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Neurobiology of Aging 35 (2014) 936.e1e936.e4

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Neurobiology of Aging

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Brief communication

Evaluating noncoding nucleotide repeat expansions in amyotrophic lateralsclerosis

Matthew D. Figley a,b, Anna Thomas a,c, Aaron D. Gitler a,*aDepartment of Genetics, Stanford University School of Medicine, Standford, CA, USAb Stanford Neuroscience Graduate Program, Stanford University School of Medicine, Stanford, CA, USAc Presentation High School, San Jose, CA, USA

a r t i c l e i n f o

Article history:Received 27 August 2013Received in revised form 16 September 2013Accepted 19 September 2013Available online 23 October 2013

Keywords:AtaxinpolyQNoncodingNucleotide repeat expansionALS

* Corresponding author at: 300 Pasteur Dr, M32294305. Tel.: þ1 650 725 6991; fax: þ1 650 725 1534.

E-mail address: agitler@stanford.edu (A.D. Gitler).

0197-4580/$ e see front matter � 2014 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.neurobiolaging.2013.09.024

a b s t r a c t

Intermediate-length polyglutamine expansions in ataxin 2 are a risk factor for amyotrophic lateralsclerosis (ALS). The polyglutamine tract is encoded by a trinucleotide repeat in a coding region of theataxin 2 gene (ATXN2). Noncoding nucleotide repeat expansions in several genes are also associated withneurodegenerative and neuromuscular diseases. For example, hexanucleotide repeat expansions locatedin a noncoding region of C9ORF72 are the most common cause of ALS. We sought to assess a potentiallarger role of noncoding nucleotide repeat expansions in ALS. We analyzed the nucleotide repeat lengthsof 6 genes (ATXN8, ATXN10, PPP2R2B, NOP56, DMPK, and JPH3) that have previously been associated withneurologic or neuromuscular disorders, in several hundred sporadic patients with ALS and healthycontrol subjects. We report no association between ALS and repeat length in any of these genes, sug-gesting that variation in the noncoding repetitive regions in these genes does not contribute to ALS.

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1. Introduction

Intermediate-length polyglutamine repeat expansions inataxin 2 are a risk factor for amyotrophic lateral sclerosis (ALS)(Elden et al., 2010) and longer expansions cause spinocerebellarataxia type 2 (SCA2) (Fischbeck and Pulst, 2011). Ataxin 2 is amember of a family of polyglutamine (polyQ) disease proteins,which includes huntingtin. The genes encoding polyQ proteins allharbor trinucleotide repeat tracts that, when expanded past acertain threshold, cause neurodegenerative disease (Orr andZoghbi, 2007). We had previously surveyed other polyQ diseasegenes in ALS patients and found no significant association be-tween polyQ length and ALS in any of the genes tested beyondataxin 2 (Lee et al., 2011).

Mutations in the C9ORF72 gene were recently identified as themost common cause of ALS and frontotemporal dementia (DeJesus-Hernandez et al., 2011; Renton et al., 2011). The pathogenicmechanism in C9ORF72-linked ALS involves the expansion of anoncoding hexanucleotide repeat, GGGGCC, located in an intron ofthe C9ORF72 gene, from a few repeats in unaffected individuals tohundreds or even thousands of copies in affected individuals(DeJesus-Hernandez et al., 2011; Renton et al., 2011). The

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mechanism through which these expansions cause disease is un-clear and may involve loss of function of C9ORF72, gain of RNAtoxicity from transcribed GGGGCC sequences that accumulate innuclear and cytoplasmic foci, or even proteotoxicity from thenonconventional translation of GGGGCC into dipeptides in multiplereading frames (Ling et al., 2013). The discovery of C9ORF72 mu-tations in ALS indicates that, in principle, noncoding repeat ex-pansions in other genes could also contribute to the disease. Indeed,besides C9ORF72-ALS, there are several other neurodegenerativeand neuromuscular diseases that are caused by expansions of re-petitive DNA in noncoding regions, including spinocerebellar ataxiatype 8, 10, 12, 31, and 36; fragile Xeassociated tremor/ataxia syn-drome; Huntington disease-like 2; and myotonic dystrophy types Iand II (Cooper et al., 2009; Li and Bonini, 2010).

In this report, we expand the analysis of nucleotide repeats inALS beyond those found in coding regions (e.g., polyQ proteins)and assess the potential role of noncoding repeats. We an-alyzed the disease-linked nucleotide repeat sequences in thefollowing genes: ATXN8dspinocerebellar ataxia type 8 (SCA8),ATXN10dSCA10, PPP2R2BdSCA12, NOP56dSCA36, JPH3dHun-tington disease-like 2 (HDL2), and DMPKdmyotonic dystrophytype I in patients with sporadic ALS and healthy control subjects.This analysis revealed no significant association between nucleo-tide repeat length and ALS in any of the genes tested, suggestingthat variation in the noncoding repetitive regions in these genesdoes not contribute to ALS.

M.D. Figley et al. / Neurobiology of Aging 35 (2014) 936.e1e936.e4936.e2

2. Materials and methods

Genomic DNA from human patients with ALS and healthycontrols was obtained from the Coriell Institute for MedicalResearch (Coriell). These genomic DNA samples were from DNApanels from the National Institute of Neurological Disorders andStroke Human Genetics Resource Center DNA and Cell Line Re-pository (http://ccr.coriell.org/ninds). The submitters thatcontributed samples are acknowledged in detailed descriptions ofeach panel: ALS (NDPT025, NDPT026, NDPT103, and NDPT106)and control (NDPT084, NDPT090, NDPT093, NDPT094). The Coriellnon-ALS control samples represent unrelated North AmericanCaucasian individuals (aged 36e48 years) who themselves werenever diagnosed with a neurologic disorder nor had a first-degreerelative with one. We used polymerase chain reaction (PCR)-basedfragment analysis to determine the repeat lengths of each geneanalyzed, using a similar protocol as in (Lee et al., 2011). PCRprimers and cycling conditions are available on request. It remainspossible that our analysis method (PCR fragment analysis) couldhave missed exceptionally long repeat expansions that are re-fractory to PCR amplification, but we think this is unlikely becausewe did not observe an increase in the frequency of apparentlyhomozygous repeat alleles in ALS cases compared with controls,except for PPP2R2B. For that gene, there was a statistically sig-nificant increase in the number of ALS samples with homozygousalleles compared with controls (p ¼ 0.01 (169 of 358 ALS and 127of 338 controls)). However, our analysis method for this genewould have detected the presence of any pathogenic expansion(55e78 repeats). We also note that both ATXN8 and NOP56contain another repetitive motif just next to the disease-causingrepetitive motif. Like previous studies, our fragment analysiscaptures the combined number of both repetitive regions(Table 1).

3. Results

To evaluate the potential contribution of noncoding nucleotiderepeat genes to ALS, we defined the nucleotide repeat length in 6noncoding repeat genes in patients with ALS and healthycontrol subjects (Table 1). We selected the following genes:ATXN8dspinocerebellar ataxia type 8 (SCA8), ATXN10dSCA10,PPP2R2BdSCA12, NOP56dSCA36, JPH3dHDL2, and DMPKdmyo-tonic dystrophy type I. For each gene, we used PCR to amplify thenucleotide repeat region, incorporating the fluorescent dye 6-FAMinto the 50 PCR primer. We determined the repeat length byresolving PCR amplicons by capillary electrophoresis, followed bysize determination with fragment analysis, compared with knownsize standards. Fig. 1A and Table 1 show the genes we analyzed andthe normal range of repeat lengths. Fig. 1BeG show the distributionof nucleotide repeats in each of the genes analyzed in both cases

Table 1Noncoding repeat genes analyzed in patients with ALS and control subjects

Gene Associateddisease

Repeatsequence

No. ALS patientsanalyzed

No healthcontrols a

ATXN8 SCA8 (CTA)n-(CTG)n 365 350ATXN10 SCA10 (ATTCT)n 356 355PPP2R2B SCA12 (CAG)n 358 338NOP56 SCA36 (GGCCTG)n-

(CGCCTG)n352 342

JPH3 HDL2 (CTG)n 360 352DMPK DM1 (CTG)n 333 329

Key: ALS, amyotrophic lateral sclerosis.a Garcia-Murias et al., 2012; Todd and Paulson 2010.

and controls. We did not observe significant differences in therepeat lengths between ALS cases and healthy controls (ATXN8[>23 repeats or >32 repeats], p ¼ 0.49 and p ¼ 0.08, respectively;ATXN10 [>14 repeats], p ¼ 0.41; PPP2R2B [>10 repeats], p ¼ 0.11;NOP56 [>9 repeats], p ¼ 0.19; JPH3 [>14 repeats], p ¼ 0.11; DMPK[>5 repeats], p ¼ 0.61). Of note, 2 ALS patients had ATXN8 CAGrepeat lengths of 70 and 85 (Fig. 1B), which border the SCA8-causing repeat range, although ATXN8 expanded repeats showincomplete penetrance (Day et al., 2000).

4. Discussion

The discoveries of nucleotide repeat expansions in ATXN2 andC9ORF72 as contributors to ALS (Ling et al., 2013) raise the pos-sibility that repeat expansions in other genes might alsocontribute to the disease. In this report, we evaluated 6 genesharboring noncoding nucleotide repeats that are expanded inneurologic and neuromuscular diseases and did not find an as-sociation with ALS. In a previous report, we assessed 7 polyQ-encoding genes and found no association with ALS (Lee et al.,2011). Other groups have also assessed additional nucleotiderepeat expansion genes in ALS. Groen et al. (2012) reported noassociation of the noncoding CGG-repeat expansions in FMR1.Blauw et al. (2012) identified an association between polyalanine-encoding GCG repeats in NIPA1 and ALS. In contrast to our pre-vious findings (Lee et al., 2011), Conforti et al. (2012) reportedincreased frequency of trinucletoide repeat expansions in theataxin 1 gene in ALS patients.

Our findings suggest two possibilities. First, the effects of repeatexpansions in ATXN2 and C9ORF72 that contribute to ALS are spe-cific to those particular genes and thus further understanding thenormal functions of these genes will provide insight into their rolein disease. Second, our analysis of selected candidate nucleotiderepeat genes here and previously (Lee et al., 2011) could certainlyhave missed other repeat-containing genes. Thus, we suggest that acomprehensive genomewide assessment of nucleotide repeat ex-pansions is warranted. Such an analysis will likely require thedevelopment and implementation of novel methods to analyzegenome sequencing data because repetitive sequences are typicallyrefractory to standard next-generation sequencing alignment ap-proaches (DeJesus-Hernandez et al., 2011; Renton et al., 2011).Maybe the difficulty in mapping long repeat expansions could beturned into an advantage and used to scour genome sequence datafor mapping discrepancies that may in fact be hidden repeatexpansions.

Disclosure statement

The authors have no actual or potential conflicts of interest.

ynalyzed

Observed repeatsize range (maximumdetectable)

Previously reportednormal repeatsize rangea

Disease repeatexpansion rangea

15e85 (100) 15e50 w71e14009e22 (32) 10e29 400e45009e25 (126) 7e32 51e786e14 (43) 3e14 w650e2500

5e29 (142) 6e28 >415e39 (100) 5e38 >50

Fig. 1. Analysis of noncoding nucleotide repeat expansions in patients with amyotrophic lateral sclerosis (ALS) and healthy control subjects. (A) Schematic of nucleotide repeatcontaining genes analyzed in this study, including the location and composition of each repeat. Distribution of repeat lengths in nucleotide repeat disease genes in patients with ALSand healthy controls: (B) ATAXN8, (C) ATAXN10, (D) PPP2R2B, (E) NOP56, (F) JPH3, (G) DMPK. The distribution of repeat lengths for the genes assessed was not different in ALS casesand controls.

M.D. Figley et al. / Neurobiology of Aging 35 (2014) 936.e1e936.e4 936.e3

M.D. Figley et al. / Neurobiology of Aging 35 (2014) 936.e1e936.e4936.e4

Acknowledgements

This workwas supported by a National Institutes of Health (NIH)Director’s New Innovator Award DP2OD004417 (A.D.G.), and NIHgrants R01NS065317 and R01NS073660 (to A.D.G.). M.D.F. is sup-ported by the Stanford Genome Training Program. Fragment sizingwas performed by the Nucleic Acid and Protein Core facility at theChildren’s Hospital of Philadelphia. We thank Katherine Carr forhelping with PCR setup and fragment analysis.

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