Delineating SPTAN1 associated phenotypes: from isolated epilepsy to encephalopathy with progressive brain atrophy

Abstract

De novo in-frame deletions and duplications in the SPTAN1 gene, encoding the non-erythrocyte αII spectrin, have been associated with severe West syndrome with hypomyelination and pontocerebellar atrophy. We aimed at comprehensively delineating the phenotypic spectrum associated with SPTAN1 mutations. Using different molecular genetic techniques, we identified 20 patients with a pathogenic or likely pathogenic SPTAN1 variant and reviewed their clinical, genetic and imaging data. SPTAN1 de novo alterations included seven unique missense variants and nine in-frame deletions/duplications of which 12 were novel. The recurrent three-amino acid duplication p.(Asp2303_Leu2305dup) occurred in five patients. Our patient cohort exhibited a broad spectrum of neurodevelopmental phenotypes, comprising six patients with mild to moderate intellectual disability, with or without epilepsy and behavioural disorders, and 14 patients with infantile epileptic encephalopathy, of which 13 had severe neurodevelopmental impairment and four died in early childhood. Imaging studies suggested that the severity of neurological impairment and epilepsy correlates with that of structural abnormalities as well as the mutation type and location. Out of seven patients harbouring mutations outside the α/β spectrin heterodimerization domain, four had normal brain imaging and three exhibited moderately progressive brain and/or cerebellar atrophy. Twelve of 13 patients with mutations located within the spectrin heterodimer contact site exhibited severe and progressive brain, brainstem and cerebellar atrophy, with hypomyelination in most. We used fibroblasts from five patients to study spectrin aggregate formation by Triton-X extraction and immunocytochemistry followed by fluorescence microscopy. αII/βII aggregates and αII spectrin in the insoluble protein fraction were observed in fibroblasts derived from patients with the mutations p.(Glu2207del), p.(Asp2303_Leu2305dup) and p.(Arg2308_Met2309dup), all falling in the nucleation site of the α/β spectrin heterodimer region. Molecular modelling of the seven SPTAN1 amino acid changes provided preliminary evidence for structural alterations of the A-, B- and/or C-helices within each of the mutated spectrin repeats. We conclude that SPTAN1-related disorders comprise a wide spectrum of neurodevelopmental phenotypes ranging from mild to severe and progressive. Spectrin aggregate formation in fibroblasts with mutations in the α/β heterodimerization domain seems to be associated with a severe neurodegenerative course and suggests that the amino acid stretch from Asp2303 to Met2309 in the α20 repeat is important for α/β spectrin heterodimer formation and/or αII spectrin function.

Keywords: West syndrome; epileptic encephalopathy; hypomyelination; myoclonic epilepsy; pontocerebellar atrophy.

Figure 1

SPTAN1 variants and domain structure of the encoded αII spectrin. Variants are given in the three-letter code above the schematic of αII spectrin. The known mutations p.(Glu2207del), p.(Asp2303_Leu2305dup), and p.(Arg2308_2309dup) identified in our study are underlined. Novel SPTAN1 mutations are indicated in black or red based on the severity of the patient’s phenotype (red: infantile epileptic encephalopathy; black: childhood onset epilepsy syndromes; Table 1). Patient numbers are indicated below each mutation. Patients for whom fibroblasts were used in this study are marked with an asterisk. α spectrin repeats (α1 to α20) are indicated as dark blue boxes and numbered. The SH3 domain (light blue box) is located within α9 and the CCC (Ca²⁺-dependent binding site for calmodulin and cleavage sites for caspases and calpain) domain (light green box) within α10. EF hands are indicated as two blue circles at the C-terminus. α19 and α20 repeats are required for α/β heterodimerization (double arrow). N = NH₂-terminus; C = COOH-terminus.

Figure 2

Progression of structural MRI abnormalities. Six representative patients [Patients 4 (A), 8 (B), 10 (C), 11 (D), 17 (E) and 18(F)] are shown of the nine patients with SPTAN1 variants who were imaged at least twice during follow-up (comparative follow-up images for Patients 5, 6 and 9 are presented in Supplementary Fig. 1). For each patient (A–F) two sets of comparative axial (left column), coronal (middle column) and sagittal (right column) images are presented, taken respectively from the initial and follow-up investigations. Images are at 1.5 to 3 T and include T₁-weighted, T₂-weighted and fluid-attenuated inversion recovery (FLAIR) sequences. Structural abnormalities include a combination of cerebellar and brainstem atrophy, dilated ventricles and subarachnoid spaces, thinning of the corpus callosum and hypomyelination that are variably distributed. Here, however, comparison of initial and follow-up images demonstrates different rates of progression from one patient to another and from one involved structure to another. For example, in Patient 4 (A), who harbours a missense variant falling outside the heterodimerization domain, from age 3 years 3 months to age 8 years 2 months, only cerebellar atrophy has really worsened; and in Patient 18 (F), who harbours an in-frame deletion also falling outside the heterodimerization domain, MRI scan at age 2 years was still normal but exhibited isolated, severe cerebellar atrophy at age 15 years. In all remaining patients, harbouring different types of mutations falling within the heterodimerization domain, follow-up images demonstrate severely progressive changes that are generalized, involving the brain, cerebellum and brainstem, although not uniformly, and more prominent in patients imaged at older ages (see for example Patients 8, 10, 11 and 17).

Figure 3

Spectrin aggregate formation in fibroblast cells of three patients with an in-frame deletion/duplication in SPTAN1. (A) Primary fibroblasts of Patients 2, 6, 10, 15 and 17 and a control individual were co-stained by mouse anti-αII spectrin and rabbit anti-βII spectrin antibodies followed by anti-mouse Alexa Fluor^® 488 (green) and anti-rabbit Alexa Fluor^® 546 (red) conjugated secondary antibodies, respectively, and embedded in mounting solution with DAPI (blue). Camera settings were the same for all images shown, and images were not further processed (see ‘Material and methods’ section). Representative images are shown. Scale bars = 20 µm. (B) Fibroblast cells of Patients 2, 6, 10, 15, and 17 and three control individuals were subjected to Triton^™ X extraction. Supernatant (S) and pellet (P) were analysed by SDS-PAGE and immunodetection using antibodies against αII spectrin and α-tubulin as a control. Representative blots of three independent experiments are shown.