and control) and the GCKR gene, and there was no significant interaction.
Logistic regression analysis was performed to evaluate the effect of the ZPR1 and APOA5 genes, but non-significant results were obtained due to the small number (there was only one patient) of the reference group (homozygous GG) for both genes.
Different models adjusted for several variables were also calculated: for disease severity (SLICC/SDI greater or less than 0), accumulated dose of corticosteroids, insulin resistance, basal glycaemia, plasma homocysteine concentrations and the dietary questionnaire score. The results of all these analyses were similar and are shown in online supplemental tables S4–11. Statistical analysis of the allelic variants of the GCKR gene and the lipid profile adjusted for lipid-lowering drugs and the results are shown in online supplemental table S12.
To date, this is the first study that strengthens knowledge of the effect of the presence of polygenic variants of hypertriglyceridaemia on the development of subclinical atherosclerosis not only in a population with SLE but also in a control group. Women with SLE had higher concentrations of triglycerides as well as a higher prevalence of carotid plaque than women without SLE. CC homozygosity for GCKR rs1260326 and an increase of 1 mmol/L in triglyceride concentrations were associated with a greater risk of carotid plaque in women with SLE.
Subclinical atherosclerosis at an early age is common in patients with SLE. The detection of atherosclerotic carotid plaque by US has been associated with a fourfold increased risk of CVD in patients with SLE.16 This degree of risk is comparable to the risk of presenting CVD in patients with diabetes mellitus.17 Furthermore, the prevalence of carotid plaque in these patients with SLE is higher than in the general population.16 In our cohort, patients with SLE had a higher prevalence of carotid plaque than control women, although the percentages in both groups were lower than those in previously published series (6.9% vs 17.0%–26.6% and 20.5% vs 16.0%−37.0%, respectively).18–21 The study of peripheral vascular disease using the ABI showed no differences between the two groups. Only three controls and five patients with SLE presented a pathological ABI, although it is considered that this test is only useful for detecting advanced atherosclerotic disease.
The pathogenesis of ASCVD in SLE is complex and involves numerous factors. Although the presence of classical CVRF in patients with SLE is associated with a worse cardiovascular prognosis, these CVRFs do not fully explain the increase in CVR compared with patients without SLE. Historically, patients with SLE have a higher prevalence of conventional CVRF than the general population.22 In the present study, hypertension, sedentary lifestyle, early menopause and a non-healthy dietary pattern were more common in women with SLE than in those in the control group.
Dyslipidaemia was also more frequent among patients with SLE. In fact, the prevalence of dyslipidaemia in SLE ranges from 36% at diagnosis to 60% or even higher after 3 years.23 The recommendations of scientific societies regarding cardiovascular prevention in patients with SLE have led to a greater use of statins in this group of patients (45.2% vs 11 %). These drugs decrease LDL-C concentrations by 25%–50% and triglyceride concentrations by 14%–29%, and increase HDL-C concentrations by 4%–10%.7 The higher use of statins in patients with SLE explains why they had lower concentrations of total cholesterol, LDL-C, apolipoprotein B and non-HDL-C than women in the control group. However, despite the higher statin use, the patients in the SLE group had higher triglyceride concentrations and also lower HDL-C concentrations than the control group, indicating a lipid metabolism disorder known as atherogenic dyslipidaemia.
The prevalence of atherogenic dyslipidaemia is high among patients with SLE.24 The causes of atherogenic dyslipidaemia in SLE are diverse, including insulin resistance and the inability of insulin to stimulate glucose metabolism.25 Insulin resistance hinders the lipolysis and storage of fatty acids in adipocytes and alters the metabolic pathway dependent on PI 3 kinases which, among other functions, contribute to the degradation of apolipoprotein B. As a result of the massive supply of fatty acids and the defect in the degradation of apolipoprotein B, hypertriglyceridaemia and the atherogenic lipoprotein phenotype appear. This pattern is also associated with a reduction in HDL-C concentrations and the loss of antioxidant capacity.26 Inadequate secretion of a set of hormones and proteins called adipokines that modify insulin sensitivity may also play a role.27 In addition, the appearance of antilipoprotein–lipase antibodies induces a decrease in the lipolytic activity of this enzyme.28 In the current study, a trend to a higher prevalence of insulin resistance assessed by the T&G index was more frequent among patients with SLE. This index is a good diagnostic tool for insulin resistance,29 even in patients with SLE and rheumatoid arthritis,30 and has been associated with a higher prevalence of CVE and subclinical atherosclerosis in the general population.31 32
Hypertriglyceridaemia and atherogenic dyslipidaemia are the result of an interaction between genetic factors, constituted by the aggregation of multiple common and rare genetic variants, and environmental factors.5 Apolipoprotein A5 activity deficiency has been linked to diabetic dyslipidaemia33 and the rs964184 (c.*724C>G) variant of the ZPR1 gene (also called ZNF259), hypertriglyceridaemia, coronary artery disease and metabolic syndrome.34 However, in the present study, there were no significant differences between the group with SLE and the control group. There were also no differences related to the allelic variants of the APOE gene.
The last gene studied was the GCKR gene that encodes the glucokinase regulatory protein (GKRP). This protein is released into the cytoplasm in the postprandial phase and stimulates de novo glycogen deposition and lipogenesis. The presence of the TT rs1260326 (p.P446L) allele of GCKR destabilises the glucokinase binding interface. In the fasting state, increased hepatic glucokinase activity results in higher triglyceride concentrations and lower glucose concentrations.35 In addition, the GCKR rs1260326 variant has been shown to be associated with an increased risk of coronary artery disease (OR per risk allele 1.02, 95% CI 1.00 to 1.04).36 In a previous study with a large number of women with SLE, it was observed that the GCKR rs1260326 variant was related to the presence of carotid atherosclerosis in these patients. In that study, CC homozygosity of the GCKR gene was independently associated with carotid plaque in patients with SLE (OR=0.026, 95% CI 0.001 to 0.473, p=0.014).15 In the current work, CC homozygosity of the GCKR rs1260326 gene (OR=0.111, 95% CI 0.015 to 0.804, p=0.030) and plasma triglyceride concentrations (OR=7.576, 95% CI 2.415 to 23.767, p=0.001) in patients with SLE were found to be independently associated with the presence of subclinical carotid plaque compared with the control group. Although hypertension did not reach statistical significance, the association of this CVRF with subclinical atherosclerosis in patients with lupus has been demonstrated in other studies.37 As observed in the different analysis models, the T&G index and insulin resistance lost value as covariates due to their relationship with triglyceride concentrations. Nonetheless, the statistical trend corroborates the usefulness of both as markers of atherogenic dyslipidaemia in patients with SLE. Likewise, the chronicity of SLE, the cumulative dose of corticosteroids and age are some of the clinically relevant CVRFs in SLE and are usually included in CVR calculators for these patients.38 However, in our study, no significant differences were obtained when they were included in the multivariate models.
The association between osteoporosis and ASCVD is well documented both in the general population and in patients with autoimmune diseases. Lack of sun exposure, a sedentary lifestyle and especially chronic kidney disease favour a decrease in plasma concentrations of calcium and vitamin D and an increase in phosphate and fibroblast growth factor 23. These modifications induce a greater secretion of PTH, which, together with the reduction of the renal synthesis of klotho, promotes the deposit of calcium at the vascular level.39 Patients in the SLE group had higher PTH concentrations than those in the control group (5.6 pmol/L vs 4.4 pmol/L, p=0.024), although plasma PTH could only be analysed in a subsample of patients and control subjects. No statistically significant differences were observed in plasma concentrations of creatinine or vitamin D, despite the latter being lower in the SLE group than in the control group (58.6 nmol/L vs 68.0 nmol/L, p=0.131). These results are consistent with the potential role of plasma PTH as a biomarker of atherosclerosis in these patients.40
The combination of conventional CVRF with other pathogenic factors that emerge in the chronic inflammatory context of SLE may explain the development of accelerated atherosclerosis. In our cohort, women with SLE had higher CRP concentrations and lower albumin concentrations than those in the control group. Similarly, patients with SLE had higher concentrations of plasma homocysteine. There were no differences in folic acid concentrations, but vitamin B12 concentrations were lower in women with SLE than women in the control group. Homocysteine is a sulfur amino acid resulting from the metabolism of methionine that requires vitamin B12 and folic acid as cofactors to be eliminated. In a recent meta-analysis including 50 studies and 4396 patients with SLE, plasma homocysteine concentrations in patients with SLE were higher compared with the population without SLE.41 Homocysteine exerts its atherogenic role through different mechanisms. It is speculated that the inhibition of endothelial nitric oxide synthase produced by homocysteine reduces the bioavailability of nitric oxide and leads to endothelial dysfunction.42 In addition, homocysteine increases the activity of HMG CoA reductase and cholesterol synthesis.43 The association of hyperhomocysteinaemia with ASCVD is well documented both in the general population and in patients with SLE,44 but in our study, this variable did not provide additional information to the model.
The main limitation of this study is the small sample size, especially with regard to the association analysis of genetic variants. On the other hand, the sample was only made up of women, although it should be taken into account that SLE predominantly affects the female sex. Furthermore, the control group was not selected from the general population census, and this may limit its representativeness. Finally, a high percentage of patients in the group of patients with SLE and 11% of the control group were treated with statins, drugs that have a moderate hypotriglyceridaemic effect. This effect could have attenuated the magnitude of the influence of allelic variants of the GCKR gene on triglyceride concentrations and atherosclerosis, but despite this, a significant relationship was observed. As observed in online supplemental table S13, there were no patients with plaque and without lipid-lowering treatment in the CC allele group. These results should be evaluated in a larger population.
Despite the aforementioned limitations, this research reaffirms the independent association of CC homozygosis of the GCKR gene with carotid atherosclerosis in patients with SLE. Therefore, our results demonstrate the relationship between the genetic variants of one of the genes related to polygenic hypertriglyceridaemia, the GCKR gene, and subclinical atherosclerosis in patients with SLE. It should be noted that the increased risk (OR=7.576) of having carotid plaque attributed to an increase of 1 mmol/L in the concentration of triglycerides occurs even with concentrations lower than 150 mg/dL or 1.7 mmol/L. There is a direct relationship between triglyceride concentrations and CVE even with triglyceride levels below 150 mg/dL,45 and in the past years, the optimal triglyceride concentration has been defined as below 100 mg/dL.46 The recommendations of the treatment of dyslipidaemia in patients with autoimmune diseases are not uniform; however, cardiovascular prevention clinical guidelines consider the presence of these diseases as a CVR-enhancing factor. More precise stratification of CVR in these patients may be possible by studying variants of the GCKR rs1260326 gene in addition to evaluating conventional CVRFs and carotid US. Moreover, the study of GCKR rs1260326 gene variants may contribute to modulating the intensity of lipid-lowering treatment in patients with SLE.
The results of this study demonstrate that CC homozygosity of the GCKR gene and plasma triglyceride concentrations are independently associated with subclinical carotid atherosclerosis in women with SLE. Women with SLE have a higher prevalence of subclinical carotid atherosclerosis. More studies are needed to define the role of triglycerides in the residual risk of ASCVD in these patients.
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information.
Patient consent for publication
This study involves human participants and was approved by the ethics committee of Hospital Universitari de Bellvitge PR179/19. The participants gave informed consent to participate in the study before taking part.
We wish to acknowledge the help provided by the Vascular Medicine Department. We are grateful for the institutional support of the CERCA Program/Generalitat de Catalunya.
Contributors Conceptualisation: MF-M, XP and BC-E; data curation: MF-M; formal analysis: EC; investigation: MF-M, VE-L and AP-M; methodology: MF-M, XP and BC-E; writing–original draft: MF-M; writing–review and editing: MF-M, XP and BC-E. Guarantor: MF-M.
Funding This study has been funded in part by the Spanish Ministry of Health (Carlos III Health Institute) through the Fondo de Investigación para la Salud, which is cofunded by the European Regional Development Fund, funded by the following grant codes: PI16/01094 and PI19/01032. Ethical approval information, institution and number(s): Bioethics Commission of the Hospital Universitari de Bellvitge: 23/05/2019 PR179/19.
Competing interests None declared.
Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Provenance and peer review Not commissioned; externally peer reviewed.
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