PolyQ Repeat Expansions in ATXN2 Associated with ALS Are CAA Interrupted Repeats

Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressive disease leading to paralysis and death. Recently, intermediate length polyglutamine (polyQ) repeats of 27–33 in ATAXIN-2 (ATXN2), encoding the ATXN2 protein, were found to increase risk for ALS. In ATXN2, polyQ expansions of ≥34, which are pure CAG repeat expansions, cause spinocerebellar ataxia type 2. However, similar length expansions that are interrupted with other codons, can present atypically with parkinsonism, suggesting that configuration of the repeat sequence plays an important role in disease manifestation in ATXN2 polyQ expansion diseases. Here we determined whether the expansions in ATXN2 associated with ALS were pure or interrupted CAG repeats, and defined single nucleotide polymorphisms (SNPs) rs695871 and rs695872 in exon 1 of the gene, to assess haplotype association. We found that the expanded repeat alleles of 40 ALS patients and 9 long-repeat length controls were all interrupted, bearing 1–3 CAA codons within the CAG repeat. 21/21 expanded ALS chromosomes with 3CAA interruptions arose from one haplotype (GT), while 18/19 expanded ALS chromosomes with <3CAA interruptions arose from a different haplotype (CC). Moreover, age of disease onset was significantly earlier in patients bearing 3 interruptions vs fewer, and was distinct between haplotypes. These results indicate that CAG repeat expansions in ATXN2 associated with ALS are uniformly interrupted repeats and that the nature of the repeat sequence and haplotype, as well as length of polyQ repeat, may play a role in the neurological effect conferred by expansions in ATXN2

Sequence of the polyQ repeat region of expanded allele of ALS patients.

<p>*familial situation,</p><p>**SNP695871 SNP695872.</p

SNP analysis by dCAPS method.

<p>Analysis of restriction fragments for SNP (A) rs695871 and (B) rs695872. For SNP rs695871 (C or G, AvaII digestion) analysis of a Q32/Q22 patient, the amplified products were Q32:359bp and Q22:329bp. If Q32 is linked to a C, AvaII will digest the product into two fragments of 313 bp and 46 bp. If Q32 is linked to a G, AvaII will not cut. If Q22 is linked to a C, AvaII will digest it into fragments of 46 bp and 283 bp. If Q22 is linked to a G, AvaII will not cut. In the case of C/G heterozygotes, some uncut hybrid product remained. For SNP rs695872 (C or T, NotI digestion) analysis of a Q32/Q22 patient, the amplified products were Q32:308bp and Q22:278bp. If Q32 is linked to a C, NotI will digest the product into two fragments of 254 bp and 64 bp. If Q32 is linked to a T, NotI will not cut. If Q22 is linked to a C, NotI will digest it into fragments of 64 bp and 214 bp. If Q22 is linked to a T, NotI will not cut. In the case of C/T heterozygotes, some uncut hybrid product remained. In the gels, the low molecular weight bands (46, 64 bp) are largely undetectable; the pattern is scored by whether the higher bands are cut or not.</p

Age of disease onset by repeat interruptions and haplotype.

<p>Age of disease onset by (A) repeat interruptions or by (B) haplotype for ALS patients bearing either 3CAA interruptions within the polyQ repeat region, versus patients with fewer than 3 CAA interruptions. The patients with 3 interruptions show an earlier disease onset, with a hazard ratio (HR) of 1.9, compared to patients with fewer interruptions. This is despite that patients with 3 CAA interruptions have an average repeat length that is lower (27.5) compared to patients with fewer interruptions (30.8). The age of disease onset is also significantly different when broken down by haplotype.</p

The spectrum of diseases associated with ATXN2 polyQ repeat expansions.

<p>Although the repeat in <i>ATXN2</i> is polymorphic normally, it is typically 22. In this cohort of controls, we found that only 1.4% (12/980 total) had repeats greater than 27 (range 27–31), whereas 4.7% of this cohort of ALS patients <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017951#pone.0017951-Elden1" target="_blank">[31]</a> had repeats of 27–33 (43/915 sporadic, 3/65 familial); the exact cut-off associated with risk for ALS may vary in different populations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017951#pone.0017951-Lee1" target="_blank">[38]</a>. The repeats of ALS and high range controls from this study are interrupted by 1–3 CAA codons. <i>ATXN2</i> repeats associated with parkinsonism range from 34 to 49, and studies to date indicate that the repeats are interrupted <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017951#pone.0017951-Kim1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017951#pone.0017951-Charles1" target="_blank">[27]</a>. Repeat expansions associated with SCA2 are greater than 34, and are typically pure CAG repeats <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017951#pone.0017951-Imbert1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017951#pone.0017951-Cancel1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017951#pone.0017951-Orr1" target="_blank">[37]</a>.</p

Sequence of the polyQ repeat region of expanded alleles of controls.

<p>**SNP695871 SNP695872.</p

Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Mutations in related RNA-binding proteins TDP-43, FUS/TLS and TAF15 have been connected to ALS. These three proteins share several features, including the presence of a bioinformatics-predicted prion domain, aggregation-prone nature in vitro and in vivo and toxic effects when expressed in multiple model systems. Given these commonalities, we hypothesized that a related protein, EWSR1 (Ewing sarcoma breakpoint region 1), might also exhibit similar properties and therefore could contribute to disease. Here, we report an analysis of EWSR1 in multiple functional assays, including mutational screening in ALS patients and controls. We identified three missense variants in EWSR1 in ALS patients, which were absent in a large number of healthy control individuals. We show that disease-specific variants affect EWSR1 localization in motor neurons. We also provide multiple independent lines of in vitro and in vivo evidence that EWSR1 has similar properties as TDP-43, FUS and TAF15, including aggregation-prone behavior in vitro and ability to confer neurodegeneration in Drosophila. Postmortem analysis of sporadic ALS cases also revealed cytoplasmic mislocalization of EWSR1. Together, our studies highlight a potential role for EWSR1 in ALS, provide a collection of functional assays to be used to assess roles of additional RNA-binding proteins in disease and support an emerging concept that a class of aggregation-prone RNA-binding proteins might contribute broadly to ALS and related neurodegenerative diseases.13 page(s

A Yeast functional screen predicts new candidate ALS disease genes

Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregation-prone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery.10 page(s