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
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
3
Q
Folate-sensitive fragile sites
A
- FRAXA=FMR1, FRAXE=FMR2, FRA12A
- CGG-repeat expansion
4
Q
BrdU-inducible
A
- FRA10B, FRA10E
- AT-rich minisatellite repeats
5
Q
Distamycin A-inducible
A
- FRA16B
- AT-rich minisatellite repeats
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
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
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)
9
Q
Fragile sites map to regions that are ____________________
A
conserved evolutionarily
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)
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
12
Q
Interspersed repeats
A
- Short direct repeats (Kearns-Sayre deletion in mtDNA), Alu sequences, low copy number long repeats
13
Q
Inverted Repeats
A
Like interspersed but some facing each other
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
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
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
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
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
