Nicola J. Rutherford

Expanded GGGGCC Hexanucleotide Repeat in Noncoding Region of C9ORF72 Causes Chromosome 9p-Linked FTD and ALS

Several families have been reported with autosomal-dominant frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), genetically linked to chromosome 9p21. Here, we report an expansion of a noncoding GGGGCC hexanucleotide repeat in the gene C9ORF72 that is strongly associated with disease in a large FTD/ALS kindred, previously reported to be conclusively linked to chromosome 9p. This same repeat expansion was identified in the majority of our families with a combined FTD/ALS phenotype and TDP-43-based pathology. Analysis of extended clinical series found the C9ORF72 repeat expansion to be the most common genetic abnormality in both familial FTD (11.7%) and familial ALS (23.5%). The repeat expansion leads to the loss of one alternatively spliced C9ORF72 transcript and to formation of nuclear RNA foci, suggesting multiple disease mechanisms. Our findings indicate that repeat expansion in C9ORF72 is a major cause of both FTD and ALS.

Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis

The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43–positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the ∼25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.

Neuroimaging signatures of frontotemporal dementia genetics: C9ORF72, tau, progranulin and sporadics.

A major recent discovery was the identification of an expansion of a non-coding GGGGCC hexanucleotide repeat in the C9ORF72 gene in patients with frontotemporal dementia and amyotrophic lateral sclerosis. Mutations in two other genes are known to account for familial frontotemporal dementia: microtubule-associated protein tau and progranulin. Although imaging features have been previously reported in subjects with mutations in tau and progranulin, no imaging features have been published in C9ORF72. Furthermore, it remains unknown whether there are differences in atrophy patterns across these mutations, and whether regional differences could help differentiate C9ORF72 from the other two mutations at the single-subject level. We aimed to determine the regional pattern of brain atrophy associated with the C9ORF72 gene mutation, and to determine which regions best differentiate C9ORF72 from subjects with mutations in tau and progranulin, and from sporadic frontotemporal dementia. A total of 76 subjects, including 56 with a clinical diagnosis of behavioural variant frontotemporal dementia and a mutation in one of these genes (19 with C9ORF72 mutations, 25 with tau mutations and 12 with progranulin mutations) and 20 sporadic subjects with behavioural variant frontotemporal dementia (including 50% with amyotrophic lateral sclerosis), with magnetic resonance imaging were included in this study. Voxel-based morphometry was used to assess and compare patterns of grey matter atrophy. Atlas-based parcellation was performed utilizing the automated anatomical labelling atlas and Statistical Parametric Mapping software to compute volumes of 37 regions of interest. Hemispheric asymmetry was calculated. Penalized multinomial logistic regression was utilized to create a prediction model to discriminate among groups using regional volumes and asymmetry score. Principal component analysis assessed for variance within groups. C9ORF72 was associated with symmetric atrophy predominantly involving dorsolateral, medial and orbitofrontal lobes, with additional loss in anterior temporal lobes, parietal lobes, occipital lobes and cerebellum. In contrast, striking anteromedial temporal atrophy was associated with tau mutations and temporoparietal atrophy was associated with progranulin mutations. The sporadic group was associated with frontal and anterior temporal atrophy. A conservative penalized multinomial logistic regression model identified 14 variables that could accurately classify subjects, including frontal, temporal, parietal, occipital and cerebellum volume. The principal component analysis revealed similar degrees of heterogeneity within all disease groups. Patterns of atrophy therefore differed across subjects with C9ORF72, tau and progranulin mutations and sporadic frontotemporal dementia. Our analysis suggested that imaging has the potential to be useful to help differentiate C9ORF72 from these other groups at the single-subject level.

Characterization of frontotemporal dementia and/or amyotrophic lateral sclerosis associated with the GGGGCC repeat expansion in C9ORF72

Numerous kindreds with familial frontotemporal dementia and/or amyotrophic lateral sclerosis have been linked to chromosome 9, and an expansion of the GGGGCC hexanucleotide repeat in the non-coding region of chromosome 9 open reading frame 72 has recently been identified as the pathogenic mechanism. We describe the key characteristics in the probands and their affected relatives who have been evaluated at Mayo Clinic Rochester or Mayo Clinic Florida in whom the hexanucleotide repeat expansion were found. Forty-three probands and 10 of their affected relatives with DNA available (total 53 subjects) were shown to carry the hexanucleotide repeat expansion. Thirty-six (84%) of the 43 probands had a familial disorder, whereas seven (16%) appeared to be sporadic. Among examined subjects from the 43 families (n = 63), the age of onset ranged from 33 to 72 years (median 52 years) and survival ranged from 1 to 17 years, with the age of onset <40 years in six (10%) and >60 in 19 (30%). Clinical diagnoses among examined subjects included behavioural variant frontotemporal dementia with or without parkinsonism (n = 30), amyotrophic lateral sclerosis (n = 18), frontotemporal dementia/amyotrophic lateral sclerosis with or without parkinsonism (n = 12), and other various syndromes (n = 3). Parkinsonism was present in 35% of examined subjects, all of whom had behavioural variant frontotemporal dementia or frontotemporal dementia/amyotrophic lateral sclerosis as the dominant clinical phenotype. No subject with a diagnosis of primary progressive aphasia was identified with this mutation. Incomplete penetrance was suggested in two kindreds, and the youngest generation had significantly earlier age of onset (>10 years) compared with the next oldest generation in 11 kindreds. Neuropsychological testing showed a profile of slowed processing speed, complex attention/executive dysfunction, and impairment in rapid word retrieval. Neuroimaging studies showed bilateral frontal abnormalities most consistently, with more variable degrees of parietal with or without temporal changes; no case had strikingly focal or asymmetric findings. Neuropathological examination of 14 patients revealed a range of transactive response DNA binding protein molecular weight 43 pathology (10 type A and four type B), as well as ubiquitin-positive cerebellar granular neuron inclusions in all but one case. Motor neuron degeneration was detected in nine patients, including five patients without ante-mortem signs of motor neuron disease. While variability exists, most cases with this mutation have a characteristic spectrum of demographic, clinical, neuropsychological, neuroimaging and especially neuropathological findings.

Common variation in the miR-659 binding-site of GRN is a major risk factor for TDP43-positive frontotemporal dementia

Loss-of-function mutations in progranulin (GRN) cause ubiquitin- and TAR DNA-binding protein 43 (TDP-43)-positive frontotemporal dementia (FTLD-U), a progressive neurodegenerative disease affecting ∼10% of early-onset dementia patients. Here we expand the role of GRN in FTLD-U and demonstrate that a common genetic variant (rs5848), located in the 3′-untranslated region (UTR) of GRN in a binding-site for miR-659, is a major susceptibility factor for FTLD-U. In a series of pathologically confirmed FTLD-U patients without GRN mutations, we show that carriers homozygous for the T-allele of rs5848 have a 3.2-fold increased risk to develop FTLD-U compared with homozygous C-allele carriers (95% CI: 1.50–6.73). We further demonstrate that miR-659 can regulate GRN expression in vitro, with miR-659 binding more efficiently to the high risk T-allele of rs5848 resulting in augmented translational inhibition of GRN. A significant reduction in GRN protein was observed in homozygous T-allele carriers in vivo, through biochemical and immunohistochemical methods, mimicking the effect of heterozygous loss-of-function GRN mutations. In support of these findings, the neuropathology of homozygous rs5848 T-allele carriers frequently resembled the pathological FTLD-U subtype of GRN mutation carriers. We suggest that the expression of GRN is regulated by miRNAs and that common genetic variability in a miRNA binding-site can significantly increase the risk for FTLD-U. Translational regulation by miRNAs may represent a common mechanism underlying complex neurodegenerative disorders.

Mutations in the colony stimulating factor 1 receptor ( CSF1R ) gene cause hereditary diffuse leukoencephalopathy with spheroids

Hereditary diffuse leukoencephalopathy with spheroids (HDLS) is an autosomal-dominant central nervous system white-matter disease with variable clinical presentations, including personality and behavioral changes, dementia, depression, parkinsonism, seizures and other phenotypes. We combined genome-wide linkage analysis with exome sequencing and identified 14 different mutations affecting the tyrosine kinase domain of the colony stimulating factor 1 receptor (encoded by CSF1R) in 14 families with HDLS. In one kindred, we confirmed the de novo occurrence of the mutation. Follow-up sequencing identified an additional CSF1R mutation in an individual diagnosed with corticobasal syndrome. In vitro, CSF-1 stimulation resulted in rapid autophosphorylation of selected tyrosine residues in the kinase domain of wild-type but not mutant CSF1R, suggesting that HDLS may result from partial loss of CSF1R function. As CSF1R is a crucial mediator of microglial proliferation and differentiation in the brain, our findings suggest an important role for microglial dysfunction in HDLS pathogenesis.

Clinical and neuropathologic heterogeneity of c9FTD/ALS associated with hexanucleotide repeat expansion in C9ORF72

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are part of a disease spectrum associated with TDP-43 pathology. Strong evidence supporting this is the existence of kindreds with family members affected by FTD, ALS or mixed features of FTD and ALS, referred to as FTD-MND. Some of these families have linkage to chromosome 9, with hexanucleotide expansion mutation in a noncoding region of C9ORF72. Discovery of the mutation defines c9FTD/ALS. Prior to discovery of mutations in C9ORF72, it was assumed that TDP-43 pathology in c9FTD/ALS was uniform. In this study, we examined the neuropathology and clinical features of 20 cases of c9FTD/ALS from a brain bank for neurodegenerative disorders. Included are six patients clinically diagnosed with ALS, eight FTD, one FTD-MND and four Alzheimer-type dementia. Clinical information was unavailable for one patient. Pathologically, the cases all had TDP-43 pathology, but there were three major pathologic groups: ALS, FTLD-MND and FTLD-TDP. The ALS cases were morphologically similar to typical sporadic ALS with almost no extramotor TDP-43 pathology; all had oligodendroglial cytoplasmic inclusions. The FTLD-MND showed predominantly Mackenzie Type 3 TDP-43 pathology, and all had ALS-like pathology in motor neurons, but more extensive extramotor pathology, with oligodendroglial cytoplasmic inclusions and infrequent hippocampal sclerosis. The FTLD-TDP cases had several features similar to FTLD-TDP due to mutations in the gene for progranulin, including Mackenzie Type 1 TDP-43 pathology with neuronal intranuclear inclusions and hippocampal sclerosis. FTLD-TDP patients were older and some were thought to have Alzheimer-type dementia. In addition to the FTD and ALS clinical presentations, the present study shows that c9FTD/ALS can have other presentations, possibly related to age of onset and the presence of hippocampal sclerosis. Moreover, there is pathologic heterogeneity not only between ALS and FTLD, but also within the FTLD group. Further studies are needed to address the molecular mechanism of clinical and pathological heterogeneity of c9FTD/ALS due to mutations in C9ORF72.

Clinical, neuroimaging and neuropathological features of a new chromosome 9p-linked FTD-ALS family

Background Frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) is a heritable form of FTD, but the gene(s) responsible for the majority of autosomal dominant FTD-ALS cases have yet to be found. Previous studies have identified a region on chromosome 9p that is associated with FTD and ALS. Methods The authors report the clinical, volumetric MRI, neuropathological and genetic features of a new chromosome 9p-linked FTD-ALS family, VSM-20. Results Ten members of family VSM-20 displayed heterogeneous clinical phenotypes of isolated behavioural-variant FTD (bvFTD), ALS or a combination of the two. Parkinsonism was common, with one individual presenting with a corticobasal syndrome. Analysis of structural MRI scans from five affected family members revealed grey- and white-matter loss that was most prominent in the frontal lobes, with mild parietal and occipital lobe atrophy, but less temporal lobe atrophy than in 10 severity-matched sporadic bvFTD cases. Autopsy in three family members showed a consistent and unique subtype of FTLD-TDP pathology. Genome-wide linkage analysis conclusively linked family VSM-20 to a 28.3 cM region between D9S1808 and D9S251 on chromosome 9p, reducing the published minimal linked region to a 3.7 Mb interval. Genomic sequencing and expression analysis failed to identify mutations in the 10 known and predicted genes within this candidate region, suggesting that next-generation sequencing may be needed to determine the mutational mechanism associated with chromosome 9p-linked FTD-ALS. Conclusions Family VSM-20 significantly reduces the region linked to FTD-ALS on chromosome 9p. A distinct pattern of brain atrophy and neuropathological findings may help to identify other families with FTD-ALS caused by this genetic abnormality.

Clinical and pathological features of familial frontotemporal dementia caused by C9ORF72 mutation on chromosome 9p

Frontotemporal dementia and amyotrophic lateral sclerosis are closely related clinical syndromes with overlapping molecular pathogenesis. Several families have been reported with members affected by frontotemporal dementia, amyotrophic lateral sclerosis or both, which show genetic linkage to a region on chromosome 9p21. Recently, two studies identified the FTD/ALS gene defect on chromosome 9p as an expanded GGGGCC hexanucleotide repeat in a non-coding region of the chromosome 9 open reading frame 72 gene (C9ORF72). In the present study, we provide detailed analysis of the clinical features and neuropathology for 16 unrelated families with frontotemporal dementia caused by the C9ORF72 mutation. All had an autosomal dominant pattern of inheritance. Eight families had a combination of frontotemporal dementia and amyotrophic lateral sclerosis while the other eight had a pure frontotemporal dementia phenotype. Clinical information was available for 30 affected members of the 16 families. There was wide variation in age of onset (mean = 54.3, range = 34-74 years) and disease duration (mean = 5.3, range = 1-16 years). Early diagnoses included behavioural variant frontotemporal dementia (n = 15), progressive non-fluent aphasia (n = 5), amyotrophic lateral sclerosis (n = 9) and progressive non-fluent aphasia-amyotrophic lateral sclerosis (n = 1). Heterogeneity in clinical presentation was also common within families. However, there was a tendency for the phenotypes to converge with disease progression; seven subjects had final clinical diagnoses of both frontotemporal dementia and amyotrophic lateral sclerosis and all of those with an initial progressive non-fluent aphasia diagnosis subsequently developed significant behavioural abnormalities. Twenty-one affected family members came to autopsy and all were found to have transactive response DNA binding protein with M(r) 43 kD (TDP-43) pathology in a wide neuroanatomical distribution. All had involvement of the extramotor neocortex and hippocampus (frontotemporal lobar degeneration-TDP) and all but one case (clinically pure frontotemporal dementia) had involvement of lower motor neurons, characteristic of amyotrophic lateral sclerosis. In addition, a consistent and relatively specific pathological finding was the presence of neuronal inclusions in the cerebellar cortex that were ubiquitin/p62-positive but TDP-43-negative. Our findings indicate that the C9ORF72 mutation is a major cause of familial frontotemporal dementia with TDP-43 pathology, that likely accounts for the majority of families with combined frontotemporal dementia/amyotrophic lateral sclerosis presentation, and further support the concept that frontotemporal dementia and amyotrophic lateral sclerosis represent a clinicopathological spectrum of disease with overlapping molecular pathogenesis.

TMEM106B regulates progranulin levels and the penetrance of FTLD in GRN mutation carriers

Objectives: To determine whether TMEM106B single nucleotide polymorphisms (SNPs) are associated with frontotemporal lobar degeneration (FTLD) in patients with and without mutations in progranulin (GRN) and to determine whether TMEM106B modulates GRN expression. Methods: We performed a case-control study of 3 SNPs in TMEM106B in 482 patients with clinical and 80 patients with pathologic FTLD–TAR DNA-binding protein 43 without GRN mutations, 78 patients with FTLD with GRN mutations, and 822 controls. Association analysis of TMEM106B with GRN plasma levels was performed in 1,013 controls and TMEM106B and GRN mRNA expression levels were correlated in peripheral blood samples from 33 patients with FTLD and 150 controls. Results: In our complete FTLD patient cohort, nominal significance was identified for 2 TMEM106B SNPs (top SNP rs1990622, pallelic = 0.036). However, the most significant association with risk of FTLD was observed in the subgroup of GRN mutation carriers compared to controls (corrected pallelic = 0.0009), where there was a highly significant decrease in the frequency of homozygote carriers of the minor alleles of all TMEM106B SNPs (top SNP rs1990622, CC genotype frequency 2.6% vs 19.1%, corrected precessive = 0.009). We further identified a significant association of TMEM106B SNPs with plasma GRN levels in controls (top SNP rs1990622, corrected p = 0.002) and in peripheral blood samples a highly significant correlation was observed between TMEM106B and GRN mRNA expression in patients with FTLD (r = −0.63, p = 7.7 × 10−5) and controls (r = −0.49, p = 2.2 × 10−10). Conclusions: In our study, TMEM106B SNPs significantly reduced the disease penetrance in patients with GRN mutations, potentially by modulating GRN levels. These findings hold promise for the development of future protective therapies for FTLD.