C9orf72 Repeat Expansion Research Articles

Relax, Don’t RAN Translate It

The (GGGGCC)n repeat expansion in C9orf72, which is the most common cause of frontotemporal dementia and amyotrophic lateral sclerosis, is translated through repeat-associated non-AUG (RAN) translation. In this issue of Neuron, Cheng etal. (2019) report that the helicase DDX3X, which unwinds (or relaxes) RNA, suppresses RAN translation and toxicity.

Circadian sleep/wake-associated cells show dipeptide repeat protein aggregates in C9orf72-related ALS and FTLD cases

Motor-, behavior- and/or cognition-related symptoms are key hallmarks in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with TDP-43 pathology (FTLD-TDP), respectively. It has been reported that these patients also experience sleep disturbances, which might implicate a disturbed circadian rhythm of the sleep/wake cycle. It remains unknown, however, whether cells involved in the circadian sleep/wake cycle are affected by ALS- and FTLD-related neuropathological changes including phosphorylated TDP-43 (pTDP-43) aggregates and dipeptide repeat protein (DPR) inclusions resulting from the C9orf72 hexanucleotide repeat expansion. Immunohistochemistry for DPR and pTDP-43 pathology was performed in post-mortem hypothalamus and pineal gland tissue of patients with ALS and/or FTLD-TDP with and without the C9orf72 repeat expansion and healthy controls. Circadian sleep/wake-associated cells, including pinealocytes and hypothalamic neurons related to the suprachiasmatic nucleus (SCN), were microscopically assessed. We observed numerous DPR inclusions (poly(GA), poly(GP), poly(GR) and poly(PR)) in the pinealocytes and few poly(GA) inclusions in the SCN-related neurons in C9orf72-related ALS and/or FTLD-TDP cases. These circadian sleep/wake-associated cells, however, were devoid of pTDP-43 pathology both in C9orf72- and nonC9orf72-related ALS and/or FTLD-TDP cases. Our neuropathological findings show that pinealocytes and, to a lesser extent, SCN-related neurons are affected by DPR pathology. This may reflect an involvement of these cells in sleep/wake disturbances observed in ALS and/or FTLD-TDP patients.

Genome sequencing for early-onset or atypical dementia: high diagnostic yield and frequent observation of multiple contributory alleles.

We assessed the results of genome sequencing for early-onset dementia. Participants were selected from a memory disorders clinic. Genome sequencing was performed along with C9orf72 repeat expansion testing. All returned sequencing results were Sanger-validated. Prior clinical diagnoses included Alzheimer's disease, frontotemporal dementia, and unspecified dementia. The mean age of onset was 54 (41–76). Fifty percent of patients had a strong family history, 37.5% had some, and 12.5% had no known family history. Nine of 32 patients (28%) had a variant defined as pathogenic or likely pathogenic (P/LP) by American College of Medical Genetics and Genomics standards, including variants in APP, C9orf72, CSF1R, and MAPT. Nine patients (including three with P/LP variants) harbored established risk alleles with moderate penetrance (odds ratios of ∼2–5) in ABCA7, AKAP9, GBA, PLD3, SORL1, and TREM2. All six patients harboring these moderate penetrance variants but not P/LP variants also had one or two APOE ε4 alleles. One patient had two APOE ε4 alleles with no other established contributors. In total, 16 patients (50%) harbored one or more genetic variants likely to explain symptoms. We identified variants of uncertain significance (VUSs) in ABI3, ADAM10, ARSA, GRID2IP, MME, NOTCH3, PLCD1, PSEN1, TM2D3, TNK1, TTC3, and VPS13C, also often along with other variants. In summary, genome sequencing for early-onset dementia frequently identified multiple established or possible contributory alleles. These observations add support for an oligogenic model for early-onset dementia.

Links Between the C9orf72 Repeat Expansion and Psychiatric Symptoms.

To present recent findings on the links between the C9orf72 expansion and psychiatric impairment. Repeat hexanucleotide expansions in the C9orf72 gene are a cause of familial frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and the mixed phenotype, FTD-ALS. Symptomatic expansion carriers display higher rates of psychotic and other psychiatric symptoms than non-carriers. Neuroanatomical associations of these symptoms have been found in cortical and subcortical areas. Family members of symptomatic carriers have higher rates of primary neuropsychiatric disorders than control populations, and the C9orf72 expansion may contribute to this association. However, the expansion does not appear to directly cause primary psychiatric disorders. While there is strong evidence associating the C9orf72 expansion with psychotic symptoms in carriers and psychiatric disorders in their kindreds, the link between these two phenomena, if any, remains unclear.

Dysregulation of AMPA receptor subunit expression in sporadic ALS post-mortem brain.

Amyotrophic lateral sclerosis (ALS) is characterised by progressive motor neuron degeneration. Although there are over 40 genes associated with causal monogenetic mutations, the majority of ALS patients are not genetically determined. Causal ALS mutations are being increasingly mechanistically studied, though how these mechanisms converge and diverge between the multiple known familial causes of ALS (fALS) and sporadic forms of ALS (sALS) and furthermore between different neuron types, is poorly understood. One common pathway that is implicated in selective motor neuron death is enhanced α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate (AMPAR)‐mediated excitoxicity. Specifically, human in vitro and pathological evidence has linked the C9orf72 repeat expansion mutation to a relative increase in the Ca2+‐permeable AMPAR population due to AMPAR subunit dysregulation. Here, we provide the first comparative quantitative assessment of the expression profile of AMPAR subunit transcripts, using BaseScope, in post‐mortem lower motor neurons (spinal cord, anterior horn), upper motor neurons (motor cortex) and neurons of the pre‐frontal cortex in sALS and fALS due to mutations in SOD1 and C9orf72. Our data indicated that AMPAR dysregulation is prominent in lower motor neurons in all ALS cases. However, sALS and mutant C9orf72 cases exhibited GluA1 upregulation whereas mutant SOD1 cases displayed GluA2 down regulation. We also showed that sALS cases exhibited widespread AMPAR dysregulation in the motor and pre‐frontal cortex, though the exact identity of the AMPAR subunit being dysregulated was dependent on brain region. In contrast, AMPAR dysregulation in mutant SOD1 and C9orf72 cases was restricted to lower motor neurons only. Our data highlight the complex dysregulation of AMPAR subunit expression that reflects both converging and diverging mechanisms at play between different brain regions and between ALS cohorts. © 2019 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Survival and Prognostic Factors in C9orf72 Repeat Expansion Carriers

The c9orf72 repeat expansion (c9 or c9orf72RE) confers a survival disadvantage in amyotrophic lateral sclerosis (ALS); its effect on prognosis in frontotemporal dementia (FTD) remains uncertain. Data on prognostic factors in c9orf72RE disorders could inform patient care, genetic counseling, and trial design. To examine prognostic factors in c9ALS, c9FTD, c9ALS-FTD, and atypical phenotypes. The MEDLINE, Embase, Amed, ProQuest, PsychINFO, CINAHL, and LILACS databases were searched between January 2011 and January 2019. Keywords used were c9orf72 and chromosome 9 open reading frame 72. Reference lists, citations of eligible studies, and review articles were also searched by hand. Studies reporting disease duration for patients with a confirmed c9orf72RE and a neurological and/or psychiatric disorder were included. A second author independently reviewed studies classified as irrelevant by the first author. Analysis began in January 2019. Data were extracted by 1 author; a further author independently extracted 10% of data. Data were synthesized in univariate and multivariable Cox regression and are displayed as hazard ratios (HR) and 95% confidence intervals. Survival after symptom onset. Overall, 206 studies reporting on 1060 patients were included from 2878 publications identified (c9ALS: n = 455; c9FTD: n = 296; c9ALS-FTD: n = 198; atypical phenotypes: n = 111); 197 duplicate cases were excluded. The median (95% CI) survival (in years) differed significantly between patients with c9ALS (2.8 [2.67-3.00]), c9FTD (9.0 [8.09-9.91]), and c9ALS-FTD (3.0 [2.73-3.27]); survival in atypical phenotypes varied substantially. Older age at onset was associated with shorter survival in c9ALS (HR, 1.03; 95% CI, 1.02-1.04; P < .001), c9FTD (HR, 1.04; 95% CI, 1.02-1.06; P < .001), and c9ALS-FTD (HR, 1.02; 95% CI, 1.004-1.04; P = .016). Bulbar onset was associated with shorter survival in c9ALS (HR, 1.64; 95% CI, 1.27-2.08; P < .001). Age at onset and bulbar onset ALS remained significant in multivariable regression including variables indicating potential diagnostic ascertainment bias, selection bias, and reporting bias. Family history, sex, study continent, FTD subtype, or the presence of additional pathogenic sequence variants were not significantly associated with survival. Clinical phenotypes in patients with neuropathologically confirmed frontotemporal lobar degeneration-TDP-43, motor neuron disease-TDP-43 and frontotemporal lobar degeneration-motor neuron disease-TDP-43 were heterogenous and impacted on survival. Several factors associated with survival in c9orf72RE disorders were identified. The inherent limitations of our methodological approach must be considered; nonetheless, the reported prognostic factors were not significantly associated with the bias indicators examined.

The C9orf72 expansion is associated with accelerated respiratory function decline in a large Amyotrophic Lateral Sclerosis cohort

Introduction: The C9orf72 hexanucleotide repeat expansion is causal in amyotrophic lateral sclerosis (ALS) and has a negative effect on prognosis. The C9orf72 repeat expansion has been associated with an accelerated deterioration of respiratory function and survival in a cohort of 372 Portuguese patients. Methods: Cases presenting to the Irish ALS clinic with both longitudinal occluded sniff nasal inspiratory pressure (SNIP) and C9orf72 testing were including in the study. Clinical variables and survival characteristics of these patients were collected. Joint longitudinal and time to event models were constructed to explore the longitudinal characteristics of the cohort by C9orf72 status. Results: In total, 630 cases were included, of which 58 (9.2%) carried the C9orf72 repeat expansion. Plots of the longitudinal trend after joint modelling revealed that those carrying the expansion had worse respiratory function throughout the course of their disease than those without. The ALS Functional Rating Scale-revised (ALSFRS-R) respiratory sub-score did not distinguish C9orf72 normal from expanded cases. Furthermore, modelling by site of onset and gender sub-groups revealed that this difference was greatest in male spinal onset cases. Joint models further indicated that occluded SNIP values were of prognostic importance. Conclusions: Our results confirm findings from Portugal that the C9orf72 repeat expansion is associated with accelerated respiratory function decline. Analysis via joint models indicate that respiratory function is of prognostic importance and may explain previous observations of poorer prognosis in male spinal onset patients carrying the C9orf72 expansion.

Impact of C9orf72 on Japanese Patients with Amytrophic Lateral Sclerosis (ALS)/Frontotemporal Dementia (FTD)

In 2011, C9orf72 hexanucleotide (GGGGCC) repeat expansion (HRE) in intron 1 was reported as the most common cause of sporadic and familial amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD) in the Caucasian population. In the Japanese population, the C9orf72 repeat expansion was found to account for 0.2% cases of sporadic ALS and 2.6% of familial ALS. Notably, among individuals in the Kii peninsula which has recorded high incidence of ALS or ALS/PDC (parkinsonism-dementia complex), the frequency of C9orf72 repeat expansion was 20% (3/15) indicating high prevalence. It is important to obtain detailed family history of ALS and FTD to understand the cause of the diseases including the C9orf72 mutation.

Theme 2 Genetics and genomics

Background: A genetic basis is found in ∼70% of familial and ∼15% of sporadic ALS, in research cohorts. Clinical trials of gene-targeted therapies are underway, heralding a new era of personalized medicine in ALS treatment. However, ALS management guidelines do not include recommendations for the offer of genetic testing. Many persons with ALS who desire genetic testing are not currently offered it, and the yield of genetic testing in clinic-based ALS populations is unknown. The ALS GAP program, sponsored by the Northeast ALS (NEALS) Consortium, provides free genetic testing for patients with ALS who have a family history of ALS or dementia. We report genetic testing outcomes in the first 142 patients tested in the program.Objectives: 1) To create a pilot ALS genetic testing program for NEALS clinics, 2) To study the rate of ALS gene identification in a US clinic-based populationMethods: Persons with ALS and a family history of ALS (fALS) or dementia (dALS) who receive care at a US NEALS clinic are eligible for testing. Patients classified as fALS (having a positive family history of ALS in a 1st, 2nd, or 3rd degree relative) are eligible for C9orf72 testing, with the option to reflex to a 5 gene (SOD1, FUS, TARDBP, TBK1, VCP) panel. Patients classified as dALS (having a positive family history of dementia of any type in a 1st or 2nd degree relative) are eligible for C9orf72 testing only.Results: Currently, 29.5% (34/115) of US NEALS clinics have participated in the program. Of 142 patients who have completed testing to date, 78 (54.9%) were classified as fALS and 64 (45.1%) as dALS. Among fALS cases, 42/78 (53.9%) tested positive, including 32/78 (41%) with a C9orf72 repeat expansion, and 10/78 (12.8%) with other pathogenic or likely pathogenic variants in SOD, FUS, TARDP or VCP. Variants of uncertain significance (VUS) in FUS were identified in 2/78 (2.6%). Among dALS cases, 12/60 (20%) tested positive for C9orf72.Discussion and conclusions: Participation in ALS-GAP indicates significant clinician and patient interest in ALS genetic testing. This program addresses several current barriers to testing access, including cost, identifying appropriate candidates for testing, and appropriate test selection. Although 38% of patients who participated in the program have thus far received a genetic diagnosis, our testing outcome data suggests that the gene identification rate in fALS cases may be lower in clinic-based patients than in research cohorts, particularly for genes other than C9orf72. This program may serve as a model for the practice of ALS genetic testing in the clinic setting. Consistent, equitable testing policies, as well as an accurate understanding of the genetic profile of clinic-based ALS populations, are needed as gene-targeted therapies reach patient care.

Low Serum High-Density Lipoprotein Cholesterol Levels Associate with the C9orf72 Repeat Expansion in Frontotemporal Lobar Degeneration Patients.

Decreased levels of serum high-density lipoprotein (HDL) cholesterol have previously been linked to systemic inflammation and neurodegenerative diseases, such as Alzheimer’s disease. Here, we aimed to analyze the lipoprotein profile and inflammatory indicators, the high-sensitivity C-reactive peptide (hs-CRP) and glycoprotein acetyls (GlycA), in sporadic and C9orf72 repeat expansion-associated frontotemporal lobar degeneration (FTLD) patients. The C9orf72 hexanucleotide repeat expansion is the most frequent genetic etiology underlying FTLD. The concentrations of different lipid measures in the sera of 67 FTLD patients (15 C9orf72 repeat expansion carriers), including GlycA, were analyzed by nuclear magnetic resonance spectroscopy. To verify the state of systemic inflammation, hs-CRP was also quantified from patient sera. We found that the total serum HDL concentration was decreased in C9orf72 repeat expansion carriers when compared to non-carriers. Moreover, decreased concentrations of HDL particles of different sizes and subclass were consistently observed. No differences were detected in the very low- and low-density lipoprotein subclasses between the C9orf72 repeat expansion carriers and non-carriers. Furthermore, hs-CRP and GlycA levels did not differ between the C9orf72 repeat expansion carriers and non-carriers. In conclusion, the HDL-related changes were linked with C9orf72 repeat expansion associated FTLD but were not seen to associate with systemic inflammation. The underlying reason for the HDL changes remains unclear.

Poly-glycine\u2013alanine exacerbates C9orf72 repeat expansion-mediated DNA damage via sequestration of phosphorylated ATM and loss of nuclear hnRNPA3

Repeat expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Expanded sense and antisense repeat RNA transcripts in C9orf72 are translated into five dipeptide-repeat proteins (DPRs) in an AUG-independent manner. We previously identified the heterogeneous ribonucleoprotein (hnRNP) A3 as an interactor of the sense repeat RNA that reduces its translation into DPRs. Furthermore, we found that hnRNPA3 is depleted from the nucleus and partially mislocalized to cytoplasmic poly-GA inclusions in C9orf72 patients, suggesting that poly-GA sequesters hnRNPA3 within the cytoplasm. We now demonstrate that hnRNPA3 also binds to the antisense repeat RNA. Both DPR production and deposition from sense and antisense RNA repeats are increased upon hnRNPA3 reduction. All DPRs induced DNA double strand breaks (DSB), which was further enhanced upon reduction of hnRNPA3. Poly-glycine–arginine and poly-proline-arginine increased foci formed by phosphorylated Ataxia Telangiectasia Mutated (pATM), a major sensor of DSBs, whereas poly-glycine–alanine (poly-GA) evoked a reduction of pATM foci. In dentate gyri of C9orf72 patients, lower nuclear hnRNPA3 levels were associated with increased DNA damage. Moreover, enhanced poly-GA deposition correlated with reduced pATM foci. Since cytoplasmic pATM deposits partially colocalized with poly-GA deposits, these results suggest that poly-GA, the most frequent DPR observed in C9orf72 patients, differentially causes DNA damage and that poly-GA selectively sequesters pATM in the cytoplasm inhibiting its recruitment to sites of DNA damage. Thus, mislocalization of nuclear hnRNPA3 caused by poly-GA leads to increased poly-GA production, which partially depletes pATM, and consequently enhances DSB.

Pharmacogenetic interactions in amyotrophic lateral sclerosis: a step closer to a cure?

Genetic mutations related to amyotrophic lateral sclerosis (ALS) act through distinct pathophysiological pathways, which may lead to varying treatment responses. Here we assess the genetic interaction between C9orf72, UNC13A, and MOBP with creatine and valproic acid treatment in two clinical trials. Genotypic data was available for 309 of the 338 participants (91.4%). The UNC13A genotype affected mortality (p = 0.012), whereas C9orf72 repeat-expansion carriers exhibited a faster rate of decline in overall (p = 0.051) and bulbar functioning (p = 0.005). A dose-response pharmacogenetic interaction was identified between creatine and the A allele of the MOBP genotype (p = 0.027), suggesting a qualitative interaction in a recessive model (HR 3.96, p = 0.015). Not taking genetic information into account may mask evidence of response to treatment or be an unrecognized source of bias. Incorporating genetic data could help investigators to identify critical treatment clues in patients with ALS.

Extensive transcriptomic study emphasizes importance of vesicular transport in C9orf72 expansion carriers

The majority of the clinico-pathological variability observed in patients harboring a repeat expansion in the C9orf72-SMCR8 complex subunit (C9orf72) remains unexplained. This expansion, which represents the most common genetic cause of frontotemporal lobar degeneration (FTLD) and motor neuron disease (MND), results in a loss of C9orf72 expression and the generation of RNA foci and dipeptide repeat (DPR) proteins. The C9orf72 protein itself plays a role in vesicular transport, serving as a guanine nucleotide exchange factor that regulates GTPases. To further elucidate the mechanisms underlying C9orf72-related diseases and to identify potential disease modifiers, we performed an extensive RNA sequencing study. We included individuals for whom frontal cortex tissue was available: FTLD and FTLD/MND patients with (n = 34) or without (n = 44) an expanded C9orf72 repeat as well as control subjects (n = 24). In total, 6706 genes were differentially expressed between these groups (false discovery rate [FDR] < 0.05). The top gene was C9orf72 (FDR = 1.41E-14), which was roughly two-fold lower in C9orf72 expansion carriers than in (disease) controls. Co-expression analysis revealed groups of correlated genes (modules) that were enriched for processes such as protein folding, RNA splicing, synaptic signaling, metabolism, and Golgi vesicle transport. Within our cohort of C9orf72 expansion carriers, machine learning uncovered interesting candidates associated with clinico-pathological features, including age at onset (vascular endothelial growth factor A [VEGFA]), C9orf72 expansion size (cyclin dependent kinase like 1 [CDKL1]), DPR protein levels (eukaryotic elongation factor 2 kinase [EEF2K]), and survival after onset (small G protein signaling modulator 3 [SGSM3]). Given the fact that we detected a module involved in vesicular transport in addition to a GTPase activator (SGSM3) as a potential modifier, our findings seem to suggest that the presence of a C9orf72 repeat expansion might hamper vesicular transport and that genes affecting this process may modify the phenotype of C9orf72-linked diseases.

Recurrent homozygous damaging mutation in TMX2, encoding a protein disulfide isomerase, in four families with microlissencephaly

BackgroundProtein disulfide isomerase (PDI) proteins are part of the thioredoxin protein superfamily. PDIs are involved in the formation and rearrangement of disulfide bonds between cysteine residues during protein folding in...

CRISPR-Cas9 Screens Identify the RNA Helicase DDX3X as a Repressor of C9ORF72 (GGGGCC)n Repeat-Associated Non-AUG Translation

Hexanucleotide GGGGCC repeat expansion in C9ORF72 is the most prevalent genetic cause ofamyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One pathogenic mechanism isthe aberrant accumulation of dipeptide repeat (DPR) proteins produced by the unconventional translation of expanded RNA repeats. Here, we performed genome-wide CRISPR-Cas9 screens for modifiers of DPR protein production in human cells.We found that DDX3X, an RNA helicase, suppresses the repeat-associated non-AUG translation of GGGGCC repeats. DDX3X directly binds to (GGGGCC)n RNAs but not antisense (CCCCGG)n RNAs. Its helicase activity is essential for the translation repression. Reduction of DDX3X increases DPR levels in C9ORF72-ALS/FTD patient cells and enhances (GGGGCC)n-mediated toxicity in Drosophila. Elevating DDX3X expression is sufficient to decrease DPR levels, rescue nucleocytoplasmic transport abnormalities, and improve survival of patient iPSC-differentiated neurons. This work identifies genetic modifiers of DPR protein production and provides potential therapeutic targets for C9ORF72-ALS/FTD.

Pathogenic Mechanisms and Therapy Development for C9orf72 Amyotrophic Lateral Sclerosis/Frontotemporal Dementia.

In 2011, a hexanucleotide repeat expansion in the first intron of the C9orf72 gene was identified as the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The proposed disease mechanisms include loss of C9orf72 function and gain of toxicity from the bidirectionally transcribed repeat-containing RNAs. Over the last few years, substantial progress has been made to determine the contribution of loss and gain of function in disease pathogenesis. The extensive body of molecular, cellular, animal, and human neuropathological studies is conflicted, but the predominance of evidence favors gain of toxicity as the main pathogenic mechanism for C9orf72 repeat expansions. Alterations in several downstream cellular functions, such as nucleocytoplasmic transport and autophagy, are implicated. Exciting progress has also been made in therapy development targeting this mutation, such as by antisense oligonucleotide therapies targeting sense transcripts and small molecules targeting nucleocytoplasmic transport, and these are now in phase 1 clinical trials.

Peripheral inflammatory markers and clinical correlations in patients with frontotemporal lobar degeneration with and without the C9orf72 repeat expansion

In this study, our aim was to evaluate potential peripheral inflammatory changes in frontotemporal lobar degeneration (FTLD) patients carrying or not the C9orf72 repeat expansion. To this end, levels of several inflammatory markers (MCP-1, RANTES, IL-10, IL-17A, IL-12p, IFN-γ, IL-1β, IL-8, and hs-CRP) and blood cells counts in plasma and/or serum of FTLD patients (N = 98) with or without the C9orf72 repeat expansion were analyzed. In addition, we evaluated whether the analyzed peripheral inflammatory markers correlated with disease progression or distinct clinical phenotypes under the heterogenous FTLD spectrum. Elevated levels of pro-inflammatory RANTES or MCP-1 and decreased levels of anti-inflammatory IL-10 were found to associate with Parkinsonism and a more rapid disease progression, indicated by longitudinal measurements of either MMSE or ADCS-ADL decline. These findings were observed in the total cohort in general, whereas the C9orf72 repeat expansion carriers showed only slight differences in IL-10 and hemoglobin levels compared to non-carriers. Furthermore, these C9orf72 repeat expansion-associated differences were observed mostly in male subjects. The females in general showed elevated levels of several pro-inflammatory markers compared to males regardless of the C9orf72 genotype. Our study suggests that pro-inflammatory changes observed in the early symptomatic phase of FTLD are associated with distinct clinical profiles and a more rapid disease progression, and that the C9orf72 repeat expansion and gender may also affect the inflammatory profile in FTLD.

The C9orf72 expansion is associated with accelerated respiratory function decline in a large Amyotrophic Lateral Sclerosis cohort.

Introduction: The C9orf72 hexanucleotide repeat expansion is causal in amyotrophic lateral sclerosis (ALS) and has a negative effect on prognosis. The C9orf72 repeat expansion has been associated with an accelerated deterioration of respiratory function and survival in a cohort of 372 Portuguese patients. Methods: Cases presenting to the Irish ALS clinic with both longitudinal occluded sniff nasal inspiratory pressure (SNIP) and C9orf72 testing were including in the study. Clinical variables and survival characteristics of these patients were collected. Joint longitudinal and time to event models were constructed to explore the longitudinal characteristics of the cohort by C9orf72 status. Results: In total, 630 cases were included, of which 58 (9.2%) carried the C9orf72 repeat expansion. Plots of the longitudinal trend after joint modelling revealed that those carrying the expansion had worse respiratory function throughout the course of their disease than those without. The ALS Functional Rating Scale-revised (ALSFRS-R) respiratory sub-score did not distinguish C9orf72 normal from expanded cases. Furthermore, modelling by site of onset and gender sub-groups revealed that this difference was greatest in male spinal onset cases. Joint models further indicated that occluded SNIP values were of prognostic importance. Conclusions: Our results confirm findings from Portugal that the C9orf72 repeat expansion is associated with accelerated respiratory function decline. Analysis via joint models indicate that respiratory function is of prognostic importance and may explain previous observations of poorer prognosis in male spinal onset patients carrying the C9orf72 expansion.

CRISPR/Cas9 does not facilitate stable expression of long C9orf72 dipeptides in mice

A C9orf72 repeat expansion is the most common cause of both frontotemporal dementia and motor neuron disease. The expansion is translated to produce dipeptide repeat proteins (DPRs), which are toxic in vivo and in vitro. However, the mechanisms underlying DPR toxicity remain unclear. Mouse models which express DPRs at repeat lengths found in human disease are urgently required to investigate this. We aimed to generate transgenic mice expressing DPRs at repeat lengths of >1000 using alternative codon sequences, to reduce the repetitive nature of the insert. We found that although these inserts did integrate into the mouse genome, the alternative codon sequences did not protect from instability between generations. Our findings suggest that stable integration of long DPR sequences may not be possible. Administration of viral vectors after birth may be a more effective delivery method for long repeats.

Subcortical TDP-43 pathology patterns validate cortical FTLD-TDP subtypes and demonstrate unique aspects of C9orf72 mutation cases.

Frontotemporal lobar degeneration with TDP-43 immunoreactive (TDP-ir) inclusions (FTLD-TDP) is sub-classified based on the pattern of neocortical pathology, with each subtype showing clinical and genetic correlations. Recent studies indicate that accurate subtyping of cases may be important to help identify genetic risk factors and develop biomarkers. Although most FTLD-TDP cases are easily classified, some do not match well to one of the existing subtypes. In particular, cases with the C9orf72 repeat expansion (C9+) have been reported to show FTLD-TDP type A, type B or a combination of A and B pathology (A + B). In our series of FTLD-TDP cases, we found that those lacking the C9orf72 mutation (non-C9) were all readily classified as type A (n = 29), B (n = 16) or C (n = 18), using current criteria and standard observational methods. This classification was validated using non-biased hierarchical cluster analysis (HCA) of neocortical pathology data. In contrast, only 14/28 (50%) of the C9+ cases were classified as either pure type A or pure type B, with the remainder showing A + B features. HCA confirmed separation of the C9+ cases into three groups. We then investigated whether patterns of subcortical TDP-ir pathology helped to classify the difficult cases. For the non-C9 cases, each subtype showed a consistent pattern of subcortical involvement with significant differences among the groups. The most distinguishing features included white matter threads, neuronal intranuclear inclusions in hippocampus and striatum, and delicate threads in CA1 in type A; glial cytoplasmic inclusions in white matter and neuronal cytoplasmic inclusions (NCI) in lower motor neurons in type B; compact NCI in striatum in type C. HCA of the C9+ cases based on subcortical features increased the number that clustered with the non-C9 type A (46%) or non-C9 type B (36%); however, there remained a C9+ group with A + B features (18%). These findings suggest that most FTLD-TDP cases can be classified using existing criteria and that each group also shows characteristic subcortical TDP-ir pathology. However, C9+ cases may be unique in the degree to which their pathology overlaps between FTLD-TDP types A and B.