Marka van Blitterswijk

Antisense transcripts of the expanded C9ORF72 hexanucleotide repeat form nuclear RNA foci and undergo repeat-associated non-ATG translation in c9FTD/ALS

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are devastating neurodegenerative disorders with clinical, genetic, and neuropathological overlap. A hexanucleotide (GGGGCC) repeat expansion in a non-coding region of C9ORF72 is the major genetic cause of both diseases. The mechanisms by which this repeat expansion causes “c9FTD/ALS” are not definitively known, but RNA-mediated toxicity is a likely culprit. RNA transcripts of the expanded GGGGCC repeat form nuclear foci in c9FTD/ALS, and also undergo repeat-associated non-ATG (RAN) translation resulting in the production of three aggregation-prone proteins. The goal of this study was to examine whether antisense transcripts resulting from bidirectional transcription of the expanded repeat behave in a similar manner. We show that ectopic expression of (CCCCGG)66 in cultured cells results in foci formation. Using novel polyclonal antibodies for the detection of possible (CCCCGG)exp RAN proteins [poly(PR), poly(GP) and poly(PA)], we validated that (CCCCGG)66 is also subject to RAN translation in transfected cells. Of importance, foci composed of antisense transcripts are observed in the frontal cortex, spinal cord and cerebellum of c9FTD/ALS cases, and neuronal inclusions of poly(PR), poly(GP) and poly(PA) are present in various brain tissues in c9FTD/ALS, but not in other neurodegenerative diseases, including CAG repeat disorders. Of note, RNA foci and poly(GP) inclusions infrequently co-occur in the same cell, suggesting these events represent two distinct ways in which the C9ORF72 repeat expansion may evoke neurotoxic effects. These findings provide mechanistic insight into the pathogenesis of c9FTD/ALS, and have significant implications for therapeutic strategies.

Evidence for an oligogenic basis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with a substantial heritable component. In pedigrees affected by its familial form, incomplete penetrance is often observed. We hypothesized that this could be caused by a complex inheritance of risk variants in multiple genes. Therefore, we screened 111 familial ALS (FALS) patients from 97 families, and large cohorts of sporadic ALS (SALS) patients and control subjects for mutations in TAR DNA-binding protein (TARDBP), fused in sarcoma/translated in liposarcoma (FUS/TLS), superoxide dismutase-1 (SOD1), angiogenin (ANG) and chromosome 9 open reading frame 72 (C9orf72). Mutations were identified in 48% of FALS families, 8% of SALS patients and 0.5% of control subjects. In five of the FALS families, we identified multiple mutations in ALS-associated genes. We detected FUS/TLS and TARDBP mutations in combination with ANG mutations, and C9orf72 repeat expansions with TARDBP, SOD1 and FUS/TLS mutations. Statistical analysis demonstrated that the presence of multiple mutations in FALS is in excess of what is to be expected by chance (P = 1.57 × 10(-7)). The most compelling evidence for an oligogenic basis was found in individuals with a p.N352S mutation in TARDBP, detected in five FALS families and three apparently SALS patients. Genealogical and haplotype analyses revealed that these individuals shared a common ancestor. We obtained DNA of 14 patients with this TARDBP mutation, 50% of whom had an additional mutation (ANG, C9orf72 or homozygous TARDBP). Hereby, we provide evidence for an oligogenic aetiology of ALS. This may have important implications for the interpretation of whole exome/genome experiments designed to identify new ALS-associated genes and for genetic counselling, especially of unaffected family members.

Association between repeat sizes and clinical and pathological characteristics in carriers of C9ORF72 repeat expansions (Xpansize-72): a cross-sectional cohort study

BACKGROUND Hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9ORF72) are the most common known genetic cause of frontotemporal dementia (FTD) and motor neuron disease (MND). We assessed whether expansion size is associated with disease severity or phenotype. METHODS We did a cross-sectional Southern blot characterisation study (Xpansize-72) in a cohort of individuals with FTD, MND, both these diseases, or no clinical phenotype. All participants had GGGGCC repeat expansions in C9ORF72, and high quality DNA was available from one or more of the frontal cortex, cerebellum, or blood. We used Southern blotting techniques and densitometry to estimate the repeat size of the most abundant expansion species. We compared repeat sizes between different tissues using Wilcoxon rank sum and Wilcoxon signed rank tests, and between disease subgroups using Kruskal-Wallis rank sum tests. We assessed the association of repeat size with age at onset and age at collection using a Spearmans test of correlation, and assessed the association between repeat size and survival after disease onset using Cox proportional hazards regression models. FINDINGS We included 84 individuals with C9ORF72 expansions: 35 had FTD, 16 had FTD and MND, 30 had MND, and three had no clinical phenotype. We focused our analysis on three major tissue subgroups: frontal cortex (available from 41 patients [21 with FTD, 11 with FTD and MND, and nine with MND]), cerebellum (40 patients [20 with FTD, 12 with FTD and MND, and eight with MND]), and blood (47 patients [15 with FTD, nine with FTD and MND, and 23 with MND] and three carriers who had no clinical phenotype). Repeat lengths in the cerebellum were smaller (median 12·3 kb [about 1667 repeat units], IQR 11·1-14·3) than those in the frontal cortex (33·8 kb [about 5250 repeat units], 23·5-44·9; p<0·0001) and those in blood (18·6 kb [about 2717 repeat units], 13·9-28·1; p=0·0002). Within these tissues, we detected no difference in repeat length between disease subgroups (cerebellum p=0·96, frontal cortex p=0·27, blood p=0·10). In the frontal cortex of patients with FTD, repeat length correlated with age at onset (r=0·63; p=0·003) and age at sample collection (r=0·58; p=0·006); we did not detect such a correlation in samples from the cerebellum or blood. When assessing cerebellum samples from the overall cohort, survival after disease onset was 4·8 years (IQR 3·0-7·4) in the group with expansions greater than 1467 repeat units (the 25th percentile of repeat lengths) versus 7·4 years (6·3-10·9) in the group with smaller expansions (HR 3·27, 95% CI 1·34-7·95; p=0·009). INTERPRETATION We detected substantial variation in repeat sizes between samples from the cerebellum, frontal cortex, and blood, and longer repeat sizes in the cerebellum seem to be associated with a survival disadvantage. Our findings indicate that expansion size does affect disease severity, which--if replicated in other cohorts--could be relevant for genetic counselling. FUNDING The ALS Therapy Alliance, the National Institute of Neurological Disorders and Stroke, the National Institute on Aging, the Arizona Department of Health Services, the Arizona Biomedical Research Commission, and the Michael J Fox Foundation for Parkinsons Research.

Angiogenin variants in Parkinson disease and amyotrophic lateral sclerosis

Several studies have suggested an increased frequency of variants in the gene encoding angiogenin (ANG) in patients with amyotrophic lateral sclerosis (ALS). Interestingly, a few ALS patients carrying ANG variants also showed signs of Parkinson disease (PD). Furthermore, relatives of ALS patients have an increased risk to develop PD, and the prevalence of concomitant motor neuron disease in PD is higher than expected based on chance occurrence. We therefore investigated whether ANG variants could predispose to both ALS and PD.

How do C9ORF72 repeat expansions cause amyotrophic lateral sclerosis and frontotemporal dementia: can we learn from other noncoding repeat expansion disorders?

PURPOSE OF REVIEW The aim of this review is to describe disease mechanisms by which chromosome 9 open reading frame 72 (C9ORF72) repeat expansions could lead to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and to discuss these diseases in relation to other noncoding repeat expansion disorders. RECENT FINDINGS ALS and FTD are complex neurodegenerative disorders with a considerable clinical and pathological overlap, and this overlap is further substantiated by the recent discovery of C9ORF72 repeat expansions. These repeat expansions are currently the most important genetic cause of familial ALS and FTD, accounting for approximately 34.2 and 25.9% of the cases. Clinical phenotypes associated with these repeat expansions are highly variable, and combinations with mutations in other ALS-associated and/or FTD-associated genes may contribute to this pleiotropy. It is challenging, however, to diagnose patients with C9ORF72 expansions, not only because of large repeat sizes, but also due to somatic heterogeneity. Most other noncoding repeat expansion disorders share an RNA gain-of-function disease mechanism, a mechanism that could underlie the development of ALS and/or FTD as well. SUMMARY The discovery of C9ORF72 repeat expansions provides novel insights into the pathogenesis of ALS and FTD and highlights the importance of noncoding repeat expansions and RNA toxicity in neurodegenerative diseases.

Distinct brain transcriptome profiles in C9orf72-associated and sporadic ALS

Increasing evidence suggests that defective RNA processing contributes to the development of amyotrophic lateral sclerosis (ALS). This may be especially true for ALS caused by a repeat expansion in C9orf72 (c9ALS), in which the accumulation of RNA foci and dipeptide-repeat proteins are expected to modify RNA metabolism. We report extensive alternative splicing (AS) and alternative polyadenylation (APA) defects in the cerebellum of c9ALS subjects (8,224 AS and 1,437 APA), including changes in ALS-associated genes (for example, ATXN2 and FUS), and in subjects with sporadic ALS (sALS; 2,229 AS and 716 APA). Furthermore, heterogeneous nuclear ribonucleoprotein H (hnRNPH) and other RNA-binding proteins are predicted to be potential regulators of cassette exon AS events in both c9ALS and sALS. Co-expression and gene-association network analyses of gene expression and AS data revealed divergent pathways associated with c9ALS and sALS.

Hexanucleotide repeat expansions in C9ORF72 in the spectrum of motor neuron diseases

Objective: To assess the frequency and phenotype of hexanucleotide repeat expansions in C9ORF72 in a large cohort of patients of Dutch descent with familial (fALS) and sporadic (sALS) amyotrophic lateral sclerosis (ALS), progressive muscular atrophy (PMA), and primary lateral sclerosis (PLS). Methods: Included were 78 patients with fALS, 1,422 with sALS, 246 with PMA, and 110 with PLS, and 768 control subjects. Repeat expansions were determined by a repeat primed PCR. Familial aggregation of dementia and Parkinson disease (PD) was examined among patients with ALS who carried the repeat expansion. Results: The expanded repeat was found in 33 (37%) of all patients with fALS, in 87 (6.1%) patients with sALS, in 4 (1.6%) patients with PMA, and in 1 (0.9%) patient with PLS. None of the controls carried the mutation. Patients with ALS with the repeat expansion had an earlier age at onset (median 59.3 vs 61.9 years, hazard ratio 1.55, p = 5 × 10−5) and shorter survival (median 2.5 vs 2.7 years, hazard ratio 1.46, p = 8 × 10−4). Dementia, but not PD, occurred nearly twice as often in relatives of patients with the expansion compared to all patients with ALS or controls (p = 9 × 10−4). Conclusions: The hexanucleotide repeat expansion in C9ORF72 is a major cause of fALS and apparently sporadic ALS in the Netherlands. Patients who carry the repeat expansion have an earlier onset, shorter survival, and familial aggregation of dementia. These results challenge the classic definition of fALS and may justify genetic testing in patients with sALS.

C9ORF72 repeat expansions in cases with previously identified pathogenic mutations

Objective: To identify potential genetic modifiers contributing to the phenotypic variability that is detected in patients with repeat expansions in chromosome 9 open reading frame 72 (C9ORF72), we investigated the frequency of these expansions in a cohort of 334 subjects previously found to carry mutations in genes known to be associated with a spectrum of neurodegenerative diseases. Methods: A 2-step protocol, with a fluorescent PCR and a repeat-primed PCR, was used to determine the presence of hexanucleotide expansions in C9ORF72. For one double mutant, we performed Southern blots to assess expansion sizes, and immunohistochemistry to characterize neuropathology. Results: We detected C9ORF72 repeat expansions in 4 of 334 subjects (1.2% [or 1.8% of 217 families]). All these subjects had behavioral phenotypes and also harbored well-known pathogenic mutations in either progranulin (GRN: p.C466LfsX46, p.R493X, p.C31LfsX35) or microtubule-associated protein tau (MAPT: p.P301L). Southern blotting of one double mutant with a p.C466LfsX46 GRN mutation demonstrated a long repeat expansion in brain (>3,000 repeats), and immunohistochemistry showed mixed neuropathology with characteristics of both C9ORF72 expansions and GRN mutations. Conclusions: Our findings indicate that co-occurrence of 2 evidently pathogenic mutations could contribute to the pleiotropy that is detected in patients with C9ORF72 repeat expansions. These findings suggest that patients with known mutations should not be excluded from further studies, and that genetic counselors should be aware of this phenomenon when advising patients and their family members.

A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories

Background The GGGGCC-repeat expansion in C9orf72 is the most frequent mutation found in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Most of the studies on C9orf72 have relied on repeat-primed PCR (RP-PCR) methods for detection of the expansions. To investigate the inherent limitations of this technique, we compared methods and results of 14 laboratories. Methods The 14 laboratories genotyped DNA from 78 individuals (diagnosed with ALS or FTD) in a blinded fashion. Eleven laboratories used a combination of amplicon-length analysis and RP-PCR, whereas three laboratories used RP-PCR alone; Southern blotting techniques were used as a reference. Results Using PCR-based techniques, 5 of the 14 laboratories got results in full accordance with the Southern blotting results. Only 50 of the 78 DNA samples got the same genotype result in all 14 laboratories. There was a high degree of false positive and false negative results, and at least one sample could not be genotyped at all in 9 of the 14 laboratories. The mean sensitivity of a combination of amplicon-length analysis and RP-PCR was 95.0% (73.9–100%), and the mean specificity was 98.0% (87.5–100%). Overall, a sensitivity and specificity of more than 95% was observed in only seven laboratories. Conclusions Because of the wide range seen in genotyping results, we recommend using a combination of amplicon-length analysis and RP-PCR as a minimum in a research setting. We propose that Southern blotting techniques should be the gold standard, and be made obligatory in a clinical diagnostic setting.

NEK1 variants confer susceptibility to amyotrophic lateral sclerosis

To identify genetic factors contributing to amyotrophic lateral sclerosis (ALS), we conducted whole-exome analyses of 1,022 index familial ALS (FALS) cases and 7,315 controls. In a new screening strategy, we performed gene-burden analyses trained with established ALS genes and identified a significant association between loss-of-function (LOF) NEK1 variants and FALS risk. Independently, autozygosity mapping for an isolated community in the Netherlands identified a NEK1 p.Arg261His variant as a candidate risk factor. Replication analyses of sporadic ALS (SALS) cases and independent control cohorts confirmed significant disease association for both p.Arg261His (10,589 samples analyzed) and NEK1 LOF variants (3,362 samples analyzed). In total, we observed NEK1 risk variants in nearly 3% of ALS cases. NEK1 has been linked to several cellular functions, including cilia formation, DNA-damage response, microtubule stability, neuronal morphology and axonal polarity. Our results provide new and important insights into ALS etiopathogenesis and genetic etiology.