Research On The Clinical Features And Pathogenesis Of A Family With Phytosterolaemia Coexisting Erythrocyte And Platelet Abnormalities

Giant platelet disorders are a heterogeneous group of diseases with thrombocytopenia,large platelets and variable hemorrhagic phenomena,which are from either primary platelet disorders or a specific phenotype of other conditions.With the development of laboratory technology,some novel platelet disorders are constantly discovered.However,to date there are a lot of cases which are not classified and identified.Recently,we found a rare case with macrothrombocytopenia,abnormal erythrocyte shape,hemolysis and increased plasma levels of phytosterols.Although rare, the further research on particular disorders with giant platelets will help clinicians correctly understand,diagnose and treat them.This work may provide a valuable reference for clinicians.ObjectiveThe combination of macrothrombocytopenia and hemolysis is an unusually and poorly understood hematological condition.Some of hematological diseases present with hemolysis and thrombocytopenia,such as Evan's syndrome,thrombotic thrombocytopenic purpura.In none of these conditions were the platelets large. Mediterranean stomatocytosis/macrothrombocytopenia was first described in individuals of Mediterranean origin in Adelaide,Australia by Ducrou in 1969.No reports arose from any other Australian centre,nor from any Mediterranean country. The exact nature of this condition that combines stomatocytic hemolysis with the presence of very large platelets has never been elucidated.Phytosterolaemia is a rare autosomal recessive disorder,first described by Bhattacharyya in 1974,characterized by hyperabsorption of plant sterols from the intestine and inability to excrete these sterols into the bile.As a consequence,affected individuals show very high levels of plant sterols.The patients suffer from tendon and tuberous xanthomas and premature atherosclerosis and coronary artery disease.Rees first reported phytosterolaemia associated stomatocytic hemolysis and macrothrombocytopenia in 2005.Recently,we found a family with phytosterolaemia from Jiangsu province,China,all children of which showed hemolysis with very large platelets,conforming to the description of Mediterranean macrothrombocytopenia.However,since the age of over10 years they had noticed enlarging tendon and tuberous xanthomas that began on the both elbows, and then in hips.To analyze the relationship between hematological abnormalities and phytosterolaemia,elucidate the molecular mechanisms of this disorder and distinguish it from other syndromes associated with giant platelets,in this study,we characterized the clinical,biochemical and molecular genetic features of a Chinese family with phytosterolaemia that mainly manifested hematological abnormalities.MethodsPartâ… :A simple,rapid and accurate method to separate and quantify serum sterols was performed by high performance liquid chromatography(HPLC).No coagulant fasting blood samples were collected from the affected patients,their parents,other two family members and 10 normal volunteers.Briefly,100μl of serum,or 10 mg/dl stigmasterol,2.5 mg/dl cholestanol and 1.5 mg/dl sitosterol standard was placed respectively in a screw-capped test-tube,and then 1ml of 1M ethanolic potassium hydroxide was added.After vigorous stirring,the saponified mixture was allowed to stand for 1h at 80℃,and then 1ml of water was added.The unsaponifiable material was extracted with two-2ml portions of hexane.The extracts were evaporated under a stream of nitrogen.The sterols in the extracts were converted into benzoate derivatives with benzoyl chloride,and then re-extracted with hexane.The hexane layer was again evaporated under nitrogen.Finally,the residue was dissolved in 0.1 ml of acetonitrile-tetrahydrofuran(1:1,v/v),and 10μl were injected into the HPLC system. The instrument was an Agilent 1100 HPLC system,equipped with a Diamonsil C₁₈ column which was maintained at 30℃.The solvent was acetonitrile-isopropylalcohol (75:25,v/v) at a flow-rate of 1 ml/min.Steryl benzoates were detected by their absorbance at 235 nm. Partâ…¡:The clinical features of the affected patients in this family were analyzed. Erythrocyte and platelet morphology was observed under light microscope.Platelet aggregation was measured turbidometrically.Platelet glycoproteins(GPs) were analyzed using flow cytometry.Their plasma sterol levels were measured by HPLC method.ABCG5 and ABCG8 genes sequencing was searched for the mutation. Restriction endonuclease analysis was used for the PCR products of exon 1 of ABCG5 and its flanking regions of the family members and 70 healthy volunteers.Partâ…¢:Erythroeyte membrane phytosterol concentrations were determined by HPLC.The changes in shape and osmotic fragility in vitro of erythrocytes in sitosterol enriched plasma were studied.ResultPartâ… :Plasma concentrations of sitosterol,stigmasterol and cholestanol were markedly elevated in the affected patients.Their parents and other two family members showed slight elevation in plasma sitosterol and cholestanol levels compared with normal subjects,but their stigmasterol levels were normal.Partâ…¡:The affected patients had obviously large platelets and abnormal erythrocytes of various shapes,such as spherocytes,stomatocytes and target cells on the blood smear.Meanwhile,the erythrocytic osmotic fragility was highly increased.Their bleeding time was prolonged;their platelets had a low agglutination in response to ristocetin.They had increased plasma sitosterol,stigmasterol and cholestanol levels,but maintained normal cholesterol levels.Sequence analysis of ABCG5 and ABCG8 genes revealed a C to T transition at nucleotide 18802 resulting in a nonsense mutation in ABCG5 exon 1,Gln22X.The affected patients were homozygous whereas their parents and other two family members were heterozygous for this defect.Allele-specific restriction enzyme analysis using Bfa I did not detect this mutation in 70 healthy individuals.Partâ…¢:Three affected patients had increased erythrocyte membrane sitosterol levels.After erythrocytes were incubated with sitosterol,the shape changes of them were abnormal.Spherocytes,stomatocytes and target cells were easily observed on the blood smear.The osmotic fragility of sitosterol-enriched erythrocytes in vitro is increased compared to that of control cellsConclusion1.This method of the quantitative analysis of serum phytosterols by HPLC was found to be simple,quick and sensitive,to provide the referenced foundation for the biochemical diagnosis of phytosterolaemia.2.The blood cells could be a "target" in the toxic effect of plasma phytosterols in some patients,and the coexisting hematological abnormalities might represent a specific phenotype of sitosterolemia,which should be included in the differential diagnosis of inherited hemolytic anemia and(or) macrothrombocytopenia.A novel nonsense mutation Gln22X in ABCG5 gene reported in this study could be useful to investigate the molecular basis of sitosterolemia with erythrocyte and platelet abnormalities.This mutation is a novel mutation of phytosterolaemia,which,to our knowledge,has not been described before.3.The patients of phytosterolaemia had not only increased plasma phytosterol levels,but also increased erythrocyte membrane phytosterol levels.The increased plasma phytosterol levels had obvious influence on morphology and osmotic fragility of erythrocytes.