16. Other repeats - polyalanine & FSHD

Question 1

What encodes polyalanine repeats?

What do they form?

Answer 1

Imperfect trinucleotide repeats (GCN)

Form beta sheets that are resistant to degradation - flexible spacer elements located between functional domains

Question 2

What is the result of polyalanine expansions? What size are the repeats?

Answer 2

> 19 repeats

Protein misfolding, degradation, mislocalisation, aggregation, LoF, GoF, dominant negative

Question 3

How do polyalanine tracts compare to polyglutamine tracts?

Answer 3

PolyA tracts are smaller than polyQ

PolyA are stable through meiosis/mitosis

PolyA are often de novo

Question 4

Describe a polyalanine repeat-associated disorder

Answer 4

OPMD (oculopharyngeal muscular dystrophy)

Late onset, neurodegenerative - dysphagia, ptosis, proximal limb weakness

Caused by polyA expansion in PABN1 exon 1 - encodes nuclear polyA binding protein, involved in polyadenylation of mRNA precursors

Usually AD

Question 5

What is the expansion mechanism in polyA tracts?

Answer 5

Unequal crossing over between 2 normal sized alleles

Question 6

What is the phenotype of FSHD?

Answer 6

Asymmetrical, progressive muscle weakness (including facial muscles), scapular winging

2 phenotypically indistinguishable sub-types (95% FSHD1)

Question 7

What is the molecular mechanism behind FSHD1?

Answer 7

Contraction of D4Z4 repeats on 4q35 sub-telomere

Normal = 11-100 repeats, pathogenic = 1-10

Each repeat contains copy of DUX4 (DUX4, DUX4-like 1 etc.)

Question 8

What is the role of DUX4 and DUX4L1, 2, 3, etc

Answer 8

DUX4 causes apoptosis of myeloblasts.

Active in early development and in testes of adults, normally inactive in most adult tissue.

DUX4-like genes - epigenetically inactivated

Question 9

What two events must occur to cause FSHD?

Answer 9

D4Z4 contraction on permissive 4qA haplotype

No disease if contraction occurs on 4qB haplotype

Polymorphism is distal to D4Z4 repeats

Question 10

What is the effect of the 4qA allele?

Answer 10

Provides a polyadenylation sequence allowing stable expression of DUX4 mRNA –> apoptosis of myoblasts

Question 11

What is the effect on a reduction in the number of D4Z4 repeats?

Answer 11

Loss of heterochromatin, reduced CpG methylation –> transcription of DUX4 from final repeat unit (if present on 4qA allele)

Question 12

What genes are involved in FSHD2?
What is the molecular mechanism?

Answer 12

Mutations in SMCHD1 or DNMT3B + at least 1 permissive 4qA allele (digenic inheritance)

SMCHD1 is an epigentic modifier of D4Z4 - LoF variants cause chromatin relaxation and hypomethylation of D4Z4 repeats –> DUX4 expression –> apoptosis of myoblasts

Question 13

What is the risk to the offspring of an individual with FSHD2?

Answer 13

25-50%, depending on number of pathogenic haplotypes in the family - due to digenic inheritance

Question 14

How is FSHD1 tested for?

Answer 14

Southern blotting - size repeats and distinguish chr 4 repeats from chr 10 repeats (not associated with FSHD)

Detection with P13E-11 probe - hybridises to ch4 and 10 repeats

Double restriction enzyme digest - cuts proximal and distal to chr4 and 10 repeats. Chr 10 repeats digested completely

4qA/B probes + HindIII digest to detect permissive haplotype

Question 15

How is FSHD2 tested for?

Answer 15

Bisulphite PCR of 4q locus + quantification of methylation by pyrosequencing

If hypomethylated –> sequence SMCHD1/DNMT3B

Question 16

How does the methylation profile differ in FSHD1 patients vs FSHD2 patients?

Answer 16

FSHD2 - D4Z4 repeats on chr 4 AND 10 are hypomethylated due to SMCHD1 LoF

FSHD1 - only D4Z4 repeats on chr 4 are hypomethylated due to contraction