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

The proband (IV:23) was referred to a memory clinic at age 56 with symptoms suggesting cognitive decline in the context of a family history of young-onset dementia. She subsequently visited a clinical genetics service, where a detailed family history was compiled including available medical records. Twelve individuals with dementia and two with ALS were identified over four generations in a pattern consistent with autosomal dominant inheritance. Approval to approach relatives for a family genetics study was granted by the Ethics Committee of the Women’s and Children’s Hospital, Adelaide, and genetic studies were approved by the Ethics Committee of Concord Hospital in Sydney. The next of kin of individuals IV:5 and IV:7 consented to an autopsy study for brain research at the time of death and tissue sections and neuropathological reports for this study were obtained from the South Australian Brain Bank. To determine final diagnoses for IV:5 and IV:7, a retrospective review of their neuropathology was performed using current diagnostic criteria for Alzheimer’s disease, dementia with Lewy bodies, FTLD, ALS, and other neurodegenerative syndromes including CBD, progressive supranuclear palsy, and vascular dementia [8, 10, 15, 17, 22, 24, 26]. Brain tissue from a previously published familial FTLD-TDP case with a R493X GRN mutation [29] and two neuropathologically confirmed CBD FTLD-tau cases, obtained from the Sydney Brain Bank, were examined for comparison.

Immunohistochemistry was performed on formalin-fixed paraffin-embedded 7–10-μm superior frontal cortex, hippocampus or spinal cord sections. We performed standard peroxidase immunohistochemistry with citrate buffer antigen retrieval and 0.5 % cresyl violet counterstaining [19]. Antibodies used were for ubiquitin (Z0458, DAKO, Denmark, diluted 1:200), phosphorylated tau (MN1020, PIERCE, USA, diluted 1:10,000), phosphorylated TDP (BC001487, PTG, USA, diluted 1:500), FUS (HPA008784, Sigma, Australia, diluted 1:1,000), α-internexin (32-3600, ZYMED Laboratories, USA, diluted 1:50) and phosphorylated 200 kD neurofilament (MAS330, Seralab, UK, diluted 1:200). Reaction specificity was confirmed by omitting primary antibodies. Double immunofluorescent labelling was performed to detect phosphorylated tau and phosphorylated TDP. After antigen retrieval, sections were treated for autofluorescence by immersion in 0.25 % potassium permanganate solution for 7 min, rinsed with water and placed in 1 % potassium metabisulphite, 1 % oxalic acid until the sections returned to their original colour. Tau and TDP immunoreactivity was visualised with Alexa Fluor 568 goat anti-rabbit and Alexa Fluor 488 goat anti-mouse secondary antibodies (Invitrogen) on a confocal microscope (C190; Nikon Corporation, Tokyo, Japan). A section without primary antibodies was included for each staining procedure as a negative control. In addition, a mixture of the secondary antibodies was applied to sections with only one primary antibody incubated on each section.

FTLD-TDP was classified into one of four subtypes [21] based on the morphology and laminar distribution of TDP-immunopositive inclusions in the affected brain regions.

Blood was collected from 13 family members and DNA extracted. DNA was screened for mutations in known dementia/ALS genes (Supplementary Table 1). A 10-cM genome-wide scan was performed on DNA from 12 individuals by the Australian Genome Research Facility (AGRF) with microsatellite markers from the ABI-400 set (ABI Prism Linkage Mapping Set, version 2.5, MD-10). Parametric pair-wise and multipoint LOD scores were calculated and simulation analyses were performed using MERLIN v1.1.2 [2]. Autosomal dominant inheritance of a single genetic locus for all clinical variants was assumed with a phenocopy rate of 0.005, a disease gene frequency of 0.001 and equal marker allele frequencies. Seven liability classes were established based on pedigree data with 1 % penetrance—age < 25 years, 8 %—between 26 and 34 years, 22 %—between 35 and 44 years, 46 %—between 45 and 54 years, 71 %—between 55 and 64 years, 91 %—between 65 and 74 years, and 95 %—age > 75 years. Individuals were assigned a liability class based on age of onset for affected cases and age at last consultation for asymptomatic cases.

High-resolution fine mapping of chromosome 16 was performed at the AGRF using microsatellite markers selected from the UCSC Human Genome Browser Gateway (http://​genome.​ucsc.​edu/​cgi-bin/​hgGateway). Primers were fluorescently labelled with 6-FAM and PCR was carried out according to standard protocols. For fine mapping, allele frequencies were derived from a cohort of European ancestry Australian normals [13], from CEPH family data (http://​www.​cephb.​fr/​en/​cephdb/​), or were assumed to be equal if information was unavailable. The novel markers 16GT and 21AC were amplified in-house and amplified products were run on the Applied Biosystems 3730 DNA Analyser at the Ramaciotti Centre, University of New South Wales. We generated allele frequencies for these markers from a panel of 24 unrelated European ancestry healthy controls.

Whole-exome sequencing was performed on one unaffected and four affected Aus-12 family members, using 100-bp paired-end sequencing on the Illumina HiSeq2000 with 40× coverage, by Macrogen (Seoul, Korea).

Family members of pedigree Aus-12 (Fig. 1) were the subjects of detailed clinical review (Table 1). The proband (IV:23) presented with memory impairments and was given a final clinical diagnosis of FTD. The proband’s mother, III:17, died at 61 with dementia; four of her nine siblings were affected with dementia and died before age 65. The proband’s grandmother and great-grandmother were affected with dementia, dying at 64 and 40, respectively. Four first cousins of the proband had presenile dementia; two died with ALS aged 45 and 49 years, respectively; and one was diagnosed with FTD-ALS. Detailed clinical notes for pedigree members IV:23, IV:5, IV:7 and IV:12 are available in Supplementary Material.

Case IV:5 The brain weighed 1,003 g. Microscopy confirmed severe fronto-temporal atrophy with diffuse neuronal loss and reactive gliosis maximal in the temporal poles where status spongiosis was present (Supplementary Table 2). The main immunocytochemical pathology was that of a tauopathy with the phospho-tau (AT8) immunostains showing prominent cytoplasmic neuronal reactivity (“pre-tangles”) and occasional neurofibrillary tangles involving subsets of hippocampal CA1 pyramidal neurons, temporal neocortex, frontal and parietal cortex in association with numerous AT8 immunoreactive neuronal threads (Fig. 2a–c). There was limited overall loss of hippocampal CA1 neurons (Fig. 2e). Many of the dentate granule cells showed phospho-tau cytoplasmic immunoreactivity (Fig. 2c). Occasional phosphorylated neurofilament and tau immunopositive ‘ballooned neurons’ were noted in the frontal and hippocampal sections but this was a rare finding. Phospho-tau immunoreactive astrocytic plaques (Fig. 2d) and coiled fibres were also present. A prominent subset of neurons in the nucleus basalis of Meynert showed AT8 immunoreactive cytoplasmic staining in association with numerous positive neuronal threads. Subsets of neurons and astrocytes in the caudate nucleus showed positive AT8 cytoplasmic immunostaining, whereas in the putamen and globus pallidus astrocyte immunoreactivity was more prominent than neuronal staining. Phospho-TDP-43 immunoreactive NCIs were found in both superficial and deep cortical laminae of the frontal (Fig. 2f, inset) and temporal cortex. A subset of the dentate granule cells showed phospho-TDP-43 immunopositive NCIs (Fig. 2f). Double labelling immunofluorescence revealed that cytoplasmic phospho-tau immunoreactivity and phospho-TDP-43 immunopositive NCIs were found together in approximately 20 % of hippocampal dentate granule cells and 5 % of cortical neurons (Supplementary Fig. 1c, f, j, k). While many neurons and glia contained AT8 immunoreactivity (Supplementary Fig. 1a, d, g), a minority of phospho-TDP-43 immunopositive NCIs were found in neurons that were not immunoreactive for phospho-tau (Supplementary Fig. 1c, k). No FUS or α-internexin immunoreactive inclusion pathology was observed. In the midbrain there was neuronal loss, depigmentation and gliosis of the substantia nigra. A subset of surviving substantia nigra neurons showed cytoplasmic AT8 immunoreactivity, as did scattered periaqueductal neurons in association with immunoreactive neurites. The AT8 immunostains also showed intracytoplasmic staining in neurons of the locus coeruleus, scattered neurons of midline raphe nuclei, dorsal motor vagal nuclei, nuclei ambiguii and reticular formation in association with immunoreactive neurites. The findings are those of CBD FTLD-tau associated with type B FTLD-TDP without significant bulbar motor neuron involvement.

Case IV:7 The brain weighed 1,160 g. There was more severe frontotemporal atrophy in this case, and the substantia nigra was severely depigmented. Microscopy showed a similar pattern and type of neuropathological features to those described above but with greater neuronal loss (Supplementary Table 2), consistent with more advanced CBD FTLD-tau and type B FTLD-TDP. Western blot analysis of the sarkosyl-insoluble fraction of frontal pole tissue revealed that the insoluble tau species present in this case was consistent with a 4-repeat tauopathy, as seen with other CBD cases [5, 34] (Supplementary Fig. 2). The spinal cord was also available in this case and segmental sections showed preservation of the corticospinal tracts (myelin stain). There was patchy loss of anterior horn cells in the cervical and thoracic spinal cord segments and ubiquitinated, neurofilament-immunoreactive motor neurons, neurites (Fig. 2g, h) and NCI (Fig. 2g, inset) were observed in these sections, along with rare phospho-tau immunoreactivity (Fig. 2i). Phospho-TDP immunoreactivity was restricted to neurites and granular staining in small neurons (Fig. 2j) and absent in anterior horn cells. No FUS or α-internexin immunoreactivity was observed in these spinal cord sections.

The phospho-tau neuropathology of Aus-12 cases closely resembled that in the CBD FTLD-tau cases examined. The TDP-43 neuropathology was similar in amount and distribution but not structural type to that observed in the GRN mutation-positive FTLD-TDP case. Greater deposition in the dentate gyrus and fewer phospho-TDP-positive neurites was observed in the Aus-12 cases (Supplementary Material). The motor neuron involvement was mild compared with classic end-stage ALS cases and was without TDP or FUS deposition, although ubiquitinated inclusions were present (as has been observed in other familial ALS cases negative for known mutations) [23].

DNA from IV:5, IV:6 and IV:24 was subjected to DNA sequence analysis of the coding regions and flanking intronic sequences for known dementia and ALS genes (Supplementary Table 1). No mutations were detected.

We undertook a genome-wide linkage analysis on 12 pedigree members, five of whom were classed as affected. Six regions generated two-point LOD scores ≥1, on chromosomes 3 (D3S1285, LOD = 1.3), 12 (D12S352, 1.0), 15 (D15S117, 1.5; D15S127, 1.1), 16 (D16S415, 1.2) and 20 (D20S107, 1.4); however, with multipoint analysis the only regions with a LOD score ≥1 were on chromosome 16 (D16S415, LOD = 2.7; D16S516, 1.1). Examination of haplotypes revealed that affected individual IV-23 was identical by state rather than by descent at D16S516: this individual harboured the same haplotype as unaffected sibling IV-24 at D16S516 and flanking markers D16S515 and D16S3091 (data not shown). In contrast, alleles at D16S415 and upstream markers D16S3046, D16S3068 and D16S3136 were shared by all affected individuals. This region on chromosome 16 was therefore fine mapped with 21 additional markers. Supplementary Table 3 details two-point LOD scores, showing a maximum of 2.4 (θ = 0) at D16S3396. Gene-dropping simulation analyses indicated that two chromosome 16 microsatellite markers exceeded the genome-wide 1 % significance threshold for 100 simulations. Multipoint analysis with MERLIN yielded a maximum LOD score of 2.9 for all markers from D16S753 to D16S2623 inclusive and excluded linkage (LOD < −2) at other FTD loci (CHMP2B, C9ORF72, VCP, GRN/MAPT) (Supplementary Fig. 3).

Haplotypes were constructed for the 16p12.1–q12.2 region in Aus-12 (Fig. 1). Recombinations in this family defined a 37.9-Mb region flanked by the markers D16S3103 and D16S489, in which a common haplotype was shared by all affected individuals (Fig. 3b). A smaller suggestive disease haplotype spanning 24.4 Mb was defined by recombination at D16S753 in elderly unaffected individual III:15. This region overlaps with a separate locus on 16q12.1–q12.2 reported in an ALS family by Abalkhail et al. [1] (Fig. 3b).

We performed whole-exome sequencing of affected individuals IV:5, IV:6, IV:17, IV:23 and elderly unaffected individual III:15. No pathogenic variants were identified in known dementia or ALS genes, including FUS on chromosome 16 (Supplementary Table 1). We identified seven variants from the exome sequencing data within the Aus-12 maximal critical region that were absent from dbSNP131, and were detected in at least 2 affected individuals and resequenced them in all 13 Aus-12 DNA samples. Four variants were present in all affected individuals and absent in the elderly unaffected individual, two of which were also absent from public databases of normal human variation (Table 2). One of these variants, g.48576222C>A, was detected in 2/934 healthy Australian individuals of European ancestry by examination of exome sequencing data (Dr Paul Leo and Prof Matthew Brown, personal communication). The second variant, g.50825515A>G, leads to the substitution of methionine to valine at amino acid position 719 of the cylindromatosis protein, CYLD. This variant is present within the region of overlap with the chromosome 16q12.1—linked ALS pedigree [1] (Table 2).

We used nine rare variants detected by whole-exome sequencing and confirmed by Sanger sequencing to repeat the linkage analysis. This generated a maximum two-point LOD score of 2.7 for markers g.31484758G>A, g.48576222C>A, g.50825515A>G and g.53721944A>G (Supplementary Table 3) and a maximum multipoint LOD score of 3.0 for markers 16GT through to g.53721944A>G inclusive (Fig. 3a).