Acanthocytosis and the c.680 A>G Mutation in the PANK2 Gene: A Study Enrolling a Cohort of PKAN Patients from the Dominican Republic.

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a form of Neurodegeneration with Brain Iron Accumulation (NBIA) associated with mutations in the pantothenate kinase 2 gene (PANK2). Pantothenate kinases catalyze the rate-limiting step of coenzyme A synthesis and Pank2 is the only pantothenate kinase isoform in humans that is localized to mitochondria. Acanthocytosis, the occurrence of spiculated erythrocytes, is observed in about 10% of the PKAN patients. Therefore PKAN is also classified together with other rare neurodegenerative diseases like Chorea Acanthocytosis (ChAc) and McLeod syndrome (MLS) into the Neuroacanthocytosis (NA) syndromes. It has not been investigated yet whether acanthocytosis in PKAN is associated with a specific subset of Pank2 mutations. In this study, we analyzed acanthocytosis of a cohort of 25 PKAN patients from the Dominican Republic that are homozygous for the c.680 A>G mutation in the PANK2 gene as compared to control donors that are heterozygous or wild-type with respect to this mutation. 3D modeling of this mutation indicated that the replacement of a tyrosine by a cysteine at position 227 in Pank2 disrupts a polar interaction within the A domain of the enzyme. Mean acanthocyte count was elevated in the cohort of patients, however, acanthocytosis varied among the patients with nearly half of them showing high (>20%) or elevated acanthocytosis and the rest showing mild (6-10%) or no (<6%) acanthocytosis. Heterozygous control donors revealed a tendency to mild acanthocytosis. Based on the insight that Pank2 is a normal constituent of red blood cells and de novo biosynthesis of coenzyme A is likely to take place in the erythrocyte cytosol we propose a hypothetical model that accounts for the variability in the occurrence of acanthocytic cells in PKAN

Mapping of the PKAN-linked Pank2 Y227C mutation onto the Pank3 structure und partial alignment of human Pank proteins.

<p>A) Pank3 dimer structure (PDB ID 2i7P) is shown in blue (chain A) and yellow (chain B), respectively. B) The magnification reveals a polar interaction (dashed line) in the WT Pank3 between Y27 (magenta, corresponding to Y227 in Pank2) and R49 (green, corresponding to R249 in Pank2). Side chains of F14, L25, L45 and I85 (cyan) are in close proximity to Y27 (less than 5 Ångström) constituting a hydrophobic environment around it. C) A partial multiple alignment of human Pank proteins, Pank2 (GI: 85838513), Pank1 (GI:23510400) and Pank3 (GI:119581908) is shown. Elements of secondary structure, helices (α) and β-strands (-) are indicated above and numbered accordingly. Known sites of PKAN missense mutations are boxed in yellow, the Pank2 Y227 site described here is boxed in magenta, the Pank2 R249 site which is in polar contact with Pank2 Y227 and is itself a known PKAN-linked Pank2 mutation is boxed in green. D) The magnification shows the effect of a Pank3 Y27C mutation (magenta) corresponding to the PKAN-linked Pank2 Y227C mutation disrupting the tyrosine-specific polar contact and local hydrophobic packing. PANK3 structure views and mutation Y27C were edited and modeled by PyMOL (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>). The contact map of Y27 was calculated using the Protein Interactions Calculator (PIC) server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125861#pone.0125861.ref024" target="_blank">24</a>].</p

Degree of acanthocytosis in patients and control donors.

<p>Acanthocyte counts of donors that are heterozygous (n = 36), homozygous (n = 23) and wild-type (22) with respect to the c.680 A>G mutation in the PANK2 gene were microscopically assessed as described in the Materials and Methods section. The samples were grouped in four classes of acanthocytosis (normal (acanthocyte count <6% of total cells), mild (6–10%), elevated (10–20%) and high (>20%)) and the number of samples of each class is shown as relative percent of the total number in each subset of donors.</p

Distribution of acanthocytosis in the patient and control samples.

<p>The distribution of the amount of acanthocytes (in % of total cells) in the samples of donors that are heterozygous (n = 36), homozygous (n = 23) and wild-type (n = 22) with respect to the c.680 A>G mutation in the PANK2 gene is shown in a Box-Whiskers blot. The circles are <i>outliers</i>, and the asterisk is a <i>far outlier</i>.</p