Heterozygous missense variants of SPTBN2 are a frequent cause of congenital cerebellar ataxia

1 INTRODUCTION

Congenital ataxias account for about 10% of non-progressive infantile encephalopathies and are characterized by severe neonatal hypotonia and developmental delay, followed by cerebellar ataxia in the first years of life. They are generally classified into pure cerebellar and syndromic forms and their inheritance can be autosomal recessive autosomal dominant or X-linked recessive (reviewed by1, 2). More than 20 genes are known to date (see Table S1). Homozygous loss of function mutations of SPTBN2 have been identified in four families with early-onset cerebellar ataxia associated with cognitive impairment, eye movements abnormalities and variable additional neurological signs, defined as autosomal recessive spinocerebellar ataxia 14 (SCAR14; MIM#615386).3-6 Heterozygous missense variants of SPTBN2 have been initially associated to autosomal dominant spinocerebellar ataxia 5 (SCA5; MIM#600224) characterized by a slowly progressive cerebellar syndrome with downbeat nystagmus and tremor, beginning in the third decade with a tendency toward anticipation in later generations.7-11 The identification of three unrelated cases of early-onset cerebellar ataxia and developmental delay carrying the same de novo pathogenic variant (p.R480W) in SPTBN2 has challenged the previous SCA5/SCAR14 genotype-phenotype correlation.12-15 We used targeted next-generation sequencing (NGS) in 100 unrelated patients with non-progressive congenital or early onset ataxia associated with cerebellar atrophy.

2 MATERIALS AND METHODS

2.1 Clinical reports

The clinical and neuroradiological features of the five probands (Figure 1, Table 1 and Data S1): congenital onset (within the first month of life), nonprogressive cerebellar symptoms (nystagmus, ataxia, hypotonia) and developmental delay, are almost constant findings. For detailed neuropsychological profiling of patients 1 and 5 (see Data S1 and Table S2).

Table 1. Summary of the clinical, genetic and neuroradiological features of the reported patients

Patient	1	2	3	4	5
Sex, age	F, 8 years	M, 18 years	M, 5 years	F, 18 years	F, 9 years
SPTBN2 variant	c.1309C > T	c.1310G > A	c.479 T > G	c.185C > T	c.888 T > C; c.6267G > A;
Protein	p.R437W	p.R437Q	p.F160C	p.T62I	p.Y272H; p.W2065*
Domain	2nd SPEC	2nd SPEC	ABD	ABD	17th SPEC, PH
Age of onset	10 months	5 months	5 months	8 months	12 months
First sign/symptom at onset	Psychomotor delay	Hypotonia	Psychomotor delay and strabismus	Psychomotor delay Microcephaly	Motor delay
Sitting unsupported	1 year	10 months	13 months	1 year	8 months
Walk (ataxic) unassisted	5 years	2 years	Not acquired	10 years	2 years 4 months
First words	2 years 6 months	1 year	Not acquired	3 years	1 year 6 months
Ocular anomalies	−	Nystagmus (horizontal)	Strabismus	Nystagmus (horizontal and vertical)	−
Congenital hypotonia	+	+	+	+	+ (mild)
Pyramidal signs	−	+ (increased OTR)	−	−	−
Tremor	−	+ (intention tremor)	−	−	−
Dystonia	−	−	−	−	−
Facial myokymia	−	−	−	−	−
Bulbar dysfunction	−	−	−	−	−
Cognitive delay	+	+	+	+	−
Behavioral problems	−	−	−	−	−
Additional findings	−	−	−	Mild bradykinesia	−
Cerebellar atrophy	+	+	+	+	+ (mild)
Progression of atrophy	NA	+	+	NA	NA
Additional MRI findings	−	−	−	−	−
Course	Non-progressive	Non-progressive	Non-progressive	Non-progressive	Non-progressive

• Abbreviations: ABD, actin-binding domain; n.d, not determined in the father, absent in the mother; OTR, osteotendineous reflexes; PH, Pleckstrin homology domain; SPEC, spectrin repeats.

3 RESULTS

3.1 Genetic data

Six novel mutations in SPTBN2 were detected. In particular, four patients harbored heterozygous missense variants (NM_006946): c.185C > T;p.T62I, c.479 T > G;p.F160C, c.1309C > T;p.R437W, and c.1310G > A;p.R437Q and one patient harbored compound heterozygous variants (a nonsense c.6267G > A; p.W2065* and a missense c.888 T > C; p.Y272H) (Figure 2, Table 1). Segregation analysis demonstrated a de novo origin of the mutations in three cases (patients 1, 2 and 3) whereas in one case (patient 4) segregation analysis could not be completed because the father was not alive. All mutations are predicted deleterious or probably damaging by in silico prediction tools. None of the six mutations have been reported in available databases (ie, dbSNP146, 1000 Genomes, ExAC and GnomAD).

3.2 Molecular modeling

The p.T62I and p.F160C pathogenic variants fall in the first Calponin-homology (CH) domain of SPTBN2 (NP_008877) affecting a highly conserved residue. The replacement of Thr62 with an isoleucine residue introduces a novel hydrophobic interaction with Trp154, which modify the interface of the CH-1 domain and actin (Figure 3B). The replacement of Phe160 (highly conserved) with a cysteine causes the loss of the hydrophobic interactions with Trp66 and Leu98 (Figure 3C), modifying also in this case the interface of the CH-1 domain with actin. The p.Y272H mutation affects a highly conserved residue and disrupts the hydrophobic interactions with nearby residues in the second CH domain of SPTBN2 (Figure 3D). Superposition of the SPTBN2 CH-2 domain with the CH2 domain of human alpha-actinin, which, like SPTBN2 also presents two CH domains arranged in tandem, suggests that the Y272H amino acid substitution likely perturbs the interface between the two CH domains and possibly their mutual three-dimensional arrangements. The p.R437W or p.R437Q mutation implies the replacement of the cationic and highly conserved arginine 437 with the aromatic and large tryptophan (W) or the polar glutamine (Q) residue in a region that has structural importance for the second spectrin repeat (Figure 3E). Both the tryptophan and glutamine residues should cause important reorganizations in the structure of the spectrin repeat 2 also affecting its arrangement with the flanking repeats (spectrin repeats 1 and 3). The p.W2065* mutation causes the loss of a functional domain of SPTBN2 (Figure 3). Based on conservation and structural analysis, all the mutations presented in this work are expected to have important impacts on functional regions of the SPTBN2 protein, providing a basis for rationalizing their pathogenic effects.

4 DISCUSSION

Spectrins are heterotetrameric cytoskeletal membrane proteins composed of two main alpha subunits (SPTA1, SPTAN1) and five beta subunits (SPTB, SPTBN1, SPTBN2, SPTBN4, SPTBN5) (reviewed by Reference 17). The non-erythrocytic beta spectrin 2 subunit (beta-III spectrin) is a 2391-amino acid protein encoded by SPTBN2 (11q13.2) and expressed throughout the soma and dendritic tree of cerebellar Purkinje cells. SPTBN2 is required for the maintenance of dendritic architecture and for the trafficking and stabilization of several membrane proteins: Ankyrin-R, cell adhesion proteins, voltage-gated sodium channels, glutamate receptors (mGluR1) and transporters (EAAT4).18 It contains the actin/ARP1 binding site at the N-terminal (calponin-homology domains), 17 spectrin repeats involved in the formation of the heterotetrameric α-β-spectrin complex and a pleckstrin homology (PH) domain which binds phosphatidylinositol lipids, at the C-terminal.19 Loss of SPTBN2 in the mouse mutant results in altered dendritic morphology and density, changes in Purkinje cells intrinsic excitability: reduced sodium currents and deficits in glutamatergic neurotransmission.20-22

To date, three cases of dominant SPTBN2 (p.R480W) mutations and four cases of homozygous recessive mutations have been reported in early-onset ataxia (Figure S1). In a recent targeted exome analysis of patients with ataxic phenotypes, de novo or dominant SPTBN2 mutations were identified in 4/88 (4.5%) two of them presenting with infantile onset ataxia and developmental delay.23 Here, we show novel pathogenic SPTBN2 variants in 5% of our cohort of patients with congenital or early-onset non-progressive ataxia; of note that only one-third (5/15) of the rare SPTBN2 variants identified in our study were confirmed to be pathogenic. The five novel pathogenic missense variants localized in the actin-binding domain or in the spectrin-repeat domains of SPTBN2 and one stop variant localized in the PH domain of the protein. Thermodynamic changes caused by different mutations may affect the overall dimerization capability of the protein. The hotspot R480W mutation, located in the second spectrin-repeat domain (as the R437Q and R437W variants identified in the present study) has been predicted to increase protein stability, whereas SCA5-related T472M found in the same domain or L253P found in the actin-binding domain were either neutral or destabilizing the protein.14 Yet, the molecular mechanisms for differences in timing of onset, progression, mode of inheritance and presence of cognitive deficits in ataxic patients, remain unknown. Interestingly, the two brothers carrying a homozygous missense mutation reported by6 and patient 5, carrying a nonsense and a missense variant, appear to have a milder phenotype with normal cognitive development (Supplementary file and Table S2) if compared with previously reported SCAR14 patients with homozygous loss of function mutations.3-5 Similar frequencies have been observed for mutations in other SCA-associated loci such as ITPR124-26 or CACNA1A,27 whereas both autosomal recessive and dominant modes of inheritance associated with neurodevelopmental and/or neurodegenerative features have been reported in KCNC328 or GRM129, 30 mutated families. The challenge will be to decipher subtle changes underpinning Purkinje cells dysfunction both in early and later stages of these various forms of ataxia. This study further delineates the clinical, mutational and epidemiological spectrum of congenital and early onset SPTBN2-related ataxias and suggest that screening of SPTBN2 should be implemented in this subgroup of disorders.

CONFLICT OF INTEREST

The authors declare no conflict of interest.