Aleksandra Wojtas

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.

TREM2 Variants in Alzheimer's Disease

BACKGROUND Homozygous loss-of-function mutations in TREM2, encoding the triggering receptor expressed on myeloid cells 2 protein, have previously been associated with an autosomal recessive form of early-onset dementia. METHODS We used genome, exome, and Sanger sequencing to analyze the genetic variability in TREM2 in a series of 1092 patients with Alzheimers disease and 1107 controls (the discovery set). We then performed a meta-analysis on imputed data for the TREM2 variant rs75932628 (predicted to cause a R47H substitution) from three genomewide association studies of Alzheimers disease and tested for the association of the variant with disease. We genotyped the R47H variant in an additional 1887 cases and 4061 controls. We then assayed the expression of TREM2 across different regions of the human brain and identified genes that are differentially expressed in a mouse model of Alzheimers disease and in control mice. RESULTS We found significantly more variants in exon 2 of TREM2 in patients with Alzheimers disease than in controls in the discovery set (P=0.02). There were 22 variant alleles in 1092 patients with Alzheimers disease and 5 variant alleles in 1107 controls (P<0.001). The most commonly associated variant, rs75932628 (encoding R47H), showed highly significant association with Alzheimers disease (P<0.001). Meta-analysis of rs75932628 genotypes imputed from genomewide association studies confirmed this association (P=0.002), as did direct genotyping of an additional series of 1887 patients with Alzheimers disease and 4061 controls (P<0.001). Trem2 expression differed between control mice and a mouse model of Alzheimers disease. CONCLUSIONS Heterozygous rare variants in TREM2 are associated with a significant increase in the risk of Alzheimers disease. (Funded by Alzheimers Research UK and others.).

Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS

Algorithms designed to identify canonical yeast prions predict that around 250 human proteins, including several RNA-binding proteins associated with neurodegenerative disease, harbour a distinctive prion-like domain (PrLD) enriched in uncharged polar amino acids and glycine. PrLDs in RNA-binding proteins are essential for the assembly of ribonucleoprotein granules. However, the interplay between human PrLD function and disease is not understood. Here we define pathogenic mutations in PrLDs of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1 in families with inherited degeneration affecting muscle, brain, motor neuron and bone, and in one case of familial amyotrophic lateral sclerosis. Wild-type hnRNPA2 (the most abundant isoform of hnRNPA2B1) and hnRNPA1 show an intrinsic tendency to assemble into self-seeding fibrils, which is exacerbated by the disease mutations. Indeed, the pathogenic mutations strengthen a ‘steric zipper’ motif in the PrLD, which accelerates the formation of self-seeding fibrils that cross-seed polymerization of wild-type hnRNP. Notably, the disease mutations promote excess incorporation of hnRNPA2 and hnRNPA1 into stress granules and drive the formation of cytoplasmic inclusions in animal models that recapitulate the human pathology. Thus, dysregulated polymerization caused by a potent mutant steric zipper motif in a PrLD can initiate degenerative disease. Related proteins with PrLDs should therefore be considered candidates for initiating and perhaps propagating proteinopathies of muscle, brain, motor neuron and bone.

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, 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.

De Novo Truncating FUS Gene Mutation as a Cause of Sporadic Amyotrophic Lateral Sclerosis

Mutations in the gene encoding fused in sarcoma (FUS) were recently identified as a novel cause of amyotrophic lateral sclerosis (ALS), emphasizing the genetic heterogeneity of ALS. We sequenced the genes encoding superoxide dismutase (SOD1), TAR DNA‐binding protein 43 (TARDBP) and FUS in 99 sporadic and 17 familial ALS patients ascertained at Mayo Clinic. We identified two novel mutations in FUS in two out of 99 (2.0%) sporadic ALS patients and established the de novo occurrence of one FUS mutation. In familial patients, we identified three (17.6%) SOD1 mutations, while FUS and TARDBP mutations were excluded. The de novo FUS mutation (g.10747A>G; IVS13‐2A>G) affects the splice‐acceptor site of FUS intron 13 and was shown to induce skipping of FUS exon 14 leading to the C‐terminal truncation of FUS (p.G466VfsX14). Subcellular localization studies showed a dramatic increase in the cytoplasmic localization of FUS and a reduction of normal nuclear expression in cells transfected with truncated compared to wild‐type FUS. We further identified a novel in‐frame insertion/deletion mutation in FUS exon 12 (p.S402_P411delinsGGGG) which is predicted to expand a conserved poly‐glycine motif. Our findings extend the mutation spectrum in FUS leading to ALS and describe the first de novo mutation in FUS.

Regional distribution of synaptic markers and APP correlate with distinct clinicopathological features in sporadic and familial Alzheimer's disease.

Recent studies suggest that subcortical structures, including striatum, are vulnerable to amyloid-β accumulation and other neuropathological features in familial Alzheimers disease due to autosomal dominant mutations. We explored differences between familial and sporadic Alzheimers disease that might shed light on their respective pathogenic mechanisms. To this end, we analysed 12 brain regions, including neocortical, limbic and subcortical areas, from post-mortem brains of familial Alzheimers disease (n = 10; age at death: 50.0 ± 8.6 years) with mutations in amyloid precursor protein (APP) or presenilin 1 (PSEN1), sporadic Alzheimers disease (n = 19; age at death: 84.7 ± 7.8 years), neurologically normal elderly without amyloid-β accumulation (normal ageing; n = 13, age at death: 82.9 ± 10.8 years) and neurologically normal elderly with extensive cortical amyloid-β deposits (pathological ageing; n = 15; age at death: 92.7 ± 5.9 years). The levels of amyloid-β₄₀, amyloid-β₄₂, APP, apolipoprotein E, the synaptic marker PSD95 (now known as DLG4), the astrocyte marker GFAP, other molecules related to amyloid-β metabolism, and tau were determined by enzyme-linked immunosorbent assays. We observed that familial Alzheimers disease had disproportionate amyloid-β₄₂ accumulation in subcortical areas compared with sporadic Alzheimers disease, whereas sporadic Alzheimers disease had disproportionate amyloid-β₄₂ accumulation in cortical areas compared to familial Alzheimers disease. Compared with normal ageing, the levels of several proteins involved in amyloid-β metabolism were significantly altered in both sporadic and familial Alzheimers disease; however, such changes were not present in pathological ageing. Among molecules related to amyloid-β metabolism, the regional distribution of PSD95 strongly correlated with the regional pattern of amyloid-β₄₂ accumulation in sporadic Alzheimers disease and pathological ageing, whereas the regional distribution of APP as well as β-C-terminal fragment of APP were strongly associated with the regional pattern of amyloid-β₄₂ accumulation in familial Alzheimers disease. Apolipoprotein E and GFAP showed negative regional association with amyloid-β (especially amyloid-β₄₀) accumulation in both sporadic and familial Alzheimers disease. Familial Alzheimers disease had greater striatal tau pathology than sporadic Alzheimers disease. In a retrospective medical record review, atypical signs and symptoms were more frequent in familial Alzheimers disease compared with sporadic Alzheimers disease. These results suggest that disproportionate amyloid-β₄₂ accumulation in cortical areas in sporadic Alzheimers disease may be mediated by synaptic processes, whereas disproportionate amyloid-β₄₂ accumulation in subcortical areas in familial Alzheimers disease may be driven by APP and its processing. Region-specific amyloid-β₄₂ accumulation might account for differences in the relative amounts of tau pathology and clinical symptoms in familial and sporadic Alzheimers disease.

TMEM106B p.T185S regulates TMEM106B protein levels: implications for frontotemporal dementia.

Frontotemporal lobar degeneration (FTLD) is the second leading cause of dementia in individuals under age 65. In many patients, the predominant pathology includes neuronal cytoplasmic or intranuclear inclusions of ubiquitinated TAR DNA binding protein 43 (FTLD‐TDP). Recently, a genome‐wide association study identified the first FTLD‐TDP genetic risk factor, in which variants in and around the TMEM106B gene (top SNP rs1990622) were significantly associated with FTLD‐TDP risk. Intriguingly, the most significant association was in FTLD‐TDP patients carrying progranulin (GRN) mutations. Here, we investigated to what extent the coding variant, rs3173615 (p.T185S) in linkage disequilibrium with rs1990622, affects progranulin protein (PGRN) biology and transmembrane protein 106 B (TMEM106B) regulation. First, we confirmed the association of TMEM106B variants with FTLD‐TDP in a new cohort of GRN mutation carriers. We next generated and characterized a TMEM106B‐specific antibody for investigation of this protein. Enzyme‐linked immunoassay analysis of progranulin protein levels showed similar effects upon T185 and S185 TMEM106B over‐expression. However, over‐expression of T185 consistently led to higher TMEM106B protein levels than S185. Cycloheximide treatment experiments revealed that S185 degrades faster than T185 TMEM106B, potentially due to differences in N‐glycosylation at residue N183. Together, our results provide a potential mechanism by which TMEM106B variants lead to differences in FTLD‐TDP risk.

Progranulin protein levels are differently regulated in plasma and CSF

Objective: We aimed to investigate the relationship between plasma and CSF progranulin (PGRN) levels. Methods: Plasma and CSF PGRN were measured in a cohort of 345 subjects from the Mayo Clinic Study of Aging by ELISA. Single nucleotide polymorphism genotyping was performed using TaqMan assays. Associations between PGRN and sex, age at sample collection, diagnosis, single nucleotide polymorphism genotypes (GRN, SORT1, and APOE), and Pittsburgh compound B score were explored separately in CSF and plasma using single variable linear regression models. Pearson partial correlation coefficient was used to estimate the correlation of PGRN in CSF and plasma. Results: Plasma (p = 0.0031) and CSF (p = 0.0044) PGRN significantly increased with age, whereas plasma PGRN levels were 7% lower (p = 0.0025) and CSF PGRN levels 5% higher (p = 0.0024) in male compared with female participants. Correcting for age and sex, higher plasma PGRN was associated with higher CSF PGRN (partial r = 0.17, p = 0.004). In plasma, both rs5848 (GRN; p = 0.002) and rs646776 (SORT1; p = 3.56E-7) were associated with PGRN, while only rs5848 showed highly significant association in CSF (p = 5.59E-14). Age, sex, rs5848 genotype, and plasma PGRN together accounted for only 18% of the variability observed in CSF PGRN. Conclusions: While some correlation exists between plasma and CSF PGRN, age, sex, and genetic factors differently affect PGRN levels. Therefore, caution should be taken when using plasma PGRN to predict PGRN changes in the brain. These findings further highlight that plasma PGRN levels may not accurately predict clinical features or response to future frontotemporal lobar degeneration therapies.

Genetic modifiers in carriers of repeat expansions in the C9ORF72 gene

BackgroundHexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9ORF72) are causative for frontotemporal dementia (FTD) and motor neuron disease (MND). Substantial phenotypic heterogeneity has been described in patients with these expansions. We set out to identify genetic modifiers of disease risk, age at onset, and survival after onset that may contribute to this clinical variability.ResultsWe examined a cohort of 330 C9ORF72 expansion carriers and 374 controls. In these individuals, we assessed variants previously implicated in FTD and/or MND; 36 variants were included in our analysis. After adjustment for multiple testing, our analysis revealed three variants significantly associated with age at onset (rs7018487 [UBAP1; p-value = 0.003], rs6052771 [PRNP; p-value = 0.003], and rs7403881 [MT-Ie; p-value = 0.003]), and six variants significantly associated with survival after onset (rs5848 [GRN; p-value = 0.001], rs7403881 [MT-Ie; p-value = 0.001], rs13268953 [ELP3; p-value = 0.003], the epsilon 4 allele [APOE; p-value = 0.004], rs12608932 [UNC13A; p-value = 0.003], and rs1800435 [ALAD; p-value = 0.003]).ConclusionsVariants identified through this study were previously reported to be involved in FTD and/or MND, but we are the first to describe their effects as potential disease modifiers in the presence of a clear pathogenic mutation (i.e. C9ORF72 repeat expansion). Although validation of our findings is necessary, these variants highlight the importance of protein degradation, antioxidant defense and RNA-processing pathways, and additionally, they are promising targets for the development of therapeutic strategies and prognostic tests.