Whispering dysphonia (DYT4 dystonia) is caused by a mutation in the TUBB4 gene

Abstract

Objective: A study was undertaken to identify the gene underlying DYT4 dystonia, a dominantly inherited form of spasmodic dysphonia combined with other focal or generalized dystonia and a characteristic facies and body habitus, in an Australian family.

Methods: Genome-wide linkage analysis was carried out in 14 family members followed by genome sequencing in 2 individuals. The index patient underwent a detailed neurological follow-up examination, including electrophysiological studies and magnetic resonance imaging scanning. Biopsies of the skin and olfactory mucosa were obtained, and expression levels of TUBB4 mRNA were determined by quantitative real-time polymerase chain reaction in 3 different cell types. All exons of TUBB4 were screened for mutations in 394 unrelated dystonia patients.

Results: The disease-causing gene was mapped to a 23cM region on chromosome 19p13.3-p13.2 with a maximum multipoint LOD score of 5.338 at markers D9S427 and D9S1034. Genome sequencing revealed a missense variant in the TUBB4 (tubulin beta-4; Arg2Gly) gene as the likely cause of disease. Sequencing of TUBB4 in 394 unrelated dystonia patients revealed another missense variant (Ala271Thr) in a familial case of segmental dystonia with spasmodic dysphonia. mRNA expression studies demonstrated significantly reduced levels of mutant TUBB4 mRNA in different cell types from a heterozygous Arg2Gly mutation carrier compared to controls.

Interpretation: A mutation in TUBB4 causes DYT4 dystonia in this Australian family with so-called whispering dysphonia, and other mutations in TUBB4 may contribute to spasmodic dysphonia. Given that TUBB4 is a neuronally expressed tubulin, our results imply abnormal microtubule function as a novel mechanism in the pathophysiology of dystonia.

FIGURE 1:

Simplified pedigree of the Australian DYT4 family including only those individuals with available DNA (L-number) and their ancestors. The shared haplotype on chromosome 19 is indicated by a black bar. Genotypes at the indicated markers are given as numbers; a question mark symbolizes that genotyping did not work. Individuals marked with a plus sign were included in the genome-wide linkage analysis; in those marked with a star, genome sequencing was performed. DYT4 dystonia-affected family members are labeled with black and unaffected with white symbols, and presumably affected ones with question marks. The proband is indicated by an arrow. Slashes indicate deceased family members. Circles = females; squares = males.

FIGURE 2:

Recent photographs of the index patient showing the characteristic facial features of atrophy most prominent in the jaw and lower facial muscle producing a triangular appearance of the lower face (left panels). The index patient and several other members of the family also exhibit bilateral eyelid ptosis. All observed affected family members have a generally thin body habitus from childhood until well before the obvious onset of DYT4 features, which invariably commences with spasmodic dysphonia. Poor dentition and eventually edentulousness is common in the more severely affected family members, especially those with extrusional tongue movements (left, middle panel). Synkinetic movements of the levator palpebrae superioris, ocular, and jaw muscles are triggered by jaw opening (left, lower panel). The characteristic dystonic “hobby horse” gait of DYT4 dystonia is seen in the right panel. [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.]

FIGURE 3:

Cranial 3T magnetic resonance imaging with representative fluid attenuated inversion recovery sequences in the axial (left), coronal (middle), and midline sagittal plane (right). Brain parenchyma and in particular the deep and cortical gray and white matter exhibited normal patterning. The subcutaneous tissues of the inferior face, predominantly comprising the muscles of facial expression, are atrophic, and the patient is edentulous. The muscles of mastication were of normal volume, and an incidental, small, left mastoid effusion was present.

FIGURE 4:

Novel mutation in the TUBB4 gene. The electropherograms show the sequence of exon 1 of TUBB4 around the start codon (boxed) for Patient L-3270. The c.4C>G mutation is highlighted by the letter S in red. (A) At the DNA level, the mutation is heterozygous with equal peak heights for both alleles. (B) At the RNA level, the wild-type allele is apparently more abundant than the mutant allele, prompting real-time quantitative polymerase chain reaction experiments.

FIGURE 5:

Expression levels of wild-type and mutant TUBB4. (A) Quantitative real-time polymerase chain reaction (PCR) done at the University of Lübeck revealed a lower level of TUBB4 in the available heterozygous mutation carrier (filled bar) compared to the mean of 13 unrelated individuals without TUBB4 mutations (open bar). GAPDH was used as a reference gene. The expression level for the controls was set to 1. Standard deviation is indicated. All measurements were performed in duplicate, and all samples were analyzed at least twice. (B) Quantitative PCR was also performed at the University of Sydney in an independently extracted blood sample (Blood) as well as in fibroblasts and human primary olfactory cell cultures (Olfactory) from the same mutation carrier (filled bars) and a related but unaffected control (open bar). The expression level for the control was set to 1, and ACTB was used as a reference gene. Standard deviation is indicated, and all measurements were performed at least in duplicate.