Mutations in the autoregulatory domain of β-tubulin 4a cause hereditary dystonia

Abstract

Dystonia type 4 (DYT4) was first described in a large family from Heacham in Norfolk with an autosomal dominantly inherited whispering dysphonia, generalized dystonia, and a characteristic hobby horse ataxic gait. We carried out a genetic linkage analysis in the extended DYT4 family that spanned 7 generations from England and Australia, revealing a single LOD score peak of 6.33 on chromosome 19p13.12-13. Exome sequencing in 2 cousins identified a single cosegregating mutation (p.R2G) in the β-tubulin 4a (TUBB4a) gene that was absent in a large number of controls. The mutation is highly conserved in the β-tubulin autoregulatory MREI (methionine-arginine-glutamic acid-isoleucine) domain, highly expressed in the central nervous system, and extensive in vitro work has previously demonstrated that substitutions at residue 2, specifically R2G, disrupt the autoregulatory capability of the wild-type β-tubulin peptide, affirming the role of the cytoskeleton in dystonia pathogenesis.

FIGURE 1

Pedigree of the dystonia type 4 family. Where mutation screening has been performed, individuals are marked with either wt (wild-type allele) or m (R2G heterozygote); exome indicates exome sequencing performed. The symbols (, , ) indicates individuals included in linkage analysis. VI-27 and VI-28 were known to have Wilson disease and were also heterozygous for the R2G variant.

FIGURE 2

Multipoint parametric linkage analysis of the kindred indicating a single linkage peak at 19p13.3 with an LOD score of 6.33. [Color figure can be viewed in the online issue, which is available at http://www.annalsofneurology.org.]

FIGURE 3

Sequence chromatogram showing (A) an unaffected family member with the wild-type sequence and (B) an affected family member with a heterozygous c.4C>G: p.R2G mutation. [Color figure can be viewed in the online issue, which is available at http://www.annalsofneurology.org.]

FIGURE 4

Graph of the expression of the TUBB4a gene in 10 brain regions from 134 normal individuals, assessed using the Affymetrix Exon 1.0 ST Array. The level of TUBB4a is given as a log scale with range bars. This showed very high expression in the cerebellum and in the brain overall. The following areas were studied: cerebellum (CRBL), frontal cortex (FCTX), hippocampus (HIPP), medulla (MEDU), occipital cortex (OCTX), putamen (PUTM), substantia nigra (SNIG), temporal cortex (TCTX), thalamus (THAL), and white matter (WHMT). [Color figure can be viewed in the online issue, which is available at http://www.annalsofneurology.org.]

FIGURE 5

Expression of TUBB4a (top row) in various human tissues was determined by reverse transcriptase polymerase chain reaction using gene-specific primers against cDNA generated from tissue-specific RNA as compared to the housekeeping gene beta2-microglobulin (bottom row). Expression was high in the brain and very low in other tissues, except for moderate expression in the testes. 1 = ladder; 2 = adipose tissue; 3 = brain; 4 = esophagus; 5 = colon; 6 = heart; 7 = liver; 8 = ovary; 9 = cervix; 10 = bladder; 11 = testes; 12 = no cDNA control.