Genotypic and phenotypic features of dyslipidemia in a sample of pediatric patients in China

Dyslipidemia is rapidly increasing in both children and adolescents, posing a threat to their health. Hypercholesterolemia, especially elevated low-density lipoprotein cholesterol (LDL-C) levels, are usually of specific focus and are a recognized risk factor for premature atherosclerotic cardiovascular disease (ASCVD) [1]. Dyslipidemia may occur for several reasons, including genetic and nongenetic factors. In pediatric patients especially for those with young age and normal weight, genetic factors probably contribute more on dyslipidemia.

Precise diagnosis is much more important for children with genetic testing, both in prognostics and treatment, comparing with that for adults. Identifying monogenic hypercholesterolemia in adult patients could be ignored as it has limited influence on the treatment [2]. Statins is the primary pharmacotherapy used to lower lipid. For the most adult patients in whom statins are indicated, the benefits outweigh the risks [3]. And ezetimibe is usually recommended to be used combined with statins in patients who have not been able to achieve 50% reduction in LDL-C level. However, treatment of pediatric dyslipidemia begins with lifestyle modifications, but primary genetic dyslipidemias may require medications [4]. Drug use in pediatric patients have strict indications and many lipid-lowering drugs were only recommended to be used in pediatric patients with homozygous FH or with elder age [4]. The use of combined drugs is even more difficult in the clinic considering the side effects and indications. Furthermore, a recent study found an association between receiving a genetic diagnosis of FH and willingness to be treated with medication, suggesting genetic diagnosis may be useful for cardiovascular prevention in children [5].

Precise diagnosis may be facilitated by the advent of next-generation sequencing (NGS) technologies, which have facilitated the identification of several specific genes for this condition including LDLR, PCSK9, APOB, STAP1, and LDLRAP1 [6]. More than 25 genes have been identified in patients with dyslipidemia [7]. However, sequencing for these genes can only explain part of the patients [8] and most of studies have focused on some specific genes linked to familiar hypercholesterolemia, such as LDLR, APOB, and PCSK9, using gene panels instead of NGS, which may underestimate the proportion of monogenic dyslipidemia/hypercholesterolemia [9]. The genetic reason for dyslipidemia is often unclear and rarely analyzed in pediatric patients [10, 11]. In addition, if physical findings are identified in a child, rarer dyslipidemias should be considered [4].

Therefore, this study was designed to confirm the molecular defects of hypercholesterolemia in pediatric patients using whole-exome sequencing (WES) rather than gene panels of FH, basing on a single-center group of children and adolescents. We collected samples from 30 pediatric patients with hypercholesterolemia and performed WES, allowing us to link the genetic and phenotypic data of 63.33% of these patients. We also evaluated the differences in lipid levels and demographic data between patients with the positive genetic results and patients with negative genetic results. It is our hope that these results will help to expand the genetic spectrum for monogenic dyslipidemia and be beneficial to precise diagnosis and treatment.

This study describes both the genotypic and phenotypic data of 30 pediatric patients with dyslipidemia, using WES to minimize bias in genetic selection. Most of the patients evaluated in our study were below ten years of age. To the best of our known, this is one of the largest studies of pediatric hypercholesterolemia in China.

WES have advantages in diagnosing dyslipidemia in pediatric patients comparing with gene panels of FH. Gene panels is cost-effective but probably lead to miss diagnosis in some patients. FH has received more attention than other types of dyslipidemia, and scientists have emphasized the importance of several genes in this condition, with many of the available drugs designed to treat this specific condition [6, 14]. The most common pathogenic mutations in FH appear within the LDLR gene [15], which was further validated by our study. Many studies sequenced specific genes for patients with clinically suspected FH [16]. Genes encoding LDLR, APOB, PCSK9 are recommended to include in the genetic testing [17]. Minicocci et,al. sequenced LDLR, APOB, PCSK9 in 78 children and adolescents with clinically diagnosed FH and identified FH-causing mutations in 50% of them [9]. Comparing with their results, we identified positive genetic results in 63.33% of the pediatric patients. 11 patients were identified LDLR variants, and eight patients were identified other pathogenic genes like ABCG5/8, LPL, LIPC, and CETP, which are usually not included in FH gene panels. WES could be an effective complement to FH gene panels, especially in patients with negative results of FH gene panels. Considering the difficulty of general use of WES as a means of screening in nationally, we recommended to sequence more genes in pediatric patients with negative genetic results.

Some of the genes identified in this study are not common in patients with hypercholesterolemia. One of them is lipoprotein lipase (LPL), which is responsible for the intravascular hydrolysis of the TG in TG-rich lipoproteins. Homozygous or compound heterozygous variants in LPL gene could result in the accumulation of TG-rich lipoproteins while heterozygotes for LPL mutations present with variable plasma TG levels, ranging from normal to very high levels (> 10 mmol/L) and decreased levels of HDL-C [18]. The patient in our study with mutations in this gene presented with mildly elevated levels of TC, TG, and LDL-C and had no tendon xanthomas or abnormal HDL-C levels. Mutations in LIPC and CETP are both associated with reduced HDL-C levels and hyperalphalipoproteinemiam [19]. However, the two patients carrying variants of LIPC or CETP did not present with elevated ApoA1 or decreased HDL-C. They only presented with abnormal levels of TC and LDL-C levels.

Pediatric patients with hypercholesterolemia could be resulted by heterozygous ABCG5/8 variants. Our study shows that ABCG5/8 could be underestimated in pediatric patients with hypercholesterolemia and NGS has an advantage in diagnosing sitosterolemia or carriers of ABCG5/8 gene comparing to gene panels of FH. ABCG5/8 is the pathogenic genes associated with sitosterolemia, characterized by increasing levels of plant sterols [20]. In our study, 26.32% (5/19) of the patients were identified variants in ABCG5/8. 50.00% (3/6) of the patients with xanthomas were confirmed having at least one ABCG5/8 variant, indicating that xanthomas are probably an indicator of ABCG5/8 variant. Mauricio, et,al. found 3.10% of the patients were diagnosed sitosterolemia, through sequencing ABCG5/8 genes in 260 patients with clinical diagnosed FH and negative genetic results [21]. However, they did not analyze carrier rate of ABCG5/8 gene [21]. Recent studies showed that carriers of ABCG5/8 gene present with elevated phytosterol levels and are at increased risk of CAD [22]. Given the difficulties associated with serum sitosterol testing in China and difference in treatment between sitosterolemia and other types of hypercholesterolemia in children, NGS has become much more important in the diagnosis of these patients, especially for those who presented with xanthomas. However, it is still controversial that if patients with heterozygous ABCG5/8 variants should be treated with medicine. And these patients should be followed-up and monitored regularly.

Compared to the patients with negative genetic results, patients with positive genetic results had significantly greater ApoB and Lp (a) levels (Table 3). ApoB, as an essential constituent of very-low-density lipoprotein and its metabolites intermediate density lipoproteins and LDLs, as well as chylomicrons and their remnants, and is crucial for the maintenance of the structural stability of various lipoproteins [23, 24]. Strong evidence shows that ApoB is a more accurate indicator of cardiovascular risk than either TC or LDL-C [24]. Our study indicates that ApoB is also a potential biomarker or therapeutic target for monogenic hypercholesterolemia.

The incidence rate of tendon xanthomas is up to 20.00% in this study, which is probably resulted by Berkson’s bias. Among the six patients presenting with tendon xanthomas, five had positive genetic results, with a diagnosis rate of 83.33%, while the diagnosis rate in patients without tendon xanthomas was 58.33% (14/24). A meta-analysis showed that age, male gender, LDL-C and TG level were associated with the presence of xanthomas and that this condition indicates an increased risk of CVD [25]. Similar studies are rare in pediatric patients. Our result suggests that xanthomas remain a strong indicator for monogenic hypercholesteremia.

However, there are also some limitations in this study. First of all, the numbers of patients enrolled in this study is small. Lack of widely screening of FH in China could partly explain the phenomenon. Children’s parents could be not aware of the exact family history of hypercholesterolemia. Also, we did not list other symptoms of hypercholesterolemia like corneal arcus as an inclusion criterion. These factors could result in that children with milder profiles may not be captured in this study. Secondly, lack of control group. We didn’t analyze rate of carries with NGS in healthy patients given the economic factors.

In conclusion, we reported genetic and phenotypic description of 30 Chinese pediatric patients presenting with hypercholesterolemia/tendon xanthomas. Genetic testing with WES is important for the prognostics and treatment of pediatric patients with hypercholesterolemia. In addition, our results suggest that heterozygous ABCG5/8 variants may be underestimated in pediatric patients with hypercholesterolemia, especially for those presented with xanthomas.