Frontotemporal dementia–amyotrophic lateral sclerosis syndrome locus on chromosome 16p12.1–q12.2: genetic, clinical and neuropathological analysis

Numerous families exhibiting both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) have been described, and although many of these have been shown to harbour a repeat expansion in C9ORF72, several C9ORF72-negative FTD-ALS families remain. We performed neuropathological and genetic analysis of a large European Australian kindred (Aus-12) with autosomal dominant inheritance of dementia and/or ALS. Affected Aus-12 members developed either ALS or dementia; some of those with dementia also had ALS and/or extrapyramidal features. Neuropathology was most consistent with frontotemporal lobar degeneration with type B TDP pathology, but with additional phosphorylated tau pathology consistent with corticobasal degeneration. Aus-12 DNA samples were negative for mutations in all known dementia and ALS genes, including C9ORF72 and FUS. Genome-wide linkage analysis provided highly suggestive evidence (maximum multipoint LOD score of 2.9) of a locus on chromosome 16p12.1–16q12.2. Affected individuals shared a chromosome 16 haplotype flanked by D16S3103 and D16S489, spanning 37.9 Mb, with a smaller suggestive disease haplotype spanning 24.4 Mb defined by recombination in an elderly unaffected individual. Importantly, this smaller region does not overlap with FUS. Whole-exome sequencing identified four variants present in the maximal critical region that segregate with disease. Linkage analysis incorporating these variants generated a maximum multipoint LOD score of 3.0. These results support the identification of a locus on chromosome 16p12.1–16q12.2 responsible for an unusual cluster of neurodegenerative phenotypes. This region overlaps with a separate locus on 16q12.1–q12.2 reported in an independent ALS family, indicating that this region may harbour a second major locus for FTD-ALS. Electronic supplementary material The online version of this article (doi:10.1007/s00401-013-1078-9) contains supplementary material, which is available to authorized users.


Western blot analysis of tau isofoms
Sarkosyl-insoluble fractions were isolated from 250 mg frontal cortex of affected Aus-12 individual IV:7, one neuropathologically confirmed CBD case (4-repeat tau dominant tauopathy) and two neuropathologically confirmed AD cases (3-and 4-repeat tauopathy) according to the protocol described by de Calignon et al [39]. Approximately 15 to 30 micrograms of total protein were electrophoresed using SDS-PAGE gels. Following transfer of proteins onto nitrocellulose membrane (Bio-Rad Laboratories, Hemel Hempstead, UK), monoclonal antibodies against either 3-repeat tau (clone 8E6/C11 at 1:2000 dilution, Merck Millipore, Kilsyth, Australia) or 4-repeat tau (clone 1E1/A6 at 1:500 dilution, Merck Millipore) were used to determine the dominant type of sarkosyl-insoluble tau species. Immunoblotting with a monoclonal antibody against !-actin (clone C4, Merck Millipore) was used to visualise the level of total protein per lane. Protein bands were visualised using enhanced chemiluminescence according to manufacturer's instructions (Supersignal West Pico Chemiluminescent Substrate, Thermo Scientific, Rockford, USA).

Supplementary material for Dobson-Stone et al, Page 3 Clinical notes for Aus-12 family
Case IV:23 -The proband, IV:23, was referred to a specialist geriatrician at age 56 because over the previous year she had begun to repeat questions, misplace items and become lost when driving. She would forget the names of friends and would fail to recognise familiar faces. She was unable to manage her previous tasks of helping with the accounts and with managing horses.
She was less fastidious about her personal appearance than before. Initially she felt she was deteriorating like her mother, but at the time of referral she denied having problems. Friends were concerned about her driving. She had become verbally aggressive at times and made inappropriate comments, which was a change from her previous personality. Despite this, she scored 27/30 on the Mini-Mental State Examination (MMSE), losing 2 points for recall and 1 for copying intersecting pentagons. On a 10-word learning task she was able to recall 5/10 after an interval. There were some difficulties with higher intellectual function including description of word similarities and differences and rather concrete proverb definition, but she had reasonable verbal fluency. Calculation was impaired. Drawings and constructional tasks were initially well preserved, and it was noted that she was easily able to copy complex figures or spontaneously sketch faces and figures. Prominent early memory symptoms and geographical disorientation, together with the strong family history of clinically diagnosed Alzheimer's disease (AD), led to an initial clinical suspicion of AD. However after a year she developed significant personality and behavioural change, with disinhibition, socially inappropriate and obsessional behaviour, and a lack of insight. This, together with continuing intact constructional skills and left frontotemporal hypoperfusion on a SPECT scan, led to a diagnosis of FTD. Her condition gradually progressed. She required residential care from age 66 for management of her behavioural and self-care deficits. Interestingly her drawing skills remained one of the few areas of preservation, implying relative preservation of parietal function. She died at age 68 years.

Supplementary material for Dobson-Stone et al, Page 4
Case IV:5 -This former forklift driver moved interstate and lived alone after a divorce. His behaviour became increasingly odd from his early to mid-fifties. A brain CT scan when he was 59 was reported as normal. His house was squalid and unsafe, with the rooms filled with garbage.
There was no running water or electricity as he had disconnected the wires. He was admitted to a psychiatric institution at 63, by which time he was largely mute. He hoarded the belongings of other residents and displayed challenging, repetitive and impulsive behaviour. Some extrapyramidal features noted at the time were thought to be related to previous treatment with haloperidol. Brain CT was reported as showing temporal lobe atrophy. The clinical diagnosis was FTD. He became less mobile, developed swallowing difficulties and pneumonia and died at age 64 years. Autopsy revealed bilateral bronchopneumonia.
Case IV:7 -This former welder had not worked since being retrenched at age 54 years. He had Paget's disease of both hips as well as ischaemic heart disease. His wife reported that he had given up many of his hobbies and interests from age 50. After a coronary bypass operation at 62 he developed progressive problems with memory, orientation and language. When seen by a neurologist at 67 he had become withdrawn and lacked emotion. There was some evidence of disinhibited behaviour. He had parkinsonian features including expressionless face, increased tone and shuffling gait together with pout and grasp reflexes and some utilisation behaviour. He

FTLD-Tau
As previously described [29], the familial FTLD-TDP case with a GRN mutation had typical type A FTLD-TDP neuropathology with numerous phospho-TDP-immunoreactive NCIs, glial cytoplasmic inclusions, short dystrophic neurites, and the occasional neuronal intranuclear inclusion. No pathological FUS or !-internexin immunoreactivity was observed in this case.
Occasional phospho-tau and neurofilament immunoreactivity was observed in the entorhinal cortex, consistent with age-associated changes. In contrast, the two CBD FTLD-tau cases had phospho-tau-immunopositive astrocytic plaques and coiled bodies as well as phospho-tau immunopositive threads and neurons (Fig. 2k-m). Ballooned neurons were also immunopositive for phosphorylated neurofilament and to a lesser extent phospho-tau, as previously described [10]. No pathological FUS or !-internexin immunoreactivity was observed in these cases, and only occasional granules of abnormal phospho-TDP were observed.

Supplementary material for Dobson-Stone et al, Page 6
The phospho-tau neuropathology of Aus-12 closely resembled the CBD FTLD-tau cases. The most similar features were the considerable dentate involvement (Fig. 2c,m) and the very prominent glial phospho-tau immunoreactivity in the white matter. The TDP-43 neuropathology on the other hand was similar in amount and distribution but not structural type to the familial FTLD-TDP case with a GRN mutation, which had phospho-TDP-immunoreactive NCI and neurites in the cortical regions examined. However, greater phospho-TDP deposition occurred in the dentate gyrus and fewer phospho-TDP-immunopositive neurites were found in family Aus-12 ( Fig. 2f) a Direct DNA sequencing of the coding regions and 50 base pairs of flanking intronic sequences was performed in one unaffected and two affected individuals, using BigDye v3.1 chemistry (Applied Biosystems, CA, USA). Primer sequences and PCR conditions available on request.
Sequencing reactions were run on the Applied Biosystems 3730 DNA Analyser at the Ramaciotti Centre, University of New South Wales, Australia. b All available family members were screened for the C9ORF72 repeat expansion, using techniques described previously [12].