Sitosterolemia is a rare autosomal recessive disease. It was first reported in 1974^[1]. The disease is related mainly to the adenosine triphosphate (ATP)-binding cassette subfamily G member 5 or member 8 (ABCG5 or ABCG8). Mutations in these two genes predispose patients to reduced biliary excretion and increased intestinal absorption of sterols^[2]. The carrier frequency of deleterious mutations in ABCG5 and/or ABCG8 in the general population may exceed 1 in 200,000 individuals^[3].

The disease typically presents with xanthomas, hematological disorders, arthralgia, atherosclerosis, splenomegaly, and markedly elevated phytosterol and low-density lipoprotein (LDL) levels^[4]. However, patients with only hematological symptoms are likely to be missed or misdiagnosed^[5]. The diagnosis of sitosterolemia depends on plasma phytosterols and/or mutations in the ABCG5/ABCG8 genes^[6].

Here, we describe a boy with sitosterolemia 1 (OMIM#210250) caused by a novel homozygous missense variant in the ABCG8 gene inherited from both parents. This variant was initially classified as a variant of uncertain significance (VUS). Minigene experiments subsequently demonstrated a greater proportion of aberrant splicing in the mutant construct, which led to the reclassification of the variant as likely pathogenic (LP)^[7].

We report a case of sitosterolemia primarily presenting with epistaxis and bicytopenia. WES revealed a novel homozygous missense mutation inherited from both parents. This variant has not been recorded in the natural population (gnomAD v4.1, https://gnomad.broadinstitute.org/), supporting the potential pathogenicity of the variant (PM2_supporting). Based on in silico predictions, this missense variant is likely to alter protein structure and function [Figure 4D; REVEL score of 0.741 (PP3)]. Splicing prediction suggested that this variant might disrupt RNA splicing [Figure 4E]. Subsequent in vitro minigene experiments indicated that the variant may affect ABCG8 mRNA splicing. This aberrant splicing may lead to protein truncation [Figure 3, Supplementary Figures 1 and 4]. Because ABCG8 is expressed at very low levels in peripheral blood, we could neither verify in vivo splicing abnormalities caused by this variant nor determine the degree of aberrant splicing. PVS1 at the supporting strength level was used (PVS1_strength (RNA)_supporting)^[9]. The results of in vivo functional experiments indicated that this variant significantly affected the metabolism of phytosterols. The hallmark phenotype of sitosterolemia is elevated plant sterol levels, a condition arising from biallelic loss-of-function variants in either the ABCG5 or ABCG8 gene. Given that WES ruled out pathogenic variants in ABCG5, the patient’s specific phenotype is most likely attributable to mutations in ABCG8 (PP4_moderate). In accordance with the SVI Recommendation for in trans Criterion (PM3) - Version 1.0, https://clinicalgenome.org/working-groups/sequence-variant-interpretation/, for a rare homozygous variant, PM3 at the Supporting strength level was applied (PM3_Supporting). In accordance with the guidelines of the American College of Medical Genetics and Genomics (ACMG), this variant is considered likely pathogenic (LP).

The human genome contains numerous potential splice sites. Single-nucleotide mutations within exons may disrupt normal splicing, resulting in consequences beyond simple amino acid substitution^[10].

The missense variant (p.Leu227Pro) is located in a helix of the ABCG8 cytoplasmic domain [Supplementary Figure 6A]. Computational modeling revealed that this substitution disrupts a critical hydrogen bond between residues Leu227 and Ile223, potentially compromising the stability of the ABCG8 protein [Figure 4D and Supplementary Figure 6B]. This cytoplasmic domain constitutes the nucleotide-binding domain of the ABCG5/ABCG8 heterodimer^[11,12]. We hypothesize that the identified variant may impair ATP binding or hydrolysis. Because transmembrane phytosterol transport is ATP-dependent, such functional perturbations could disrupt sterol efflux^[12].

In silico prediction suggested that this variant may result in aberrant splicing. In vitro studies showed that both the wild-type and mutant exhibited normal and abnormal splicing, suggesting a naturally weak splicing site in this region. However, minigene assays can assess only the splicing patterns of specific gene fragments and may not fully recapitulate in vivo expression dynamics. Further validation is needed to determine whether this variant functions primarily as a missense mutation or by disrupting mRNA splicing. If the splicing effect is the primary mechanism, truncation may trigger nonsense-mediated mRNA decay (NMD), resulting in loss of function of the ABCG8 protein. Otherwise, the missense mutation may produce a full-length but functionally abnormal protein.

Patients who present solely with bleeding or anemia are at risk of being overlooked or misdiagnosed. Peripheral blood morphology analysis plays a crucial role in raising clinical suspicion for such disorders. The majority of affected patients present with hypercholesterolemia and xanthomas^[4,13]. However, a distinct subset of patients exhibit primarily hematological symptoms^[14,15]. The exact mechanism of stomatocytosis in sitosterolemia is unclear. Incorporation of excess phytosterols into RBC membranes disrupts lipid composition and enhances membrane rigidity. This loss of membrane flexibility impairs RBC deformability, which may promote stomatocyte formation^[16].

Previous studies have reported that the accumulation of phytosterols in platelet plasma membranes during sitosterolemia may induce platelet activation, promote microparticle generation, and subsequently lead to platelet dysfunction^[17]. Recent research has indicated that thrombocytopenia is caused neither by a lack of megakaryocytes nor by abnormal proteins in the platelets themselves. Notably, platelet count increases as phytosterol levels decrease^[18]. Consistent with the findings of earlier studies, the proportion of platelet-producing megakaryocytes in our patient fell within the normal reference range.

The patient presented with a normal lipid profile and impaired growth, without cutaneous xanthomas. We hypothesize that his phenotype may be attributed to his dietary pattern characterized by low fat intake. Previous studies have shown that LDL cholesterol levels in sitosterolemia patients may be either elevated or normal^[4,19,20]. His growth retardation may be attributable to picky eating and malnutrition.

Despite exhibiting normal cholesterol levels, patients with sitosterolemia frequently develop premature coronary artery disease^[16]. Growing evidence shows a strong association between sitosterolemia and cardiovascular morbidity^[16,21]. Early therapeutic intervention can significantly improve both clinical manifestations and biochemical parameters. Patients who maintain rigorous dietary compliance can achieve near-normalization of their phenotypic and metabolic profiles^[22]. Ezetimibe, as a first-line drug, is an effective inhibitor of plant sterol and cholesterol uptake and can improve plant sterol tolerance. The addition of ezetimibe to dietary control further reduced plasma plant sterol levels and was beneficial for increasing platelet count^[4,23]. Unfortunately, none of the effective treatments return plant sterols to normal^[24].

These findings suggest that heterozygous carriers may have a biochemical phenotype. They are also at risk of developing atherosclerotic cardiovascular disease (ASCVD)^[16]. For carriers, dietary intervention and clinical follow-up may reduce the risk of adverse outcomes. From a genetic counseling perspective, if a couple has more children, there is a 25% chance that their offspring will be affected. For his sisters, carrier screening of their spouses is recommended to reduce the risk of having affected children.

In short, we identified a novel homozygous missense variant (ABCG8: p.Leu227Pro) in a child with sitosterolemia. The nonspecific manifestations of anemia and bleeding may result in diagnostic confusion. The presence of stomatocytes and large platelets in the blood smear provides valuable clues for disease diagnosis. WES technology enables the detection of disease-associated genetic variants, while markedly elevated phytosterol levels serve as critical diagnostic biomarkers for sitosterolemia.