A chorea-acanthocytosis patient with novel mutations in the VPS13A gene without acanthocyte.

Chorea-acanthocytosis (ChAc) is a rare clinical genetic disorder of the nervous system, which is characterized by choreiform movement disorder, cognitive decline, and psychiatric disorders. ChAc is mostly diagnosed based on its typical clinical manifestations and the increased number of acanthocytes in peripheral blood smears. Here, we report a patient, who has the characteristic clinical manifestations of ChAc with limb choreiform movements, involuntary lip and tongue bites, seizures, and emotional instability. However, her blood smear was negative for acanthocytes with scanning electron microscopy. We later identified two novel pathogenic mutations in the patient's vacuolar protein sorting homolog 13 A (VPS13A) on chromosome 9q21 by targeted gene sequencing, and she was definitively diagnosed with "ChAc." After treatment with carbamazepine, haloperidol, the patient's symptoms gradually improved. We consider that an acanthocyte negative blood smear cannot rule out ChAC diagnosis, and genetic testing is the "gold standard" for the diagnosis. Through a review of previous research, it is rare for a patient to have a clear diagnosis of ChAc by genetic testing, but whose blood smear is negative for acanthocytes with electron microscopy. In addition, in this report, we discovered two novel pathogenic mutations, which have not been reported previously, and extended the genetic characteristics of ChAc.

Erythrocyte and Biochemical Abnormalities as Diagnostic Markers in Dogs With Hemangiosarcoma Related Hemoabdomen.

OBJECTIVE: To investigate: 1) acanthocytosis and presence of acanthocytes in peritoneal fluid as a diagnostic marker for hemangiosarcoma (HSA) in dogs with non-traumatic hemoabdomen; and 2) the association between other erythrocyte, biochemical, and hematologic abnormalities as a mean of differentiating HSA from other disease. STUDY DESIGN: Prospective double-blinded cohort study. ANIMALS: Dogs (n = 40) with non-traumatic hemoabdomen. METHODS: Dogs diagnosed with hemoabdomen (January 2012 to May 2013) had cytologic evaluation of abdominal effusion and peripheral blood smears. Peripheral blood CBC, PT, and aPTT, as well as blood and effusion acanthocytes, keratocytes, schistocytes, lactate, glucose, PCV, and TP results were compared using the paired t-test or Fisher's exact test. Based on histologic confirmation of HSA, dogs were divided into 2 groups (HSA, non-HSA) and variables compared. RESULTS: There was no significant difference in erythrocyte morphology in abdominal effusion or peripheral blood between dogs with HSA or non-HSA related hemoabdomen. Platelet concentration and peripheral blood PCV were significantly lower in the HSA group. CONCLUSIONS: A reliable preoperative biochemical or cytologic test to differentiate between HSA and non-HSA related hemoabdomen was not identified.

Abnormal red cell structure and function in neuroacanthocytosis.

BACKGROUND: Panthothenate kinase-associated neurodegeneration (PKAN) belongs to a group of hereditary neurodegenerative disorders known as neuroacanthocytosis (NA). This genetically heterogeneous group of diseases is characterized by degeneration of neurons in the basal ganglia and by the presence of deformed red blood cells with thorny protrusions, acanthocytes, in the circulation. OBJECTIVE: The goal of our study is to elucidate the molecular mechanisms underlying this aberrant red cell morphology and the corresponding functional consequences. This could shed light on the etiology of the neurodegeneration. METHODS: We performed a qualitative and semi-quantitative morphological, immunofluorescent, biochemical and functional analysis of the red cells of several patients with PKAN and, for the first time, of the red cells of their family members. RESULTS: We show that the blood of patients with PKAN contains not only variable numbers of acanthocytes, but also a wide range of other misshapen red cells. Immunofluorescent and immunoblot analyses suggest an altered membrane organization, rather than quantitative changes in protein expression. Strikingly, these changes are not limited to the red blood cells of PKAN patients, but are also present in the red cells of heterozygous carriers without neurological problems. Furthermore, changes are not only present in acanthocytes, but also in other red cells, including discocytes. The patients' cells, however, are more fragile, as observed in a spleen-mimicking device. CONCLUSION: These morphological, molecular and functional characteristics of red cells in patients with PKAN and their family members offer new tools for diagnosis and present a window into the pathophysiology of neuroacanthocytosis.

Alterations of red cell membrane properties in neuroacanthocytosis.

Neuroacanthocytosis (NA) refers to a group of heterogenous, rare genetic disorders, namely chorea acanthocytosis (ChAc), McLeod syndrome (MLS), Huntington's disease-like 2 (HDL2) and pantothenate kinase associated neurodegeneration (PKAN), that mainly affect the basal ganglia and are associated with similar neurological symptoms. PKAN is also assigned to a group of rare neurodegenerative diseases, known as NBIA (neurodegeneration with brain iron accumulation), associated with iron accumulation in the basal ganglia and progressive movement disorder. Acanthocytosis, the occurrence of misshaped erythrocytes with thorny protrusions, is frequently observed in ChAc and MLS patients but less prevalent in PKAN (about 10%) and HDL2 patients. The pathological factors that lead to the formation of the acanthocytic red blood cell shape are currently unknown. The aim of this study was to determine whether NA/NBIA acanthocytes differ in their functionality from normal erythrocytes. Several flow-cytometry-based assays were applied to test the physiological responses of the plasma membrane, namely drug-induced endocytosis, phosphatidylserine exposure and calcium uptake upon treatment with lysophosphatidic acid. ChAc red cell samples clearly showed a reduced response in drug-induced endovesiculation, lysophosphatidic acid-induced phosphatidylserine exposure, and calcium uptake. Impaired responses were also observed in acanthocyte-positive NBIA (PKAN) red cells but not in patient cells without shape abnormalities. These data suggest an "acanthocytic state" of the red cell where alterations in functional and interdependent membrane properties arise together with an acanthocytic cell shape. Further elucidation of the aberrant molecular mechanisms that cause this acanthocytic state may possibly help to evaluate the pathological pathways leading to neurodegeneration.

Computational identification of phospho-tyrosine sub-networks related to acanthocyte generation in neuroacanthocytosis.

Acanthocytes, abnormal thorny red blood cells (RBC), are one of the biological hallmarks of neuroacanthocytosis syndromes (NA), a group of rare hereditary neurodegenerative disorders. Since RBCs are easily accessible, the study of acanthocytes in NA may provide insights into potential mechanisms of neurodegeneration. Previous studies have shown that changes in RBC membrane protein phosphorylation state affect RBC membrane mechanical stability and morphology. Here, we coupled tyrosine-phosphoproteomic analysis to topological network analysis. We aimed to predict signaling sub-networks possibly involved in the generation of acanthocytes in patients affected by the two core NA disorders, namely McLeod syndrome (MLS, XK-related, Xk protein) and chorea-acanthocytosis (ChAc, VPS13A-related, chorein protein). The experimentally determined phosphoproteomic data-sets allowed us to relate the subsequent network analysis to the pathogenetic background. To reduce the network complexity, we combined several algorithms of topological network analysis including cluster determination by shortest path analysis, protein categorization based on centrality indexes, along with annotation-based node filtering. We first identified XK- and VPS13A-related protein-protein interaction networks by identifying all the interactomic shortest paths linking Xk and chorein to the corresponding set of proteins whose tyrosine phosphorylation was altered in patients. These networks include the most likely paths of functional influence of Xk and chorein on phosphorylated proteins. We further refined the analysis by extracting restricted sets of highly interacting signaling proteins representing a common molecular background bridging the generation of acanthocytes in MLS and ChAc. The final analysis pointed to a novel, very restricted, signaling module of 14 highly interconnected kinases, whose alteration is possibly involved in generation of acanthocytes in MLS and ChAc.

Chorein-sensitive polymerization of cortical actin and suicidal cell death in chorea-acanthocytosis.

Chorea-acanthocytosis is an inevitably lethal genetic disease characterized by a progressive hyperkinetic movement disorder and cognitive and behavioral abnormalities as well as acanthocytosis. The disease is caused by loss-of-function mutations of the gene encoding vacuolar protein sorting-associated protein 13A (VPS13A) or chorein, a protein with unknown function expressed in various cell types. How chorein deficiency leads to the pathophysiology of chorea-acanthocytosis remains enigmatic. Here we show decreased phosphoinositide-3-kinase (PI3K)-p85-subunit phosphorylation, ras-related C3 botulinum toxin substrate 1 (Rac1) activity, and p21 protein-activated kinase 1 (PAK1) phosphorylation as well as depolymerized cortical actin in erythrocytes from patients with chorea-acanthocytosis and in K562-erythrocytic cells following chorein silencing. Pharmacological inhibition of PI3K, Rac1, or PAK1 similarly triggered actin depolymerization. Moreover, in K562 cells, both chorein silencing and PAK1 inhibition with IPA-3 decreased phosphorylation of Bad, a Bcl2-associated protein, promoting apoptosis by forming mitochondrial pores, followed by mitochondrial depolarization, DNA fragmentation, and phosphatidylserine exposure at the cell surface, all hallmarks of apoptosis. Our observations reveal chorein as a novel powerful regulator of cytoskeletal architecture and cell survival, thus explaining erythrocyte misshape and possibly neurodegeneration in chorea-acanthocytosis.

Drug-induced hemolytic anemia and thrombocytopenia associated with alterations of cell membrane lipids and acanthocyte formation.

CXCR3 is a chemokine receptor, upregulated upon activation of T cells and expressed on nearly 100% of T cells in sites of inflammation. SCH 900875 is a selective CXCR3 receptor antagonist. Thrombocytopenia and severe hemolytic anemia with acanthocytosis occurred in rats at doses of 75, 100, and 150 mg/kg/day. Massively enlarged spleens corresponded histologically to extramedullary hematopoiesis, macrophages, and hemosiderin pigment and sinus congestion. Phagocytosed erythrocytes and platelets were within splenic macrophages. IgG and/or IgM were not detected on erythrocyte and platelet membranes. Ex vivo increased osmotic fragility of RBCs was observed. Lipid analysis of the RBC membrane revealed modifications in phosphatidylcholine, overall cholesterol, and/or sphingomyelin. Platelets exhibited slender filiform processes on their plasma membranes, analogous to those of acanthocytes. The presence of similar morphological abnormalities in acanthocytes and platelets suggests that possibly similar alterations in the lipid composition of the plasma membrane have taken place in both cell types. This phenotype correlated with alterations in plasma lipids (hypercholesterolemia and low triglycerides) that occurred after SCH 900875 administration, although other factors cannot be excluded. The increased cell destruction was considered triggered by alterations in the lipid profile of the plasma membranes of erythrocytes and platelets, as reflected morphologically.

The McLeod syndrome without acanthocytes.

A 45-year-old man developed chorea, behavioural changes, moderate amyotrophy and polyneuropathy. Hypertrophic cardiomyopathy and increased serum lactate dehydrogenase and creatine kinase (CK) were found. Acanthocytes were not detected. The absence of XK protein and faintly expressed Kell antigens on erythrocytes were found. Genetic test revealed a R133X mutation of the XK gene, confirming the McLeod syndrome. After 7 years he suddenly developed delirium followed by severe hypoglycaemia, hyperthermia, rhabdomyolysis, hepatic and renal failure. Malignant arrhythmia caused death.

Dynamics of the structural and electrical characteristics of erythrocytes in men with metastatic adenocarcinoma of the prostate before and after plastic orchiectomy.

The changes of electrophoretic mobility of erythrocytes in the practically healthy men and in men with metastatic prostate cancer before and after castration were studied. The electrophoretic mobility of erythrocytes was investigated in laboratory conditions by Kharamanenko and Abramson micro methods. Experimental data were processed by means of standard variation statistics MINITAB (Basic statistic) p

Identification of two new members, XPLAC and XTES, of the XK family.

XK, a putative membrane transporter, is a component of the XK/Kell complex of the Kell blood group system. XK's substrate is unknown but absence of the protein, as occurs in the McLeod phenotype, is associated with red cell acanthocytosis and late onset central nervous system and neuromuscular abnormalities known as the McLeod syndrome. We have cloned two cDNAs, XPLAC (GenBank accession no. AY589511) and XTES (GenBank accession no. AY989815), which are closely related to XK and define them together as the XK family. XPLAC has a 2.9 kb cDNA that encodes 462 amino acids and XTES has a 1.6 kb cDNA coding 459 amino acids. The predicted molecular weights are 53.6 kDa for XPLAC and 53.4 kDa for XTES, which are similar to that of XK, which is 50.9 kDa. Unlike XK which is ubiquitously expressed XPLAC is expressed mostly in placenta and adrenal gland while XTES is exclusively expressed in primate testis. XPLAC has 37% and XTES has 31% amino acid identity with XK protein and they are predicted to have a similar topology to XK. XPLAC, like XK, has 3 exons and is located on X chromosome at q22.1, while XTES has 4 exons and is located at 22q11.1. Phylogenetic analysis shows that there are at least 5 additional vertebrate genes that are evolutionarily distantly related to the XK family. A domain with consensus sequences (ced-8 domain) for the extended family is described.

A basis of the acanthocytosis in inherited and acquired disorders.

Acanthocytosis refers to the transformation of the normal biconcave disc erythrocyte into one with a few irregularly shaped external projections distributed unevenly at its membrane surface. It is associated with a variety of inherited and acquired disorders. A relationship between the acanthocytosis in chorea-acanthocytosis and an alteration of Band 3, the anion exchange protein, has been previously suggested. We have previously proposed a mechanism of erythrocyte shape control in which decrease and increase of the ratio of the outward-facing (Band 3(o)) and inward-facing (Band 3(i)) conformations of Band 3 contracts and relaxes the membrane skeleton, thus promoting echinocytosis and stomatocytosis, respectively. The equilibrium Band 3(o)/Band 3(i) ratio is determined by the Donnan equilibrium ratio of anions and protons, increasing with the increase of the Donnan ratio. Based on the evidence suggesting that the acanthocyte and echinocyte are interrelated, the mechanism could explain by a decrease of the Donnan ratio the occurrence of acanthocytes in pyruvate kinase and pyrimidine 5'-nucleotidase deficiencies, dehydrated hereditary stomatocytocytosis, In(Lu) phenotype, chorea-acanthocytosis, and McLeod phenotype. Consistent with the proposed mechanism indicating that the membrane skeleton is an important determinant of the erythrocyte shape, is the alteration of its conformation in chorea-acanthocytosis, McLeod phenotype and Fanconi's anemia. In agreement with the proposed mechanism indicating that Band 3 conformation controls the erythrocyte shape are the occurrence of an acanthocytosis in individuals expressing the rare Band 3 HT variant and of alterations of Band 3 properties in chorea-acanthocytosis and In(Lu) phenotype. The observations that the lipid composition or organization are normal in chorea-acanthocytosis and McLeod phenotype are supportive of the proposed mechanism since it postulates that the lipid bilayer has a secondary role in determining the erythrocyte shape. The acanthocytoses in alcoholic cirrhosis and abetalipoproteinemia are accompanied by significant increases of the cholesterol level and of the ratio of sphingomyelin and glycerophospholipids, respectively. However, they could occur by a change of the Band 3 conformation since cholesterol binds specifically to Band 3 and inhibits its anion transport activity, and that sphingomyelin potentiates this inhibition. Thus, the acanthocytosis could involve an alteration of the Band 3 conformation.

Abetalipoproteinemia: a case report.

Abetalipoproteinemia is a rare autosomal recessive disorder characterized by steatorrhea, poor weight gain, acanthocytosis and retinitis pigmentosa. Here we peresent a six-month-old patient with abetaliporoteinemia. He had a history of chronic diarrhea from the first month of life. He was cachectic and his motor development was delayed. Microscopic examination of the stool revealed fat. Mild anemia with reticulocytosis, acanthocytosis, low triglyceride, low cholesterol, low-density lipoprotein, high-density lipoprotein, and apolipoprotein A and B were detected. Ophthalmological examination was normal. Peroral jejunal capsule biopsy revealed normal villi and significant lipid deposition in the cytoplasm of affected cells. The patient was given large doses of vitamins E and A.

Case study: childhood-onset tardive dyskinesia versus choreoacanthocytosis.

A male adolescent presented with aggression, depressed mood, and severe movement disorder that developed at age 5 after exposure to neuroleptics. Evaluation revealed acanthocytes in his blood, which led to a differential diagnosis of tardive dyskinesia versus choreoacanthocytosis. However, this is unusually young age of onset for these conditions. Differential diagnosis, medication management of the movement disorder, and pathophysiological mechanisms in tardive dyskinesia and choreoacanthocytosis are discussed.

Band 3 HT, a human red-cell variant associated with acanthocytosis and increased anion transport, carries the mutation Pro-868-->Leu in the membrane domain of band 3.

1. We have studied band 3 HT, a human red-cell band 3 variant with increased M(r), which is associated with abnormal red-cell shape (acanthocytosis) and increased anion-transport activity. 2. We have shown that the increased M(r) does not result from the presence of the band 3 Memphis mutation, and that the variant band 3 is covalently labelled by 4,4'-di-isothiocyanato-1,2-diphenylethane-2,2'-disulphonic acid (H2DIDS) less readily than normal. 3. cDNA cloning studies show that band 3 HT results from the mutation Pro-868-->Leu, and the possible significance of the mutation in the altered anion-transport activity and cytoskeleton binding properties of band 3 HT is discussed.

[Studies on the erythrocyte membrane skeleton in a patient with chorea-acanthocytosis--theoretical speculation on the mechanism of neurological involvement].

Patients with chorea-acanthocytosis exhibit symptoms of self-biting, choreic movement, and acanthocytosis, but not dementia. The mechanism of choreic movements is still unknown. In order to clarify the etiologic mechanism underlying these movements, we evaluated the erythrocyte membrane in one patient with chorea-acanthocytosis. A 35-year-old female was admitted to Saitama Medical School Hospital because of involuntary movements. She was alert, well-oriented, and had no gross memory defects. She had slurred speech, choreic movements and lip biting. Laboratory examination showed acanthocytes in her peripheral red blood cells, normal serum lipid values, and caudate atrophy on her brain CT scan. In analyzing the acanthocytes, we initially evaluated the size of the acanthocyte population by incubating her red blood cells with plasma. The cell population approximately doubled after 2 hours incubation. Next we examined the protein composition of erythrocyte ghost by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). There was no significant difference between the patient's erythrocyte ghosts and those of a control. Then we investigated morphological changes in the patient's erythrocyte by scanning and transmission electron microscopy (SEM and TEM). SEM showed the typical acanthocyte shape. The quick-freeze, freeze-substitution method confirmed that the routine TEM section was not artifactual, and was in fact in accurate reflection of the actual features of acanthocytes. TEM of the sections prepared from erythrocyte ghosts demonstrated that spectrin tended to be accumulated in the thorn region. Furthermore, TEM of quick-freeze, deep-etched replica of the ghost revealed more clearly a spectrin network densely packed on the inner hydrophilic surface.(ABSTRACT TRUNCATED AT 250 WORDS)