Sitosterolemia: Four Cases of an Uncommon Cause of Hemolytic Anemia (Mediterranean Stomatocytosis with Macrothrombocytopenia)

Abstract

Sitosterolemia is a rare autosomal recessively inherited lipid metabolic disorder that is characterized by hyper absorption of plant sterols from the intestinal mucosa leading to toxic levels in the blood. Four patients of age ranging from 11 to 29 years presented to the outpatient department with clinical features of hemolytic anemia. There were no features of hypercholesterolemia in any of the patients. Peripheral smear examination of all four patients showed stomatocytes and macrothrombocytopenia. Qualitative testing for plant sterols was performed in one case. Next generation sequencing revealed a compound heterozygous mutation in ABCG5 gene (c.1222C>T and c.1255C>T) in one case and homozygous mutations in ABCG5 gene (c.727C>T), (c.332G>A (p.G111E)), (c.1222C>T) in the other three cases. Ezetimibe (10 mg/day) was administered in one case, with complete resolution of symptoms. All patients were advised a low plant sterol diet and regular monitoring of hemoglobin and lipid profile. Our cases highlight a rare but important cause of hemolytic anemia that can be suspected from careful peripheral blood examination but only conclusively established by molecular genetic diagnosis.

Introduction

Sitosterolemia or Phytosterolemia is a rare autosomal recessively inherited lipid metabolic disorder that is characterized by hyperabsorption of plant sterols from the intestinal mucosa leading to toxic levels in the blood and is caused by pathogenic variants in the genes encoding ATP—binding cassette transporter G5 (ABCG5) or G8 (ABCG8) [1–3]. These genes encode sterol efflux transporters (sterolin-1 and sterolin-2) that pump sterols into bile or intestinal lumen [1, 4]. The true prevalence of this condition is unknown due underdiagnosis [5]. It has a heterogenous clinical presentation often making it difficult for the clinicians to diagnose [6, 7]. Stomatocytic hemolysis, macrothrombocytopaenia, splenomegaly and abnormal bleeding can be the only clinical signs of the disease [2, 31]. Here we describe four cases of sitosterolemia with isolated hematologic abnormalities.

Methodology

Next generation sequencing: DNA was extracted and checked for quantity and quality using a bioanalyzer. Library was prepared using enrichment probes for a focused exome containing nearly 5000 genes of clinical relevance. Sequencing was done using Illumina HiSeq sequencer to generate 2 × 150 bp sequence reads at ≥ 100 × depth. The bioinformatic analysis was carried out using the GATK pipeline with Varminer, a bioinformatic variant interpretation tool developed by Medgenome labs.

Case 1

A 15-year-old girl, 2nd born to non-consanguineous parents from Bihar (Eastern India) presented to the outpatient department with complaints of fatigue, exertional breathlessness, yellowish discoloration of sclera and intermittent epistaxis (ISTH -BAT score 2) for 4 years. She required regular packed cell transfusions, once in 4 months with threshold hemoglobin of 6 g/dL. Antenatal, birth and neonatal period were uneventful. She had attained age appropriate developmental milestones. There was no family history of a similar illness. Physical examination revealed icterus, no significant lymphadenopathy, and a moderate hepatosplenomegaly (Liver and spleen were palpable 4 cm below right and left costal margin respectively). There were no xanthelasma or tendon xanthomas.

Her hematological and biochemical parameters are shown in Table 1.

Table 1.

Clinical, haematological and biochemical parameters

Clinical and lab features	Case 1	Case 2	Case 3	Case 4
Age at onset (years)	11	23	29	7
Sex	Female	Male	Female	Male
Hepatosplenomegaly	Yes	Yes	Yes	Isolated splenomegaly
Xanthelesma, tendon xanthomas	No	No	No	No
Hemoglobin (g/dL)	5	8.1	10.1	8.5
Reticulocyte count (%)	18.63	16.01	6.89	12.32
Mean corpuscular volume (fL)	115.3	102.8	83.4	90.2
Total white blood cell count (per cu mm)	5500	6000	5500	9300
Differential count
Neutrophils (%)	70	70	70	66
Lymphocytes (%)	22	21	22	20
Monocytes (%)	6	9	6	8
Eosinophils (%)	2	0	2	6
Basophils (%)	0	0	0	0
Platelet count on coulter (per cu mm)	12,000	46,000	13,000	6000
Manual platelet count (per cu mm)	60,000- 75,000	45,000–60,000	75,000–90,000	90,000–105,000
MPV (fL)	12.5	17.6	13.6	Not available
LDH (IU/L)	713	667	667	449
Direct Coomb’s test	Negative	Negative	Negative	Negative
Stomatocytes and macrothrombocytes on peripheral blood smear	Yes	Yes	Yes	Yes
LFT (mg/dL)
Total bilirubin	2.41	1.56	1.39	2.36
Direct bilirubin	0.71	0.65	0.61	0.68

The peripheral blood smear was reviewed by a pathologist and showed polychromasia, numerous stomatocytes and giant platelets (platelet estimated on smear to be 60,000–75,000/cu mm). Direct Coombs test was negative.

Plant sterols were detected in the peripheral blood using spectrophotometery at 625 nm (Liebermann–Burchard reaction) [8]. Quantitative analysis of plant sterol levels were not done.

The results of NGS are shown in Table 2.

Table 2.

Next generation sequencing

Case No:	cDNA	Zygosity	Protein	Prediction	Novel/reported
1	c.727C>T	Homo	p.R243X	Pathogenic	Reported
2	c.1222C>T	Het	p.R408X	Pathogenic	Reported
	c.1255C>T	Het	p.R419C	Likely Pathogenic	Reported
3	c.332G>A	Homo	p.G111E	Pathogenic	Novel (not found in EXAC/1000G)
4	c.1222C>T	Homo	p.R408X	Pathogenic	Reported

Case 2

A 23-year-old male born to non consanguineous parents from Bihar presented with complaints of easy fatiguability and pain in the left hypochondrium for 1 year. On evaluation he was found to have anemia and thrombocytopenia; however, there was no history of bleeding manifestations and he had never been transfused. There was no history of similar illness in the family. Physical examination showed pallor and hepatosplenomegaly. There were no stigmata of hypercholesterolemia.

Investigations were suggestive of hemolytic anemia (Table 1) with stomatocytes and macrothrombocytes seen in the peripheral smear (Fig. 1). Direct Coombs test was negative.

Fig. 1.

Peripheral blood smear from Case 2 showing macrothrombocytes (black arrow) and stomatocytes (red arrow) (color figure online)

The results of NGS are shown in Table 2.

Case 3

A 29-year-old female born to non consanguineous parents from Assam presented to the outpatient department with complaints of right hypochondriac pain for 2 months. There were no symptoms suggestive of anemia. She did not have any bleeding manifestations. Physical examination revealed pallor and hepatosplenomegaly (Liver was palpable 2 cm below the right costal margin and spleen 4 cm below left costal margin). There were no features suggestive of hyperlipidemia.

Investigations were suggestive of hemolytic anemia (Table 1). Blood picture showed stomatocytes and macrothrombocytes, with a smear count of 75,000–90,000/cu mm.

Direct Coombs test was negative.

Ultrasonography of the abdomen showed hepatosplenomegaly with mixed intensity solid lesions in the bilateral adrenal glands, and biopsy of these lesions showed extramedullary hematopoiesis.

The results of NGS are shown in Table 2.

Case 4

A 11-year-old boy, 2nd born to non-consanguineous parents from Bihar, presented with complaints of recurrent episodes of low-grade intermittent fever for 4 years. There was no localizing symptoms, no loss of appetite or loss of weight. Antenatal, birth and neonatal period were uneventful. He attained developmental milestones appropriate for age and was adequately immunized. He had received 2 packed red cell transfusions. Physical examination revealed pallor, icterus and splenomegaly (6 cm below left costal margin). There were no tendon or soft tissue xanthomas.

Investigations were suggestive of hemolytic anemia (Table 1). Peripheral blood picture showed stomatocytes and giant platelets with an estimated smear count of 90,000–105,000/cu mm.

Direct coombs test was negative.

Table 1 shows salient history, clinical and laboratory findings of the four cases. The results of NGS are shown in Table 2. The pathogenicity of each variant was presumed was based on previously published literature and clinical databases (ClinVar) for the reported variants. For the remaining variant it was considered to be pathogenic based on bioinformatic analysis [13/13 tools predicted the variant to be deleterious] and the strong clinical correlation.

Discussion

Dietary cholesterol and plant sterols are absorbed into the brush border epithelium of small intestinal mucosa by Niemann-Pick C1-like 1 (NPC1L1), a sterol influx transporter [9]. These lipids are esterified into chylomicrons and are transported to the liver by the portal venous system. These are finally excreted in the bile by hepatic ABCG5/ABCG8. The unesterified lipid component is effluxed into the intestinal lumen by the intestine ABCG5/ABCG8 [9]. Thus, a variant in the ABCG5/ABCG8 leads to increased absorption of sterols from the intestine and decreased excretion in the bile thereby increasing their levels in the blood. Tang,W et al. showed that genetic inactivation of NPC1L1 protected against sitosterolemia in murine models lacking ABCG5/ABCG8 [10].

The clinical features of sitosterolemia encompasses a spectrum ranging from being totally asymptomatic [11, 12] to involvement of multiple organ systems. Hematologic abnormalities in sitosterolemia include hemolytic anemia due to stomatocytosis, and bleeding episodes as a result of dysfunctional macrothrombocytes [6, 13, 14]. Kanaji et al. [15] showed that platelet hyperreactivity due to accumulation of plant sterols in the platelet membrane causes macrothrombocytopenia and bleeding phenotype in murine models of sitosterolemia. Other clinical features include xanthomas [16–19] including but not limited to tendons, intertriginous and planar areas. These occur as a result of hypercholesterolemia and foam cell formation, leading to accelerated atherosclerosis and sometimes sudden cardiac death [20–24].

Peripheral smear examination plays an important role in the diagnosis of hemolytic anemia. Numerous stomatocytes and macrothrombocytopenia should raise a suspicion of sitosterolemia [30] and patient should be further investigated by measuring blood plant sterol levels. Macrothrombocytopenia was also seen in at least 12 inherited disorders and also in some acquired immune-mediated hematological disorders [25]. Next generation sequencing identifying the pathogenic variants in the genes encoding ATP—binding cassette transporter G5 (ABCG5) or G8 (ABCG8) will confirm the diagnosis [4, 26, 27]. Lipid profile, 2D echocardiogram and carotid doppler would be ideal to look for evidence of atherosclerosis and to assess current cardiac status.

The treatment of sitosterolemia aims at reducing the plant sterols (campesterol, stigmatosterol and beta-sitosterol) levels in the blood. Patients are prescribed a diet low in plant sterol levels. Ezetimibe, taken orally at a dose of 10 mg/day has been shown to be effective in the management of sitosterolemia [11, 27, 28]. It inhibits cholesterol absorption by binding to Niemann-Pick C1-like 1 (NPC1L1), a polytopic transmembrane protein in the epithelium of small intestine, thereby reducing the level of cholesterol and related phytosterols in the blood [11, 29, 31]. Othman et al. [29] showed that Ezetimibe increased platelet counts and decreased mean platelet volume, plasma and RBC plant sterol levels, thereby decreasing bleeding episodes. Other therapeutic strategies include bile acid binding resins, HMG-CoA reductase inhibitors, dietary sitostanol and plasmapheresis [27, 31], which can be used in adjunct to Ezetimibe. Monitoring of hemoglobin, lipid profiles at regular intervals aid in long term management of the disease.

The protean manifestations of this disease make it a challenge for clinicians. The cases described here represent the largest case series of sitosterolemia from Indian subcontinent. Only two cases of sitosterolemia were reported previously from India [32, 33]. A total of 12 cases of this disorder with isolated hematological abnormalities have been reported of which genetic diagnosis using NGS was done in 11 cases [13, 14, 34–37].

Conclusion

Sitosterolemia is a rare, yet an important differential diagnosis in patients presenting with Coombs negative hemolytic anemia. Peripheral blood smear showing stomatocytes and macrothrombocytopenia should raise the suspicion of this disorder and warrants further investigation by NGS to confirm Sitosterolemia. Ezetimibe and a diet low in plant sterols is effective in reducing hemolysis and is recommended in the management of sitosterolemia.

Funding

Nil.

Compliance with Ethical Standards

Conflict of interest

There are no conflicts of interest.

Informed Consent

A written consent has been obtained to use medical information from each of the above patients.