Identification of novel and hotspot mutations in the channel domain of ITPR1 in two patients with Gillespie syndrome

Abstract

ITPR1 encodes an intracellular receptor for inositol 1,4,5-trisphosphate (InsP3) which is highly expressed in the cerebellum and is involved in the regulation of Ca2+ homeostasis. Missense mutations in the InsP3-binding domain (IRBIT) of ITPR1 are frequently associated with early onset cerebellar atrophy. Gillespie syndrome is characterized by congenital ataxia, mild to moderate intellectual disability and iris hypoplasia. Dominant or recessive ITPR1 mutations have been recently associated with this form of syndromic ataxia. We performed next generation sequencing in two simplex families with Gillespie syndrome and identified de novo pathological mutations localized in the C-terminal channel domain of ITPR1 in both patients: a recurrent deletion (p.Lys2596del) and a novel missense mutation (p.Asn2576Ile) close to a point of constriction in the Ca²⁺ pore. Our study expands the mutational spectrum of ITPR1 and confirms that ITPR1 screening should be implemented in patients with congenital cerebellar ataxia with or without iris hypoplasia.

Keywords: Cerebellar atrophy; Inositol 1,4,5 tri-phosphate receptor (InsP3) type 1 (ITPR1); Intellectual disability; Partial aniridia.

Fig. 1

A- Neuroimaging studies: T1 weighted mid-sagittal section (I, III) and T2 weighted coronal section (II, IV) of the patients at the age of 5 (patient 1) and 2 years (patient 2), showing moderate cerebellar atrophy, predominant in the vermis. T2 weighted axial image of patient 2 showing mild periventricular hyperintensities adjacent to frontal and occipital horns (white arrowheads) (V). B-Sanger sequencing chromatograms in the two families. C-Protein sequence alignment of human ITPR1 with its paralogs. D-Linear representation of the ITPR1 protein and Gillespie related mutations identified to date. Amino acid numbering is based on GenBank: NP_001161744.1 (Q14643-2; ENSP00000306253.8). Previously reported recessive inactivating mutations are highlighted in black; dominant negative (de novo) mutations are highlighted in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Structural consequences of the Gillespie syndrome-causing p.Asn2576Leu substitution. A: model of the human ITPR1 channel in a closed state showing the location of the site affected by the p.Asn2576Ile mutation and the Ca^2 + pore (the four monomers are in different colors). B: detailed view around Asn2576 (please note that all the four Asn2576 residues contributed by each ITPR1 monomer are proximal to a constriction point in the Ca^2 + pore). C: view through the pore of a modelled p.Asn2576Ile ITPR1 channel mutant (the two upper helices contributing to the pore are wild type and the two lower helices mutated). In the wild type ITPR1 monomers the Asn2576 side chains are bent away from the Ca^2 + path by hydrogen bonding (black dotted lines) with the backbone of Leu2577. Conversely, in the p.Asn2576Ile mutant monomers, an analogous hydrogen bond cannot be formed by the hydrophobic side chain of the replacing Ile2576, causing steric hindrance (highlighted by red circle with arrows) in the Ca^2 + path. The channel closed state is contributed by the side chain of Phe2579 that directly lays in the pore and obstructs it. In the p.Asn2576Ile mutant, conformational changes of Phe2579 to reverse this obstruction (as needed for channel switching to the open state) are impeded owing to hindrance by the Ile2576 side chain. Amino acid numbering is according to the NCBI entry NP_001161744.1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)