VPS13A disease (formerly known as chorea-acanthocytosis) and XK disease (formerly McLeod syndrome) are the conditions historically known as the core neuroacanthocytosis (NA) syndromes (Walker et al., 2023; Walker and Danek, 2021; Peikert et al., 2002; Jung et al., 2004). The percentage of acanthocytes in both VPS13A disease and XK disease is variable (5–50%) under the course of disease, > 6.3% acanthocytes in peripheral blood is considered abnormal. To reduce the risk for false positive and negative cases, acanthocytosis is assessed with a specific protocol (Storch et al., 2005). The red blood cells membrane properties are determined by membrane structure, which in turn, identifies membrane deformability, mechanical stability and permeability. These rheological properties can be measured by osmotic gradient ektacytometry. Data on ektacytometry in NA syndromes is still very limited (De Franceschi et al., 2014; Cluitmans et al., 2015; Lazari et al., 2020; Ballas et al., 1990; Hernández et al., 2024).

Investigations included clinical assessments, neuroimaging studies, laboratory analyses, blood smears, ektacytometry, psychometric evaluation, and genetic analyses. A Western blot analysis for chorein, with two different antibodies (Anti-VPS13A, HPA021662, Sigma-Aldrich, rabbit and Anti-VPS13A, PA5-54483, Invitrogen, rabbit), was performed according to the protocol described by Dobson-Stone et al. (2004). Briefly, one antibody targets an epitope located in middle of the protein (Invitrogen), and the second one targets an epitope in the C-terminus (Sigma). Absence of bands with both antibodies reflects lack of chorein expression, even of a truncated protein. Blood samples from a man with genetically proven VPS13A disease previously reported by Paucar et al. (2015), from a man with XK disease harboring the recurrent variant c.397C > T; p.R133X in XK (Dotti et al., 2000; Klempír et al., 2008; Murakami et al., 2019) and from a woman with liver failure and acanthocytosis were also included for comparison.

In all cases, the presence of acanthocytes was assessed according to the protocol for wet blood smears proposed by Storch et al. (2005). Erythrocytes were also studied using new generation osmotic gradient ektacytometry (LoRRca^® Maxis; RR Mechatronics). By mixing low and high osmolar solutions, an osmotic gradient was established, and the laser diffraction pattern was recorded. Few microliters of blood were suspended in 5 ml of isotonic Polyvinylpyrrolidone solution (PVP) (RR Mechatronics) and mixed carefully. Osmoscan curve was performed, and the following variables were obtained: the minimal osmolality (O-min), where 50% of RBC are lysed in a hypoosmotic environment and its corresponding minimal elongation index (EI-min), the maximal elongation index (EI-max) at optimal osmolality (O-max), the hyperosmotic osmolality (O-hyper), where half of the maximal elongation index (EI-hyper) is reached, and the area under the osmoscan curve (AUC) was calculated.

Massive parallel sequencing (MPS) of the patient was performed using a 30× PCR-free paired-end WGS protocol on an Illumina NovaSeq 6000 platform as described previously (Magnusson et al., 2020). A gene panel of 956 genes associated with movement disorders and neuromuscular disease was analyzed. The variants were prioritized based on conservation, frequency in internal and public databases, and pattern of inheritance. The ranked variants were then visualized in the Scout analysis platform (Stranneheim et al., 2021). Both VPS13A variants, confirmed in the patient by PCR and Sanger sequencing, were segregated in the parents. The Sanger sequencing was performed by standard methods on an ABI 3730 PRISM^® DNA Analyzer. Primer sequences available upon request. The homozygous RFC1 expansion was confirmed by PCR amplification covering the repeat region.

This is the first time a patient of Finnish origin is reported as affected by a VPS13A disease. Both VPS13A variants reported here are new in the context of VPS13A disease. Homozygous nucleotide expansions in RFC1 are the underlying cause of cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS). In this case, the only feature suggesting a RFC1-related disorder was incipient neuropathy. On the other hand, neuropathy is a common trait among patients with VPS13A disease. Long-term follow up is warranted to determine any synergistic effect of this dual pathology. Feeding dystonia, a hallmark of VPS13A disease, is particularly challenging to treat (Bader et al., 2010) as reported here. The risk for cardiomyopathy and ventricular arrhythmias in XK disease is well known, recently a study demonstrated that cardiomyopathy can also be a feature in VPS13A disease (Quick et al., 2021). The index case’s late brother who died suddenly and unexpectedly did not have any known neurological symptoms according to his relatives, a genetic investigation was not possible to carry out. Normal CK level in our patient is of note since this protein is elevated in the majority of patients with VPS13A disease (Peikert et al., 2002).

The morphology of erythrocytes is maintained by membrane lipids, proteins, and spectrin–actin membrane-skeleton. Cellular membranes comprise lipid bilayers that consist of glycerophospholipids, sphingolipids and cholesterol where phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine (PtdSer) are among the major glycerophospholipids as well as phosphatidylserine (PtdIns) as one of the relatively minor glycerophospholipids (Sakuragi and Nagata, 2023).

The VPS13 protein family members (A–D) have been recently identified to be located at membrane contact sites acting in bulk lipid transfer (Leonzino et al., 2021). Mutations in the corresponding genes for the VPS13 protein family members are of clinical importance as mutations in each protein lead to a specific neurological disorder (Peikert et al., 2002). However, acanthocytosis has only been identified in VPS13A disease giving rise to remarkable phenotypic similarities with XK disease, that is a genetically distinct disorder (Walker et al., 2023). XK on the other hand, was described as a transmembrane protein linked to the Kell protein by a disulfide bond in erythrocytes. XK and Kell seem, though, not linked in both brain and skeletal muscle (Spesivtseva et al., 2023). XKr8, however, was shown to function as a lipid scramblase and its role in apoptotic PtdSer exposure was subsequently identified (Suzuki et al., 2014). The reduced levels of PtdSer subspecies in the inner membrane leaflet of McLeod erythrocytes (with absent XK protein) had been already described by Redman et al. (1989). Further, VPS13A participates in regulating the phosphorylation of PtdIns on the plasma membrane of the erythrocytes. The level of PtdIns regulates the interaction between the plasma membrane and membrane-skeleton (Park et al., 2015).

Studying the pathophysiology of diseases due to mutations of the VPS13 protein family members, other lipid transfer proteins, and proteins related by a common function, such as XK, may indicate that these are all part of a group of disorders with a common mechanism of impaired bulk lipid transport (Walker et al., 2023).

The mechanism/s of the abnormal red cell membrane structure that results in acanthocytosis in VPS13A disease and XK disease is/are not fully understood. Several hypotheses are postulated among which is the disturbances in the phosphorylation-controlled binding between the integral membrane protein complexes and the membrane-skeleton (De Franceschi et al., 2014) and of depolymerized cortical actin due to absence or significant reduction in chorein encoded by VPS13A (Föller et al., 2012).

However, it now appears likely that VPS13 and XK proteins are functionally linked. At the plasma membrane, VPS13A interacts with the scramblase XK. This complex may be involved in lipid transport between the endoplasmic reticulum and plasma membrane as well as lipid scrambling between the two leaflets of the plasma membrane (Guillén-Samander et al., 2022). Erythrocytes lack organelles when circulating in peripheral blood, however, it is possible that abnormal lipid metabolism and organellar transfer during maturation (erythropoiesis) affects their membrane composition.

This complex function in membrane lipid homeostasis and survival resulting perhaps from deficient PtdSer and/or PtdIns exposure at the plasma membrane may explain the phenotypic similarities between the two disorders including formation of acanthocytes (Walker et al., 2023).

Ektacytometry is a widely used tool for assessing erythrocyte deformability and hydration status, giving a different pattern for each type of erythrocyte membrane defects (Da Costa et al., 2016; Andolfo et al., 2016). The significant change in ektacytometry data was mostly observed in the left shift of the O-hyper point, which reflects the stiffness of erythrocytes. A mild decrease in deformability was also noted in index patient as well as patient with neuroacanthocytosis with similar acanthocyte percentage in peripheral blood. The changes seen were closely related to the number of acanthocytes identified in patients with VPS13A disease and XK disease. These changes were, however, not reflected on hematological parameters. Rheologic abnormalities on four patients with XK disease reported by Ballas et al. (1990) are similar to our findings. Reichel et al. (2022) also found reduced erythrocyte deformability in VPS13A disease by means of microfluidic techniques. Data on ektacytometry for VPS13A disease and pantothenate kinase-associated neurodegeneration (PKAN), another condition also associated with acanthocytosis, is also limited (De Franceschi et al., 2014; Cluitmans et al., 2015). A report on ektacytometry of neuroacanthocytosis cases did not provide details on underlying genotype (Lazari et al., 2020). In contrast, spur cell anemia in liver failure is thought to be due to exogenous factors not related to membrane structure since transfused cells tend to undergo similar morphological alterations as well (Shah et al., 2023). That explains the fact that changes observed in ektacytometry displayed a completely different pattern in acanthocytosis caused by liver failure.

Ektacytometry is an objective tool to evaluate erythrocyte deformability. Our report, despite cross-sectional single measurements of few patients, adds on the utility of this tool for assessment of patients with VPS13A and XK diseases (Walker and Danek, 2021). This tool may constitute a promising complement especially when blood smears are inconclusive. Taken together, our data suggest an ektacytometry-based fingerprint for VPS13A disease, but larger studies are required to validate our findings.

The patient with VPS13A disease reported here provided oral and written consent for this work in the context of research approved by the Swedish Ethical Review Authority. The course of disease is progressive and feeding dystonia is highly disabling. Gastrostomy in a near future, is a reasonable and necessary option for this patient. The other patients whom blood was tested for acanthocytosis did also provide oral and written consent.