Patients and methods

Case reports
Patient 1, Pt1 (subject II-2 in Figure 1A) is now 68 years old. Her insidious disease onset started at 55 years, and was first characterized by psychiatric symptoms, initially as a bipolar disorder with depression alternated by hypomanic behavior (compulsive gambling), and eventually as a cognitive deterioration with apathy, neglect of personal care, and memory loss. Shortly thereafter, she manifested an ataxic gait with frequent falls, followed by progressive dysarthria, dysphagia to liquids, drooling, and fluctuating palatal myoclonus. An Electroencephalography at 61 showed unspecific irritative abnormalities; visual evoked potentials were altered. The neurological examination disclosed a moderate ataxic gait requiring a can, dysarthria, palatal myoclonus, and hypotonia (right > left), increased tendon reflexes, a positive Babinski sign at the right foot, mild dyskinesias, mild distal dystonia. Eye movements were normal. A Mini Mental State Examination scored 16/30. The syndrome slowly progressed, with worsening of cognitive deterioration, dysarthria and dysphagia, and onset of urinary incontinence. Several Electromyography (EMG) examinations have consistently been normal over time.
Figure 1  Pedigree and radiological features of the patients. A: Pedigree of the family. Black symbols indicate affected patients 1 and 2 (Pt1, Pt2). Genotypes of each tested individual are indicated under the corresponding symbols (GFAP G: wt; GFAP A: mutant; HDAC6 C: wt; HDAC6 T: mutant). I-3 died of colon cancer at 62 years of age; I-1died of unknown causes when he was over 80; II-3 died of a cerebral stroke at 60 years of age. B: Brain MRI findings of Pt1 (a-f) and Pt2 (g). Atrophy of the medulla is present, with signal abnormalities of the pyramidal tract and medial lemniscus (a, arrows). In the cerebellum, the hylum of the dentate nucleus is bilaterally hyperintense (a, thick arrows). At midbrain level (b,c), substantia nigra and medial lemniscus are hyperintense (b, arrows); a sub-pial rim of high signal intensity is present in the FLAIR image (c). Symmetrical signal abnormalities involve the pallida, particularly at the interface with the posterior limb of the internal capsule (d, arrows). Hyperintensitiy is present in the periventricular white matter, pre- and post-central gyri (e, arrows on the right) and subcortical frontobasal areas (f, arrows). The typical tadpole appearance of the brainstem and cervical spinal cord is seen in the midline sagittal section of Pt2 (g). Pt2 (II-4 in Figure 1A), now 60 years old, was first referred to us at 52, for insidiously progressive walking difficulties, initiated at 46 years with stiffness and weakness at the right lower limb, followed within 3-5 years by involvement of the right upper, and then left lower and upper limbs. He also reported symptoms consistent with nocturnal lower-limb myoclonus. The neurological examination at 52 years showed spastic tetraparesis, more prominent on the right side and lower limbs, bilateral pes equinovarus, normal strength, bilateral Babinski sign. His gait was paraparetic with bilateral thigh adduction; however he could still walk unassisted. He showed no muscle wasting, with the exception of bilateral atrophy of the temporalis muscle. He was diagnosed as having “primary lateral sclerosis” and started riluzole and baclofen, with no tangible benefit. Over the subsequent two years he developed mild spastic hypophonia, and moderate dysphagia for liquids, with worsening of the limb spasticity. At 56 he became wheelchair-bound, severely dysphonic and dysphagic, with severe tetra-spasticity, flexed posture, bilateral ankle clonus, bilateral Babinski, bilateral hypotrophy of temporalis, interosseus and tibialis anterior muscles. Sensory examination and neurovegetative tests were normal, as were the eye movements. The EMG showed neurogenic abnormalities, without spontaneous fibrillation. Nerve conduction studies showed motor axonal neuropathy at the lower limbs, whereas the peripheral sensory conduction was normal. Taken together, these findings indicate severe motor-neuron disease (MND) of limb and bulbar districts. Symptoms have slowly progressed over time. The patient has no cognitive deterioration.
The MRI findings of these patients were very similar and consistent with the diagnosis of AOAD (Figure 1B). Atrophy of the medulla oblongata and cervical spinal cord (“tadpole” appearance) and signal abnormalities were present in the brainstem, dentate nuclei and supratentorial periventricular white matter. Additional findings, peculiar to our patients, were mild atrophy of the midbrain with T2 hyperintensity of the substantia nigra and medial lemniscus, pallida, and subcortical white matter in the pre- and post-central gyri and frontobasal areas. Interestingly, Pt1, who had more marked cognitive impairment, had slightly more extensive supratentorial white matter involvement.
To quantitatively express the different clinical features of the two siblings, we used the Kurtzke scale [8] (Additional file 1), that scores several functional systems (motor, cerebellar, brainstem, urinary, visual, and cognitive) usually involved in white matter disease, including leukodystrophies. The scores were obtained 13 years after disease onset for Pt1 and 14 years after onset for Pt2. The global functional impairment, as assessed by the final EDSS score, [9] was 3/10 for Pt1 (able to walk, moderate ataxia and cognitive impairment, not requiring institutionalization) and 8.5/10 for Pt2 (confined to bed but with some residual upper limb function). For Pt2, the source of the severe disability was predominantly due to pyramidal dysfunction: we thus assessed both patients by also using the ALS-Severity scale, [10] which scored 33/40 for pt1 (speech 7, deglutition 6, upper limbs 10, lower limbs 10), and 17/40 for pt2 (speech 3, deglutition 8, upper limbs 4, lower limbs 2). The results of instrumental examinations are reported in Table 1.
Table 1  Clinical and instrumental assessments
    Pt1   Pt2
Current age  68  60
Age at onset  55  46
Disease duration at the time of examinations, years  13  14
Instrumental assessment *
Cognitive: MMSE score  16/30  30/30
EMG  0  Mild motor axonal neuropathy (1)
MEPs Bulbar/UL/LL  N.A./0/0  1/1/3
SEPs UL/LL  1/1  0/1
BAEPs  NA  NA
VEPs  0  0
Autonomic testing  0  0
Clinical scoring **
Dysarthria/dysphagia  2  2
Gait abnormalities  1  3
Spasticity  0  3
Axial Ataxia  1  0
Limb dysmetria  2  0
Limb weakness  0  2
Muscle wasting  0  1
Sphincter function  2  0
* For neurophysiological assessments, values are graded as 0 (normal), 1 (abnormality not exceeding 25% of upper/lower normal ranges), 2 (between 25 and 50%), and 3 (beyond 50%).
** For clinical dysfunctions, abnormalities are graded as 0 (normal), 1 (only objective signs), 2 (mild dysfunction, not interfering with activities), 3 (severe dysfunction interfering with walking, feeding, or social interactions).
MMSE: mini–mental state examination; EMG: Electromyography; MEPs: Motor evoked potentials; SEPs: Sensory evoked potentials; UL/LL: upper/lower limbs; BAEPs: Brainstem Auditory Evoked Potentials; VEPs: visual evoked potentials.

Molecular analyses
Informed consent for participation in this study was obtained from all family members, in agreement with the Declaration of Helsinki and approved by the Ethical Committee of the Fondazione Istituto Neurologico – IRCCS, Milan, Italy.
Genomic DNA was extracted by standard methods from peripheral blood samples (I-2, II-2, II-4, II-6, II-7, III-1, III-3) and from skin fibroblasts (II-2, II-4). Whole-exome and Sanger’s sequencing were performed as described [11]. Total RNA was isolated from fibroblasts (RNeasy kit, Qiagen) and then transcribed to cDNA (Cloned AMV first-strand cDNA synthesis kit, Invitrogen). Quantitative Real-time PCR (QRtPCR) was assayed on an ABI Prism 7000 apparatus (Applied Biosystems). Additional file 2 reports primers and conditions for PCR amplifications of relevant exons of human GFAP and HDAC6 and for QRtPCR of HDAC6 cDNA.
Additional file 3 reports URLs for biocomputational analysis.
A GFP tagged GFAP cDNA (Origene RG225707) was modified by using Quick-change Site-directed mutagenesis kit (Stratagene) to introduce either the c.1289G > A or the c.1288C > T nucleotide change in the RG225707 clone, using primers listed in Additional file 2.

Cellular experiments
Cell culture, transient transfections, western-blot analysis, and immunocytochemistry were performed as described, [12-15] using antibodies against α-tubulin (Life Science) and acetylated α-tubulin (Sigma). Patients’ fibroblasts and adult control fibroblasts were grown under the same conditions, and analyzed among culture passages 5 and 8. As a positive control for tubulin acetylation, fibroblasts were pre-incubated with the specific HDAC6 inhibitor Tubacin (0, 0.2 μM and 2.5 μM) (Sigma) for 24 h [16]. Immunohistochemistry was carried out on 2 μm thick sections from pellets of Pt1, Pt2 and control fibroblasts, fixed in glutaraldehyde 2.5% (Electron Microscopy Science - EMS), in 0.05 M PBS pH 7.4, dehydrated in graded acetone, and embedded in Spurr (Epoxy resin, EMS).
Transfection of U251-MG by electroporation was performed in triplicate according to the manufacturer’s protocol (GenePulserII-Biorad), and about 100 cells were analyzed blindly for each experiment (a total of 324 cells for GFP-GFAP-ϵwt and 285 for GFP-GFAP-ϵR430H in a first experiment, and 460 cells for either GFP-GFAP-ϵwt or GFP-GFAP-ϵR430C in a second experiment).