Clinical syndromes in family VSM-20
The clinical features of 10 family members who met research criteria for bvFTD and/or ALS are summarised in . The family is of Irish ancestry. The mean age of onset was 45.7±7.0 years. The mean duration of symptoms was 5.4±3.0 years. Three individuals had a bvFTD phenotype with no clear evidence of motor impairments, while two family members had bvFTD with mild Parkinsonism (primarily elevated tone) on physical exam. Two individuals had a pure limb-onset ALS phenotype with minimal cognitive or behavioural impairments. Three individuals displayed a combination of bvFTD symptoms and ALS, with one individual presenting with apraxia and Parkinsonism consistent with a corticobasal syndrome (CBS, III-7). Detailed descriptions of examples of each of the three main phenotypes are included as supplementary case descriptions
Clinical heterogeneity in affected members of family VSM-20
Patterns of brain atrophy in VSM-20 compared with sporadic bvFTD and controls
Lobar brain volumes were derived in five affected family members (four predominantly bvFTD (II-8, III-2, III-3, and III-15), one predominantly ALS (III-9)) as compared with 10 sporadic bvFTD cases, matched for CDR-SB scores to the family members, and 13 normal control subjects. Atrophy was more extensive in VSM-20 members with isolated bvFTD (II-8, ) as compared with the family member with pure ALS (III-9, ).
Figure 2 Patterns of brain atrophy in two VSM-20 clinical phenotypes: coronal T1-weighted MRI section at MNI coordinate y=14, and Freesurfer-derived cortical thickness maps in (A) a 54-year-old behavioural-variant frontotemporal dementia subject (II-8) as compared (more ...)
Cortical volumes were reduced in the VSM-20 family members and the sporadic bvFTD group as compared with controls (, ). This volume loss was reflected in decreased cortical grey- and white-matter volume in VSM-20, but only decreased grey-matter volume in the sporadic bvFTD group. The patterns of cortical volume loss were also different in the VSM-20 members as compared with the sporadic bvFTD group. Whereas both groups displayed bilateral frontal lobe atrophy as compared with controls, only the sporadic bvFTD displayed temporal lobe atrophy. In contrast, only the VSM-20 family members displayed subtle parietal and right occipital lobe atrophy as compared with controls ().
Patterns of brain atrophy in affected VSM-20 family members
A detailed summary of neuropathological data from the three affected family members (II-7, II-8 and III-7) is provided in supplemental table S3
. All three cases showed similar pathology that varied only in severity. The brain weights were reduced (mean weight=1050 g) and showed consistent atrophy of the frontal lobes but variable involvement of temporal and parietal regions ().
Figure 3 Post-mortem neuropathology in affected members of VMS-20. Cerebral atrophy was most consistent in the frontal lobes (A, case II-8). The corticospinal tracts showed loss of myelin staining (B, case III-7). Ubiquitin- and TDP-43-immunoreactive neuronal (more ...)
Affected regions of cerebral cortex showed chronic degenerative changes with neuronal loss, reactive gliosis and superficial laminar spongiosis. The corticospinal tracts showed decreased myelin staining at all levels of brainstem and spinal cord, most notably in case III-7 (). IHC for ubiquitin, p62 and TDP-43 demonstrated various types of neuronal cytoplasmic inclusions (NCI) in all neocortical layers (). The predominant morphology was dense round bodies, approximately the size of the nucleus, with either a smooth contour or an appearance of aggregated coarse granules (). In addition, layer II neocortex contained many neurons with diffuse cytoplasmic granular reactivity for TDP-43 (preinclusions) (). Swollen dystrophic neurites were moderately common in deeper cortical layers and were better demonstrated with ubiquitin and p62 IHC than with TDP-43 (). Rare TDP-43-immunoreactive glial cytoplasmic inclusions (GCI) were present in the subcortical white matter (). These pathological changes were present in all neocortical regions examined, with the frontal and temporal lobes more affected than parietal and occipital.
Both compact and granular ubiquitin/p62/TDP-43-positive NCI were numerous in dentate granule cells of all cases (). Hippocampal pyramidal neurons often contained ill-defined aggregates of fine granules that were ubiquitin- and p62-immunoreactive but only rarely TDP-43-positive (). Lower motor neurons in the brainstem and spinal cord frequently contained NCI that were reactive for ubiquitin, p62 and TDP-43 (). Finally, the granule cell layer of the cerebellar cortex contained numerous small round NCI and short neurites that were only demonstrated with ubiquitin and p62 IHC (). Neuronal intranuclear inclusions were not identified in any of the cases.
IHC for tau, Aβ, α-synuclein and FUS proteins did not show any specific pathological changes beyond those expected for patient age. Specifically, ubiquitin/p62-immunoreactive, TDP-43-negative NCI and neurites did not label for any of these other proteins.
Genome-wide linkage analysis and identification of minimal linked region
Genome-wide linkage analysis in family VSM-20 identified only one region of suggestive linkage at chromosome 9p, with a maximum two-point LOD-score of 2.59 at D9S1870 (θ=0). A total of six flanking STR markers from the genome scan also showed LOD-scores >1. To fine-map the candidate region at 9p, we analysed six STR markers from the genome scan and an additional 10 STR markers in a 33.1 cM interval flanked by markers D9S1808 and D9S1817 in all family members and reanalysed genotypes. At this time, individual III-15 was re-evaluated and found to meet clinical criteria for bvFTD and therefore was considered affected in all subsequent analysis. The highest two-point LOD-score, 3.01, was obtained with marker D9S1870 in the absence of recombinants (). A disease haplotype was present in all patients, and obligate recombinants defined a candidate region of 28.3 cM corresponding to 14.2 Mb between markers D9S1808 and D9S251 (). This candidate region overlaps with the previously reported region linked to the combined phenotype of FTD and ALS on chromosome 9p10–15
and significantly reduces the candidate region to a 3.7 Mb interval between D9S169 and D9S251, containing 10 known and predicted genes ().
Two-point LOD scores for 9p markers in family VSM-20
Figure 4 Disguised linkage pedigree of family VSM-20. Black symbols represent patients affected with behavioural-variant frontotemporal dementia (left side filled), amyotrophic lateral sclerosis (right side filled) or both. White symbols represent unaffected individuals (more ...)
Figure 5 Schematic presentation of chromosome 9p FTD-ALS locus. Grey bars indicate the minimal candidate regions on chromosome 9p in family VSM-20 and all other previously reported 9p-linked FTD-ALS families. Together, these studies define a 3.7 Mb interval between (more ...)
Mutation analysis of candidate genes
Bioinformatic analysis of the 3.7 Mb minimum candidate interval identified five protein coding genes (c9orf11
), two micro RNA genes (miR-873 and miR-876), a large non-protein coding RNA gene (NCRNA00032) and two predicted genes (AK074231 and RBMXP2
) (). No mutations were identified by direct gDNA sequencing in the exons (coding and non-coding), flanking intronic sequences and 3′ untranslated regions of the five protein coding genes or in the predicted exons of the other five genes. Furthermore, cDNA splicing analysis of the two genes with multiple coding exons and expression in cerebellum (MOBKL2B
) did not identify altered splicing patterns or small deletions or duplications in the RT-PCR products. Mutations in the coding exons of an additional 13 genes located within the larger candidate region identified in family VSM-20 alone were also excluded by gDNA sequencing (supplemental table S4
). Pathogenic mutations in GRN
were also excluded.
Analysis of three affected members of family VSM-20 on the Affymetrix genome-wide human SNP array 6.0 did not identify differences in gene dosage within the chromosome 9p linked region. In addition, complete genomic deletions or multiplications of c9orf11, MOBKL2B, IFNK, c9orf72 and LINGO2 were excluded by quantitative real-time PCR.