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Chromosomes Exam 2 > 10/31 - Fragile Sites > Flashcards

10/31 - Fragile Sites Flashcards

1
Q

Fragile Sites

A

Non-staining gaps in chromosomes under certain culture conditions or after treatment with specific chemicals

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2
Q

Rare Fragile Sites

A
  • Seen in s on)
  • 46,Y,fra(X)(q27.3)
  • Increased breakage is usually caused by expansion of trinucleotide repeats
  • Folate-sensitive
  • BrdU-inducible
  • Distamycin A-inducible
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3
Q

Folate-sensitive fragile sites

A
  • FRAXA=FMR1, FRAXE=FMR2, FRA12A

- CGG-repeat expansion

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4
Q

BrdU-inducible

A
  • FRA10B, FRA10E

- AT-rich minisatellite repeats

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5
Q

Distamycin A-inducible

A
  • FRA16B

- AT-rich minisatellite repeats

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6
Q

What does Sutherland say?

A
  • he says all but FRAXA and FRAXE which are associated with intellectual disabilit, and FRA11B which is associated with Jacobsen syndrome (variable deletions of 11q) are harmless
  • BUT GOLLIN DISAGREES
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7
Q

Fragile X Syndrome

A
  • Moderate to severe intellectual disability, macroorchidism, large ears, prominent jaw, and high-pitched, repetitive speech.
  • Folate-sensitive fragile site, FRAXA at Xq27.3
  • Second most important genetic cause of intellectual/cognitive disabilities after Down syndrome
  • Most common familial cause of intellectual disability
  • The premutational state is associated with a risk for premature ovarian failure in females and degenerative neurological syndrome in late middle-age males
  • FRAXA located in the 5’ region of the FMR1 gene that encodes a protein FMRP which binds to mRNA and is necessary for normal brain development and function, having a role in either synaptic function or dendrite growth
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8
Q

Common Fragile Sites

A
  • Inducible by aphidicolin, a DNA polymerase inhibitor that results in replication stress
  • Sites of DNA double strand breaks resulting from replication for stalling and collapse
  • Hotspots for chromosomal rearrangements, gaps, and breaks
  • Likely responsible for chromosome rearrangements (deletions, translocations) in cancer cells
  • FRA3B is the most ‘fragile’ site in the genome
  • The ATR-dependent DNA damage checkpoint pathway is critical for maintenance of fragile site stability
  • Many common fragile sites lie within large tumor suppressor genes involved in the cellular response to stress
  • Common fragile sites are conserved throughout mammalian evolution
  • Many microRNA-coding loci lie within common or rare fragile sites
  • Viral integration sites are often at chromosomal fragile sites (e.g., HPV, HBV, HIV)
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9
Q

Fragile sites map to regions that are ____________________

A

conserved evolutionarily

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10
Q

Moderately repetitive DNA

A
  • (10-10^5 copies/genome): 30-35%
  • Microsatellites - Simple Tandem Repeats (STRs)
    • Di, tri, tetra, penta nucleotide length variants
    • Large numbers, many alleles (informative), easily assayed
  • Minisatellites (Variable Number Tandem Repeats - VNTRs)
    • Generally larger: 10 to several hundred nucleotides
    • Informative, more difficult to assay technically
  • Dispersed repetitive DNA (LINES/SINES), e.g. L1/Alu
  • Multiple ‘redundant’ genes (e.g. ribosomal RNA, histones)
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11
Q

Highly Repetitive DNA

A
  • > 10^6 copies/genome - 10%
    • 5 to 100 bp, variable length motif in long tracts, up to >10 Mb
    • Alpha satellite DNA in centromeric regions, telomeric regions
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12
Q

Interspersed repeats

A
  • Short direct repeats (Kearns-Sayre deletion in mtDNA), Alu sequences, low copy number long repeats
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13
Q

Inverted Repeats

A

Like interspersed but some facing each other

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14
Q

Unstable repeats

A
  • Most are trinucleotides (sometimes called trinucleotide repeat disorders, dynamic mutations or repeat expansion disorders)
    • 10 possible repeating combinations
    • AAC/GTT, AAG/CTT, AAT/ATT, AGG/CCT, ATC/GAT, ACC/GGT, ACG/CGT, ACT/AGT, *CAG/CTG, CCG/CGG
    • Some tetra and even pentanucleotide expansions
  • Very useful for mapping and studies requiring informative loci (linkage, engraftment analysis
  • Mutiple alleles evolve over time via ‘slippage’ during DNA replication
  • -Risk for larger scale expansions
  • -Some of these are associated with disease
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15
Q

Trinucleotide Repeat Disorders: Large Expansions in noncoding regions

A
  • Interference with gene expression (loss of function, interference in RNA processing)
    • Rare point mutations are sometimes seen
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16
Q

Trinucleotide Repeat Disorders: Smaller expansions within coding regions

A
  • Toxic effects are associated with intranuclear protein aggregation in most (but not all) disorders - ‘gain of function;’ host proteins are felt to play a role
  • Incomplete penetrance at low levels of expansion
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17
Q

Trinucleotide Repeat Disorders in both

A
  • Parent-of-origin effects: meiotic/mitotic mechanisms
  • Age of onset inversely related to expansion size - can’t predict individual patients!
  • Sherman paradox (anticipation) - disease more severe in successive generations - first described for Fragile X
  • -Progressive expansion of trinucleotide sequence
  • -Pre-mutation carriers
  • -Expansions or contractions can occur, but the bias is toward expansion
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18
Q

Fragile-X site A (FRAXA)

A
  • Large Expansions of repeats outside coding sequences
  • Mode of inheritance: X
  • FMR1 gene
  • Location of gene: Xq27.3
  • Location of repeat within gene: 5’ untranslated region
  • Repeat sequence: (CGG)n
  • Stable repeat number: 4-54
  • Unstable repeat number: 200-1000+
19
Q

Myotonic dystorphy 1

A
  • Large Expansions of repeats outside coding sequences
  • Mode of inheritance: Autosomal Dominant
  • DMPK gene
  • Location of gene: 19q13
  • Location of repeat within gene: 3’ untranslated region
  • Repeat sequence: (CTG)n
  • Stable repeat number: 5-37
  • Unstable repeat number: 50-10,000
20
Q

Huntington Disease

A
  • Moderate expansions of repeats outside coding sequences
  • Mode of inheritance: Autosomal Dominant
  • HD gene
  • Location of gene: 4p16.3
  • Location of repeat within gene: Coding
  • Repeat sequence: (CAG)n
  • Stable repeat number: 6-34
  • Unstable repeat number: 36-100+
21
Q

Kennedy Disease

A
  • Moderate expansions of repeats outside coding sequences
  • Mode of inheritance: X
  • AR gene
  • Location of gene: Xq12
  • Location of repeat within gene: coding
  • Repeat sequence: (CAG)n
  • Stable repeat number: 9-35
  • Unstable repeat number: 38-62
22
Q

Fragile X Syndrome

A
  • Symptoms can be quite variable, but a common issue is mental retardation; clinical reasons for testing are usually ‘developmental delay’
  • Macro-orchidism (enlarged testes) in adult males
  • Mild macrocephaly with prominent forehead, jaw, and ‘long’ ears
  • Hyperextensible joints and mitral valve prolapse
  • Incidence: 1 in 2,500 - 4,000 males, similar or perhaps slightly fewer females whose symptoms are milder (X chromosome inactivation - Lyonization)
  • ‘Milder’ effects in adult males (tremor/ataxia) and women (premature ovarian failure) with premutations; behavioral changes in teens/young adults.
  • -Probably related to INCREASED levels of FMR1 mRNA!
23
Q

Fragile sites are inducible when _____________

A

exposed to drugs (e.g. aphidicolin, camptothecin, distamycin A) or gown in media lacking folic acid or thymidine

24
Q

Types of fragile sites

A
  • Common (small % cells, FRA3B)
  • Rare: Mendelian inheritance, FRAXA
  • Most have no clinical effect, even in homozygous conditions
25
Q

Fragile Sites appear to be associated with what…

A
  • Chromosomal breakage (FRA11B - Jacobsen 11q - deletion syndrome)
  • Late-replicating DNA
26
Q

Inducible-fragile sites were the initial ____________

A
  • Diagnostic approach for patients with developmental delay

- These are demanding studies using special media; done primarily on a research basis now

27
Q

What do we do today for patients with developmental delay?

A
  • Targeting nucleic acid studies = diagnostic tool of choice
  • Karyotype, and even more so today, array CGH or SNP/CNV arrays are important studies to do in such patients to look for chromosomal basis of developmental delay
28
Q

Genetics of Fragile X Syndrome

A
  • FRAXA site (FMR1 gene), Xq27.3
  • -FMR1 protein involved in RNA processing
  • Trinucleotide repeat (CGG expansion)
  • -Normal: 200 repeats
  • Full mutation CGG expansion is usually associated with methylation and reduced expression of FMR1 protein, FMRP; (premutation alleles may be associated with increased stability of FMR1 mRNA)
29
Q

Decreased translation of FMRP leads to ____________

A
  • Exaggerated responses via metabotropic glutamate receptors and excess dendrites
30
Q

The ‘Gray Zone’ of Fragile X

A

~3% of all FMR1 alleles

  • A range of terms by study:
  • -intermediate, borderline, inconclusive, indeterminant, ambiguous
  • Expansion AND contraction may occur
  • -Minimal instability, generally one to a few CGG repeats, maximum up to 12 for alleles in the 50s
  • -Alleles do NOT expand to full mutation status
  • No convincing evidence for any phenotypic effect
  • -We use 45-55 in the clinical laboratory although evidence indicates >50 more likely to expand
31
Q

Fragile X Syndrome - Other issues

A
  • Mosaicism: mitotic in full mutation carriers vs. small % of cells with expansion
  • ‘Silent’ carriers - due to partial methylation
  • Effects on other mRNAs - FMRP associates with RNA and ribosomes and is important in mRNA processing
  • Premutation Alleles - studies of relatives of affected individuals to evaluate tremor/ataxia symptoms in middle-aged males and perhaps behavioral changes in male teens and young adults; also, premature ovarian failure in females
  • Interruption of CGG expansion by AGG motif: every 9-10 repeats, usually 2-4 AGG repeats, seems important to stabilize CGG repeats; ~35 uninterrupted CGGs surpasses threshold and allows subsequent expansion
32
Q

Approach to clinical analysis - large expansion trinucleotide repeat syndromes (eg. Fragile X)

A
  • OLD(ER) - Southern blot to look for expansion or not (and rare mosaicism)
  • -Small enough ‘germline’ fragment to easily assess if there are premutation or gray-zone alleles
  • -if normal sign out
  • -if expanded, do methylation studies to look for partial (or rarely lack of) methylation
  • NEW(ER) - PCR amplification
33
Q

Southern Blot

A
  • Increase/decrease in size of restriction fragment, expansions, translocations
  • Dosage assessment (quantitative) Deletions, duplications
34
Q

New(er) testing methods for Fragile X Syndrome

A
  • Repeat-primed PCR
  • -Eliminates need for Southern Blots
  • -Currently investigators are working on how to assess methylation status without using Southern blots
  • Next generation sequencing
  • -Unrecognized fine structural changes which compromise FMR1 protein
35
Q

Repeat-primed PCR

A
  • Repeats into high hundreds

- Accuracy +/- in premutation range

36
Q

Myotonic Dystrophy

A
  • Two major forms
  • -DM1 (myotonin kinase, 19), 3’ UTR CTG expansion
  • -DM2 (ZNF9, 3), intron 1 CCG expansion, expansions lack “interruptions” of repeat sequence - like Fragile X
  • Weakness, pain, myotonia (involuntary muscle contraction and delayed relaxation, hyperexcitability)
  • -Cataracts, arrhythmias, testicular failure, insulinemia, retardation (DM1 only)
  • Premutation, full mutation, and congenital forms
  • -Expansions only, no point mutations described
  • ‘Trans-dominant’ binding by expansions of proteins requried for normal splicing of other transcripts, including muscle-specific chloride channels; alternative splice changes and possible repressive effects on adjacent genes
  • Intergenerational and somatic instability
37
Q

Myotonin Gene Expansion

A
  • Normal: 5-37 repeats
  • Mildly Affected: 50-150 repeats
  • Classic DM: 100-1000 repeats
  • Congenital onset: >2000 repeats
38
Q

Huntington Disease

A
  • Adult-onset neurodegenerative disease leading to choreoathetotic movements and dementia
  • Linkage to 4q established in 1980s; IT15 (Huntingtin) gene isolated 1994
  • Mutational mechanisms, CAG expansion (polyglutamine tract) within exon 1
  • Preferential expansion of alleles in males germline; intermediate zone 27-35 repeats
39
Q

Approach to clinical analysis - modest expansion trinucleotide repeat syndromes (e.g. Huntington Disease)

A
  • PCR amplification to size alleles
  • -Must account for other length polymorphisms which could confuse interpretation of results
  • Southern blot analysis for large expansion (>100 repeats) if results are unusual or clinical history warrants
40
Q

Kennedy Disease (X-linked SBMA)

A
  • Adult onset neurodegenerative syndrome affecting lower motor neurons
  • CAG repeat expansion in exon 1 (first N-terminal domain of androgen receptor gene on X chromosome
  • -Age of onset/severity inversely related to repeat size
  • -Female carriers do not have disease
  • -The only CAG repeat expansion that is X-linked
  • -Normal = 9-34 repeats
  • -Reduced Penetrance (males) = 35-37 repeats
  • -Full Penetrance (males) = 38+ repeats
  • Appears not to be a toxic effect per se
  • -Male expression seems to be induced by androgens; rare homozygous expanded females do NOT show disease
41
Q

Trinucleotide Repeat Disorders: Issues associated with presymptomatic laboratory diagnosis

A
  • Informed consent
  • -Inferred diagnosis via testing of other family members
  • -Variability among patients with same expansion levels
  • -Issues of discrimination, stigmatization
  • Testing of children at parental insistence
  • While generally raised in the context of HD, these issues apply to many other trinucleotide repeat expansions
42
Q

Inducible fragile sites on karyotype are sometimes associated with __________

A
  • Chromosome breakage and repetitive DNA; a few (FRAXA/FRAXE) are linked to disease-causing trinucleotide repeat expansions
43
Q

Two major groups of trinucleotide repeat expansions

A
  • Large expansions (>several hundred) compromise gene expression - ‘loss of gene function’ or trans-dominant interference with splicing
  • Modest expansions (40-100) are associated with ‘gain of funciton’ polyglutamine tracts in proteins toxic to cells
44
Q

What is associated with trinucleotide expansion in generations?

A

Anticipation

Chromosomes Exam 2 (11 decks)

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