From Bernard-Soulier syndrome to sitosterolemia: the role of genetic analysis in bleeding diathesis

Abstract

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Bleeding diathesis’ diagnosis can be challenging due to the high number of disorders with hemorrhagic symptomatology. Sitosterolemia is a rare disease characterized by increased sterols plasma levels and cardiovascular, cutaneous, articular, and hematological manifestations, including anemia and macrothrombocytopenia. The disorder is caused by ABCG5 and ABCG8 mutations.

We present a case of a patient with bleeding diathesis, macrothrombocytopenia, a moderate defect of primary hemostasis and a pathological platelet aggregation analysis, with an initial diagnosis of Bernard-Soulier variant syndrome. After performing a genetic study using an exome analysis, the patient had two ABCG8 gen variants, one pathogenic (NP_071882.1:p.Trp536Ter (NM_022437.2:c.1608G > A) variant, ClinVar ID: 499930) and the other one probably pathogenic (NP_071882.1:p.Leu465Arg (NM_022437.2:c.1394T > G) variant), changing the diagnosis to sitosterolemia, which has its own therapeutic approach.

This case report shows the importance of the genetic analysis. Sitosterolemia should be suspected in the presence of macrothrombocytopenia, stomatocytes in the blood smear and hemolytic anemia, performing a genetic study including ABCG5 and ABCG8 gene variants.

Background

Sitosterolemia is a rare inherited autosomal recessive disorder of lipid metabolism, characterized by an important plasma elevation of plant sterols levels. Patients with sitosterolemia develop xanthomas, premature atherosclerosis, arthritis, and hepatic and hematologic alterations such as thrombocytopenia and hemolysis. An effect of sitosterolemia in platelets function has also been proposed [1, 2]. We present a case report of a patient with platelet abnormalities related to sitosterolemia.

Case presentation

The patient was a 40-year-old woman with bleeding diathesis: she had menorrhagia since she was 15. She had a blood count with mild anemia: hemoglobin 109 g/L, platelets 135 × 10⁹/L, leukocytes 4,38 × 10⁹/L. The blood smear showed giant platelets with normal granulation, also known as macrothrombocytes and numerous stomatocytes (Fig. 1). The patient presented a pathological platelet function analysis (PFA-100) in several determinations, showing a moderate defect of primary hemostasis, with normal VIII factor and von Willebrand factor (vWF) levels, only with a vW ristocetin cofactor / vW antigen ratio of 0,76.

Fig. 1

Peripheral blood smears with macrothrombocytes and stomatocytes: (A) May–Grünwald–Giemsa x400, (B) May–Grünwald–Giemsa x1000

A platelet aggregation study was performed: it was no aggregation with ristocetin (RIPA) at low concentrations (0,5 and 0,2 mg/mL) and a at 1 mg/mL, with an only wave with final aggregation of 86–88% at 1,2 mg/mL concentration; an only wave with final aggregation of 90% induced by ADP and a double wave with at 1/5 and 1/10 concentrations; a slow double wave with final aggregation of 88% induced by adrenaline and normal latency time (20 s) with subsequent aggregation of 81% with collagen. The conclusion of the study was that the patient had a macrothrombocytopenia with a moderate defect of primary hemostasis and aggregation compatible with Bernard-Soulier variant syndrome (type 2), so a platelets flow cytometry and a genetic analysis of related-genes were performed.

Platelets flow cytometry showed a drop in median fluorescence intensity (MIF) of Ib (CD42b) and Ia (CD49) glycoproteins and a mild drop of IX (CD42a) glycoprotein (Fig. 2).

Fig. 2

(A) Different histograms showing median fluorescence intensity (MFI) of glycoprotein IX, Ia, IIb/Ia and Ib on platelets by flow cytometry (patient in red, healthy donor in blue). (B) Diagram of CD42a against CD49b showing platelets population of patient (red) versus healthy donor (blue)

The genetic study was an exome analysis using Next Generation Sequencing (NGS) NEXTseq 1000 sequencing system (Illumina). The identified variants are cross-referenced with different public databases, and amino acid changes are evaluated using prediction programs that assess the potential impact on the resulting protein (PolyPhen-2, MutationTaster, and/or VarSome). After analyzing the numerous identified variants, those with a frequency higher than 1% in the general population are excluded (unless they are related to the associated clinical features), according to the polymorphism database (http://www.ncbi.nlm.nih.gov/projects/SNP/), as well as those that appear with high frequency in the cohort of patients subjected to this study technique.

The patient presented two ABCG8 gen variants, one pathogenic (NP_071882.1:p.Trp536Ter (NM_022437.2:c.1608G > A) variant, ClinVar ID: 499930) and the other one probably pathogenic (NP_071882.1:p.Leu465Arg (NM_022437.2:c.1394T > G) variant), that could be related to patient’s phenotype. The second mutation is a novel one and it has not been reported in publicly available databases yet, whereas the first mutation is well-documented in the literature [1,2,3,4,5]. Patient had no children or alive relatives that could be tested as well.

Discussion and conclusions

The patient had hemorrhagic symptomatology and after performing a platelet aggregation study the initial suspicious diagnosis was Bernard Soulier syndrome (BSS). It is a genetic disorder caused by GPIb-alpha (GPIBA), GPIB-beta (GPIBB), and GPIX (GP9) mutations, which are subunits of the GPIb-IX-V complex (a platelet receptor complex that binds with vWF). The syndrome is characterized by giant platelets in the peripheral blood smear, thrombocytopenia, and prolonged bleeding time [6], which was consistent with patient’s manifestations. The platelet flow cytometry and the aggregation pattern also suggested this diagnosis.

However, once the genetic analysis was performed, no BSS mutations were found. The patient had two ABCG8 mutations instead, one pathogenic and the other probably pathogenic, which could lead to sitosterolemia diagnosis. Sitosterolemia is a disorder with increased levels of plasma plant sterols such as stigmasterol, campesterol and sitosterol, caused by mutations in two genes named adenosine triphosphate-binding cassette (ABC) subfamily G members 5 and 8 (ABCG5 and ABCG8). These genes are involved in the absorption and excretion of sitosterol and cholesterol, participating in the sterol transport across the cell membrane. Its mutations cause hyperabsorption and increased sterol plasma levels [7]. Sitosterolemia is a rare disorder with an unknow frequency, probably around 1 in 200 000 people in general population. Up to 80 ABCG5 and ABCG8 variants have been related to the disease, with no genotype-phenotype correlation at this point [7]. To support the diagnosis of sitosterolemia, patient’s sterol plasma levels were analyzed, and they presented increased levels: her β-Sitosterol levels were 417,02 µmol/L, with a normal range of 7,28 ± 5,82 µmol/L (Table 1).

Table 1 Patient’s increased sterol plasma levels, compatible with diagnosis of sitosterolemia

Patients with sitosterolemia develop different clinical symptoms and signs, including macrothrombocytopenia. Studies performed in bone marrow transplanted mice proposed that macrothrombocytopenia is related to increased sterol plasma levels and not directly with the genetic ABCG5/ABCG8 defect [8, 9]. It would cause direct plant sterol incorporation in the platelet membrane and premature clearance, leading to the thrombocytopenia, although a direct lipotoxicity on blood components, including platelets, has also been proposed [6]. They also showed that in mice with sitosterolemia there was an accumulation of plant sterols in the platelet plasma membrane, causing platelet hyperactivation: the fibrinogen binding to the αIIbβ3 integrin platelet receptor is increased, the αIIbβ3 complex is internalized, platelet-derived microparticles are generated and filamin is altered. This hyperactivation and its consequences lead to macrothrombocytopenia, loss of ristocetin-induced agglutination and defects in thrombus formation [5]. The case patient presents all these characteristics. The flow cytometry, which was initially related with BSS, was also compatible with sitosterolemia’s alterations, since reduced levels of platelet receptors by flow cytometry has also been described in sitosterolemia [9,10,11].

Sitosterolemia’s treatment includes a sterol-free diet and the use of ezetimibe, a sterol absorption inhibitor. A study by Del Castillo et al. showed that patients presented lower sitosterol levels and increased platelet counts a year after initiating treatment [6]. Reducing sterol plasma levels has also an important clinical impact due to the premature cardiovascular disease associated with sitosterolemia.

In conclusion, this case report shows the importance of the genetic analysis. Bleeding symptomatology, giant platelets and aggregation pattern’s alterations can be caused by different disorders and in some cases the only way to get the diagnosis is analyzing the exome or the genome. Sitosterolemia is a rare disease with different tissue and organs’ damage and a specific therapeutic approach. It should be suspected in the presence of macrothrombocytopenia, stomatocytes in the blood smear and hemolytic anemia, performing a genetic study including ABCG5 and ABCG8 gene variants. One of this patient’s variant had not been previously reported as pathogenic, so performing further genetic analysis would probably lead to describe more variants.