Genetic spectrum of familial hypertriglyceridemia from the southeastern region of Türkiye

Abstract

AimThe disorders of lipid metabolism that cause primary hypertriglyceridemia result from genetic defects in triglyceride synthesis and metabolism. Although primary causes are rare in hypertriglyceridemia, they should be considered in severe hypertriglyceridemia cases. Identified genetic mutations are LPL, APOC2, APOA5, LMF1 and GPIHBP1 mutations.MethodsThis descriptive cross-sectional study was conducted in Diyarbakir Children’s Hospital pediatric metabolism clinic on 60 patients from 41 unrelated families who were followed and diagnosed with severe hypertriglyceridemia based on clinical presentation, neurological parameters, biochemical measurements, and molecular analysis. The LPL, APOC2, APOA5, LMF1, GPIHBP1 genes were sequenced in 60 patients. Patients with initial triglyceride levels >885mg/dL were included in the study. Patients with a secondary cause were excluded from the study.ResultsRare DNA sequence variants were identified in 49 patients (81.66%), including variants LPL (n = 15), APOC2 (n = 32), and APOA5 (n = 2). No mutations were found in 11 patients (21%). The mean initial triglyceride level was 4322.8 ± 4483mg/dL. Acute pancreatitis occurred in 38.33% (n = 23) of the patients. The incidence of eruptive xanthoma was 28.33%, organomegaly was 23.33%, and failure to thrive was 21.66%. 69.23% of the patients with failure to thrive were patients with pancreatitis. Two different variants, c.55+6T>G and c.55+1G>C were detected in the APOC2 gene, seven different variants one of which is novel, c.557G>A, c.953A>G, c.296T>C, c.662T>C, c.1262G>A, c.644G>A and c.679G>C, were detected in the LPL gene, and two different variants one of which is novel, c.334_399dup65bp and c.16_39del24bp were detected in the APOA5 gene. Six patients were homozygous for both c.557G>A and c.953A>G variants.ConclusionThe frequency of mutations in APOC2 was 50%, LPL was 25% and APOA5 was 3.33%. The relatively high prevalence of APOC2 mutations in our cohort may be due to regional frequency. The development of new therapeutic options for this rare disease requires awareness and screening among these patients. These findings highlight the need for molecular analysis in patients with severe HTG. It is anticipated to guide future individualized therapeutic strategies.

Keywords

Introduction

Severe hypertriglyceridemia is characterized by plasma triglyceride levels > 885mg/dL (>10.0 mmol/L) in the fasting state (>12 hours).¹ It is known that the genetic etiology is extremely complex, and both common and rare variants are effective. Moderate elevation of triglyceride (177-885 mg/dL or 2.0-10.0 mmol/L) may be a condition resulting from the polygenic effect of multiple genes and secondary causes.² However, it has been determined that mutations in six genes (LPL, APOC2, APOA5, LMF1, GPIHBP1 and GPD1) show severe hypertriglyceridemia (HTG) due to disruption of chylomicron removal pathways. Dietary fats are absorbed from the intestine and transported as triglycerides (TG) in chylomicrons.³ Chylomicrons that enter the blood begin to degrade when the APOC2 they carry is recognized by lipoprotein lipase (LPL, EC 3.1.1.34). When this process is impaired or insufficient, chylomicron particles accumulate in the plasma and cause hypertriglyceridemia.⁴ It has been determined that familial hypertriglyceridemia occurs in the presence of biallelic mutations in the LPL, APOC2, APOA5, LMF1 and GPIHBP1 genes. Clinical features include recurrent pancreatitis, organomegaly, growth retardation, eruptive xanthomas and lipemia retinalis.² General circulating persistence of chylomicron is associated with free fatty acid toxicity and, together with its proinflammatory properties, is a trigger for pancreatitis.⁵ Pancreatitis due to hypertriglyceridemia is more serious and has a higher complication rate compared to other causes.⁴ Lipoprotein electrophoresis was not performed due to technical incompetence. Also, Fredrickson’s classification is not useful in daily practice.
In patients with severe HTG, omega-3, medium chain triglyceride (MCT) and fenofibrates are used in the treatment.⁶ However, since therapeutic interventions to reduce TG levels are often ineffective, individualized therapeutic strategies targeting its molecular basis are being developed. Therefore, we aim to evaluate the coexistence of severe HTG and pancreatitis and to define the variants that cause monogenic HTG in our center.

Materials and Methods

Study Design and Data AcquisitionThis descriptive cross-sectional study was conducted at Diyarbakır Children’s Hospital pediatric metabolism clinic on 60 patients from 41 unrelated families who were followed and diagnosed with severe hypertriglyceridemia based on clinical presentation, neurological parameters, biochemical measurements, and molecular analysis. Patients with initial triglyceride levels >885 mg/dL were included in the study. Patients with body weight <-2 SDS under 2 years of age, and patients with body mass index <-2 SDS over 2 years of age were considered as failure to thrive. Patients with a secondary cause were excluded from the study. Inclusion criteria were rare biallelic variants in LPL, APOC2, APOA5, LMF1, GPIHBP1 genes classified as likely pathogenic or pathogenic according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines.Molecular AnalysesAll exons and exon-intron junctions of the genes were evaluated by the next-generation sequencing method. Genomic DNA was extracted from peripheral blood samples using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Standardized PCR pools were prepared using NexteraXT sample preparation kit for next-generation sequencing analysis with the Miseq device (Illumina, Inc.). Sanger sequencing of genomic variants identified by exome sequencing or targeted gene sequencing was performed for all patients and their families. Sanger sequencing was used to validate pathogenic variants within families on 3500 genetic analyzer (Applied Biosystems, Foster City, USA). The sequencing results were analyzed using CLC genomic workbench software. For clinical interpretation of variants, allele frequency data were obtained from various databases, including gnomAD (http://gnomad.broadinstitute.org/) and ExAc (http://exac. broadinstitute.org/). The pathogenicity of variants was assessed using in silico prediction tools, such as PolyPhen-2 (http://genetics.bwh. harvard.edu/pph2), SIFT (http://sift.jcvi.org), and MutationTaster (http: // www. mutationtaster.org) and Human Splicing Foundation (http://www.umd.be/hsf/).Ethical ApprovalThe study involving the use of human subjects was conducted in accordance with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration and has been approved by the the local Diyarbakır Gaziyaşargil Research and Training Hospital Ethics Committee (Date: 31.12.2021, Decision No: 966).Statistical AnalysisStatistical analyses of the data were performed using the SPSS software package for Windows software package (ver.18.0; SPSS Inc., Chicago, IL, USA). As descriptive statistics, numbers, and percentages for categorical variables, mean ± standard deviation or median (minimum-maximum) were used for numerical variables. The distribution of data was evaluated using the Shapiro-Wilk test. For numerical comparisons, Student’s t-test or Mann-Whitney U- test was used to assess differences between two groups according to the normal distribution of the measured parameters.

Results

A total of 60 patients, 26 female and 34 male, from 45 different families were included in the study. None of the patients have been previously published. The consanguinity rate was 96.66%. The mean age at the time of the data collection was 5.31 ± 4.37 years (min: 6 months max: 14 years). The mean age at diagnosis was 2.62 ± 3.90 years (min: 7 days max: 13 years). The total duration of follow-up of the cohort was three years, individually ranging from 4 to 46 (median = 12.1) months.
Rare DNA sequence variants were identified in 49 patients (81.66%), including variants LPL (n = 15), APOC2 (n = 32), APOA5 (n = 2). No mutation was detected in 11 patients (21%). The mean age at diagnosis of APOC2 patients was 2.95 ± 4.18 years, LPL patients was 2.97 ± 4.33 years. There was no significant difference between the mean age at diagnosis between genetic defects. 15/23 patients had recurrent pancreatitis. Initial TG levels in patients with APOC2 were significantly higher than in those with LPL (p<0.05). The clinical and laboratory characteristics according to the gene defects are shown in Table 1. The mean age of the patients with pancreatitis in patients with pancreatitis was 2.62 ± 3.81 years, and of patients without pancreatitis it was 2.73 ± 4.03 years. Triglyceride levels of the patients with pancreatitis at the time of diagnosis were found significantly higher (p<0.05). Failure to thrive was observed more frequently in patients with pancreatitis. The clinical and laboratory characteristics of patients with pancreatitis and without pancreatitis are presented in Table 2. Dietary treatment was applied in all patients. The recommended fat intake for patients was 10–15% of their total caloric intake. MCT oil was recommended to be 50-80% of total fat. Low-fat, medium chain triglyceride (MCT) oil-rich formula was used in infants under 1 year old. Lipid restriction and MCT oil supplementation were performed in patients older than 1 year. Omega 3 was given to 54 patients older than three months old. Fenofibrate was received by six patients. When the mean undertreatment TG levels were evaluated, no significant difference was found between the groups. A total of 47 pancreatitis episodes were recorded. Therapeutic apheresis was performed in six patients due to severe pancreatic episodes. Three sessions of TA were performed in 2/6 patients, and one session in 4/6 patients. Triglyceride levels were measured after each TA and continued until <1000 mg/dL. Necrotizing pancreatitis was observed in two patients. In the follow-up, pancreatic enzyme supplementation was started due to exocrine pancreatic insufficiency. Two different variants, c.55+6T>G and c.55+1G>C were detected in the APOC2 gene. A c.55+6T>G (IVS2+6T>G) intronic variant was detected in 22 patients from 11 families. This variant has so far been considered a “variant of uncertain significance (VUS)” because it is extremely rare (PM2) in healthy population databases and deleterious (PP3) in in-silico prediction tools. The c.55+1G>C (IVS2+1G>C) splice-site variant was detected in the APOC2 gene in 10 patients. Seven different variants, c.557G>A, c.953A>G, c.296T>C, c.662T>C, c.1262G>A, c.644G>A and c.679G>C were detected in the LPL gene. Both c.557G>A p.Gly186Glu (G186E) and c.953A>G p.Asn318Ser (N318S) (double homozygous variants) variants in the LPL gene were homozygous in six patients. In the F30, c.644G>A p.Gly215Glu(G215E) heterozygous and c.679G>C p.Val227Leu(V227L) heterozygous variants were found as compound in two siblings. c.679G>C p.Val227Leu variant is novel. In P48, the c.334_399dup p.Ala112_Thr133dup variant was homozygous in the APOA5 gene. This variant is a novel variant. Molecular analysis results are shown in Table 3.

Discussion

Familial hypertriglyceridemia is a rare cause of severe triglyceride elevation, which is seen with a prevalence of 1 in 1.000.000.² The LPL, APOC2, APOA5, LMF1, GPIHBP1 genes have been identified as the causative genes of monogenic chylomicronemia.⁷ These genes are necessary for the normal functioning of the LPL enzyme. Loss-of-function mutations in the LPL pathway could be detected in less than 30-40% of familial HTG patients.^8-9 In patients without genetic mutation, the underlying cause may be the development of autoantibodies against proteins in the LPL pathway or additional genetic factors. In our study group, LPL, APOC2, APOA5, LMF1 and GPIHBP1 genes were analyzed in 60 patients. A total of 11 different variants were detected in 81.66% (n = 49) of patients, including two novel variants. The reason why this rate is higher than in other studies may be due to very high consanguinity rates (96.66%) or very strict inclusion criteria.
In familial HTG, the LPL gene was the most commonly affected gene in the literature.^8-10 The most commonly affected gene was APOC2 in our study group and the prevalence of APOC2 variants was high compared to previous observations.^8-9 The c.55+6T>G (IVS2+6T>G) intronic variant was detected in 22 patients in our study. This variant has so far been considered a “variant of unsignificant (VUS)” because it is extremely rare (PM2) in healthy population databases and deleterious (PP3) in in-silico prediction tools. This mutation was detected in a Turkish infant and three Turkish adult patients with hypertriglyceridemia.^11-12 Since it was detected in 22 familial HTG cases in our study, it was thought that it would be appropriate to classify it as a “likely pathogenic/pathogenic”. Additionally, the higher proportion of cases with this variant in our Turkish cohorts could reflect ascertainment bias or possible founder effect of this variant.
In our study, the LPL gene was the second most affected gene. The c.557G>A p.Gly186Glu (G186E) and c.953A>G p.Asn318Ser (N318S) variants were found double homozygous in the LPL gene in six patients. The c.557G>A p.Gly186Glu variant is a pathogenic variant that has been previously reported.¹³ The c.953A>G p.Asn318Ser variant has been reported in the ClinVar database with different classifications as pathogenic, VUS, and benign. Although, this variant was evaluated as a polymorphism but associated with increased cardiovascular risk. In addition, this variant has also been shown to reduce LPL activity.^14-15 The c.953A>G p.Asn318Ser variant, which was detected as a double homozygous mutation in six patients, the clinical significance of this variant cannot be commented on since the c.557G>A p.Gly186Glu variant is pathogenic. The c.644G>A variant was previously reported as pathogenic.^15-16 The c.1262G>A p.Trp421Ter (W421*), c.644G>A p.Gly215Glu (G215E) variants in the LPL gene has also been reported previously and are classified as pathogenic.¹³ The c.679G>C p.Val227Leu variant is novel. The amino acid valine at position 227 is a conserved amino acid in protein; previously reported as c.679G>T p.Val227Phe (V227F) in a patient as a pathogenic in the form of different amino acid conversion with different nucleotide change. In another patient, it was reported as likely pathogenic as c.680T>C p.Val227Ala conversion to a different aminoacids (ClinVar#2441229). The c.334_399dup p.Ala112_Thr133dup variant in APOA5 gene is novel variant. This variant was classified as likely pathogenic because the patient had hypertriglyceridemia, although it was evaluated as VUS in databases such as Varsome and Franklin, but not found in healthy population databases. The c.16_39del p.Ala6_Ala13del (A6_A13del) variant in the APOA5 gene was previously reported. Initial TG levels of patients with APOC2 gene defect were significantly higher than those with LPL gene defect (p<0.05). The significant difference in mean initial triglyceride levels of the APOC2 and LPL genes may be due to the different sample size and small number of subgroups. Severe HTG is an independent risk factor for pancreatitis. TG level ≥1000 mg/dL is usually indicated as the threshold for the development of pancreatitis.¹⁷ However, some studies argue that TG>500mg/dL increases the risk of pancreatitis.⁶ Acute pancreatitis was observed in 38.33% of the patients, and recurrent pancreatitis was observed in 65.21% of them. Triglyceride levels of the patients with pancreatitis at the time of diagnosis were found significantly higher than the without pancreatitis patients (p<0.05). Consistent with the literature, the frequency of pancreatitis was higher in patients with higher triglyceride levels in our study. To prevent pancreatitis, therapeutic plasmapheresis has been included in the guidelines as an option.^18-20 We also performed TA in six patients because of severe pancreatitis unresponsive to medical treatment and poor general condition. In our study, triglyceride levels were measured after each TA and continued until <1000 mg/dL. In most of our patients, triglyceride levels decreased after one session. MCT-rich low-fat diet, fenofibrates are not effective enough to reduce TG levels in patients with severe HTG.²¹ Recently, LPL gene therapy (Alipogene tiparvovec), APOC3 inhibitors (Volanesorsen), ANGPTL3 and ANGPTL4 inhibitors have been developed and evaluated in clinical trials.²² The development of new therapeutic options for this rare disease requires awareness and screening among these patients. These findings highlight the need for molecular analysis in patients with severe HTG. It is anticipated to guide future individualized therapeutic strategies.

Declarations

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