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1

Fragile Sites

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

2

Rare Fragile Sites

- 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

Folate-sensitive fragile sites

- FRAXA=FMR1, FRAXE=FMR2, FRA12A
- CGG-repeat expansion

4

BrdU-inducible

- FRA10B, FRA10E
- AT-rich minisatellite repeats

5

Distamycin A-inducible

- FRA16B
- AT-rich minisatellite repeats

6

What does Sutherland say?

- 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

Fragile X Syndrome

- 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

Common Fragile Sites

- 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

Fragile sites map to regions that are ____________________

conserved evolutionarily

10

Moderately repetitive DNA

- (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

Highly Repetitive DNA

- >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

Interspersed repeats

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

13

Inverted Repeats

Like interspersed but some facing each other

14

Unstable repeats

- 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

Trinucleotide Repeat Disorders: Large Expansions in noncoding regions

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

16

Trinucleotide Repeat Disorders: Smaller expansions within coding regions

- 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

Trinucleotide Repeat Disorders in both

- 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

Fragile-X site A (FRAXA)

- 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

Myotonic dystorphy 1

- 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

Huntington Disease

- 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

Kennedy Disease

- 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

Fragile X Syndrome

- 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

Fragile sites are inducible when _____________

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

24

Types of fragile sites

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

25

Fragile Sites appear to be associated with what...

- Chromosomal breakage (FRA11B - Jacobsen 11q - deletion syndrome)
- Late-replicating DNA

26

Inducible-fragile sites were the initial ____________

- Diagnostic approach for patients with developmental delay
- These are demanding studies using special media; done primarily on a research basis now

27

What do we do today for patients with developmental delay?

- 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

Genetics of Fragile X Syndrome

- 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

Decreased translation of FMRP leads to ____________

- Exaggerated responses via metabotropic glutamate receptors and excess dendrites

30

The 'Gray Zone' of Fragile X

~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