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Archived Comments for: Neuroacanthocytosis associated with a defect of the 4.1R membrane protein

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  1. Has a new type of neuroacanthocytosis been discovered?

    Ruth Walker, Bronx VA Medical Center

    30 March 2007

    We were highly intrigued by the recent article by Orlacchio et al. “Neuroacanthocytosis associated with a defect of the 4.1 R membrane protein” [1]. Such a condition would represent a new type of neuroacanthocytosis in addition to those associated with mutations in the VPS13A gene (chorea-acanthocytosis), the XK gene (McLeod syndrome), the JPH3 gene (Huntington’s disease-like 2; HDL2), and the PANK2 gene (pantothenate kinase-associated neurodegeneration; PANK), as recently reviewed [2]. In support of this, the authors present evidence of the absence of mutations in VPS13A, a large gene that they reportedly had sequenced completely in their patients, as well as to normality of Kell antigen expression which argues against a diagnosis of McLeod syndrome. The two core syndromes of neuroacanthocytosis thus appear excluded in their cases, and from their clinical descriptions, the other 2 syndromes (HDL2 and PANK) appear to be unlikely. The establishment of an association with a defect in a protein with relatively well-known function and cellular distribution would be extremely welcome for further elucidating the pathophysiology of neuroacanthocytosis conditions.

    We were disappointed, however, by the lack of evidence for a defect in the 4.1 R protein.

    (1) The conclusion that the content of 4.1R is decreased in the patients’ erythrocyte membranes is not supported by the evidence presented. The protein content was estimated by densitometric analysis. However, the linearity between the Coomassie Blue signal for 4.1 and the signal has not been evaluated, so there is no evidence that variations in amount are detected as variations in signal in the patients’ samples as compared with the control samples. In addition, it was not mentioned if 4.1R was measured as 4.1a together with 4.1b, or the separate forms. It is possible that variations in deamidation causing the doublet may affect staining and thus obscure content. Also, there is no evidence for any specificity, i.e. it was not mentioned if other proteins had been evaluated and/or used as internal controls. For example, Figure 2 seems to suggest that there are differences in band 3 content between lanes a and b (control), and between lanes d and e and lane f (control).

    (2) The Coomassie Blue patterns shown in Figure 2 suggest that there may be qualitative differences between 4.1R of the patients; for example, the 4.1 band of patient IV:6 RM13 (lane a) seems to have a higher Mr than that of the control. Again, this does not seem to be restricted to the 4.1 protein; differences in mobility can be noted between 4.2 in lane a and lane b, and bands are seen in lane c that are much less visible than in lane f. Thus, differences between the membrane proteins of erythrocytes from these patients do not seem to be restricted and/or specific for defects in the 4.1R protein.

    (3) Similarly, the conclusion regarding any differences in the content of spectrin dimers and tetramers is not supported by the data. In addition to the reasons stated above, variation in especially cytoskeletal proteins such as spectrin and ankyrin can easily be caused by random processes such as proteolytic breakdown. This may be the case since the buffer in which the erythrocytes were lysed and that was used to prepare the membranes, was not suitable to prevent activation of the broad spectrum of proteases that are activated upon lysis. Moreover, the data presented may also be compatible with hereditary red cell membrane disorders, such as hereditary spherocytosis or elliptocytosis, which are due to partial or complete lacking in membrane or cytoskeleton red cell proteins [3, 4]. In addition, whenever a defect in the membrane anchoring sites to the cytoskeleton is present, it is possible to observe abnormalities in spectrin content or in its structural organization as reported here in Fig. 3 [3]. In human 4.1R deficiency the hematological phenotype is characterized by abnormal red cell morphology and loss of red cell membrane mechanical stability which is associated with mild to severe hemolytic anemia [5, 6]. Here, no hematological data are shown.

    (4) A major omission is that Western blots are not shown for unaffected relatives, leaving us unable to determine to what extent this feature, if it truly exists, segregates with acanthocytosis, and the manner in which it might be inherited.

    In addition, the values for 4.1R shown in Table 1 are, if anything, slightly lower in the relatives with acanthocytosis than in the subjects with hyperkinesias, which argues against a causative role for a deficiency of this protein in the movement disorder.

    In the cited mouse model knockout for the 4.1R red cell membrane protein the red cell morphology is mainly characterized by microspherocytes, which have not been noted as a hematological phenotype in neuroacanthocytosis [7, 8]. Thus, we think it is crucial to conduct a complete hematological screening for hereditary red cell membrane disorders before jumping to the conclusion regarding the relationship between band 4.1 deficiency and neuroacanthocytosis.

    From the clinical point of view the neurological phenotype of the four patients appears rather heterogeneous and difficult to classify. It is of note that one case (patient IV:I [RM16]) had previously been reported with self-limited hyperkinesia in the context of blood glucose dysregulation associated with transient basal ganglia MRI signal abnormalities [9] although this reference is not cited in the current paper.

    The emphasis of this earlier paper is acanthocytosis as a predisposing factor for diabetic hemichorea, rather than a genetic abnormality of band 4.1R as the etiology. The blood smear in that earlier publication of this case, however, is difficult to evaluate since acanthocytosis affects almost the complete red cell population shown, whereas commonly acanthocytes account for 10 to 30% of the red cells in neuroacanthocytosis. This high proportion suggests that this finding is an artefect of smear preparation, as does the blood film illustrated in this paper appears to show spiculated echinocytes rather than true acanthocytes, which are typically more contracted and irregular with larger protrusions.

    Patient IV:3 (RM15) is reported as having orofaciolingual dyskinesias and distal right arm dystonia. He had been treated with tricyclic antidepressants and selective serotonin-reuptake inhibitors, both of which have been reported to cause tardive dyskinesia, again making the diagnosis of a neuroacanthocytosis syndrome questionable.

    Their case IV:6 (RM13) most closely resembles other patients with a diagnosis of chorea-acanthocytosis (ChAc) and it must be borne in mind that there are a great variety of mutations in ChAc, some of which may escape detection in spite even of sophisticated VPS13A mutation analysis [10].

    In summary, in our opinion the authors did not provide clear data in support of the hypothesis that the neurological findings in the patients presented are specifically associated with a defect in 4.1R. The suggestion that this might represent a new type of neuroacanthocytosis thus is not supported and it will take considerable additional effort to arrive at definite diagnoses for the intriguing case observations presented by Orlacchio and collaborators.

    Giel Bosman

    Lucia de Franceschi

    Adrian Danek

    Ruth H. Walker

    Reference List

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    2. Walker RH, Danek A, Dobson-Stone C, Guerrini R, Jung HH, Lafontaine A-L, Rampoldi L, Andermann E: Developments in neuroacanthocytosis: Expanding the spectrum of choreatic syndromes. Mov Disord 2006, 21:1794-1905.

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    5. Tchernia G, Mohandas N, Shohet SB: Deficiency of Skeletal Membrane-Protein Band 4.1 in Homozygous Hereditary Elliptocytosis - Implications for Erythrocyte-Membrane Stability. J Clin Invest 1981, 68:454-460.

    6. Takakuwa Y, Tchernia G, Rossi M, Benabadji M, Mohandas N: Restoration of Normal Membrane Stability to Unstable Protein-4.1-Deficient Erythrocyte-Membranes by Incorporation of Purified Protein-4.1. J Clin Invest 1986, 78:80-85.

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    8. Shi ZT, Afzal V, Coller B, Patel D, Chasis JA, Parra M, Lee G, Paszty C, Stevens M, Walensky L, Peters LL, Mohandas N, Rubin E, Conboy JG: Protein 4.1R-deficient mice are viable but have erythroid membrane skeleton abnormalities. J Clin Invest 1999, 103:331-340.

    9. Pisani A, Diomedi M, Rum A, Cianciulli P, Floris R, Orlacchio A, Bernardi G, Calabresi P: Acanthocytosis as a predisposing factor for non-ketotic hyperglycaemia induced chorea-ballism. J Neurol Neurosurg Psychiatry 2005, 76:1717-1719.

    10. Dobson-Stone C, Danek A, Rampoldi L, Hardie RJ, Chalmers RM, Wood NW, Bohlega S, Dotti MT, Federico A, Shizuka M, Tanaka M, Watanabe M, Ikeda Y, Brin M, Goldfarb LG, Karp BI, Mohiddin S, Fananapazir L, Storch A, Fryer AE, Maddison P, Sibon I, Trevisol-Bittencourt PC, Singer C, Caballero IR, Aasly JO, Schmierer K, Dengler R, Hiersemenzel LP, Zeviani M, Meiner V, Lossos A, Johnson S, Mercado FC, Sorrentino G, Dupre N, Rouleau GA, Volkmann J, Arpa J, Lees A, Geraud G, Chouinard S, Nemeth A, Monaco AP: Mutational spectrum of the CHAC gene in patients with chorea-acanthocytosis. Eur J Hum Genet 2002, 10:773-781.

    Competing interests