Advertisement

On the wrong DNA track: Molecular mechanisms of repeat-mediated genome instability

Open AccessPublished:February 14, 2020DOI:https://doi.org/10.1074/jbc.REV119.007678
      Expansions of simple tandem repeats are responsible for almost 50 human diseases, the majority of which are severe, degenerative, and not currently treatable or preventable. In this review, we first describe the molecular mechanisms of repeat-induced toxicity, which is the connecting link between repeat expansions and pathology. We then survey alternative DNA structures that are formed by expandable repeats and review the evidence that formation of these structures is at the core of repeat instability. Next, we describe the consequences of the presence of long structure-forming repeats at the molecular level: somatic and intergenerational instability, fragility, and repeat-induced mutagenesis. We discuss the reasons for gender bias in intergenerational repeat instability and the tissue specificity of somatic repeat instability. We also review the known pathways in which DNA replication, transcription, DNA repair, and chromatin state interact and thereby promote repeat instability. We then discuss possible reasons for the persistence of disease-causing DNA repeats in the genome. We describe evidence suggesting that these repeats are a payoff for the advantages of having abundant simple-sequence repeats for eukaryotic genome function and evolvability. Finally, we discuss two unresolved fundamental questions: (i) why does repeat behavior differ between model systems and human pedigrees, and (ii) can we use current knowledge on repeat instability mechanisms to cure repeat expansion diseases?

      Introduction

      In 1905, British ophthalmologist Edward Nettleship made a peculiar observation: children that suffered from certain degenerative genetic diseases exhibited pathological symptoms earlier than their parents. Nettleship termed this phenomenon “genetic anticipation” (
      • Nettleship E.
      On heredity in the various forms of cataract.
      ). This concept was further substantiated by Bruno Fleischer in his genetic studies of myotonic dystrophy (DM)
      The abbreviations used are: DM
      myotonic dystrophy
      HD
      Huntington's disease
      SBMA
      spinal and bulbar muscular atrophy
      RED
      repeat expansion disease
      SCA
      spinocerebellar ataxia
      DRPLA
      dentatorubral-pallidoluysian atrophy
      XDP
      X-linked dystonia parkinsonism
      FAME3
      familial adult myoclonic epilepsy 3
      FRDA
      Friedreich's ataxia
      HFG
      hand-foot-genital
      polyQ
      polyglutamine
      RAN
      repeat-associated non-ATG
      G4
      G-quadruplex
      DUE
      DNA-unwinding element
      DSB
      double-strand break
      RIM
      repeat-induced mutagenesis
      Pol
      polymerase
      ssDNA
      single-stranded DNA
      RPA
      replication protein A
      BIR
      break-induced replication
      SSA
      single-strand annealing
      EJ
      end-joining
      NHEJ
      nonhomologous EJ
      MMEJ
      microhomology-mediated EJ
      MMR
      mismatch repair
      BER
      base excision repair
      TC-NER
      transcription-coupled NER
      CSB
      Cockayne syndrome group B
      IVF
      in vitro fertilization
      PGD
      preimplantation genetic diagnosis.
      with cataracts (
      • Fleischer B.
      Über myotonische Dystrophie mit Katarakt.
      ).
      For most of the 20th century, the idea of genetic anticipation was highly controversial due to its adoption, misinterpretation, and abuse by eugenicists. Most notably, Nazi officials used the concept to justify the sterilization of people who exhibited mild symptoms of mental disorders, believing that these symptoms were harbingers of severe psychopathology in later generations (
      • Friedman J.E.
      Anticipation in hereditary disease: the history of a biomedical concept.
      ).
      The post-war political backlash against eugenics spelled the near demise for genetic anticipation as a serious scientific concept. The scientific community firmly rejected the existence of non-Mendelian inheritance of this type. Nonetheless, a growing body of evidence began to show that patterns of inheritance in certain types of human diseases, such as DM or Huntington's disease (HD) are hard to explain using conventional genetics (
      • Friedman J.E.
      Anticipation in hereditary disease: the history of a biomedical concept.
      ). Furthermore, in 1985, Stephany Sherman convincingly demonstrated that penetrance and expressivity of fragile X syndrome alleles increase as the disease alleles pass through generations. This phenomenon became known as the Sherman paradox (
      • Sherman S.L.
      • Jacobs P.A.
      • Morton N.E.
      • Froster-Iskenius U.
      • Howard-Peebles P.N.
      • Nielsen K.B.
      • Partington M.W.
      • Sutherland G.R.
      • Turner G.
      • Watson M.
      Further segregation analysis of the fragile X syndrome with special reference to transmitting males.
      ).
      In 1991, two disease-causing genes were cloned: the fragile X gene FMR1 in the laboratories of Stephen Warren, Robert Richards, and Jean-Louis Mandel (
      • Yu S.
      • Pritchard M.
      • Kremer E.
      • Lynch M.
      • Nancarrow J.
      • Baker E.
      • Holman K.
      • Mulley J.C.
      • Warren S.T.
      • Schlessinger D.
      • Warren S.T.
      • Schlessinger D.
      Fragile X genotype characterized by an unstable region of DNA.
      ,
      • Fu Y.H.
      • Kuhl D.P.
      • Pizzuti A.
      • Pieretti M.
      • Sutcliffe J.S.
      • Richards S.
      • Verkerk A.J.
      • Holden J.J.
      • Fenwick Jr., R.G.
      • Warren S.T.
      Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox.
      ,
      • Oberlé I.
      • Rousseau F.
      • Heitz D.
      • Kretz C.
      • Devys D.
      • Hanauer A.
      • Boué J.
      • Bertheas M.F.
      • Mandel J.L.
      Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome.
      ,
      • Verkerk A.J.
      • Pieretti M.
      • Sutcliffe J.S.
      • Fu Y.H.
      • Kuhl D.P.
      • Pizzuti A.
      • Reiner O.
      • Richards S.
      • Victoria M.F.
      • Zhang F.P.
      Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome.
      ,
      • Kremer E.J.
      • Pritchard M.
      • Lynch M.
      • Yu S.
      • Holman K.
      • Baker E.
      • Warren S.T.
      • Schlessinger D.
      • Sutherland G.R.
      • Richards R.I.
      Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n.
      ) and the X-linked spinal and bulbar muscular atrophy (SBMA) gene AR in the laboratory of Kenneth Fischbeck (
      • La Spada A.R.
      • Wilson E.M.
      • Lubahn D.B.
      • Harding A.E.
      • Fischbeck K.H.
      Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy.
      ). These feats revealed the molecular basis of the genetic anticipation. It turned out that fragile X syndrome results from an expansion of the CGG repeats in the 5′-untranslated region (UTR) of the FMR1 gene (
      • Yu S.
      • Pritchard M.
      • Kremer E.
      • Lynch M.
      • Nancarrow J.
      • Baker E.
      • Holman K.
      • Mulley J.C.
      • Warren S.T.
      • Schlessinger D.
      • Warren S.T.
      • Schlessinger D.
      Fragile X genotype characterized by an unstable region of DNA.
      ,
      • Fu Y.H.
      • Kuhl D.P.
      • Pizzuti A.
      • Pieretti M.
      • Sutcliffe J.S.
      • Richards S.
      • Verkerk A.J.
      • Holden J.J.
      • Fenwick Jr., R.G.
      • Warren S.T.
      Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox.
      ,
      • Oberlé I.
      • Rousseau F.
      • Heitz D.
      • Kretz C.
      • Devys D.
      • Hanauer A.
      • Boué J.
      • Bertheas M.F.
      • Mandel J.L.
      Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome.
      ,
      • Verkerk A.J.
      • Pieretti M.
      • Sutcliffe J.S.
      • Fu Y.H.
      • Kuhl D.P.
      • Pizzuti A.
      • Reiner O.
      • Richards S.
      • Victoria M.F.
      • Zhang F.P.
      Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome.
      ,
      • Kremer E.J.
      • Pritchard M.
      • Lynch M.
      • Yu S.
      • Holman K.
      • Baker E.
      • Warren S.T.
      • Schlessinger D.
      • Sutherland G.R.
      • Richards R.I.
      Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n.
      ). The number of CGG repeats expands during parental transmission, leading to increased disease severity. These observations resolved Sherman’s paradox and provided a molecular explanation for genetic anticipation. Similarly to fragile X, SBMA was found to be caused by an expansion of CAG repeats in the coding region of the AR gene (
      • La Spada A.R.
      • Wilson E.M.
      • Lubahn D.B.
      • Harding A.E.
      • Fischbeck K.H.
      Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy.
      ). These discoveries were rapidly followed by the recognition of repeat expansion as the cause of DM1 (
      • Harley H.G.
      • Rundle S.A.
      • Reardon W.
      • Myring J.
      • Crow S.
      • Brook J.D.
      • Harper P.S.
      • Shaw D.J.
      Unstable DNA sequence in myotonic dystrophy.
      ) and HD (
      • The Huntington's Disease Collaborative Research Group
      A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes.
      ). In the blink of an eye, the infamous concept of genetic anticipation was reestablished as a valid scientific phenomenon (
      • Friedman J.E.
      Anticipation in hereditary disease: the history of a biomedical concept.
      ).
      Today, we know 13 different types of tandem repeats whose expansions cause various human diseases (Table 1). The majority of these repeat expansion diseases (REDs) are neither curable nor preventable at present. The most common cause of REDs is an expansion of CAG repeats (or complementary CTG repeats), which are responsible for 16 conditions, including HD and multiple spinocerebellar ataxias (SCAs). CGG repeat expansions cause six different conditions, including fragile X syndrome. Next, two disorders are caused by GAA repeat expansion. In addition to the expansions of these trinucleotide repeats, expansions of one tetranucleotide (CCTG), five pentanucleotide (ATTCT, TGGAA, TTTTA, TTTCA, and AAGGG), three hexanucleotide (GGCCTG, CCCTCT, and GGGGCC), and one dodecanucleotide (CCCCGCCCCGCG) repeat cause 13 other diseases. A separate class of REDs, so-called polyalanine (poly(A)) diseases, are caused by an in-frame expansion of imperfect GCN repeats. This expansion results in abnormally long stretches of alanine in the corresponding proteins. Altogether, nearly 50 REDs are currently known. Beyond these monogenic diseases, some specific repeat expansions might contribute to the etiology of various complex polygenic psychiatric and brain disorders, such as autism spectrum disorder (
      • Metsu S.
      • Rainger J.K.
      • Debacker K.
      • Bernhard B.
      • Rooms L.
      • Grafodatskaya D.
      • Weksberg R.
      • Fombonne E.
      • Taylor M.S.
      • Scherer S.W.
      • Kooy R.F.
      • FitzPatrick D.R.
      A CGG-repeat expansion mutation in ZNF713 causes FRA7A: association with autistic spectrum disorder in two families.
      ), bipolar spectrum disorders, schizophrenia, and others (reviewed in Ref.
      • Hannan A.J.
      Tandem repeat polymorphisms: modulators of disease susceptibility and candidates for “missing heritability”.
      ). The number of known REDs will likely grow, as more than 100 human genes contain DNA repeats that are known to expand in some REDs. These repeats, therefore, can a priori expand, causing a disease (database of genes related to REDs RRID:SCR_018086).
      Table 1Currently known REDs
      Repeat unitGene and position of the repeat in the geneDiseaseYear of discoveryHighlight paperInheritance patternHealthy range, no. of repeatsSymptomatic range, no. of repeats
      ATTCTATXN10, intronSpinocerebellar ataxia 10 (SCA10)2000
      • Matsuura T.
      • Yamagata T.
      • Burgess D.L.
      • Rasmussen A.
      • Grewal R.P.
      • Watase K.
      • Khajavi M.
      • McCall A.E.
      • Davis C.F.
      • Zu L.
      • Achari M.
      • Pulst S.M.
      • Alonso E.
      • Noebels J.L.
      • Nelson D.L.
      • et al.
      Large expansion of the ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10.
      Autosomal dominant10–22500–4500
      DAB1; intronSpinocerebellar ataxia 37 (SCA37)2017
      • Seixas A.I.
      • Loureiro J.R.
      • Costa C.
      • Ordóñez-Ugalde A.
      • Marcelino H.
      • Oliveira C.L.
      • Loureiro J.L.
      • Dhingra A.
      • Brandão E.
      • Cruz V.T.
      • Timóteo A.
      • Quintáns B.
      • Rouleau G.A.
      • Rizzu P.
      • Carracedo A.
      • Bessa J.
      • Heutink P.
      • Sequeiros J.
      • Sobrido M.J.
      • Coutinho P.
      • Silveira I.
      A pentanucleotide ATTTC repeat insertion in the non-coding region of DAB1, mapping to SCA37, causes spinocerebellar ataxia.
      Autosomal dominant<3046–71
      CAGATXN1, coding sequenceSpinocerebellar ataxia1 (SCA1)1993
      • Orr H.T.
      • Chung M.Y.
      • Banfi S.
      • Kwiatkowski Jr., T.J.
      • Servadio A.
      • Beaudet A.L.
      • McCall A.E.
      • Duvick L.A.
      • Ranum L.P.
      • Zoghbi H.Y.
      Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia type 1.
      Autosomal dominant6–3941–83
      ATXN2, coding sequenceSpinocerebellar ataxia2 (SCA2)1996
      • Imbert G.
      • Saudou F.
      • Yvert G.
      • Devys D.
      • Trottier Y.
      • Garnier J.M.
      • Weber C.
      • Mandel J.L.
      • Cancel G.
      • Abbas N.
      • Dürr A.
      • Didierjean O.
      • Stevanin G.
      • Agid Y.
      • Brice A.
      Cloning of the gene for spinocerebellar ataxia 2 reveals a locus with high sensitivity to expanded CAG/glutamine repeats.
      ,
      • Pulst S.M.
      • Nechiporuk A.
      • Nechiporuk T.
      • Gispert S.
      • Chen X.N.
      • Lopes-Cendes I.
      • Pearlman S.
      • Starkman S.
      • Orozco-Diaz G.
      • Lunkes A.
      • DeJong P.
      • Rouleau G.A.
      • Auburger G.
      • Korenberg J.R.
      • Figueroa C.
      • Sahba S.
      Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2.
      Autosomal dominant<3133–200
      ATXN3, coding sequenceSpinocerebellar ataxia3 (SCA3)1994
      • Kawaguchi Y.
      • Okamoto T.
      • Taniwaki M.
      • Aizawa M.
      • Inoue M.
      • Katayama S.
      • Kawakami H.
      • Nakamura S.
      • Nishimura M.
      • Akiguchi I.
      CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1.
      Autosomal dominant<4452–86
      CACNA1A, coding sequenceSpinocerebellar ataxia6 (SCA6)1997
      • Zhuchenko O.
      • Bailey J.
      • Bonnen P.
      • Ashizawa T.
      • Stockton D.W.
      • Amos C.
      • Dobyns W.B.
      • Subramony S.H.
      • Zoghbi H.Y.
      • Lee C.C.
      Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the α 1A-voltage-dependent calcium channel.
      Autosomal dominant<1820–33
      TPB, coding sequenceSpinocerebellar ataxia 17 (SCA17)2001
      • Nakamura K.
      • Jeong S.Y.
      • Uchihara T.
      • Anno M.
      • Nagashima K.
      • Nagashima T.
      • Ikeda S.
      • Tsuji S.
      • Kanazawa I.
      SCA17, a novel autosomal dominant cerebellar ataxia caused by an expanded polyglutamine in TATA-binding protein.
      Autosomal dominant25–4447–63
      ATN1, coding sequenceDentatorubral-pallidoluysian atrophy, Naito-Oyanagi disease (DRPLA)1994
      • Koide R.
      • Ikeuchi T.
      • Onodera O.
      • Tanaka H.
      • Igarashi S.
      • Endo K.
      • Takahashi H.
      • Kondo R.
      • Ishikawa A.
      • Hayashi T.
      Unstable expansion of CAG repeat in hereditary dentatorubral-pallidoluysian atrophy (DRPLA).
      ,
      • Nagafuchi S.
      • Yanagisawa H.
      • Sato K.
      • Shirayama T.
      • Ohsaki E.
      • Bundo M.
      • Takeda T.
      • Tadokoro K.
      • Kondo I.
      • Murayama N.
      Dentatorubral and pallidoluysian atrophy expansion of an unstable CAG trinucleotide on chromosome 12p.
      Autosomal dominant6–3549–88
      HTT, coding sequenceHuntington disease (HD)1993
      • The Huntington's Disease Collaborative Research Group
      A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes.
      Autosomal dominant9–2936–121
      ATXN8, coding sequenceSpinocerebellar ataxia8 (SCA8)1999
      • Koob M.D.
      • Moseley M.L.
      • Schut L.J.
      • Benzow K.A.
      • Bird T.D.
      • Day J.W.
      • Ranum L.P.
      An untranslated CTG expansion causes a novel form of spinocerebellar ataxia (SCA8).
      Autosomal dominant15–5071–1300
      PPP2R2B, 5′ UTRSpinocerebellar ataxia 12 (SCA12)1999
      • Holmes S.E.
      • O'Hearn E.E.
      • McInnis M.G.
      • Gorelick-Feldman D.A.
      • Kleiderlein J.J.
      • Callahan C.
      • Kwak N.G.
      • Ingersoll-Ashworth R.G.
      • Sherr M.
      • Sumner A.J.
      • Sharp A.H.
      • Ananth U.
      • Seltzer W.K.
      • Boss M.A.
      • Vieria-Saecker A.M.
      • Epplen J.T.
      • Riess O.
      • Ross C.A.
      • Margolis R.L.
      Expansion of a novel CAG trinucleotide repeat in the 5′ region of PPP2R2B is associated with SCA12.
      Autosomal dominant7–3251–78
      ATXN7, coding sequenceSpinocerebellar ataxia7 (SCA7)1997
      • David G.
      • Abbas N.
      • Stevanin G.
      • Dürr A.
      • Yvert G.
      • Cancel G.
      • Weber C.
      • Imbert G.
      • Saudou F.
      • Antoniou E.
      • Drabkin H.
      • Gemmill R.
      • Giunti P.
      • Benomar A.
      • Wood N.
      • Ruberg M.
      • Agid Y.
      • Mandel J.L.
      • Brice A.
      Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion.
      Autosomal dominant7–1738–130
      AR, coding sequenceSpinal and bulbar muscular atrophy (SBMA)1991
      • La Spada A.R.
      • Wilson E.M.
      • Lubahn D.B.
      • Harding A.E.
      • Fischbeck K.H.
      Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy.
      X-linked recessive<34>38
      GLS, 5′-UTRGlutaminase deficiency (GD)2019
      • van Kuilenburg A.B.P.
      • Tarailo-Graovac M.
      • Richmond P.A.
      • Drögemöller B.I.
      • Pouladi M.A.
      • Leen R.
      • Brand-Arzamendi K.
      • Dobritzsch D.
      • Dolzhenko E.
      • Eberle M.A.
      • Hayward B.
      • Jones M.J.
      • Karbassi F.
      • Kobor M.S.
      • Koster J.
      • et al.
      Glutaminase deficiency caused by short tandem repeat expansion in GLS.
      Autosomal recessive8–16400–1500
      CCCCGCCCCGCGCSTB, 5′-UTRProgressive myoclonus epilepsy of the Unverricht--Lundborg type (EPM1)1997
      • Lalioti M.D.
      • Scott H.S.
      • Buresi C.
      • Rossier C.
      • Bottani A.
      • Morris M.A.
      • Malafosse A.
      • Antonarakis S.E.
      Dodecamer repeat expansion in cystatin B gene in progressive myoclonus epilepsy.
      Autosomal recessive2–338–77
      CCCTCTTAF1, intronX-linked dystonia parkinsonism (XDP)2019
      • Westenberger A.
      • Reyes C.J.
      • Saranza G.
      • Dobricic V.
      • Hanssen H.
      • Domingo A.
      • Laabs B.H.
      • Schaake S.
      • Pozojevic J.
      • Rakovic A.
      • Grütz K.
      • Begemann K.
      • Walter U.
      • Dressler D.
      • Bauer P.
      • et al.
      A hexanucleotide repeat modifies expressivity of X-linked dystonia parkinsonism.
      X-linked recessiveN/A30–60
      CCTGCNBP, intronMyotonic mystrophy2 (DM2)2001
      • Liquori C.L.
      • Ricker K.
      • Moseley M.L.
      • Jacobsen J.F.
      • Kress W.
      • Naylor S.L.
      • Day J.W.
      • Ranum L.P.
      Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9.
      Autosomal dominant<3075–11,000
      CTGDM1, 3′-UTRMyotonic mystrophy1 (DM1)1992
      • Harley H.G.
      • Rundle S.A.
      • Reardon W.
      • Myring J.
      • Crow S.
      • Brook J.D.
      • Harper P.S.
      • Shaw D.J.
      Unstable DNA sequence in myotonic dystrophy.
      Autosomal dominant5–3750–5,000
      JPH3, 3′-UTRHuntington disease-like 22001
      • Holmes S.E.
      • O'Hearn E.
      • Rosenblatt A.
      • Callahan C.
      • Hwang H.S.
      • Ingersoll-Ashworth R.G.
      • Fleisher A.
      • Stevanin G.
      • Brice A.
      • Potter N.T.
      • Ross C.A.
      • Margolis R.L.
      A repeat expansion in the gene encoding junctophilin-3 is associated with Huntington disease-like 2.
      Autosomal dominant6–28>41
      ATXN8, intronSpinocerebellar ataxia8 (SCA8)1999
      • Koob M.D.
      • Moseley M.L.
      • Schut L.J.
      • Benzow K.A.
      • Bird T.D.
      • Day J.W.
      • Ranum L.P.
      An untranslated CTG expansion causes a novel form of spinocerebellar ataxia (SCA8).
      Autosomal dominant15–3490–250
      TCF4, intronFuchs endothelial corneal dystrophy2012
      • Wieben E.D.
      • Aleff R.A.
      • Tosakulwong N.
      • Butz M.L.
      • Highsmith W.E.
      • Edwards A.O.
      • Baratz K.H.
      A common trinucleotide repeat expansion within the transcription factor 4 (TCF4, E2–2) gene predicts Fuchs corneal dystrophy.
      Autosomal dominant<40>50
      GAADMD, intronDuchenne muscular dystrophy (DMD)2016
      • Kekou K.
      • Sofocleous C.
      • Papadimas G.
      • Petichakis D.
      • Svingou M.
      • Pons R.M.
      • Vorgia P.
      • Gika A.
      • Kitsiou-Tzeli S.
      • Kanavakis E.
      A dynamic trinucleotide repeat (TNR) expansion in the DMD gene.
      X-linked recessive11–3359–82
      FXN, intronFriedreich ataxia 1 (FRDA)1996
      • Campuzano V.
      • Montermini L.
      • Moltó M.D.
      • Pianese L.
      • Cossée M.
      • Cavalcanti F.
      • Monros E.
      • Rodius F.
      • Duclos F.
      • Monticelli A.
      • Zara F.
      • Cañizares J.
      • Koutnikova H.
      • Bidichandani S.I.
      • Gellera C.
      • et al.
      Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.
      Autosomal recessive5–30>70
      CGGXYLT1, 5′-UTRBaratela--Scott syndrome (BSS)2019
      • LaCroix A.J.
      • Stabley D.
      • Sahraoui R.
      • Adam M.P.
      • Mehaffey M.
      • Kernan K.
      • Myers C.T.
      • Fagerstrom C.
      • Anadiotis G.
      • Akkari Y.M.
      • Robbins K.M.
      • Gripp K.W.
      • Baratela W.A.R.
      • Bober M.B.
      • Duker A.L.
      • et al.
      GGC repeat expansion and exon 1 methylation of XYLT1 is a common pathogenic variant in Baratela-Scott syndrome.
      Autosomal recessive9–20100–800
      FMR2, 5′-UTRMental retardation, X-linked, associated with fragile site FRAXE1993
      • Knight S.J.
      • Flannery A.V.
      • Hirst M.C.
      • Campbell L.
      • Christodoulou Z.
      • Phelps S.R.
      • Pointon J.
      • Middleton-Price H.R.
      • Barnicoat A.
      • Pembrey M.E.
      Trinucleotide repeat amplification and hypermethylation of a CpG island in FRAXE mental retardation.
      X-linked recessive4–39>200
      DIP2B, 5′-UTRMental retardation, associated with fragile site FRA12A2007
      • Winnepenninckx B.
      • Debacker K.
      • Ramsay J.
      • Smeets D.
      • Smits A.
      • FitzPatrick D.R.
      • Kooy R.F.
      CGG-repeat expansion in the DIP2B gene is associated with the fragile site FRA12A on chromosome 12q13.1.
      Autosomal dominant12–26>150
      FMR1, 5′-UTRFragile X mental retardation syndrome1991
      • Yu S.
      • Pritchard M.
      • Kremer E.
      • Lynch M.
      • Nancarrow J.
      • Baker E.
      • Holman K.
      • Mulley J.C.
      • Warren S.T.
      • Schlessinger D.
      • Warren S.T.
      • Schlessinger D.
      Fragile X genotype characterized by an unstable region of DNA.
      • Fu Y.H.
      • Kuhl D.P.
      • Pizzuti A.
      • Pieretti M.
      • Sutcliffe J.S.
      • Richards S.
      • Verkerk A.J.
      • Holden J.J.
      • Fenwick Jr., R.G.
      • Warren S.T.
      Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox.
      ,
      • Oberlé I.
      • Rousseau F.
      • Heitz D.
      • Kretz C.
      • Devys D.
      • Hanauer A.
      • Boué J.
      • Bertheas M.F.
      • Mandel J.L.
      Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome.
      ,
      • Verkerk A.J.
      • Pieretti M.
      • Sutcliffe J.S.
      • Fu Y.H.
      • Kuhl D.P.
      • Pizzuti A.
      • Reiner O.
      • Richards S.
      • Victoria M.F.
      • Zhang F.P.
      Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome.
      • Kremer E.J.
      • Pritchard M.
      • Lynch M.
      • Yu S.
      • Holman K.
      • Baker E.
      • Warren S.T.
      • Schlessinger D.
      • Sutherland G.R.
      • Richards R.I.
      Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n.
      X-linked dominant6–52231–2000
      CBL, 5′-UTRJacobsen syndrome1998
      • Michaelis R.C.
      • Velagaleti G.V.
      • Jones C.
      • Pivnick E.K.
      • Phelan M.C.
      • Boyd E.
      • Tarleton J.
      • Wilroy R.S.
      • Tunnacliffe A.
      • Tharapel A.T.
      Most Jacobsen syndrome deletion breakpoints occur distal to FRA11B.
      Not inherited11>100
      NOTCH2NLCNeuronal intranuclear inclusion disease (NIID)2019
      • Sone J.
      • Mitsuhashi S.
      • Fujita A.
      • Mizuguchi T.
      • Hamanaka K.
      • Mori K.
      • Koike H.
      • Hashiguchi A.
      • Takashima H.
      • Sugiyama H.
      • Kohno Y.
      • Takiyama Y.
      • Maeda K.
      • Doi H.
      • Koyano S.
      • et al.
      Long-read sequencing identifies GGC repeat expansions in NOTCH2NLC associated with neuronal intranuclear inclusion disease.
      Autosomal dominant13–3060–959
      GCN (polyAla)RUNX2, coding sequenceCleidocranial dysplasia1997
      • Mundlos S.
      • Otto F.
      • Mundlos C.
      • Mulliken J.B.
      • Aylsworth A.S.
      • Albright S.
      • Lindhout D.
      • Cole W.G.
      • Henn W.
      • Knoll J.H.
      • Owen M.J.
      • Mertelsmann R.
      • Zabel B.U.
      • Olsen B.R.
      Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia.
      Autosomal dominant1727
      SOX3, coding sequenceMental retardation, X-linked2002
      • Laumonnier F.
      • Ronce N.
      • Hamel B.C.
      • Thomas P.
      • Lespinasse J.
      • Raynaud M.
      • Paringaux C.
      • Van Bokhoven H.
      • Kalscheuer V.
      • Fryns J.P.
      • Chelly J.
      • Moraine C.
      • Briault S.
      Transcription factor SOX3 is involved in X-linked mental retardation with growth hormone deficiency.
      X-linked recessive1115–26
      PRDM12, coding sequenceCongenital insensitivity to pain (CIP)2015
      • Chen Y.C.
      • Auer-Grumbach M.
      • Matsukawa S.
      • Zitzelsberger M.
      • Themistocleous A.C.
      • Strom T.M.
      • Samara C.
      • Moore A.W.
      • Cho L.T.
      • Young G.T.
      • Weiss C.
      • Schabhüttl M.
      • Stucka R.
      • Schmid A.B.
      • Parman Y.
      • et al.
      Transcriptional regulator PRDM12 is essential for human pain perception.
      Autosomal recessive1218–19
      PABPN1, coding sequenceOculopharyngeal muscular dystrophy1998
      • Brais B.
      • Bouchard J.P.
      • Xie Y.G.
      • Rochefort D.L.
      • Chrétien N.
      • Tomé F.M.
      • Lafrenière R.G.
      • Rommens J.M.
      • Uyama E.
      • Nohira O.
      • Blumen S.
      • Korczyn A.D.
      • Heutink P.
      • Mathieu J.
      • Duranceau A.
      • et al.
      Short GCG expansions in the PABP2 gene cause oculopharyngeal muscular dystrophy.
      Autosomal dominant67–17
      HOXD13, coding sequenceSynpolydactyly 11996
      • Muragaki Y.
      • Mundlos S.
      • Upton J.
      • Olsen B.R.
      Altered growth and branching patterns in synpolydactyly caused by mutations in HOXD13.
      ,
      • Akarsu A.N.
      • Stoilov I.
      • Yilmaz E.
      • Sayli B.S.
      • Sarfarazi M.
      Genomic structure of HOXD13 gene: a nine polyalanine duplication causes synpolydactyly in two unrelated families.
      Autosomal dominant1522–29
      HOXA13, coding sequenceHand-foot-genital (HFG) syndrome2000
      • Goodman F.R.
      • Bacchelli C.
      • Brady A.F.
      • Brueton L.A.
      • Fryns J.P.
      • Mortlock D.P.
      • Innis J.W.
      • Holmes L.B.
      • Donnenfeld A.E.
      • Feingold M.
      • Beemer F.A.
      • Hennekam R.C.
      • Scambler P.J.
      Novel HOXA13 mutations and the phenotypic spectrum of hand-foot-genital syndrome.
      Autosomal dominant1824–26
      ARX, coding sequenceEpileptic encephalopathy, early infantile, 12007
      • Guerrini R.
      • Moro F.
      • Kato M.
      • Barkovich A.J.
      • Shiihara T.
      • McShane M.A.
      • Hurst J.
      • Loi M.
      • Tohyama J.
      • Norci V.
      • Hayasaka K.
      • Kang U.J.
      • Das S.
      • Dobyns W.B.
      Expansion of the first PolyA tract of ARX causes infantile spasms and status dystonicus.
      ,
      • Kato M.
      • Saitoh S.
      • Kamei A.
      • Shiraishi H.
      • Ueda Y.
      • Akasaka M.
      • Tohyama J.
      • Akasaka N.
      • Hayasaka K.
      A longer polyalanine expansion mutation in the ARX gene causes early infantile epileptic encephalopathy with suppression-burst pattern (Ohtahara syndrome).
      X-linked recessive10–1617–23
      PHOX2B, coding sequenceCentral hypoventilation syndrome, congenital2003
      • Weese-Mayer D.E.
      • Berry-Kravis E.M.
      • Zhou L.
      • Maher B.S.
      • Silvestri J.M.
      • Curran M.E.
      • Marazita M.L.
      Idiopathic congenital central hypoventilation syndrome: analysis of genes pertinent to early autonomic nervous system embryologic development and identification of mutations in PHOX2b.
      Autosomal dominant2024–33
      ZIC2, coding sequenceHoloprosencephaly 52001
      • Brown L.Y.
      • Odent S.
      • David V.
      • Blayau M.
      • Dubourg C.
      • Apacik C.
      • Delgado M.A.
      • Hall B.D.
      • Reynolds J.F.
      • Sommer A.
      • Wieczorek D.
      • Brown S.A.
      • Muenke M.
      Holoprosencephaly due to mutations in ZIC2: alanine tract expansion mutations may be caused by parental somatic recombination.
      Autosomal dominant1525
      FOXL2, coding sequenceBlepharophimosis, ptosis, and epicanthus inversus syndrome2001
      • De Baere E.
      • Dixon M.J.
      • Small K.W.
      • Jabs E.W.
      • Leroy B.P.
      • Devriendt K.
      • Gillerot Y.
      • Mortier G.
      • Meire F.
      • Van Maldergem L.
      • Courtens W.
      • Hjalgrim H.
      • Huang S.
      • Liebaers I.
      • Van Regemorter N.
      • et al.
      Spectrum of FOXL2 gene mutations in blepharophimosis-ptosis-epicanthus inversus (BPES) families demonstrates a genotype–phenotype correlation.
      Autosomal dominant1422–24
      GGCCTGNOP56, intronSpinocerebellar ataxia 36 (SCA36)2011
      • Kobayashi H.
      • Abe K.
      • Matsuura T.
      • Ikeda Y.
      • Hitomi T.
      • Akechi Y.
      • Habu T.
      • Liu W.
      • Okuda H.
      • Koizumi A.
      Expansion of intronic GGCCTG hexanucleotide repeat in NOP56 causes SCA36, a type of spinocerebellar ataxia accompanied by motor neuron involvement.
      Autosomal dominant3–14650–2500
      GGGGCCC9orf72, intronAmyotrophic lateral sclerosis and frontotemporal degeneration (ALS/FTD)2011
      • Renton A.E.
      • Majounie E.
      • Waite A.
      • Simón-Sánchez J.
      • Rollinson S.
      • Gibbs J.R.
      • Schymick J.C.
      • Laaksovirta H.
      • van Swieten J.C.
      • Myllykangas L.
      • Kalimo H.
      • Paetau A.
      • Abramzon Y.
      • Remes A.M.
      • Kaganovich A.
      • et al.
      A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD.
      ,
      • DeJesus-Hernandez M.
      • Mackenzie I.R.
      • Boeve B.F.
      • Boxer A.L.
      • Baker M.
      • Rutherford N.J.
      • Nicholson A.M.
      • Finch N.A.
      • Flynn H.
      • Adamson J.
      • Kouri N.
      • Wojtas A.
      • Sengdy P.
      • Hsiung G.Y.
      • Karydas A.
      • et al.
      Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.
      Autosomal dominant2–19250–1600
      TGGAABEAN1, intronSpinocerebellar ataxia 31 (SCA31)2009
      • Sato N.
      • Amino T.
      • Kobayashi K.
      • Asakawa S.
      • Ishiguro T.
      • Tsunemi T.
      • Takahashi M.
      • Matsuura T.
      • Flanigan K.M.
      • Iwasaki S.
      • Ishino F.
      • Saito Y.
      • Murayama S.
      • Yoshida M.
      • Hashizume Y.
      • et al.
      Spinocerebellar ataxia type 31 is associated with “inserted” penta-nucleotide repeats containing (TGGAA)n.
      Autosomal dominant26500–760
      TTTCA/TTTTASAMD12, intronFamilial adult myoclonic epilepsy1 (FAME1)2018
      • Ishiura H.
      • Doi K.
      • Mitsui J.
      • Yoshimura J.
      • Matsukawa M.K.
      • Fujiyama A.
      • Toyoshima Y.
      • Kakita A.
      • Takahashi H.
      • Suzuki Y.
      • Sugano S.
      • Qu W.
      • Ichikawa K.
      • Yurino H.
      • Higasa K.
      • et al.
      Expansions of intronic TTTCA and TTTTA repeats in benign adult familial myoclonic epilepsy.
      ,
      • Zeng S.
      • Zhang M.Y.
      • Wang X.J.
      • Hu Z.M.
      • Li J.C.
      • Li N.
      • Wang J.L.
      • Liang F.
      • Yang Q.
      • Liu Q.
      • Fang L.
      • Hao J.W.
      • Shi F.D.
      • Ding X.B.
      • Teng J.F.
      • et al.
      Long-read sequencing identified intronic repeat expansions in SAMD12 from Chinese pedigrees affected with familial cortical myoclonic tremor with epilepsy.
      Autosomal dominant7–20440–3680
      TNRC6A, intronFamilial adult myoclonic epilepsy6 (FAME6)2018
      • Ishiura H.
      • Doi K.
      • Mitsui J.
      • Yoshimura J.
      • Matsukawa M.K.
      • Fujiyama A.
      • Toyoshima Y.
      • Kakita A.
      • Takahashi H.
      • Suzuki Y.
      • Sugano S.
      • Qu W.
      • Ichikawa K.
      • Yurino H.
      • Higasa K.
      • et al.
      Expansions of intronic TTTCA and TTTTA repeats in benign adult familial myoclonic epilepsy.
      ,
      • Zeng S.
      • Zhang M.Y.
      • Wang X.J.
      • Hu Z.M.
      • Li J.C.
      • Li N.
      • Wang J.L.
      • Liang F.
      • Yang Q.
      • Liu Q.
      • Fang L.
      • Hao J.W.
      • Shi F.D.
      • Ding X.B.
      • Teng J.F.
      • et al.
      Long-read sequencing identified intronic repeat expansions in SAMD12 from Chinese pedigrees affected with familial cortical myoclonic tremor with epilepsy.
      Autosomal dominant18>22
      RAPGEF2, intronFamilial adult myoclonic epilepsy7 (FAME7)2018
      • Ishiura H.
      • Doi K.
      • Mitsui J.
      • Yoshimura J.
      • Matsukawa M.K.
      • Fujiyama A.
      • Toyoshima Y.
      • Kakita A.
      • Takahashi H.
      • Suzuki Y.
      • Sugano S.
      • Qu W.
      • Ichikawa K.
      • Yurino H.
      • Higasa K.
      • et al.
      Expansions of intronic TTTCA and TTTTA repeats in benign adult familial myoclonic epilepsy.
      ,
      • Zeng S.
      • Zhang M.Y.
      • Wang X.J.
      • Hu Z.M.
      • Li J.C.
      • Li N.
      • Wang J.L.
      • Liang F.
      • Yang Q.
      • Liu Q.
      • Fang L.
      • Hao J.W.
      • Shi F.D.
      • Ding X.B.
      • Teng J.F.
      • et al.
      Long-read sequencing identified intronic repeat expansions in SAMD12 from Chinese pedigrees affected with familial cortical myoclonic tremor with epilepsy.
      Autosomal dominant18>22
      MARCH6, intronFamilial adult myoclonic epilepsy3 (FAME3)2019
      • Florian R.T.
      • Kraft F.
      • Leitão E.
      • Kaya S.
      • Klebe S.
      • Magnin E.
      • van Rootselaar A.F.
      • Buratti J.
      • Kühnel T.
      • Schröder C.
      • Giesselmann S.
      • Tschernoster N.
      • Altmueller J.
      • Lamiral A.
      • Keren B.
      • et al.
      Unstable TTTTA/TTTCA expansions in MARCH6 are associated with familial adult myoclonic epilepsy type 3.
      Autosomal dominant9–20791–1035
      STARD7, intronFamilial adult myoclonic epilepsy2 (FAME2)2019
      • Corbett M.A.
      • Kroes T.
      • Veneziano L.
      • Bennett M.F.
      • Florian R.
      • Schneider A.L.
      • Coppola A.
      • Licchetta L.
      • Franceschetti S.
      • Suppa A.
      • Wenger A.
      • Mei D.
      • Pendziwiat M.
      • Kaya S.
      • Delledonne M.
      • et al.
      Intronic ATTTC repeat expansions in STARD7 in familial adult myoclonic epilepsy linked to chromosome 2.
      Autosomal dominant5–3540–1000
      AAGGGRFC1, intronCerebellar ataxia, neuropathy and vestibular areflexia syndrome2019
      • Cortese A.
      • Simone R.
      • Sullivan R.
      • Vandrovcova J.
      • Tariq H.
      • Yau W.Y.
      • Humphrey J.
      • Jaunmuktane Z.
      • Sivakumar P.
      • Polke J.
      • Ilyas M.
      • Tribollet E.
      • Tomaselli P.J.
      • Devigili G.
      • Callegari I.
      • et al.
      Biallelic expansion of an intronic repeat in RFC1 is a common cause of late-onset ataxia.
      Autosomal recessive11400–2000
      The majority of REDs share two common features. First, the number of inherited repeats typically positively correlates with disease severity and negatively correlates with age of onset. Diseases with strong correlations between the number of repeats and age of onset are SCA7 (
      • Gouw L.G.
      • Castañeda M.A.
      • McKenna C.K.
      • Digre K.B.
      • Pulst S.M.
      • Perlman S.
      • Lee M.S.
      • Gomez C.
      • Fischbeck K.
      • Gagnon D.
      • Storey E.
      • Bird T.
      • Jeri F.R.
      • Ptácek L.J.
      Analysis of the dynamic mutation in the SCA7 gene shows marked parental effects on CAG repeat transmission.
      ,
      • David G.
      • Abbas N.
      • Stevanin G.
      • Dürr A.
      • Yvert G.
      • Cancel G.
      • Weber C.
      • Imbert G.
      • Saudou F.
      • Antoniou E.
      • Drabkin H.
      • Gemmill R.
      • Giunti P.
      • Benomar A.
      • Wood N.
      • Ruberg M.
      • Agid Y.
      • Mandel J.L.
      • Brice A.
      Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion.
      ), SCA3 (
      • Kawaguchi Y.
      • Okamoto T.
      • Taniwaki M.
      • Aizawa M.
      • Inoue M.
      • Katayama S.
      • Kawakami H.
      • Nakamura S.
      • Nishimura M.
      • Akiguchi I.
      CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1.
      ), SCA2 (
      • Giuffrida S.
      • Lanza S.
      • Restivo D.A.
      • Saponara R.
      • Valvo S.C.
      • Le Pira F.
      • Trovato Salinaro A.
      • Spinella F.
      • Nicoletti A.
      • Condorelli D.F.
      Clinical and molecular analysis of 11 Sicilian SCA2 families: influence of gender on age at onset.
      ,
      • Cancel G.
      • Dürr A.
      • Didierjean O.
      • Imbert G.
      • Burk K.
      • Lezin A.
      • Belal S.
      • Benomar A.
      • Abada-Bendib M.
      • Vial C.
      • Guimaraes J.
      • Chneiweiss H.
      • Stevanin G.
      • Yvert G.
      • Abbas N.
      • et al.
      Molecular and clinical correlations in spinocerebellar ataxia 2: a study of 32 families.
      ,
      • Imbert G.
      • Saudou F.
      • Yvert G.
      • Devys D.
      • Trottier Y.
      • Garnier J.M.
      • Weber C.
      • Mandel J.L.
      • Cancel G.
      • Abbas N.
      • Dürr A.
      • Didierjean O.
      • Stevanin G.
      • Agid Y.
      • Brice A.
      Cloning of the gene for spinocerebellar ataxia 2 reveals a locus with high sensitivity to expanded CAG/glutamine repeats.
      ), SCA37 (
      • Seixas A.I.
      • Loureiro J.R.
      • Costa C.
      • Ordóñez-Ugalde A.
      • Marcelino H.
      • Oliveira C.L.
      • Loureiro J.L.
      • Dhingra A.
      • Brandão E.
      • Cruz V.T.
      • Timóteo A.
      • Quintáns B.
      • Rouleau G.A.
      • Rizzu P.
      • Carracedo A.
      • Bessa J.
      • Heutink P.
      • Sequeiros J.
      • Sobrido M.J.
      • Coutinho P.
      • Silveira I.
      A pentanucleotide ATTTC repeat insertion in the non-coding region of DAB1, mapping to SCA37, causes spinocerebellar ataxia.
      ), HD (
      • Ranen N.G.
      • Stine O.C.
      • Abbott M.H.
      • Sherr M.
      • Codori A.M.
      • Franz M.L.
      • Chao N.I.
      • Chung A.S.
      • Pleasant N.
      • Callahan C.
      Anticipation and instability of IT-15 (CAG)n repeats in parent-offspring pairs with Huntington disease.
      ,
      • Trottier Y.
      • Biancalana V.
      • Mandel J.L.
      Instability of CAG repeats in Huntington's disease: relation to parental transmission and age of onset.
      ,
      • Vital M.
      • Bidegain E.
      • Raggio V.
      • Esperon P.
      Molecular characterization of genes modifying the age at onset in Huntington's disease in Uruguayan patients.
      ,
      • Andrew S.E.
      • Goldberg Y.P.
      • Kremer B.
      • Telenius H.
      • Theilmann J.
      • Adam S.
      • Starr E.
      • Squitieri F.
      • Lin B.
      • Kalchman M.A.
      The relationship between trinucleotide (CAG) repeat length and clinical features of Huntington's disease.
      ), DM1 (
      • Morales F.
      • Couto J.M.
      • Higham C.F.
      • Hogg G.
      • Cuenca P.
      • Braida C.
      • Wilson R.H.
      • Adam B.
      • del Valle G.
      • Brian R.
      • Sittenfeld M.
      • Ashizawa T.
      • Wilcox A.
      • Wilcox D.E.
      • Monckton D.G.
      Somatic instability of the expanded CTG triplet repeat in myotonic dystrophy type 1 is a heritable quantitative trait and modifier of disease severity.
      ,
      • Morales F.
      • Vásquez M.
      • Santamaría C.
      • Cuenca P.
      • Corrales E.
      • Monckton D.G.
      A polymorphism in the MSH3 mismatch repair gene is associated with the levels of somatic instability of the expanded CTG repeat in the blood DNA of myotonic dystrophy type 1 patients.
      ), dentatorubral-pallidoluysian atrophy (DRPLA) (
      • Koide R.
      • Ikeuchi T.
      • Onodera O.
      • Tanaka H.
      • Igarashi S.
      • Endo K.
      • Takahashi H.
      • Kondo R.
      • Ishikawa A.
      • Hayashi T.
      Unstable expansion of CAG repeat in hereditary dentatorubral-pallidoluysian atrophy (DRPLA).
      ,
      • Nagafuchi S.
      • Yanagisawa H.
      • Sato K.
      • Shirayama T.
      • Ohsaki E.
      • Bundo M.
      • Takeda T.
      • Tadokoro K.
      • Kondo I.
      • Murayama N.
      Dentatorubral and pallidoluysian atrophy expansion of an unstable CAG trinucleotide on chromosome 12p.
      ), X-linked dystonia parkinsonism (XDP) (
      • Westenberger A.
      • Reyes C.J.
      • Saranza G.
      • Dobricic V.
      • Hanssen H.
      • Domingo A.
      • Laabs B.H.
      • Schaake S.
      • Pozojevic J.
      • Rakovic A.
      • Grütz K.
      • Begemann K.
      • Walter U.
      • Dressler D.
      • Bauer P.
      • et al.
      A hexanucleotide repeat modifies expressivity of X-linked dystonia parkinsonism.
      ), familial adult myoclonic epilepsy 3 (FAME3) (
      • Florian R.T.
      • Kraft F.
      • Leitão E.
      • Kaya S.
      • Klebe S.
      • Magnin E.
      • van Rootselaar A.F.
      • Buratti J.
      • Kühnel T.
      • Schröder C.
      • Giesselmann S.
      • Tschernoster N.
      • Altmueller J.
      • Lamiral A.
      • Keren B.
      • et al.
      Unstable TTTTA/TTTCA expansions in MARCH6 are associated with familial adult myoclonic epilepsy type 3.
      ), and Friedreich’s ataxia (FRDA) (
      • Al-Mahdawi S.
      • Ging H.
      • Bayot A.
      • Cavalcanti F.
      • La Cognata V.
      • Cavallaro S.
      • Giunti P.
      • Pook M.A.
      Large interruptions of GAA repeat expansion mutations in Friedreich ataxia are very rare.
      ,
      • Monrós E.
      • Moltó M.D.
      • Martínez F.
      • Cañizares J.
      • Blanca J.
      • Vílchez J.J.
      • Prieto F.
      • de Frutos R.
      • Palau F.
      Phenotype correlation and intergenerational dynamics of the Friedreich ataxia GAA trinucleotide repeat.
      ). For some disorders, the correlation between the number of repeats and symptom manifestation is less clear, although there typically is a marginally significant trend (
      • Fournier C.
      • Barbier M.
      • Camuzat A.
      • Anquetil V.
      • Lattante S.
      • Clot F.
      • Cazeneuve C.
      • Rinaldi D.
      • Couratier P.
      • Deramecourt V.
      • Sabatelli M.
      • Belliard S.
      • Vercelletto M.
      • Forlani S.
      • Jornea L.
      • et al.
      Relations between C9orf72 expansion size in blood, age at onset, age at collection and transmission across generations in patients and presymptomatic carriers.
      ,
      • Nordin A.
      • Akimoto C.
      • Wuolikainen A.
      • Alstermark H.
      • Jonsson P.
      • Birve A.
      • Marklund S.L.
      • Graffmo K.S.
      • Forsberg K.
      • Brännstrom T.
      • Andersen P.M.
      Extensive size variability of the GGGGCC expansion in C9orf72 in both neuronal and non-neuronal tissues in 18 patients with ALS or FTD.
      ,
      • Renton A.E.
      • Majounie E.
      • Waite A.
      • Simón-Sánchez J.
      • Rollinson S.
      • Gibbs J.R.
      • Schymick J.C.
      • Laaksovirta H.
      • van Swieten J.C.
      • Myllykangas L.
      • Kalimo H.
      • Paetau A.
      • Abramzon Y.
      • Remes A.M.
      • Kaganovich A.
      • et al.
      A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD.
      ,
      • Wiethoff S.
      • O'Connor E.
      • Haridy N.A.
      • Nethisinghe S.
      • Wood N.
      • Giunti P.
      • Bettencourt C.
      • Houlden H.
      Sequencing analysis of the SCA6 CAG expansion excludes an influence of repeat interruptions on disease onset.
      ,
      • Hyppönen J.
      • Äikiä M.
      • Joensuu T.
      • Julkunen P.
      • Danner N.
      • Koskenkorva P.
      • Vanninen R.
      • Lehesjoki A.E.
      • Mervaala E.
      • Kälviäinen R.
      Refining the phenotype of Unverricht-Lundborg disease (EPM1): a population-wide Finnish study.
      ). Second, expansion of one disease-causing repeat does not promote expansions of other repeats in the patient’s genome. In other words, each RED patient typically has only a single repeat type expanded (
      • Ida C.M.
      • Butz M.L.
      • Lundquist P.A.
      • Dawson D.B.
      C9orf72 repeat expansion frequency among patients with Huntington disease genetic testing.
      ,
      • Aydin G.
      • Dekomien G.
      • Hoffjan S.
      • Gerding W.M.
      • Epplen J.T.
      • Arning L.
      Frequency of SCA8, SCA10, SCA12, SCA36, FXTAS and C9orf72 repeat expansions in SCA patients negative for the most common SCA subtypes.
      ).
      Other characteristics differ among REDs. First, even though repeat expansion is necessary for some RED manifestation, there are REDs in which mutations other than repeat expansions can cause the same disease. Second, the RED mode of inheritance can be either autosomal recessive, dominant, or X-linked. Third, REDs can demonstrate a clear pattern of genetic anticipation (
      • Harley H.G.
      • Rundle S.A.
      • Reardon W.
      • Myring J.
      • Crow S.
      • Brook J.D.
      • Harper P.S.
      • Shaw D.J.
      Unstable DNA sequence in myotonic dystrophy.
      ,
      • Ranen N.G.
      • Stine O.C.
      • Abbott M.H.
      • Sherr M.
      • Codori A.M.
      • Franz M.L.
      • Chao N.I.
      • Chung A.S.
      • Pleasant N.
      • Callahan C.
      Anticipation and instability of IT-15 (CAG)n repeats in parent-offspring pairs with Huntington disease.
      ) or a complete lack thereof (
      • Monrós E.
      • Moltó M.D.
      • Martínez F.
      • Cañizares J.
      • Blanca J.
      • Vílchez J.J.
      • Prieto F.
      • de Frutos R.
      • Palau F.
      Phenotype correlation and intergenerational dynamics of the Friedreich ataxia GAA trinucleotide repeat.
      ). Fourth, expandable repeats can be located in the coding part of a gene, in an intron, or in the 5′- or 3′-UTR. Fifth, the size of repeat expansion sufficient to cause pathological symptoms ranges from several repeat units for poly(A) diseases (
      • Mundlos S.
      • Otto F.
      • Mundlos C.
      • Mulliken J.B.
      • Aylsworth A.S.
      • Albright S.
      • Lindhout D.
      • Cole W.G.
      • Henn W.
      • Knoll J.H.
      • Owen M.J.
      • Mertelsmann R.
      • Zabel B.U.
      • Olsen B.R.
      Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia.
      ,
      • Laumonnier F.
      • Ronce N.
      • Hamel B.C.
      • Thomas P.
      • Lespinasse J.
      • Raynaud M.
      • Paringaux C.
      • Van Bokhoven H.
      • Kalscheuer V.
      • Fryns J.P.
      • Chelly J.
      • Moraine C.
      • Briault S.
      Transcription factor SOX3 is involved in X-linked mental retardation with growth hormone deficiency.
      ,
      • Chen Y.C.
      • Auer-Grumbach M.
      • Matsukawa S.
      • Zitzelsberger M.
      • Themistocleous A.C.
      • Strom T.M.
      • Samara C.
      • Moore A.W.
      • Cho L.T.
      • Young G.T.
      • Weiss C.
      • Schabhüttl M.
      • Stucka R.
      • Schmid A.B.
      • Parman Y.
      • et al.
      Transcriptional regulator PRDM12 is essential for human pain perception.
      ,
      • Brais B.
      • Bouchard J.P.
      • Xie Y.G.
      • Rochefort D.L.
      • Chrétien N.
      • Tomé F.M.
      • Lafrenière R.G.
      • Rommens J.M.
      • Uyama E.
      • Nohira O.
      • Blumen S.
      • Korczyn A.D.
      • Heutink P.
      • Mathieu J.
      • Duranceau A.
      • et al.
      Short GCG expansions in the PABP2 gene cause oculopharyngeal muscular dystrophy.
      ,
      • Muragaki Y.
      • Mundlos S.
      • Upton J.
      • Olsen B.R.
      Altered growth and branching patterns in synpolydactyly caused by mutations in HOXD13.
      ,
      • Akarsu A.N.
      • Stoilov I.
      • Yilmaz E.
      • Sayli B.S.
      • Sarfarazi M.
      Genomic structure of HOXD13 gene: a nine polyalanine duplication causes synpolydactyly in two unrelated families.
      ,
      • Goodman F.R.
      • Bacchelli C.
      • Brady A.F.
      • Brueton L.A.
      • Fryns J.P.
      • Mortlock D.P.
      • Innis J.W.
      • Holmes L.B.
      • Donnenfeld A.E.
      • Feingold M.
      • Beemer F.A.
      • Hennekam R.C.
      • Scambler P.J.
      Novel HOXA13 mutations and the phenotypic spectrum of hand-foot-genital syndrome.
      ,
      • Guerrini R.
      • Moro F.
      • Kato M.
      • Barkovich A.J.
      • Shiihara T.
      • McShane M.A.
      • Hurst J.
      • Loi M.
      • Tohyama J.
      • Norci V.
      • Hayasaka K.
      • Kang U.J.
      • Das S.
      • Dobyns W.B.
      Expansion of the first PolyA tract of ARX causes infantile spasms and status dystonicus.
      ,
      • Kato M.
      • Saitoh S.
      • Kamei A.
      • Shiraishi H.
      • Ueda Y.
      • Akasaka M.
      • Tohyama J.
      • Akasaka N.
      • Hayasaka K.
      A longer polyalanine expansion mutation in the ARX gene causes early infantile epileptic encephalopathy with suppression-burst pattern (Ohtahara syndrome).
      ,
      • Weese-Mayer D.E.
      • Berry-Kravis E.M.
      • Zhou L.
      • Maher B.S.
      • Silvestri J.M.
      • Curran M.E.
      • Marazita M.L.
      Idiopathic congenital central hypoventilation syndrome: analysis of genes pertinent to early autonomic nervous system embryologic development and identification of mutations in PHOX2b.
      ,
      • Brown L.Y.
      • Odent S.
      • David V.
      • Blayau M.
      • Dubourg C.
      • Apacik C.
      • Delgado M.A.
      • Hall B.D.
      • Reynolds J.F.
      • Sommer A.
      • Wieczorek D.
      • Brown S.A.
      • Muenke M.
      Holoprosencephaly due to mutations in ZIC2: alanine tract expansion mutations may be caused by parental somatic recombination.
      ,
      • De Baere E.
      • Dixon M.J.
      • Small K.W.
      • Jabs E.W.
      • Leroy B.P.
      • Devriendt K.
      • Gillerot Y.
      • Mortier G.
      • Meire F.
      • Van Maldergem L.
      • Courtens W.
      • Hjalgrim H.
      • Huang S.
      • Liebaers I.
      • Van Regemorter N.
      • et al.
      Spectrum of FOXL2 gene mutations in blepharophimosis-ptosis-epicanthus inversus (BPES) families demonstrates a genotype–phenotype correlation.
      ) to thousands for DM1 (
      • Harley H.G.
      • Rundle S.A.
      • Reardon W.
      • Myring J.
      • Crow S.
      • Brook J.D.
      • Harper P.S.
      • Shaw D.J.
      Unstable DNA sequence in myotonic dystrophy.
      ) and DM2 (
      • Liquori C.L.
      • Ricker K.
      • Moseley M.L.
      • Jacobsen J.F.
      • Kress W.
      • Naylor S.L.
      • Day J.W.
      • Ranum L.P.
      Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9.
      ). Sixth, the origin of expanded alleles varies across repeats. For example, single common ancestors had a pathogenic amplification of ATTCT repeats in SCA10 (
      • Gheno T.C.
      • Furtado G.V.
      • Saute J.A.M.
      • Donis K.C.
      • Fontanari A.M.V.
      • Emmel V.E.
      • Pedroso J.L.
      • Barsottini O.
      • Godeiro-Junior C.
      • van der Linden H.
      • Ternes Pereira E.
      • Cintra V.P.
      • Marques Jr., W.
      • de Castilhos R.M.
      • Alonso I.
      • et al.
      Spinocerebellar ataxia type 10: common haplotype and disease progression rate in Peru and Brazil.
      ,
      • Almeida T.
      • Alonso I.
      • Martins S.
      • Ramos E.M.
      • Azevedo L.
      • Ohno K.
      • Amorim A.
      • Saraiva-Pereira M.L.
      • Jardim L.B.
      • Matsuura T.
      • Sequeiros J.
      • Silveira I.
      Ancestral origin of the ATTCT repeat expansion in spinocerebellar ataxia type 10 (SCA10).
      ,
      • Landrian I.
      • McFarland K.N.
      • Liu J.
      • Mulligan C.J.
      • Rasmussen A.
      • Ashizawa T.
      Inheritance patterns of ATCCT repeat interruptions in spinocerebellar ataxia type 10 (SCA10) expansions.
      ), CCTG repeats in DM2 (
      • Bachinski L.L.
      • Udd B.
      • Meola G.
      • Sansone V.
      • Bassez G.
      • Eymard B.
      • Thornton C.A.
      • Moxley R.T.
      • Harper P.S.
      • Rogers M.T.
      • Jurkat-Rott K.
      • Lehmann-Horn F.
      • Wieser T.
      • Gamez J.
      • Navarro C.
      • et al.
      Confirmation of the type 2 myotonic dystrophy (CCTG)n expansion mutation in patients with proximal myotonic myopathy/proximal myotonic dystrophy of different European origins: a single shared haplotype indicates an ancestral founder effect.
      ), and TTTTA/TTTCA repeats in FAME3 (
      • Florian R.T.
      • Kraft F.
      • Leitão E.
      • Kaya S.
      • Klebe S.
      • Magnin E.
      • van Rootselaar A.F.
      • Buratti J.
      • Kühnel T.
      • Schröder C.
      • Giesselmann S.
      • Tschernoster N.
      • Altmueller J.
      • Lamiral A.
      • Keren B.
      • et al.
      Unstable TTTTA/TTTCA expansions in MARCH6 are associated with familial adult myoclonic epilepsy type 3.
      ). Contrastingly, de novo repeat expansions were reported for the CAG repeat in HD (
      • De Rooij K.E.
      • De Koning Gans P.A.
      • Skraastad M.I.
      • Belfroid R.D.
      • Vegter-Van Der Vlis M.
      • Roos R.A.
      • Bakker E.
      • Van Ommen G.J.
      • Den Dunnen J.T.
      • Losekoot M.
      Dynamic mutation in Dutch Huntington's disease patients: increased paternal repeat instability extending to within the normal size range.
      ), the CGG repeat in fragile X syndrome (
      • Maia N.
      • Loureiro J.R.
      • Oliveira B.
      • Marques I.
      • Santos R.
      • Jorge P.
      • Martins S.
      Contraction of fully expanded FMR1 alleles to the normal range: predisposing haplotype or rare events?.
      ), and the GCN repeat in hand-foot-genital (HFG) syndrome (
      • Owens K.M.
      • Quinonez S.C.
      • Thomas P.E.
      • Keegan C.E.
      • Lefebvre N.
      • Roulston D.
      • Larsen C.A.
      • Stadler H.S.
      • Innis J.W.
      Analysis of de novo HOXA13 polyalanine expansions supports replication slippage without repair in their generation.
      ). Last, the mechanisms of repeat toxicity vary among different repeats and include both a loss and gain of function.
      The goal of this review is to depict the molecular mechanisms implicated in REDs. We first describe models of repeat-induced pathogenicity. We then discuss how repeat length instability and the propensity to induce various types of mutations compromises genome integrity. The focal point of the review is the description of the molecular mechanisms responsible for repeat instability and repeat-induced mutagenesis (RIM). Whereas these mechanisms were primarily established in model experimental systems, we discuss their relevance to human REDs whenever it is possible. Then we reflect on the existence of expandable repeats from the standpoints of genome function and evolvability. As for future directions, we discuss possible reasons for the differences in expandable repeats' behavior in model systems versus human pedigrees as well as how we could use our current knowledge about characteristics and instability of expandable repeats to develop therapies for REDs.

      From expanded DNA repeats to disease

      Repeat expansions induce changes in cell metabolism that become detrimental to the function of specific tissues. What are these changes? First, an expanded repeat located within a gene may lead to a transcription defect and consequently a loss of function of the carrier gene. Such a loss-of-function mechanism predicts an autosomal recessive or X-linked mode of inheritance. It also predicts that in a subset of patients, an inactivation of the same gene could happen due to a missense, indel, or frameshift mutation, instead of repeat expansion. Both of these predictions are true for autosomal recessive FRDA (
      • Campuzano V.
      • Montermini L.
      • Moltó M.D.
      • Pianese L.
      • Cossée M.
      • Cavalcanti F.
      • Monros E.
      • Rodius F.
      • Duclos F.
      • Monticelli A.
      • Zara F.
      • Cañizares J.
      • Koutnikova H.
      • Bidichandani S.I.
      • Gellera C.
      • et al.
      Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.
      ), progressive myoclonus epilepsy of the Unverricht–Lundborg type (EPM1) (
      • Lalioti M.D.
      • Scott H.S.
      • Antonarakis S.E.
      Altered spacing of promoter elements due to the dodecamer repeat expansion contributes to reduced expression of the cystatin B gene in EPM1.
      ), Congenital insensitivity to pain (
      • Chen Y.C.
      • Auer-Grumbach M.
      • Matsukawa S.
      • Zitzelsberger M.
      • Themistocleous A.C.
      • Strom T.M.
      • Samara C.
      • Moore A.W.
      • Cho L.T.
      • Young G.T.
      • Weiss C.
      • Schabhüttl M.
      • Stucka R.
      • Schmid A.B.
      • Parman Y.
      • et al.
      Transcriptional regulator PRDM12 is essential for human pain perception.
      ), X-linked Duchenne muscular dystrophy (
      • Kekou K.
      • Sofocleous C.
      • Papadimas G.
      • Petichakis D.
      • Svingou M.
      • Pons R.M.
      • Vorgia P.
      • Gika A.
      • Kitsiou-Tzeli S.
      • Kanavakis E.
      A dynamic trinucleotide repeat (TNR) expansion in the DMD gene.
      ), and fragile X syndrome (
      • Zink A.M.
      • Wohlleber E.
      • Engels H.
      • Rødningen O.K.
      • Ravn K.
      • Heilmann S.
      • Rehnitz J.
      • Katzorke N.
      • Kraus C.
      • Blichfeldt S.
      • Hoffmann P.
      • Reutter H.
      • Brockschmidt F.F.
      • Kreiss-Nachtsheim M.
      • Vogt P.H.
      • et al.
      Microdeletions including FMR1 in three female patients with intellectual disability—further delineation of the phenotype and expression studies.
      ,
      • Hammond L.S.
      • Macias M.M.
      • Tarleton J.C.
      • Shashidhar Pai G.
      Fragile X syndrome and deletions in FMR1: new case and review of the literature.
      ).
      The majority of REDs exhibit an autosomal dominant mode of inheritance and are caused exclusively by repeat expansions. Moreover, an expansion of a particular repeat may have the same detrimental consequences regardless of the gene in which this expansion happens. For example, expansion of (CAG)n repeats in 13 different genes causes SCAs. Similarly, recently discovered expansions of (TTTTA)n(TTTCA)m repeats in the introns of five different genes are all linked to FAME (
      • Florian R.T.
      • Kraft F.
      • Leitão E.
      • Kaya S.
      • Klebe S.
      • Magnin E.
      • van Rootselaar A.F.
      • Buratti J.
      • Kühnel T.
      • Schröder C.
      • Giesselmann S.
      • Tschernoster N.
      • Altmueller J.
      • Lamiral A.
      • Keren B.
      • et al.
      Unstable TTTTA/TTTCA expansions in MARCH6 are associated with familial adult myoclonic epilepsy type 3.
      ,
      • Ishiura H.
      • Doi K.
      • Mitsui J.
      • Yoshimura J.
      • Matsukawa M.K.
      • Fujiyama A.
      • Toyoshima Y.
      • Kakita A.
      • Takahashi H.
      • Suzuki Y.
      • Sugano S.
      • Qu W.
      • Ichikawa K.
      • Yurino H.
      • Higasa K.
      • et al.
      Expansions of intronic TTTCA and TTTTA repeats in benign adult familial myoclonic epilepsy.
      ,
      • Zeng S.
      • Zhang M.Y.
      • Wang X.J.
      • Hu Z.M.
      • Li J.C.
      • Li N.
      • Wang J.L.
      • Liang F.
      • Yang Q.
      • Liu Q.
      • Fang L.
      • Hao J.W.
      • Shi F.D.
      • Ding X.B.
      • Teng J.F.
      • et al.
      Long-read sequencing identified intronic repeat expansions in SAMD12 from Chinese pedigrees affected with familial cortical myoclonic tremor with epilepsy.
      ,
      • Corbett M.A.
      • Kroes T.
      • Veneziano L.
      • Bennett M.F.
      • Florian R.
      • Schneider A.L.
      • Coppola A.
      • Licchetta L.
      • Franceschetti S.
      • Suppa A.
      • Wenger A.
      • Mei D.
      • Pendziwiat M.
      • Kaya S.
      • Delledonne M.
      • et al.
      Intronic ATTTC repeat expansions in STARD7 in familial adult myoclonic epilepsy linked to chromosome 2.
      ). Therefore, researchers have proposed toxic gain-of-function mechanisms, which could be either at the RNA or protein levels.

      Loss of function of a gene with an expanded repeat tract

      Long repeat tracts can impede transcription of their corresponding gene (
      • Soragni E.
      • Herman D.
      • Dent S.Y.
      • Gottesfeld J.M.
      • Wells R.D.
      • Napierala M.
      Long intronic GAA*TTC repeats induce epigenetic changes and reporter gene silencing in a molecular model of Friedreich ataxia.
      ) and therefore lead to decreased production of an essential protein (
      • Westenberger A.
      • Reyes C.J.
      • Saranza G.
      • Dobricic V.
      • Hanssen H.
      • Domingo A.
      • Laabs B.H.
      • Schaake S.
      • Pozojevic J.
      • Rakovic A.
      • Grütz K.
      • Begemann K.
      • Walter U.
      • Dressler D.
      • Bauer P.
      • et al.
      A hexanucleotide repeat modifies expressivity of X-linked dystonia parkinsonism.
      ,
      • Campuzano V.
      • Montermini L.
      • Moltó M.D.
      • Pianese L.
      • Cossée M.
      • Cavalcanti F.
      • Monros E.
      • Rodius F.
      • Duclos F.
      • Monticelli A.
      • Zara F.
      • Cañizares J.
      • Koutnikova H.
      • Bidichandani S.I.
      • Gellera C.
      • et al.
      Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.
      ,
      • Li Y.
      • Lu Y.
      • Polak U.
      • Lin K.
      • Shen J.
      • Farmer J.
      • Seyer L.
      • Bhalla A.D.
      • Rozwadowska N.
      • Lynch D.R.
      • Butler J.S.
      • Napierala M.
      Expanded GAA repeats impede transcription elongation through the FXN gene and induce transcriptional silencing that is restricted to the FXN locus.
      ). This process could occur in several ways. Formation of RNA-DNA hybrids (R-loops) potentially combined with formation of an unusual secondary structure within the expanded repeats may physically stall transcription (
      • Groh M.
      • Lufino M.M.
      • Wade-Martins R.
      • Gromak N.
      R-loops associated with triplet repeat expansions promote gene silencing in Friedreich ataxia and fragile X syndrome.
      ). For example, in FRDA, RNA polymerase fails to successfully transcribe through expanded GAA repeats located in the intron of the FXN gene (
      • Campuzano V.
      • Montermini L.
      • Moltó M.D.
      • Pianese L.
      • Cossée M.
      • Cavalcanti F.
      • Monros E.
      • Rodius F.
      • Duclos F.
      • Monticelli A.
      • Zara F.
      • Cañizares J.
      • Koutnikova H.
      • Bidichandani S.I.
      • Gellera C.
      • et al.
      Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion.
      ,
      • Li Y.
      • Lu Y.
      • Polak U.
      • Lin K.
      • Shen J.
      • Farmer J.
      • Seyer L.
      • Bhalla A.D.
      • Rozwadowska N.
      • Lynch D.R.
      • Butler J.S.
      • Napierala M.
      Expanded GAA repeats impede transcription elongation through the FXN gene and induce transcriptional silencing that is restricted to the FXN locus.
      ), likely because of the formation of R-loop–containing structures (
      • Ohshima K.
      • Montermini L.
      • Wells R.D.
      • Pandolfo M.
      Inhibitory effects of expanded GAA.TTC triplet repeats from intron I of the Friedreich ataxia gene on transcription and replication in vivo.
      ,
      • Grabczyk E.
      • Mancuso M.
      • Sammarco M.C.
      A persistent RNA.DNA hybrid formed by transcription of the Friedreich ataxia triplet repeat in live bacteria, and by T7 RNAP in vitro.
      ), such as an H-loop (
      • Neil A.J.
      • Liang M.U.
      • Khristich A.N.
      • Shah K.A.
      • Mirkin S.M.
      RNA-DNA hybrids promote the expansion of Friedreich's ataxia (GAA)n repeats via break-induced replication.
      ). This transcription hindrance leads to the appearance of repressive chromatin marks at and around an expanded repeat, ultimately leading to local heterochromatinization and gene repression (reviewed in Ref.
      • Kumari D.
      • Usdin K.
      Is Friedreich ataxia an epigenetic disorder?.
      ) (Fig. 1). Reduced FXN expression causes mitochondrial dysfunction, which eventually leads to cell death (
      • Koutnikova H.
      • Campuzano V.
      • Foury F.
      • Dollé P.
      • Cazzalini O.
      • Koenig M.
      Studies of human, mouse and yeast homologues indicate a mitochondrial function for frataxin.
      ,
      • Anzovino A.
      • Lane D.J.
      • Huang M.L.
      • Richardson D.R.
      Fixing frataxin: “ironing out” the metabolic defect in Friedreich’s ataxia.
      ). Similarly, R-loop formation within expanded CGG (
      • Groh M.
      • Lufino M.M.
      • Wade-Martins R.
      • Gromak N.
      R-loops associated with triplet repeat expansions promote gene silencing in Friedreich ataxia and fragile X syndrome.
      ,
      • Loomis E.W.
      • Sanz L.A.
      • Chédin F.
      • Hagerman P.J.
      Transcription-associated R-loop formation across the human FMR1 CGG-repeat region.
      ) repeats in the 5′-UTR of the FMR1 gene leads to promoter DNA methylation at the repeat and the adjacent promoter (
      • Pretto D.I.
      • Mendoza-Morales G.
      • Lo J.
      • Cao R.
      • Hadd A.
      • Latham G.J.
      • Durbin-Johnson B.
      • Hagerman R.
      • Tassone F.
      CGG allele size somatic mosaicism and methylation in FMR1 premutation alleles.
      ), followed by histone and DNA methylation, resulting in massive heterochromatinization that can spread up to 1.8 Mb (reviewed in Ref.
      • Kumari D.
      • Gazy I.
      • Usdin K.
      Pharmacological reactivation of the silenced FMR1 gene as a targeted therapeutic approach for fragile X syndrome.
      ) (Fig. 1). It is, in fact, this constitutive heterochromatinization that slows down DNA replication through this region, leading to the characteristic fragile X phenotype (
      • Hansen R.S.
      • Canfield T.K.
      • Lamb M.M.
      • Gartler S.M.
      • Laird C.D.
      Association of fragile X syndrome with delayed replication of the FMR1 gene.
      ).
      Figure thumbnail gr1
      Figure 1Loss-of-function mechanisms caused by expanded repeats exemplified by GAA repeats in FXN and CGG repeats in FMR1 genes. Repeats are shown in blue and red, and the flanking DNA sequences are shown in black. Top, when repeats are short, transcription is unimpeded. Bottom left, expansion of GAA repeats leads to formation of an R-loop at the repeat, which triggers histone deacetylation and the presence of repressive histone marks (such as histone H3 Lys-9 methylation), heterochromatization, and gene silencing (reviewed in Ref.
      • Kumari D.
      • Usdin K.
      Is Friedreich ataxia an epigenetic disorder?.
      ). Bottom right, expansion of CGG repeats leads to formation of an R-loop at the repeat, which triggers methylation of the repeat as well as of the CpG islands upstream of the FMR1 promoter, followed by histone methylation. This results in heterochromatization and, ultimately, gene silencing (reviewed in Ref.
      • Kumari D.
      • Gazy I.
      • Usdin K.
      Pharmacological reactivation of the silenced FMR1 gene as a targeted therapeutic approach for fragile X syndrome.
      ).
      Expansion of the dodecamer CCCCGCCCCGCG repeat in the 5′-UTR of the CSTB gene seems to decrease CSTB gene expression by a mechanism different from that discussed above. This repeat is located between two cis-regulatory elements that activate CSTB transcription. Expansion of the repeat increases the distance between the two regulatory elements such that it prevents efficient transcription initiation of the CSTB gene (
      • Lalioti M.D.
      • Scott H.S.
      • Antonarakis S.E.
      Altered spacing of promoter elements due to the dodecamer repeat expansion contributes to reduced expression of the cystatin B gene in EPM1.
      ).

      Toxic gain of function at the level of RNA

      Growing evidence suggests that some expanded repeats exhibit cellular toxicity on their own, rather than in a context of their carrier gene. One example is the expansion of CCTG repeats in an intron of the CNBP gene (in DM2). This expansion does not directly affect gene expression: it changes neither the methylation pattern of the gene promoter (
      • Santoro M.
      • Fontana L.
      • Maiorca F.
      • Centofanti F.
      • Massa R.
      • Silvestri G.
      • Novelli G.
      • Botta A.
      Expanded [CCTG]n repetitions are not associated with abnormal methylation at the CNBP locus in myotonic dystrophy type 2 (DM2) patients.
      ) nor the splicing pattern of CNBP mRNA nor the CNBP protein level (
      • Botta A.
      • Caldarola S.
      • Vallo L.
      • Bonifazi E.
      • Fruci D.
      • Gullotta F.
      • Massa R.
      • Novelli G.
      • Loreni F.
      Effect of the [CCTG]n repeat expansion on ZNF9 expression in myotonic dystrophy type II (DM2).
      ). Instead, expanded CCTG repeats are transcribed into toxic repetitive RNA. The toxicity of expanded RNA repeats is likely associated with their ability to fold into unusual RNA secondary structures. The list of known repetitive RNAs with confirmed toxicity includes CUG, CCUG, CGG, CAG, AUUCU, UGGAA, AUUUC, GGCCUG, and GGGGCC repeats (
      • Seixas A.I.
      • Loureiro J.R.
      • Costa C.
      • Ordóñez-Ugalde A.
      • Marcelino H.
      • Oliveira C.L.
      • Loureiro J.L.
      • Dhingra A.
      • Brandão E.
      • Cruz V.T.
      • Timóteo A.
      • Quintáns B.
      • Rouleau G.A.
      • Rizzu P.
      • Carracedo A.
      • Bessa J.
      • Heutink P.
      • Sequeiros J.
      • Sobrido M.J.
      • Coutinho P.
      • Silveira I.
      A pentanucleotide ATTTC repeat insertion in the non-coding region of DAB1, mapping to SCA37, causes spinocerebellar ataxia.
      ,
      • Qawasmi L.
      • Braun M.
      • Guberman I.
      • Cohen E.
      • Naddaf L.
      • Mellul A.
      • Matilainen O.
      • Roitenberg N.
      • Share D.
      • Stupp D.
      • Chahine H.
      • Cohen E.
      • Garcia S.M.D.A.
      • Tabach Y.
      Expanded CUG repeats trigger disease phenotype and expression changes through the RNAi machinery in C. elegans.
      ,
      • Yu Z.
      • Teng X.
      • Bonini N.M.
      Triplet repeat-derived siRNAs enhance RNA-mediated toxicity in a Drosophila model for myotonic dystrophy.
      ,
      • Hu J.
      • Rong Z.
      • Gong X.
      • Zhou Z.
      • Sharma V.K.
      • Xing C.
      • Watts J.K.
      • Corey D.R.
      • Mootha V.V.
      Oligonucleotides targeting TCF4 triplet repeat expansion inhibit RNA foci and mis-splicing in Fuchs' dystrophy.
      ,
      • Meola G.
      • Cardani R.
      Myotonic dystrophies: an update on clinical aspects, genetic, pathology, and molecular pathomechanisms.
      ,
      • Cerro-Herreros E.
      • Chakraborty M.
      • Pérez-Alonso M.
      • Artero R.
      • Llamusí B.
      Expanded CCUG repeat RNA expression in Drosophila heart and muscle trigger myotonic dystrophy type 1-like phenotypes and activate autophagocytosis genes.
      ,
      • Pascual M.
      • Vicente M.
      • Monferrer L.
      • Artero R.
      The Muscleblind family of proteins: an emerging class of regulators of developmentally programmed alternative splicing.
      ,
      • Wojciechowska M.
      • Krzyzosiak W.J.
      Cellular toxicity of expanded RNA repeats: focus on RNA foci.
      ,
      • Niimi Y.
      • Takahashi M.
      • Sugawara E.
      • Umeda S.
      • Obayashi M.
      • Sato N.
      • Ishiguro T.
      • Higashi M.
      • Eishi Y.
      • Mizusawa H.
      • Ishikawa K.
      Abnormal RNA structures (RNA foci) containing a penta-nucleotide repeat (UGGAA)n in the Purkinje cell nucleus is associated with spinocerebellar ataxia type 31 pathogenesis.
      ,
      • Kobayashi H.
      • Abe K.
      • Matsuura T.
      • Ikeda Y.
      • Hitomi T.
      • Akechi Y.
      • Habu T.
      • Liu W.
      • Okuda H.
      • Koizumi A.
      Expansion of intronic GGCCTG hexanucleotide repeat in NOP56 causes SCA36, a type of spinocerebellar ataxia accompanied by motor neuron involvement.
      ,
      • Yuva-Aydemir Y.
      • Almeida S.
      • Gao F.B.
      Insights into C9ORF72-related ALS/FTD from Drosophila and iPSC models.
      ).
      What are the molecular mechanisms of this toxicity? Expanded repetitive RNAs can engage in multivalent base pairing, which leads to their gelation and aggregation into visible nuclear foci (
      • Jain A.
      • Vale R.D.
      RNA phase transitions in repeat expansion disorders.
      ). As a consequence, these RNAs remain in the nucleus and might sequester RNA-binding proteins. In the best-studied cases of DM1 and DM2 diseases, expanded RNA repeats sequester Muscleblind (Mbnl) proteins, whose normal function is to regulate splicing of muscle-specific genes (
      • Miller J.W.
      • Urbinati C.R.
      • Teng-Umnuay P.
      • Stenberg M.G.
      • Byrne B.J.
      • Thornton C.A.
      • Swanson M.S.
      Recruitment of human muscleblind proteins to (CUG)n expansions associated with myotonic dystrophy.
      ) (Fig. 2). This mechanism is evidenced by the fact that disruption of the MBNL1 gene leads to the same symptoms as the expression of expanded repeats alone (
      • Kanadia R.N.
      • Johnstone K.A.
      • Mankodi A.
      • Lungu C.
      • Thornton C.A.
      • Esson D.
      • Timmers A.M.
      • Hauswirth W.W.
      • Swanson M.S.
      A muscleblind knockout model for myotonic dystrophy.
      ). For other repeats, the precise toxicity mechanism is less understood. Expression of r(GGGGCC)exp causes length-dependent cognitive impairment in mice (
      • Jiang J.
      • Zhu Q.
      • Gendron T.F.
      • Saberi S.
      • McAlonis-Downes M.
      • Seelman A.
      • Stauffer J.E.
      • Jafar-Nejad P.
      • Drenner K.
      • Schulte D.
      • Chun S.
      • Sun S.
      • Ling S.C.
      • Myers B.
      • Engelhardt J.
      • et al.
      Gain of toxicity from ALS/FTD-linked repeat expansions in C9ORF72 is alleviated by antisense oligonucleotides targeting GGGGCC-containing RNAs.
      ), but the mechanistic connection between RNA expression and symptoms remains unclear. This repetitive RNA folds into G-quadruplexes (
      • Reddy K.
      • Zamiri B.
      • Stanley S.Y.
      • Macgregor Jr., R.B.
      • Pearson C.E.
      The disease-associated r(GGGGCC)n repeat from the C9orf72 gene forms tract length-dependent uni- and multimolecular RNA G-quadruplex structures.
      ), forms nuclear foci (
      • Jiang J.
      • Zhu Q.
      • Gendron T.F.
      • Saberi S.
      • McAlonis-Downes M.
      • Seelman A.
      • Stauffer J.E.
      • Jafar-Nejad P.
      • Drenner K.
      • Schulte D.
      • Chun S.
      • Sun S.
      • Ling S.C.
      • Myers B.
      • Engelhardt J.
      • et al.
      Gain of toxicity from ALS/FTD-linked repeat expansions in C9ORF72 is alleviated by antisense oligonucleotides targeting GGGGCC-containing RNAs.
      ,
      • DeJesus-Hernandez M.
      • Mackenzie I.R.
      • Boeve B.F.
      • Boxer A.L.
      • Baker M.
      • Rutherford N.J.
      • Nicholson A.M.
      • Finch N.A.
      • Flynn H.
      • Adamson J.
      • Kouri N.
      • Wojtas A.
      • Sengdy P.
      • Hsiung G.Y.
      • Karydas A.
      • et al.
      Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.
      ), sequesters important RNA-binding proteins (
      • Conlon E.G.
      • Lu L.
      • Sharma A.
      • Yamazaki T.
      • Tang T.
      • Shneider N.A.
      • Manley J.L.
      The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains.
      ,
      • Shen J.
      • Zhang Y.
      • Zhao S.
      • Mao H.
      • Wang Z.
      • Li H.
      • Xu Z.
      Purα repaired expanded hexanucleotide GGGGCC repeat noncoding RNA-caused neuronal toxicity in Neuro-2a cells.
      ,
      • Xu Z.
      • Poidevin M.
      • Li X.
      • Li Y.
      • Shu L.
      • Nelson D.L.
      • Li H.
      • Hales C.M.
      • Gearing M.
      • Wingo T.S.
      • Jin P.
      Expanded GGGGCC repeat RNA associated with amyotrophic lateral sclerosis and frontotemporal dementia causes neurodegeneration.
      ,
      • Mori K.
      • Lammich S.
      • Mackenzie I.R.
      • Forné I.
      • Zilow S.
      • Kretzschmar H.
      • Edbauer D.
      • Janssens J.
      • Kleinberger G.
      • Cruts M.
      • Herms J.
      • Neumann M.
      • Van Broeckhoven C.
      • Arzberger T.
      • Haass C.
      hnRNP A3 binds to GGGGCC repeats and is a constituent of p62-positive/TDP43-negative inclusions in the hippocampus of patients with C9orf72 mutations.
      ), and impairs mRNA transport (
      • Burguete A.S.
      • Almeida S.
      • Gao F.B.
      • Kalb R.
      • Akins M.R.
      • Bonini N.M.
      GGGGCC microsatellite RNA is neuritically localized, induces branching defects, and perturbs transport granule function.
      ) likely by damaging the nuclear pore complex (
      • Freibaum B.D.
      • Lu Y.
      • Lopez-Gonzalez R.
      • Kim N.C.
      • Almeida S.
      • Lee K.H.
      • Badders N.
      • Valentine M.
      • Miller B.L.
      • Wong P.C.
      • Petrucelli L.
      • Kim H.J.
      • Gao F.B.
      • Taylor J.P.
      GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport.
      ). However, it is not yet clear which of these features of the r(GGGGCC)exp repeats is primarily responsible for toxicity.
      Figure thumbnail gr2
      Figure 2Toxic gain-of-function mechanisms in REDs exemplified by an expansion of a CTG repeat. Left, transcription of expanded CTG repeats produces long r(CUG)n RNA species that fold into RNA secondary structures, aggregate, and sequester the Muscleblind protein. This is the main source of toxicity in DM1 disease (reviewed in Ref.
      • Sznajder Ł.J.
      • Swanson M.S.
      Short tandem repeat expansions and RNA-mediated pathogenesis in myotonic dystrophy.
      ). Right, RAN translation of r(CUG)n. Expanded RNA repeats recruit ribosomes. This recruitment is likely mediated by the formation of an RNA secondary structure. Translation of r(CUG)n results in accumulation of toxic repetitive polypeptides in all three reading frames (reviewed in Refs.
      • Cleary J.D.
      • Pattamatta A.
      • Ranum L.P.W.
      Repeat-associated non-ATG (RAN) translation.
      • Cleary J.D.
      • Ranum L.P.
      New developments in RAN translation: insights from multiple diseases.
      • Swinnen B.
      • Robberecht W.
      • Van Den Bosch L.
      RNA toxicity in non-coding repeat expansion disorders.
      ). Note that antisense transcription of the same repeat might also undergo RAN translation (not shown).

      Toxic gain of function at the level of protein

      A group of REDs are caused by expansions of GCN or CAG repeats located in coding parts of various genes. Translation of these in-frame repeats results in abnormally long poly(A) or polyglutamine poly(Q) tracts in their corresponding proteins. These long tracts could abrogate function of a protein and/or exhibit toxic gain of function.
      Poly(A) tracts are common protein motifs that were suggested to promote protein interactions (
      • Lavoie H.
      • Debeane F.
      • Trinh Q.D.
      • Turcotte J.F.
      • Corbeil-Girard L.P.
      • Dicaire M.J.
      • Saint-Denis A.
      • Pagé M.
      • Rouleau G.A.
      • Brais B.
      Polymorphism, shared functions and convergent evolution of genes with sequences coding for polyalanine domains.
      ) or serve as nuclear export signals (
      • Li L.
      • Ng N.K.
      • Koon A.C.
      • Chan H.Y.
      Expanded polyalanine tracts function as nuclear export signals and promote protein mislocalization via eEF1A1 factor.
      ). At the molecular level, poly(A) peptides undergo a conformational change as their length increases. Whereas short poly(A) tracts preferably form monomeric α-helices, longer tracts fold into polymeric β-sheets (reviewed in Ref.
      • Messaed C.
      • Rouleau G.A.
      Molecular mechanisms underlying polyalanine diseases.
      ) and into coiled coils (
      • Pelassa I.
      • Corà D.
      • Cesano F.
      • Monje F.J.
      • Montarolo P.G.
      • Fiumara F.
      Association of polyalanine and polyglutamine coiled coils mediates expansion disease-related protein aggregation and dysfunction.
      ). These conformational changes can lead to a toxic gain of function by introducing novel types of protein interactions, aggregation, mislocalization, and sequestration of other essential proteins. Commonly, proteins with poly(A) expansions co-aggregate with their WT versions, leading to effective haploinsufficiency, which explains their dominant inheritance patterns (reviewed in Ref.
      • Messaed C.
      • Rouleau G.A.
      Molecular mechanisms underlying polyalanine diseases.
      ).
      Similar to expanded poly(A) tracts, long poly(Q) tracts misfold into β-sheet structures that aggregate into toxic inclusion bodies in neurons. These structures might act as so-called “polar zippers,” which exhibit nonspecific affinity to various regulatory proteins in a cell (
      • Perutz M.F.
      • Johnson T.
      • Suzuki M.
      • Finch J.T.
      Glutamine repeats as polar zippers: their possible role in inherited neurodegenerative diseases.
      ). Accumulation of poly(Q) runs is believed to cause neurodegeneration (reviewed in Ref.
      • Takeuchi T.
      • Nagai Y.
      Protein misfolding and aggregation as a therapeutic target for polyglutamine diseases.
      ). First discovered in 1997 for CAG repeats in HD (
      • DiFiglia M.
      • Sapp E.
      • Chase K.O.
      • Davies S.W.
      • Bates G.P.
      • Vonsattel J.P.
      • Aronin N.
      Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain.
      ), poly(Q) accumulation was used to explain symptoms of SCAs and other diseases caused by expansions of translated CAG repeats. However, there was one mysterious exception: SCA8. Unlike other SCAs, SCA8 is caused by an expansion of transcribed CTG rather than CAG repeats. Nonetheless, its symptoms resemble other SCAs (
      • Koob M.D.
      • Moseley M.L.
      • Schut L.J.
      • Benzow K.A.
      • Bird T.D.
      • Day J.W.
      • Ranum L.P.
      An untranslated CTG expansion causes a novel form of spinocerebellar ataxia (SCA8).
      ). This conundrum was partially resolved after the discovery of bidirectional transcription of the SCA8 locus and subsequent translation of antisense transcripts (
      • Moseley M.L.
      • Zu T.
      • Ikeda Y.
      • Gao W.
      • Mosemiller A.K.
      • Daughters R.S.
      • Chen G.
      • Weatherspoon M.R.
      • Clark H.B.
      • Ebner T.J.
      • Day J.W.
      • Ranum L.P.
      Bidirectional expression of CUG and CAG expansion transcripts and intranuclear polyglutamine inclusions in spinocerebellar ataxia type 8.
      ). Yet one question remained unanswered: how can CAG repeats from antisense transcripts be translated without a start codon?
      The discovery of an unusual phenomenon called repeat-associated non-ATG (RAN) translation shed some light on this mystery (
      • Zu T.
      • Gibbens B.
      • Doty N.S.
      • Gomes-Pereira M.
      • Huguet A.
      • Stone M.D.
      • Margolis J.
      • Peterson M.
      • Markowski T.W.
      • Ingram M.A.
      • Nan Z.
      • Forster C.
      • Low W.C.
      • Schoser B.
      • Somia N.V.
      • et al.
      Non-ATG-initiated translation directed by microsatellite expansions.
      ). RAN translation requires neither the canonical nor an alternative start codon for initiation and results in production of repetitive proteins in all possible reading frames. Taking into consideration bidirectional transcription, one expanded repeat may produce up to six repetitive polypeptides. To illustrate, RAN translation of the GGGGCC repeat sense transcript, r(GGGGCC)exp, results in poly(GA), poly(GP), and poly(AR) peptides, all of which were detected in ALS/FTD patient–derived cells (
      • Mori K.
      • Weng S.M.
      • Arzberger T.
      • May S.
      • Rentzsch K.
      • Kremmer E.
      • Schmid B.
      • Kretzschmar H.A.
      • Cruts M.
      • Van Broeckhoven C.
      • Haass C.
      • Edbauer D.
      The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS.
      ). The antisense transcription of the same repeat leads to accumulation of polyGP, polyAP, and polyPR peptides (
      • Gendron T.F.
      • Bieniek K.F.
      • Zhang Y.J.
      • Jansen-West K.
      • Ash P.E.
      • Caulfield T.
      • Daughrity L.
      • Dunmore J.H.
      • Castanedes-Casey M.
      • Chew J.
      • Cosio D.M.
      • van Blitterswijk M.
      • Lee W.C.
      • Rademakers R.
      • Boylan K.B.
      • et al.
      Antisense transcripts of the expanded C9ORF72 hexanucleotide repeat form nuclear RNA foci and undergo repeat-associated non-ATG translation in c9FTD/ALS.
      ,
      • Mori K.
      • Arzberger T.
      • Grässer F.A.
      • Gijselinck I.
      • May S.
      • Rentzsch K.
      • Weng S.M.
      • Schludi M.H.
      • van der Zee J.
      • Cruts M.
      • Van Broeckhoven C.
      • Kremmer E.
      • Kretzschmar H.A.
      • Haass C.
      • Edbauer D.
      Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins.
      ). These polypeptides aggregate into high-molecular weight insoluble clusters (
      • Ash P.E.
      • Bieniek K.F.
      • Gendron T.F.
      • Caulfield T.
      • Lin W.L.
      • Dejesus-Hernandez M.
      • van Blitterswijk M.M.
      • Jansen-West K.
      • Paul 3rd, J.W.
      • Rademakers R.
      • Boylan K.B.
      • Dickson D.W.
      • Petrucelli L.
      Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS.
      ) and seem to be neurotoxic (
      • Jiang J.
      • Zhu Q.
      • Gendron T.F.
      • Saberi S.
      • McAlonis-Downes M.
      • Seelman A.
      • Stauffer J.E.
      • Jafar-Nejad P.
      • Drenner K.
      • Schulte D.
      • Chun S.
      • Sun S.
      • Ling S.C.
      • Myers B.
      • Engelhardt J.
      • et al.
      Gain of toxicity from ALS/FTD-linked repeat expansions in C9ORF72 is alleviated by antisense oligonucleotides targeting GGGGCC-containing RNAs.
      ,
      • Freibaum B.D.
      • Taylor J.P.
      The role of dipeptide repeats in C9ORF72-related ALS-FTD.
      ), although it is not yet clear to what extent different polypeptides contribute to overall toxicity (
      • Lopez-Gonzalez R.
      • Lu Y.
      • Gendron T.F.
      • Karydas A.
      • Tran H.
      • Yang D.
      • Petrucelli L.
      • Miller B.L.
      • Almeida S.
      • Gao F.B.
      Poly(GR) in C9ORF72-related ALS/FTD compromises mitochondrial function and increases oxidative stress and DNA damage in iPSC-derived motor neurons.
      ).
      Length-dependent RAN translation has been reported for CAG, CGG, GGGGCC, GGCCUG, and TGGAA repeats (reviewed in Refs.
      • Cleary J.D.
      • Pattamatta A.
      • Ranum L.P.W.
      Repeat-associated non-ATG (RAN) translation.
      ,
      • Cleary J.D.
      • Ranum L.P.
      New developments in RAN translation: insights from multiple diseases.
      ,
      • Swinnen B.
      • Robberecht W.
      • Van Den Bosch L.
      RNA toxicity in non-coding repeat expansion disorders.
      ). Nonetheless, we still lack a good understanding of RAN’s mechanistic details. One hypothesis is that formation of unusual secondary RNA structures, such as hairpins (
      • Koob M.D.
      • Moseley M.L.
      • Schut L.J.
      • Benzow K.A.
      • Bird T.D.
      • Day J.W.
      • Ranum L.P.
      An untranslated CTG expansion causes a novel form of spinocerebellar ataxia (SCA8).
      ) or G-quadruplexes (G4s) (
      • Fay M.M.
      • Lyons S.M.
      • Ivanov P.
      RNA G-quadruplexes in biology: principles and molecular mechanisms.
      ), might somehow recruit ribosomes and trigger this peculiar mode of translation (Fig. 2).
      Taken together, mechanisms by which expanded repeats lead to cellular toxicity include loss of function of a protein and a toxic gain of function on the RNA or protein levels. These mechanisms might take place in combination with each other. Unfortunately, for many REDs, there is still no consensus on what mechanism plays a major role in disease progression. For example, accumulation of toxic poly(Q) proteins was historically used to explain HD’s phenotype. However, evidence accumulated in the last decade suggests that gain of function at the RNA level might also contribute to disease manifestation. This is supported by experiments in model systems where CAG repeats interrupted by the CAA codon express decreased cellular toxicity when compared with pure CAG repeat tracts (reviewed in Ref.
      • Martí E.
      RNA toxicity induced by expanded CAG repeats in Huntington's disease.
      ). This is unexpected because both CAA and CAG codons code for glutamine. As such, the length of transcribed poly(Q) tracts does not depend on the presence of a CAA interruption. Additionally, it was recently documented that HD age of onset is better predicted by the length of uninterrupted CAG repeat tracts rather than by the sheer number of consecutive glutamines in the Htt protein (
      • Genetic Modifiers of Huntington's Disease (GeM-HD) Consortium and Genetic Modifiers of Huntington's Disease (GeM-HD) Consortium
      CAG repeat not polyglutamine length determines timing of Huntington's disease onset.
      ,
      • Wright G.E.B.
      • Collins J.A.
      • Kay C.
      • McDonald C.
      • Dolzhenko E.
      • Xia Q.
      • Bečanović K.
      • Drögemöller B.I.
      • Semaka A.
      • Nguyen C.M.
      • Trost B.
      • Richards F.
      • Bijlsma E.K.
      • Squitieri F.
      • Ross C.J.D.
      • et al.
      Length of uninterrupted CAG, independent of polyglutamine size, results in increased somatic instability, hastening onset of Huntington disease.
      ,
      • Ciosi M.
      • Maxwell A.
      • Cumming S.A.
      • Hensman Moss D.J.
      • Alshammari A.M.
      • Flower M.D.
      • Durr A.
      • Leavitt B.R.
      • Roos R.A.C.
      • Track-HD team, Enroll-HD team
      • Holmans P.
      • Jones L.
      • Langbehn D.R.
      • Kwak S.
      • et al.
      A genetic association study of glutamine-encoding DNA sequence structures, somatic CAG expansion, and DNA repair gene variants, with Huntington disease clinical outcomes.
      ). However, there exists an alternative explanation of the same phenomenon: the disease age of onset might depend on somatic expansions of CAG repeats during the patient’s lifetime, a process that requires repeat integrity (see below).
      Overall, due to technical obstacles, it is highly challenging to decipher the precise role of various toxicity mechanisms in a specific RED (
      • Swinnen B.
      • Robberecht W.
      • Van Den Bosch L.
      RNA toxicity in non-coding repeat expansion disorders.
      ). Therefore, we have a good understanding of possible mechanisms of expanded repeats' toxicity, even though the precise mechanisms are established for only few REDs.

      Dynamic DNA structures as the key to repeat instability

      Tandem repeats are abundant in the human genome (
      • Treangen T.J.
      • Salzberg S.L.
      Repetitive DNA and next-generation sequencing: computational challenges and solutions.
      ), particularly in centromeres and telomeres, as well as in regulatory regions (
      • Gemayel R.
      • Vinces M.D.
      • Legendre M.
      • Verstrepen K.J.
      Variable tandem repeats accelerate evolution of coding and regulatory sequences.
      ). High variability of tandem repeats between different individuals (
      • Tautz D.
      Hypervariability of simple sequences as a general source for polymorphic DNA markers.
      ) makes analysis of tandem repeat polymorphisms useful in forensics and paternity testing (
      • Panneerchelvam S.
      • Norazmi M.N.
      Forensic DNA profiling and database.
      ). Nonetheless, most of the known tandem repeats do not expand as dramatically as disease-causing repeats, which may gain up to thousands of repeat units in affected individuals (
      • Harley H.G.
      • Rundle S.A.
      • Reardon W.
      • Myring J.
      • Crow S.
      • Brook J.D.
      • Harper P.S.
      • Shaw D.J.
      Unstable DNA sequence in myotonic dystrophy.