Abstract
Phytosterolemia is a rare autosomal recessive disease of plant sterol metabolism, the pathophysiological features of which are high plasma levels of plant sterols and xanthomatosis caused by mutations of ABCG5 and ABCG8 genes, and the combination of hemolysis and macrothrombocytopenia is an unusual clinical manifestation. All the patients of the 3 unrelated phytosterolemia first presented with prominent macrothrombocytopenia and stomatocytosis. They were either homozygous or compound heterozygous for ABCG5/ABCG8 gene mutations and had significantly elevated serum plant sterols levels quantified using high-performance liquid chromatography. The in vitro study demonstrated that sitosterol can cause changes in shape and osmotic fragility of red blood cells. These findings suggest that macrothrombocytopenia and stomatocytosis could be initial and main features in some patients with phytosterolemia and that serum phytosterols and relevant genes should be analyzed in patients whose macrothrombocytopenia and/or stomatocytosis are unexplained, especially whose parents are of consanguineous marriage.
Introduction
Phytosterolemia (sitosterolemia) is a rare autosomal recessive disease of plant sterol metabolism causing significantly elevated plasma levels of plant sterols in the form of sitosterol, cholestanol, and stigmasterol. 1 To date, there are only about 100 known cases worldwide. 2 Phytosterolemia has been confirmed to be caused by mutations in genes of adenosine triphosphate-binding cassette (ABC) subfamily G members 5 and 8 (ABCG5 and ABCG8) encoding sterolin-1 and sterolin-2, respectively, which preferentially excrete plant sterols out of intestinal cells into the gut lumen and out of liver cells into the bile ducts, thereby decreasing sterol absorption.3 –5
The most common clinical features of phytosterolemia are tendinous and cutaneous xanthomas, premature coronary atherosclerosis, arthritis, and arthralgia.1,6-10 In addition, a number of associated hematological abnormalities, including hemolysis, iron deficiency anemia, thrombocytopenia, large platelets, and splenomegaly, have been reported in some patients.1,11 In 2005, Rees and his coworkers, 10 in their attempts to genetically map hereditary stomatocytosis, identified 5 pedigrees with specific macrothrombocytopenia/stomatocytosis, which had significantly elevated levels of plant sterols in the blood and mutations in ABCG5 and ABCG8, diagnostic of phytosterolemia. A year later, we also reported the clinical, biochemical, and molecular genetic features of 3 siblings of a Chinese family with phytosterolemia characterized by remarkable macrothrombocytopenia/stomatocytosis. We then suggested that blood cells could be a target for the toxic effect of elevated plant sterols in circulation. 9 In the present study, we describe 6 patients from 3 unrelated families of phytosterolemia who presented with a highly specific coexistence of macrothrombocytopenia and stomatocytosis and investigated the effect of high level of sitosterol on erythrocytes in in vitro study to demonstrate the clinical significance of hematological abnormalities in this condition.
Methods
Patients
Three family trees were consistent with autosomal recessive inheritance (Figure 1 ). All the affected patients presented similar clinical problems to various degrees: thrombocytopenia, anemia, xanthomas, and splenomegaly. All the patients had an otherwise normal physical development with no obvious evidence of premature atherosclerosis or cardiovascular disease. Their blood smears showed large platelets, some of which were as large as erythrocytes, and abnormal erythrocyte shapes, mainly as stomatocytes (Figure 2 ). Bone marrow examination showed normoplasia with normal megakaryocytic series. The platelet aggregation in response to adenosine diphosphate and ristocetin were generally normal. Platelet glycoproteins Ib/IX and IIb/IIIa measured by flow cytometry were also normal. The patients' erythrocytes showed an increased osmotic fragility. Hemolysis began at concentrations of 0.48% to 0.64% of saline solution and was complete at 0.32% to 0.40%. The results of other hemolytic tests, including Coombs test, Ham test, glucose-6-phosphate dehydrogenase, glucose phosphate isomerase, and pyruvate kinase activity, were either negative or within normal ranges. The plasma levels of total cholesterol, low-density lipoprotein cholesterol, and apolipoprotein (apo)B in all patients were slightly higher than those in normal individuals, whereas the levels of high-density lipoprotein cholesterol and apoA were not changed (Table 1 ).
Pedigrees of 3 families: filled square and circle, affected patients; half filled square and circle, heterozygotes; double line, consanguineous union.
Wright-stained blood films on 3 probands. A-II-2, B-II-1, C-II-4 (original magnification ×1000), macroplatelets (mp) are prominent, some of which are as large as erythrocytes; stomatocytes (st) have slit-shaped central pallor.
Routine Hematology and Lipids Analysis of Probandsa
Abbreviations: Plt, platelet count; Hb, hemoglobin; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; apo-B, apolipoprotein B; LP, lipoprotein; imp, impedance method; opt, optical method; HDL-C, high-density lipoprotein cholesterol.
a Routine hematological data were collected by Sysmex XE-2100. The impedance mode uses fixed gated, tends to underestimate platelet counts in the presence of large platelets, and optical method is preferable. The Hb was 40 to 50 g/L before their large spleen had been removed. The TC, LDL-C, and apoB in patients were higher, whereas the levels of HDL-C and apoA were not changed.
Apart from the common features mentioned above, the proband of family A (A-II-2), whose parents are first cousins, had frequent gingival bleeding and menorrhagia. After splenectomy, at the age of 12 because of thrombocytopenia and splenomegaly, xanthomas started to emerge on her bilateral eyelids and gradually expanded. Her aspartate transaminase and alanine transaminase were slightly elevated, and she had evidence of cholelithiasis. She underwent hysterectomy because of severe postpartum hemorrhage at the age of 21. In family B, the proband (B-II-1) was once diagnosed as Evans' syndrome due to thrombocytopenia and hemolytic anemia. After her large spleen had been removed, her anemia was markedly improved, but she noticed xanthomas that gradually grew on eyelids and right shoulder. The patient had no significant bleeding history, and intermarriage had not occurred in her recent generations. In family C, the proband (C-II-4) and her 2 brothers (C-II-2 and C-II-3) had very similar case histories. At the ages of 10 to 12, they had thrombocytopenia, anemia, and splenomegaly. They all underwent splenectomy at about 15 years old. C-II-2 had large fibromas (6 × 8 cm and 7.5 × 10 cm) in the 2 cheeks of his face. C-II-4 had iron deficiency anemia caused by menorrhagia. The oldest sibling (C-II-1) showed only mild anemia, small xanthelasma, and a palpable spleen. Their parents are not consanguineous. This study was approved by our institutional ethical committee.
Quantitative Analysis of Serum Sterol Levels
Blood samples were collected without anticoagulants from all patients and their family members as well as 15 normal individuals. The serum was obtained by centrifuging at 2000 g for 20 minutes and then the serum was stored at −80°C until quantitative analysis of sterols. Serum plant sterols were quantified using high-performance liquid chromatography (HPLC), as described by Kasama et al. 12 The sitosterol, cholestanol, and stigmasterol standards which were employed were purchased from Sigma (St. Louis, Masachusetts).
Sequence Analysis of ABCG5 and ABCG8 Genes
Genomic DNA was isolated from whole blood using the QIAamp DNA Blood Midi Kit (QIAGEN, Hilden, Germany). In order to amplify the 26 coding exons including all intron–exon boundaries as well as the promoter region of ABCG5 (NG 008883.1) and ABCG8 (NG 008884.1), 26 primer pairs were designed as previously described. 9 The polymerase chain reaction (PCR) products were then electrophoresed on 1.5% agarose gel, purified by QIAquick Gel Extraction Kit (QIAGEN). They were reacted with Big Dye terminator Cycle Sequencing Reagent (Applied Biosystems, Foster City, California) according to the manufacturer’s protocol and then directly sequenced on an automated sequencer (ABI 3100; Perkin-Elmer, Foster City, California).
Thymine Adenine Cloning
The PCR product of exon 12 of 13 of ABCG8 from family B was subcloned in the TA cloning vector. After the transfection of T carrier with PCR fragment into the competent cells (DH5α), 8 positive colonies were sequenced as described above.
Restriction Fragment Length Polymorphism of the PCR Product
In order to analyze A20883G of family C, PCR products of exon 10 of ABCG5 containing A20883G fragment from the patients, their families, and 50 normal individuals were digested with restriction enzyme BmrI (New England Biolabs, Ipswich, Masachusetts). The wild-type amplification product was completely cleaved into 2 fragments (203 and 80 bp), whereas the product of heterozygote into 3 fragments (283, 203, and 80 bp). Samples were electrophoresed on 3% agarose gels and then stained with ethidium bromide.
Osmotic Fragility of Erythrocytes in the Presence of Sitosterol
The Na2EDTA-anticoagulated blood from 10 normal individuals were separately incubated in methyl β-cyclodextrin solution containing appropriate or high dose of cholesterol (final concentrations were 560 and 1210 μmol/L, respectively) or sitosterol (608 μmol/L) at 37°C for 50 minutes to promote cellular membrane sterol flux. Then 30 μL of samples was each incubated in 1 mL of a series of phosphate-buffered sodium chloride solutions from 0.24% to 0.74% NaCl for 2 hours. After that the erythrocyte osmotic lysis was determined. At the same time, the erythrocyte morphologic change was observed under light microscope.
Results
Serum and Erythrocyte Membrane Sterol Levels
Serum concentrations of main phytosterols (stigmasterol, cholestanol, and sitosterol) were significantly elevated in all the affected patients in comparison with normal individuals, which could be increased more than 30 times (Table 2 ). The concentrations of these phytosterols were slightly increased without statistically significant difference in heterozygotes (data not shown). There was a correspondence between serum phytosterols levels and erythrocyte morphologic changes.
Serum Sterols Levels of Phytosterolemia Patients in 3 Families
a For serum plant sterol measurements, the data are expressed as mean ± standard deviation. The serum sterol levels of all patients were increased in comparison with those of normal controls.
Gene Mutations
Families A and C were caused by mutations in ABCG5. The proband of family A was a homozygote for a C20896T nonsense mutation (R446X) which had been discovered. 13 The 4 patients of family C were compound heterozygous for C20896T (R446X) and A20883G. The novel A20883G mutation occurs in classical splice site in intron 9, and deletes a recognition site for restriction enzyme BmrI. The amplification products from 50 normal individuals could completely be cleaved with the specific enzyme, excluding the possibility that the mutation is a polymorphism of ABCG5 gene. The proband of family B showed compound heterozygous for complex del43683-43724 and del43866C-43867G/ins43866T mutations in ABCG8, which were confirmed by TA cloning sequencing, and they could not be found in 50 normal individuals by direct sequencing, indicating they are novel mutations rather than polymorphisms. In addition, several neutral polymorphisms, such as Q604E of ABCG5, C54Y, T400K, and A632V of ABCG8 which had been previously described were also found in this study (not shown).
Osmotic Fragility of Erythrocytes in the Presence of Sitosterol
The results of osmotic fragility of incubated erythrocytes were shown in Table 3 . The erythrocyte lysis began at 0.46% ± 0.01% NaCl in the group of appropriate cholesterol dose (560 µmol/L, corresponding to normal serum level). The osmotic fragility was decreased in the high-cholesterol group. In contrary, the osmotic fragility was markedly increased in the sitosterol group with erythrocyte lysis beginning at 0.60% ± 0.01% NaCl.
The Osmotic Fragility of Erythrocytes Incubated in the Presence of Sitosterol or High Cholesterol
a Significantly different from control (P < .01). The osmotic fragility of sitosterol-treated erythrocytes was remarkably increased, while the osmotic fragility of the high-cholesterol group was decreased.
Blood smears showed normal red cell morphology in the group of appropriate cholesterol dose. In the high-cholesterol group, there was a slight change in the red cell morphology with some spur cells appearing. However, various abnormal shapes of red cells were noted in the sitosterol group, mainly as stomatocyte, and some as target cell, spherocyte, and irregularly appearing erythrocyte, just like the findings in peripheral blood smears of the patients with phytosterolemia.
Discussion
All the affected individuals reported here showed specific hematological problems as well as extremely high plasma levels of plant sterols ascertained by HPLC, which are the biochemical hallmark of phytosterolemia. The identified homozygous or compound heterozygous mutations in ABCG5 and ABCG8 genes further confirmed the diagnosis of phytosterolemia in these patients.
The coexistence of macrothrombocytopenia and stomatocytic hemolysis is an unusual condition, similar clinical feature was previously found only in individuals with Mediterranean macrothrombocytopenia/stomatocytosis, originally described in Mediterranean migrants living in Australia.14,15 However, the related reports ceased over a 5-year period, indicating that the condition was not persistent. 16 In a later investigation also in Australian residents of Mediterranean migrants, no more evidence of such blood cell changes could be detected. 14 Conversely, similar hematologic characteristics have been found in some patients of phytosterolemia reported by Rees 10 and by us. 9 And this study showed 3 other pedigrees with same specific features. It should be noted that there are several important differences between these 2 disorders: (1) Mediterranean macrothrombocytopenia/stomatocytosis occurred only in small population of Australian immigrants from Italy and Greece, 14 whereas phytosterolemia appears on no ethnic prevalence; (2) in Mediterranean macrothrombocytopenia/stomatocytosis, no evidence of inheritance was suspected, 17 while phytosterolemia has been confirmed to be an inherited disease; and (3) Mediterranean stomatocytosis/macrothrombocytopenia was seen in otherwise healthy individuals, while patients with phytosterolemia often present with various relevant symptoms or signs. Therefore, Mediterranean macrothrombocytopenia/stomatocytosis seems unlikely to be a separate entity but perhaps due to the diet rich in plant oil, especially olive oil. 18 Although both clinical and laboratory features are rather variable among the patients with phytosterolemia, the results of this study, together with the 2 previous reports, demonstrate that macrothrombocytopenia/stomatocytosis could be the most prominent or initial clinical features in some patients. The precise mechanisms underlying this interesting link between defective plant sterol metabolism and hematological abnormalities remain unclear so far. The ABCG5 and ABCG8 genes are not expressed in hematologic cells, but an excess of sterols can be detected in both platelets and erythrocytes. 1 In addition, the in vitro experiment also showed an increase in osmotic fragility with obvious dysmorphism when normal erythrocytes were incubated in the presence of sitosterol. In animal model experiments, Kruit et al 19 observed overt macrothrombocytopenia in a Abcg5-deficient phytosterolemia mice. Wild-type mice transplanted with bone marrow from Abcg5-deficient mice appeared normal, whereas Abcg5-deficient mice transplanted with wild-type bone marrow still showed macrothrombocytopenia. In addition, Chase et al 20 described the spontaneous mouse Abcg5 mutation characterized by macrothrombocytopenia. Crosses with mice doubly transgenic for the human ABCG5 and ABCG8 genes rescued platelet counts and volumes. Both of these mice models demonstrated that accumulation of plant sterol is responsible for development of macrothrombocytopenia, although hemolytic stress was not or only mildly affected because of species difference. Therefore, all these results support our previous hypothesis that the hematological abnormalities would be due to the toxic effect of plant sterols on blood cells, rending them more prone to dysmormophism and rupture. 9
The question why only a few, but not all, patients with phytosterolemia prominently or initially present with the specific hematological abnormalities is unanswered. It could probably be due to other background genetic factors, or it might be contributable to various environmental influence on hematologic cells. These presumptions need further more investigation.
During the past 5 years, we have found 5 pedigrees of phytosterolemia in our single institute via hematology, including the one previously reported 9 and another one, the gene mutation of which is to be identified. This fact indicates that this disease is certainly not as rare as originally thought.
Of note, the usual assays for cholesterol levels cannot differentiate between cholesterol and plant sterols, and plant sterol measurement is not yet routinely available. In fact, many of the patients originally diagnosed with pseudohomozygous familial hypercholesterolemia were subsequently found to have phytosterolemia.3,7 Therefore, it is essential that plasma plant sterols should be measured in patients with unexplained stomatocytosis and/or macrothrombocytopenia especially their parents are of consanguineous marriage, in order to determine if they have phytosterolemia. This is clinically important for early treatment with diet low in plant sterols, bile acid-binding resins,6,21 and a new sterol absorption inhibitor, ezetimibe8,22 which have been proved to improve the prognosis and then probably to decrease the risk of early mortality. Statins are of no use because endogenous cholesterol syntesis is suppressed by the exogenous load.
In conclusion, we identified 3 new phytosterolemia families with specific hematological abnormalities which are caused by the toxic effect of plant sterol on blood cells and identified 3 novel mutations of AGCG5 and ABCG8 genes (A20883G of ABCG5, del43683-43724 and del43866C-43867G/ins43866T of ABCG8). We suggest macrothrombocytopenia/stomatocytosis associated with phytosterolemia would represent a distinguishing subtype of phytosterolemia.
Footnotes
Authors’ Note
Gaifeng Wang and Lijuan Cao contributed equally to this work.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: supported in part by a grant to ZYW from the Nature Science Foundation of China number, (81070395) Beijing, China; and by A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.