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DNA REPEAT SEQUENCES & DISEASE ⁷

Repeat Sequences: Principles ¹

• Human genome repeat sequences
  • Nucleotides: Adenine; Thymine; Guanine; Cytosine
  • Genome contains many repeated DNA sequences
  • Some repeat sequences have increased frequency in primates
  • Repeat sequence length: Varies from 1 nucleotide to whole gene
    • Highly repetitive DNA is found in some untranslated regions
      • 6 to 10 base pair sequences may be repeated 100,000 to 1,000,000 times
    • Whole genes may exist as tandem clusters of multiple copies (50 to 10,000)
      • Multiple copy genes include histones, ribosomal RNA, tRNA, SMN
      • ? Allow more gene product to accumulate per unit time
      • Meet high biosynthetic requirements during early development
      • Repeat induced gene silencing: Via methylation & chromatin compaction
        • 1 gene copy at locus expressed more effectively than
          multiple copies in arrays
        • More repression when multiple copies of gene on 1 chromosome than
          with dispersion over several chromosomes
  • Some DNA repeats present in numerous places & genes in genome
  • Some DNA repeats are mutation "hot spots"
    • Cause mismatching during DNA replication, e.g. Alu
    • Form fragile chromosomal break points, e.g. CCG repeats
    • Number of repeats may be unstable during DNA replication, e.g. CAG repeats
• DNA Repeat types: Tandemly repeated DNA & Interspersed repetitive DNA
  • Tandemly repeated DNA
    • Repeats often associated with disease syndromes
    • Telomeres
      • Contain long arrays of TTAGGG repeats
      • Repeats form nucleoprotein complex: Associate with TERF proteins
      • Repeat function
        • Protection of chromosome ends
        • Replication of chromosome ends
        • Control of telomere length
  • Interspersed repetitive DNA
    • Short Interspersed Nuclear Elements (SINEs)
      • Alu
        • GC rich
        • Length: ~ 280 base pairs
        • Location: Untranslated intronic regions
        • Species: Primate-specific
        • Methylation: Maternal
        • Function: ?
        • Polymorphism in: Myotonic dystrophy CTG repeats
        • Mutations involving Alu elements: Occasional patient
          • CCFDN: Only mutation identified
          • LGMD 2A
          • Mental retardation with epilepsy, rostral ventricular enlargement
          • ACE polymorphism
          • Dystrophin-related cardiomyopathy
      • Mariner (Mariner-like) elements
        • Flanked sides by TA dinucleotide
        • Length: ~80 bp
        • Sequence structure
          • 2 perfect inverted repeat sequences of 37 base pairs
          • Separated by six unique base pairs (GAAAGT)
        • ? related to production of mutations in CMT 1A
    • Long Interspersed Nuclear Elements (LINEs)
      • L1 element (Kpn repeat)
      • AT rich regions
      • Length: 6-8 kb
      • LINEs contain internal promotors for RNA polymerase III
      • Methylation: Paternal
      • Species: Mammals
    • Transposable elements with Long Terminal Repeats
      • Length: 1.5 - 10 kbp
      • Encode reverse transcriptase
      • Flanked by 300 - 1000 bps terminal repeats
      • Regulation
        • Methylation: Paternal
        • Repressive chromatin structures
      • Include non-functional human endogenous retroviruses (hERV)
    • DNA Transposons
      • Single intron-less open reading frame
      • Encode transposase
      • Two short inverted repeat sequences flanking the reading frame
  • Transposable repeat elements: Possible adverse effects
    • Direct insertional mutagenesis
      • ~1 in 500 new germ line mutations 2" transposable elements
    • Recombination between non-allelic repeats
      • Can cause translocations and other re-arrangements
      • See CMT 1A
    • Presence of strong promotor regions
      • Can cause inappropriate protein production
    • Anti-sense production
    • Demethylation in tumors
      • Dysfunctional transposon activity
      • ? Inappropriate gene expression
      • ? Increased Oncogene function
    • Inserted within genes
      • Effect: Reduced function
      • See Fukuyama congenital muscular dystrophy
• Trinucleotide repeats
  • 10 possible sequence motifs
  • Further functional variation depending on reading frame
  • Nomenclature:
    • Same nucleotide repeats can be written in different ways
      • CCG, CGG, GCC are identical
        
      • AGC, CAG are identical
    • AGC repeat in exon reading frame: Translated from CAG to polyglutamine

Repeat Sequences: Disease Associations

• General Features
  • Arise from: Normally existing polymorphic repeats
  • Expansion sizes
    • Unstable
    • May change size: As mosaic in individual, or Across generations
  • Longer repeats: Often associated with more severe, earlier-onset disease
  • Clinical anticipation (Earlier-onset, more severe disease in successive generations): Common
  • Phenotype: May be variable & reflect differences in repeat size
  • Mutation effects
    • Toxic gain of function
      • Mutant protein: AUG initiation (Amino acid, Ribosome, tRNA)
        • CAG/polyQ diseases: SBMA
        • Spinocerebellar ataxias types 1, 2, 3, 6, 7, 17
        • Oculopharyngeal muscular dystrophy
        • Huntington disease-like 2
      • RNA translation: Non-AUG initiation
        • c9orf72: Frontotemporal dementia/Amyotrophic lateral sclerosis
        • Myotonic dystrophy types 1 & 2
        • Spinocerebellar ataxias types 8, 10, 12, 31, 36, 37
        • Fragile X tremor ataxia syndrome
        • Fuchs endothelial corneal dystrophy 3
        • OPDM
    • Loss of function
      • Mechanisms
        • Repeat induced: Transcription silencing; R-loop formation
        • RBP sequestration
      • Disorders
        • Friedreich ataxia
        • Fragile X syndrome
        • Myoclonic epilepsy (Unverricht.Lundborg)
        • CANVAS: RFC1
• Trinucleotide repeats: Disease correlations
  • Repeat sizes
    • Normal: Polymorphic range within a defined "usual" size range
      • DNA replication may change repeat size in normals, but rarely out of usual range
    • Intermediate
      • Clinical: No clinical features
      • Genetic
        • "Premutation" range
        • May predispose to mutations into large disease-related range
    • Borderline: May produce disease syndromes with
      • Reduced penetrance (Huntington's)
      • Recessive inheritance (OPMD)
    • Large "full" mutation
      • Usually associated with disease expression
      • May be stable or predispose to further expansion or contraction mutations
    • When repeats are not translated into protein
      • Larger range of normal and more repeats in disease state
  • Repeat character
    • Normals
      • Usually slight variation within repeat sequence
      • Contains interruptions (variations in nucleotides) within repeats
    • Mutants
      • Larger than normal range
      • Often more perfect repeat sequence
      • Mutant (Large) repeat sequences not perfect: SCA 3 & Huntington
  • Mutational process: Increase in number of copies of repeat
    • Often more likely if repeat sequence is perfect
    • Initial mutation
      • ? loss of interruption leading to perfect repeat
      • Premutation repeat size in intermediate range
    • May be affected by polymorphism at 3' end of CAG repeat in normal allele
      • Intergenerational instability: > with GGG than with CGG (in SCA 3)
    • More prominent in humans than sub-primates
    • Transgenic mice
      • Huntington: Higher paternal age ® More likelihood of CAG expansion
      • SCA1: Higher maternal age ® Greater frequency & size of CAG instability
  • Trinucleotide repeats with disease associations: CCG; CAG; AAG; CTG; GCG
  • Parental bias
    • Fragile X, Type A
      • Full mutation only after maternal transmission of repeat
    • Myotonic dystrophy
      • Female transmission: Size of repeat unconstrained
        
      • Male transmission: Large repeats tend to regress in size
    • CAG repeats
      • Paternal inheritance: Larger repeat & earlier disease onset
• Types of trinucleotide (& other length) repeat disorders
  
  

  	Repeat translated into protein
  	Yes	No
  Nucleotide repeats	CAG; GCG; Other	AAG; CAG; CCG; CGG; Other
  Level of instability	Moderate	High
  Mutation length	Moderate (< 100)	Long (100 to 2,000)
  Pathology	Specific neurons	Multisystem
  Postulated disease mechanism	Protein: Gains toxic function	Altered expression: Protein or RNA Proteins bind to nucleotide repeat
  Disorders	Bulbospinal neuronopathy SCA 1, 2, 3, 6, 7 & 17 OPMD; FSP2; DRPLA Huntington 1 & 2	Baltic myoclonus Fragile chromosome sites Friedreich Ataxia Ataxia, Tremor & Cognitive decline Myotonic Dystrophy: 1; 2 SCA: 8; 10; 12 OPDM (CGG) FTDALS1 (GGGGCC)

  
  
• _p(CCG)_n repeats
  • Frequency: Not uncommon in human genome
  • Location: Usually in 5' untranslated regions of genes
  • Repeat sizes
    • Normal: Polymorphic range (25 to 55)
      
    • Large: Full mutation (> ~230)
  • Large mutation: Mechanisms of disease
    • Chromosome susceptible to breakage near site
    • May inactivate gene: ? Inhibits transcription of gene
      • Expanded repeat
      • Cytosine methylation of mutation & adjacent CpG residues
      • ? Interaction of CGG binding protein with expanded repeat sequence
  • Diseases
    • Fragile X syndrome
    • Ataxia with Tremor & Cognitive decline
    • Fragile X, folic acid type (FRAXE)
  • Inheritance: X-linked or dominant
  
  
• _p(CAG)_n repeats
  • Location
    • In exons of genes throughout genome
    • ? Exception for SCA12
  • Amino acid coding: Glutamine
  • Repeat sizes
    • Normal: Upper limit 32 to 40
    • Large: Lower limit 32 to 62
    • Exception is SCA6: Normal 4 to 18; Large 21 to 30
    • Reduced disease penetrance: HD 36 to 41 repeats; SCA2 32 to 34 repeats
    • Intermediate alleles: CAG size
      • From which new mutations arise
      • > than usual in general population, < than in patients with disease
      • Found clinically in some normal persons
      • Huntington: 29 to 35 repeats
    • Size range: Normal vs. disease
      • Mutually exclusive disease & normal range: SBMA; SCA 3, 6 & 7; DRPLA
      • No gap between normal & disease ranges: Huntington; SCA1 & 2
  • CAG repeat related diseases & Interruptions in CAG sequences

  Disease	Protein	Protein location	Normal/ Disease Repeats	Normal CAG sequence	Disease CAG sequence	Aggregates
  Huntington   (HD)	Huntingtin	Cytoplasm	6-35/ 38-180	Interruption   Penultimate CAA	Interruption   Penultimate CAA	Nuclear
  Huntington 2   (HDL2)	Junctophilin-3	Membrane	6-27/ 35-57	?	Pure CAG	?
  SCA1	Ataxin-1	Nucleus	6-39/ 40-88	Interruptions   1-3 CAT	Pure CAG	Nuclear
  SCA2	Ataxin-2	Cytoplasm	14-32/ 33-77	Interruptions   CAA	Pure CAG	Cytoplasm
  SCA3	Ataxin-3	Nucleus	12-40/ 55-86	Interruptions   1 GGG or CGG	Interruptions   1 CGG (90%)	Nuclear
  SCA6	P/Q Ca ++ Channel	Membrane	4-18/ 21-31	?	?	None
  SCA7	Ataxin-7	Nucleus	7-17/ 34-200	?	?	Nuclear
  SCA12	PPP2R2B		7-32/ 55-93	?	Pure CAG	?
  SCA 17	TATA-binding   protein	Nucleus	25-42/ 41-63	Interruptions   CAA	Interruptions   CAA	Nuclear
  SCA 51	THAP11	Nucleus	20-38/ 45-100	Interruptions	Interruptions ↓	Cytoplasm
  SBMA	Androgen Receptor	Nucleus Cytoplasm	9-36/ 38-65	Pure CAG	Pure CAG	Nuclear
  DRPLA	Atrophin-1	Cytoplasm	3-36/ 49-88	Pure CAG	Pure CAG	Nuclear
  Schizophrenia	KCNN3	Membrane	12-28/ Long alleles over- represented
  Male Infertility	POLG1	Mitochondria	10/0

  
  
  • Inheritance of disorders: Dominant
    • Exception: SBMA is X-linked recessive
  • Anticipation: Maternal > Paternal
  • Possible effect of extra CAG repeats (Huntington's & SCA-1 disease models):
    "Toxic gain of function" - Mutant protein with large glutamate repeat sequence becomes toxic
    • Extra length polyglutamine repeats ® translated into protein
    • Expanded protein undergoes conformational change
      • ? Self aggregation of sites in polyglutamine stretch
        • Hairpin formation via anti-parallel hydrogen bonds
        • "Polar zipper"
    • Expanded protein more susceptible to proteolytic cleavage
    • Fragments of degraded protein transported to nucleus
      • Degradation via caspase-3 & other enzymes
      • N-terminus transported to nucleus & binds to nuclear matrix
      • Degradation of mutant protein to shorter fragment ® Transport to nucleus more abundant
        • Expanded ataxin-1 binds to Leucine-rich acidic nuclear protein (LANP):
          ? Explains selective cell vulnerability
    • Formation of aggregates
      • Protein fragment in nucleus ® Forms nidus for more molecules to aggregate
      • Aggregates include
        • Ubiquitin
        • Proteasome (20S) components
        • Chaperone HDJ-2/HDSJ (Hsp40 family)
      • ? Aggregation involves transglutaminase reactions
        • Reduced by
          • Transglutaminase inhibitors: Cystamine & Monodansyl cadaverine
          • Over expression of chaperone HDJ-2/HDSJ
      • Aggregation forms β-pleated sheet
      • Aggregate is "solidified" by ubiquitination
      • Cells with nuclear aggregates more susceptible to apoptosis
      • Similar aggregates may form in cytoplasm & cause dystrophic neurites
      • Similar events when polyglutamine expressed in normally non-pathogenic protein
    • ? Nuclear inclusions related to disease
      • For
        • Present before disease phenotype
        • Often localized to regions of disease
        • Nuclear expression of polyglutamate proteins implicated in disease pathogenesis
      • Against
        • Poor correlation between neuronal death and abundance of inclusions
        • Some diseases without nuclear inclusiions: SCA2 & SCA6
  • Fewer CAG repeats
    • Prostate cancer: < 18 in Androgen receptor
      • Increased frequency, especially @ young age
      • Extraprostatic extension, distant metastases, or high histologic grade
  • Extra CAG repeats without disease (Polymorphisms)
    • ERDA1 : Chromosome 17q21.3
      • Frequency of large (gt; 40) CAG repeats: Common in Asians; Least common in Africans
      • Repeat length change with transmission
        • Reduced with paternal
        • Increased with maternal
      • Large repeat size
        • Increased mutation rate
        • No change in size of change
    • SEF2-1 : Chromosome 18q21.1
  
  
• _p(CTG)_n repeats
  • Location: 3' untranslated region
  • Repeat sizes
    • Normal: 5 to 35 copies
    • Intermediate range: 35 to 50
    • Mildly affected: 50 to 80
    • Severely affected: Up to 4,000 copies
  • Diseases: Myotonic Dystrophy; ? SCA8
  • Inheritance: Dominant
  • Postulated disease mechanisms
    • ? effect on RNA stability or processing
      
    • Protein binding to large trinucleotide repeats
    • Altered expression of flanking DNA into protein (DMAHP)
  
  
• _p(GCG)_n repeats⁶
  • Nucleotide composition of repeats
    • Imperfect
    • GCN triplets: Over-representation by GCG triplet
  • Location: Exon
  • Amino acid coding: Alanine
  • Stable during meiosis & mitosis
  • Normal proteins: Poly-Alanine repeats
    • Frequent in eucaryotic cells: Especially common in trtanscription factors
    • Poorly conserved in vertebrates
    • Commonly located
      • Outside of other functional domains
      • N-terminal end of proteins
  • Diseases: Poly-alanine repeat disorders
    • General
      • Disease types: Congenital malformations
        • Especially skeletal & nervous system disorders
      • Protein
        • Functions: All transcription factor genes, except OPMD
        • Mutant: Present; Stable
      • Onset age: Young, except OPMD
      • Comparison with other mutation types in same gene
        • Tend to produce milder disorders
        • May not produce same disease manifestations
      • Longer repeat sequences: Some disorders more severe
      • Inheritance
        • Dominant: Usual
        • Recessive: Infantile spasm syndrome
        • Dosage effect: Homozygous more severe than heterozygous
      • Mutation mechanism: Unequal allelic homologous recombination
      • Disease mechanism
        • Toxic gain of function
        • Protein aggregation: Cytosol or Nucleus
    • Oculopharyngeal Muscular Dystrophy (OPMD)
      • Repeat sizes (OPMD)
        • Normal: 6 repeats
        • Recessive OPMD: Homozygous 7 repeats
        • Dominant OPMD: 8 to 13 repeats
    • Neural disorders
      • Congenital hypoventilation syndrome (CCHS) : Paired mesoderm homeo box 2B (PHOX2B)
        • Normal 20 repeats; Disease 25 to 29 repeats
      • Congenital hypoventilation syndrome : Hash-1
        • Normal 13 repeats; Disease 5 to 8 repeats
      • Holoprosencephaly : ZIC2
      • Infantile spasm syndrome : Aristaless-related homeobox, X-linked (ARX)
        • Normal 10 to 12 repeats; Disease 17 to 20 repeats
        • Other features: Mental retardation; Lissencephaly; Abnormal genitalia
      • Mental retardation, X-linked, with isolated Growth hormone deficiency: SOX3
        • Normal 9 repeats; Disease 20 to 31 repeats
    • Skeletal disorders
      • Cleidocranial dysplasia (CCD)(Dominant) : α1 core-binding factor (RUNX2)
        • Alanine tract expansion: Normal 11 to 17 repeats; Disease 27 repeats
      • Synpolydactyly : HOXD13
      • Hand-Foot-Genital syndrome : HOXA13
        • Normal 18 repeats; Disease 26 repeats
      • Blepharophimosis/Ptosis/Epicanthus inversus syndrome (BPEIS) : FOXL2
  
  
• _p(AAG)_n repeats
  • Location: Intron
  • Repeat sizes
    • Normal: Polymorphic range (7 to 22)
      
    • Large: Full mutation (200 to 900)
  • Disease: Friedreich Ataxia
  • Inheritance: Recessive
  • Disease mechanism: Reduced protein production

• Other disease associated repeat sequences
  • Instability of simple tandem repeat sequences: Neoplasms
    • TGF-β receptor, type II gene
      • Repeat sequences (GT)₃ & (A)₁₀
        
      • Sequences are targets for copy # mutation
        
      • Mutations produce frame shift & loss of receptor
        
      • Eliminates TGF-β control of epithelial cell growth
  • Replications: Disorders can be associated with replications of a small or large DNA fragment within the gene
    • SCA 10
      • Mutated gene: SCA 10
      • Pentanucleotide repeat ATTCT in intron 9
      • Normal repeat range: 10 to 22
      • Clinical: Larger number of repeats correlates with younger onset age of SCA 10
    • Baltic myoclonus
      • Mutated gene: Cystatin B
      • 12 base pair repeat in 5' promoter region
      • Repeat range
        • Normal: 2 or 3 sequences
        • Disease: ~50 to 75
    • Prion protein: Contains 24 base pair repeat coding for an octapeptide
      • Normal phenotype: 4 ± 1 copies
      • Neurological disease of varying severity: > 4 extra copies
    • SCA36: Intronic GGCCTG repeat, 1500-2500
    • SCA37: Non-coding ATTTC repeat insertion. 31 to 75,
    • Other examples
      • CMT 1A; MEN 2A (ret gene) ; Duchenne muscular dystrophy
      • CAG & GCG repeats
  • Disease expansions in transcribed but untranslated DNA
    • DM1 (CTG)
    • DM2/PROMM (CCTG)
    • SCA8 (CTG)
    • SCA10 (ATTCT)
    • ALS-FTD 2 (GGGGCC)
  • Tendency to produce deletions in repeat sequences
    • CMT 1A - HNPP
    • Facioscapulohumeral dystrophy
  • Repeat-induced gene silencing
    • Increased gene copy # ® More Gene methylation & chromatin compaction
    • More Repression of gene expression
  
  
• Repeat disorders: Muscle disease
  • DM1 (CTG)
  • DM2/PROMM (CCTG)
  • OPMD (GCG)
  • FSH (Deletion Kpnl repeat)