C9ORF72 repeat expansion in Australian and Spanish frontotemporal dementia patients

A hexanucleotide repeat expansion in C9ORF72 has been established as a common cause of frontotemporal dementia (FTD). However, the minimum repeat number necessary for disease pathogenesis is not known. The aims of our study were to determine the frequency of the C9ORF72 repeat expansion in two FTD patient collections (one Australian and one Spanish, combined n = 190), to examine C9ORF72 expansion allele length in a subset of FTD patients, and to examine C9ORF72 allele length in ‘non-expansion’ patients (those with <30 repeats). The C9ORF72 repeat expansion was detected in 5–17% of patients (21–41% of familial FTD patients). For one family, the expansion was present in the proband but absent in the mother, who was diagnosed with dementia at age 68. No association was found between C9ORF72 non-expanded allele length and age of onset and in the Spanish sample mean allele length was shorter in cases than in controls. Southern blotting analysis revealed that one of the nine ‘expansion-positive’ patients examined, who had neuropathologically confirmed frontotemporal lobar degeneration with TDP-43 pathology, harboured an ‘intermediate’ allele with a mean size of only ~65 repeats. Our study indicates that the C9ORF72 repeat expansion accounts for a significant proportion of Australian and Spanish FTD cases. However, C9ORF72 allele length does not influence the age at onset of ‘non-expansion’ FTD patients in the series examined. Expansion of the C9ORF72 allele to as little as ~65 repeats may be sufficient to cause disease.Carol Dobson-Stone, Marianne Hallupp, Clement T. Loy, Elizabeth M. Thompson, Eric Haan, Carolyn M. Sue, Peter K. Panegyres, Cristina Razquin, Manuel Seijo-Martínez, Ramon Rene, Jordi Gascon, Jaume Campdelacreu, Birgit Schmoll, Alexander E. Volk, William S. Brooks, Peter R. Schofield, Pau Pastor, John B. J. Kwo

Multivariate Repeat Measure Mixed Linear Model Regression analyses for the effects of disease status and <i>MAPT</i> transcript levels.

<p>Multivariate Repeat Measure Mixed Linear Model Regression analyses for the effects of disease status and <i>MAPT</i> transcript levels.</p

Role of the Long Non-Coding RNA <i>MAPT-AS1</i> in Regulation of <i>Microtubule Associated Protein Tau (MAPT)</i> Expression in Parkinson's Disease

<div><p>Studies investigating the pathogenic role of the microtubule associated protein tau (<i>MAPT</i>) gene in Parkinson’s disease (PD) have indicated that DNA methylation of the promoter region is aberrant in disease, leading to dysregulated <i>MAPT</i> expression. We examined two potential regulators of <i>MAPT</i> gene expression in respect to PD, a promoter-associated long non-coding RNA <i>MAPT-AS1</i>, and DNA methyltransferases (DNMTs), enzymes responsible for new and maintenance of DNA methylation. We assessed the relationship between expression levels of <i>MAPT</i> and the candidate <i>MAPT-AS1</i>, <i>DNMT1</i>, <i>DNMT3A</i> and <i>DNMT3B</i> transcripts in four brain regions with varying degrees of cell loss and pathology (putamen, anterior cingulate cortex, visual cortex and cerebellum) in N = 10 PD and N = 10 controls. We found a significant decrease in <i>MAPT-AS1</i> expression in PD (p = 7.154 x 10⁻⁶). The transcript levels of both <i>MAPT-AS1</i> (p = 2.569 x 10⁻⁴) and <i>DNMT1</i> (p = 0.001) correlated with those of <i>MAPT</i> across the four brain regions, but not with each other. Overexpression of <i>MAPT-AS1</i> decreased <i>MAPT</i> promoter activity by ∼2.2 to 4.3 fold in an <i>in vitro</i> luciferase assay performed in two cell lines (p ≤ 2.678 x 10⁻⁴). Knock-down expression of <i>MAPT-AS1</i> led to a 1.3 to 6.3 fold increase in methylation of the endogenous <i>MAPT</i> promoter (p ≤ 0.011) and a 1.2 to 1.5 fold increased expression of the 4-repeat <i>MAPT</i> isoform transcript (p ≤ 0.013). In conclusion, <i>MAPT-AS1</i> and <i>DNMT1</i> have been identified as potential epigenetic regulators of <i>MAPT</i> expression in PD across four different brain regions. Our data also suggest that increased <i>MAPT</i> expression could be associated with disease state, but not with PD neuropathology severity.</p></div

Disease-specific expression levels of <i>MAPT</i>, <i>DNMT1</i>, <i>DNMT3A</i> and <i>DNMT3B</i> across four brain regions.

<p>Putamen (black circle), ACC (dark grey circle), visual cortex (light grey circle) and cerebellum (open circle). Data points are derived from estimated marginal means after adjusting for significant demographic predictors apart from disease status.</p

Effect of <i>MAPT-AS1</i> over- and knock-down expression on <i>MAPT</i> expression in HEK293 and SK-N-MC cells.

<p>A) H1 (white columns) and H2 (black columns) haplotype <i>MAPT</i> promoter-driven luciferase activity. Luciferase activity is normalized to each control transfection levels. B) Endogenous transcript levels of either total <i>MAPT</i> (light grey columns) or 4 repeat <i>MAPT</i> transcript (dark grey columns). Transcript levels are normalized to each control transfection levels C) Endogenous haplotype-specific DNA methylation with H1 (white columns) and H2 (black columns) specific data indicated. Methylation levels from <i>MAPT-AS1</i> over- or under-expression are normalized to control transfection levels. Error bars indicate standard error of the mean from 5 independent experiments. *, p < 0.05; *** p < 0.0001.</p

Comparison of demographics and <i>MAPT</i> haplotype frequency in brain tissue cohort.

<p>Comparison of demographics and <i>MAPT</i> haplotype frequency in brain tissue cohort.</p

Retiring the term FTDP-17 as MAPT mutations are genetic forms of sporadic frontotemporal tauopathies

In many neurodegenerative disorders, familial forms have provided important insights into the pathogenesis of their corresponding sporadic forms. The first mutations associated with frontotemporal lobar degeneration (FTLD) were found in the microtubule-associated protein tau (MAPT) gene on chromosome 17 in families with frontotemporal degeneration and parkinsonism (FTDP-17). However, it was soon discovered that 50% of these families had a nearby mutation in progranulin. Regardless, the original FTDP-17 nomenclature has been retained for patients with MAPT mutations, with such patients currently classified independently from the different sporadic forms of FTLD with tau-immunoreactive inclusions (FTLD-tau). The separate classification of familial FTLD with MAPT mutations implies that familial forms cannot inform on the pathogenesis of the different sporadic forms of FTLD-tau. To test this assumption, this study pathologically assessed all FTLD-tau cases with a known MAPT mutation held by the Sydney and Cambridge Brain Banks, and compared them to four cases of four subtypes of sporadic FTLD-tau, in addition to published case reports. Ten FTLD-tau cases with a MAPT mutation (K257T, S3035S, P301L, IVS10+16, R406W) were screened for the core differentiating neuropathological features used to diagnose the different sporadic FTLD-tau subtypes to determine whether the categorical separation of MAPT mutations from sporadic FTLD-tau is valid. Compared with sporadic cases, FTLD-tau cases with MAPT mutations had similar mean disease duration but were younger at age of symptom onset (55 ± 4 years versus 70 ± 6 years). Interestingly, FTLD-tau cases with MAPT mutations had similar patterns and severity of neuropathological features to sporadic FTLD-tau subtypes and could be classified into: Pick's disease (K257T), corticobasal degeneration (S305S, IVS10+16, R406W), progressive supranuclear palsy (S305S) or globular glial tauopathy (P301L, IVS10+16). The finding that the S305S mutation could be classified into two tauopathies suggests additional modifying factors. Assessment of our cases and previous reports suggests that distinct MAPT mutations result in particular FTLD-tau subtypes, supporting the concept that they are likely to inform on the varied cellular mechanisms involved in distinctive forms of sporadic FTLD-tau. As such, FTLD-tau cases with MAPT mutations should be considered familial forms of FTLD-tau subtypes rather than a separate FTDP-17 category, and continued research on the effects of different mutations more focused on modelling their impact to produce the very different sporadic FTLD-tau pathologies in animal and cellular models

C9ORF72 Repeat Expansion in Australian and Spanish Frontotemporal Dementia Patients

A hexanucleotide repeat expansion in C9ORF72 has been established as a common cause of frontotemporal dementia (FTD). However, the minimum repeat number necessary for disease pathogenesis is not known. The aims of our study were to determine the frequency of the C9ORF72 repeat expansion in two FTD patient collections (one Australian and one Spanish, combined n = 190), to examine C9ORF72 expansion allele length in a subset of FTD patients, and to examine C9ORF72 allele length in 'non-expansion' patients (those with <30 repeats). The C9ORF72 repeat expansion was detected in 5-17% of patients (21-41% of familial FTD patients). For one family, the expansion was present in the proband but absent in the mother, who was diagnosed with dementia at age 68. No association was found between C9ORF72 non-expanded allele length and age of onset and in the Spanish sample mean allele length was shorter in cases than in controls. Southern blotting analysis revealed that one of the nine 'expansion-positive' patients examined, who had neuropathologically confirmed frontotemporal lobar degeneration with TDP-43 pathology, harboured an 'intermediate' allele with a mean size of only similar to 65 repeats. Our study indicates that the C9ORF72 repeat expansion accounts for a significant proportion of Australian and Spanish FTD cases. However, C9ORF72 allele length does not influence the age at onset of 'non-expansion' FTD patients in the series examined. Expansion of the C9ORF72 allele to as little as similar to 65 repeats may be sufficient to cause disease

Analysis of <i>C9ORF72</i> expansion allele size.

<p>A, Southern blot analysis of five unrelated probands and four Aus-14 family members with the <i>C9ORF72</i> repeat expansion, plus an expansion-negative control (N). Wild-type (wt) and expansion (exp) alleles are indicated; the smaller expansion allele in case 100 is arrowed. A band running at ∼3.8 kb was also observed (asterisk); this probably arises from unspecific probe binding. B, Scatter plot of age of onset of disease versus estimated mean number of <i>C9ORF72</i> hexanucleotide repeats in expansion allele carriers.</p