Recessive mutations in SPTBN2 implicate β-III spectrin in both cognitive and motor development

Abstract

β-III spectrin is present in the brain and is known to be important in the function of the cerebellum. Heterozygous mutations in SPTBN2, the gene encoding β-III spectrin, cause Spinocerebellar Ataxia Type 5 (SCA5), an adult-onset, slowly progressive, autosomal-dominant pure cerebellar ataxia. SCA5 is sometimes known as "Lincoln ataxia," because the largest known family is descended from relatives of the United States President Abraham Lincoln. Using targeted capture and next-generation sequencing, we identified a homozygous stop codon in SPTBN2 in a consanguineous family in which childhood developmental ataxia co-segregates with cognitive impairment. The cognitive impairment could result from mutations in a second gene, but further analysis using whole-genome sequencing combined with SNP array analysis did not reveal any evidence of other mutations. We also examined a mouse knockout of β-III spectrin in which ataxia and progressive degeneration of cerebellar Purkinje cells has been previously reported and found morphological abnormalities in neurons from prefrontal cortex and deficits in object recognition tasks, consistent with the human cognitive phenotype. These data provide the first evidence that β-III spectrin plays an important role in cortical brain development and cognition, in addition to its function in the cerebellum; and we conclude that cognitive impairment is an integral part of this novel recessive ataxic syndrome, Spectrin-associated Autosomal Recessive Cerebellar Ataxia type 1 (SPARCA1). In addition, the identification of SPARCA1 and normal heterozygous carriers of the stop codon in SPTBN2 provides insights into the mechanism of molecular dominance in SCA5 and demonstrates that the cell-specific repertoire of spectrin subunits underlies a novel group of disorders, the neuronal spectrinopathies, which includes SCA5, SPARCA1, and a form of West syndrome.

Figure 1. Genetic analysis of family with ataxia and cognitive impairment.

A. Pedigree of family. B. Sanger sequencing of the mutation c. 1881C>A; p.C627X in normal, parents of V3 (IV3 and IV4) and affecteds, V1, V2, V3.

Figure 2. Neuroimaging of patients.

A. Sagittal T1w MRI in subject V1 age 21 demonstrating clear cerebellar atrophy. B. Sagittal T1w MRI in subject V2 at age 6. Sagittal T1w MRI in subject V2 age 16 shows clear atrophy of the cerebellum. C. Sagittal T1w MRI in subject V3 showing hypoplasia of posterior corpus callosum (white arrow).

Figure 3. SNP Zygosity data for chromosome 11 from affecteds V1, V2, V3, and V3's parents IV3 and IV4.

Red lines correspond to homozygous SNPs and blue lines to heterozygous SNPs (the gap represents the centromere).

Figure 4. Abnormal dendritic morphology in β-III spectrin −/− mouse compared to wild type.

A. Sagittal sections immunostained for MAP2 show irregular reactivity throughout prefrontal cortical layers and caudate putamen/striatum of 6-week-old β-III spectrin knockout (−/−) mice compared to wild type (+/+) but normal staining within hippocampus (N = 3 each genotype; Bar, 20 µm). B. Top, Representative images of pyramidal neurons in layer 2/3 prefrontal cortex from 8-week-old WT and β-III spectrin knockout mice filled with Alexa 568 (Bar, 20 µm). Bottom, Neuronal 3-D reconstruction over laid using NeuronStudio software. C. Quantification of basal dendrite morphological parameters measured from reconstructed images shows greater distal thinning of dendrites in cells from β-III spectrin knockout mice (open circles; N = 9) compared with WT cells (filled squares; N = 8). All data are mean ± SEM (* denotes significance between groups and # significance within a group.) D. High magnification image of single basal dendrite showing distal thinning in β-III spectrin knockout compared to WT but presence of normal spines (Bar, 5 µm). E. High magnification image of Golgi impregnated pyramidal neuron from prefrontal cortex of WT and β-III spectrin knockout mice (Bar, 10 µm).

Figure 5. Absence of hypoplasia of posterior corpus callosum in β-III spectrin knockout mice.

Sagittal sections from 8-week old WT (+/+) and knockout (−/−) animals stained with cresyl violet (A, Bar 200 µm) and anti-tau antibody (B, Bar 500 µm) with arrow pointing to posterior corpus callosum. C. Coronal sections immunostained for MBP (Bar 200 µm).

Figure 6. β-III spectrin knockout mice display deficits in some object recognition tasks.

Diagram of task and performance of WT (+/+) and β-III spectrin knockout mice (−/−) in the four object recognition tasks. Two-novel object recognition (A), four-novel object recognition (B), object-in-place (C) and object location task (D). All data are mean ± SEM (N = 6–9; * P<0.05).

Figure 7. Diagram of β-III spectrin/α-II spectrin tetramer.

This is composed of 2 β-III spectrin and 2 α-II spectrin molecules and the location of the homozygous stop codon C627X in SPTBN2 causing SPARCA1 relative to dominant mutations in SPTBN2 and SPTAN1. Mutations are only shown in one of the two molecules. Loss or truncation of β-III is likely to prevent formation of normal tetramers. The glutamate transporter, EAAT4, binds near the C terminus of β-III.