Bi-allelic variants in CHKA cause a neurodevelopmental disorder with epilepsy and microcephaly

Abstract

The Kennedy pathways catalyse the de novo synthesis of phosphatidylcholine and phosphatidylethanolamine, the most abundant components of eukaryotic cell membranes. In recent years, these pathways have moved into clinical focus because four of ten genes involved have been associated with a range of autosomal recessive rare diseases such as a neurodevelopmental disorder with muscular dystrophy (CHKB), bone abnormalities and cone-rod dystrophy (PCYT1A) and spastic paraplegia (PCYT2, SELENOI). We identified six individuals from five families with bi-allelic variants in CHKA presenting with severe global developmental delay, epilepsy, movement disorders and microcephaly. Using structural molecular modelling and functional testing of the variants in a cell-based Saccharomyces cerevisiae model, we determined that these variants reduce the enzymatic activity of CHKA and confer a significant impairment of the first enzymatic step of the Kennedy pathway. In summary, we present CHKA as a novel autosomal recessive gene for a neurodevelopmental disorder with epilepsy and microcephaly.

Keywords: Kennedy pathway; choline kinase alpha; epilepsy; exome sequencing; neurodevelopmental disorder.

Figure 1

Muscle biopsy of Individual 4 and an aged-matched control. The histochemical analysis revealed a prominent mitochondrial patterning. Scale bar = 20 µm. (A) Haematoxylin and eosin staining: in the sarcoplasm basophilic dots indicate dense and enlarged mitochondria in the affected individual. (B) Cytochrome c oxidase staining: the mitochondria are slightly increased in size compared to the control. (C) Succinate dehydrogenase staining: mitochondria are evenly distributed. (D) Electron microscope (without aged-matched control), scale bar = 300 nm. Mitochondria show dense matrix and regular cristae. Because of the high electron density, the mitochondria appear prominent. The arrows indicate the broadened cristae by material of higher density.

Figure 2

Overview on location of variants, structural modelling and functional testing in an S. cerevisiae model. (A) Location of variants in CHKA with respect to reported domain structure. (B) Structural effect of CHKA sequence variants. (i) Arg141 forms stabilizing hydrogen bonds (black dotted lines) with Pro130 and Thr133 in the vicinity of the ADP binding site. Interacting residues and ADP are shown in stick presentation (atom-type colouring) and are labelled. The CSKH protein backbone is depicted as a cyan ribbon. (ii) Trp141 cannot form the stabilizing hydrogen bonds to Pro130/Thr133 and adopts a different sidechain orientation. Colour coding as in B(i). (iii) Pro194 (grey) is located in a turn close to the ADP binding site. (iv) The Ser194 sidechain causes steric problems (indicated as a red arrow) with the adjacent Ile209, thereby destabilizing the structure. (v) Phe341 (grey) is part of a hydrophobic cluster close to the binding site of phosphocholine (PC; shown as sticks). Residues of the hydrophobic cluster are shown in space-filled presentation. (vi) The presence of the non-aromatic Leu341 results in a loss of hydrophobic interactions (denoted as a red circle). (C) Western blot of human CHKA expressed in yeast demonstrates that each allele was expressed at a similar level. Pgk1 is the loading control. Choline kinase activity of each allele, normalized to the level of CHKA expressed, was determined. It was determined that each patient-derived allele possessed reduced choline kinase activity.