Testing for Triplet Repeat Expansions

1. Testing for Triplet Repeat Expansions James Polke, Institute of Neurology, London 4th September 2009

3. Techniques • PCR • Long PCR • Triplet-primed (FRDA and DM) • GC-rich (FRAX) • Analysis • Capillary electrophoresis (<~800 bp) • Agarose • Southern blot • Large expansions (FRAX and DM1) • Sequencing / Restriction digest (SCA1)

4. Main Challenges • Accurate sizing • Especially close to allele size range boundaries (HD, FRAX) • Detecting large expansions • Methylation (FRAX) • Allele drop-out • SNPs under primers • Structure of repeat / interruptions (TP-PCR)

5. Sizing in HD • Size Ranges: • Normal: <26 • Intermediate: 27-35 (prone to expansion) • Pathogenic: 36-250 • Reduced Penetrance: 36-39 • Accuracy most important at 35/36 and 39/40 repeats • Importance of controls • Cloned, sequenced standards are available to validate assays – each set-up will be different • Quote accuracy on reports (e.g. +/- 2 repeats)

6. European HD Disease Network Data • 1311 cases throughout Europe (Jan 2004 – Jun 2009), accurately sized in central lab, Milan • Upper allele sizing: • 49.5% accurate • 31% 1 repeat discrepant • 12% 2 repeats discrepant • 7.5% more than 2 repeats discrepant • UK performed better than Europe as a whole • 35/36 boundary: • UK: No inaccuracies • Other European: x2 decreased to <36 • 39/40 boundary: • UK: x 1 increased to >40; x2 decreased to <40 • Other European: x34 increased to >40; x7 decreased <40

7. Detecting large expansions • Possible false negatives if large expansions fail to amplify in PCR • If only 1 allele seen (or none in FRAX males): • May be guided by phenotype (e.g. age of onset in HD and DM1, SCAs) • Large expansion positive controls • Alternative primers e.g. in HD, second RVS primer that covers polymorphic region • Long PCR, TP-PCR • Southern blot • Large expansions not sized on capillaries • Agarose gel - somatic instability: smear

8. Southern blotting • For large expansions • Routine in DM1 and FRAX, rarely used in other diseases • Methylation-specific in FRAX • Size of fragment: • Large expansions liable to somatic instability, less smearing with large fragments, allows better sizing • DM1: PstI for visualising small expansions (~1.1kb) EcoR1 for large expansions (~10kb) • Particularly important for FRAX prenatals, methylation pattern can't be interpreted, need to distinguish pre and full mutations by size.

9. Allele Drop-out • SNPs under PCR primers • False negatives in TP-PCR • SNPs under primers have caused false negative results • Interruptions in expansions can prevent amplification (DM1 – Musova et al 2009) • Primer mix? • Recommended to PCR from both ends of the expansion Musova et al (2009) Am J Med Genet Part A p.1365-1374

10. Repeat Structure / Interruptions • Many triplet repeats include common interruptions (SCA1, 2, FMR-1 and others) that most notably effect repeat length stability, but rarely diagnosis • SCA1:Normal: 6-38 CAG (+/- 1-3 CAT repeats) Pathogenic: 39+ uninterrupted CAG Intermediate: 36-38 uninterrupted CAG • (CAT)n create a LweI restriction site, alleles from 36-39 are digested with LweI to check for the interruption. In practice, uninterrupted repeats give a 'hedgehog' pattern on the trace. • SCA 2: CAG repeat with CAA interruption, CAA also codes for glutamine, so still a polyQ disease, but CAA provides stability at low repeat ranges (? Instability at high repeat range?)

11. Reporting • Quote sizes on reports? • HD – we only quote pathogenic or intermediate sizes – clinicians have requested • Others – we don't quote sizes (FRDA, SCAs, SBMA – best practice) • Quote allele size ranges • Quote accuracy of analysis • ? Reporting FXTAS and POF in FRAX premutations • Point mutations • FRDA • FMR1