Table 7. [RFC1 Technical Considerations]. - GeneReviews®

Technical Issue	Comment [Reference]
Sequence of repeat	AAAAG (normal) & AAGGG (expanded pathogenic). However, expansions may be AAAAG, AAAGG, AAGAG, AGAGG, ACAGG or AAGGG; imperfect repeats w/interruptions are also possible.
Methods to detect expanded allele	Conventional PCR, repeat-primed PCR (RP-PCR) [Cortese et al 2019, Akçimen et al 2019], & Southern blotting [Cortese et al 2019] have been described. The presence of biallelic AAGGG pentanucleotide expansions is suggested by the following: Absence of PCR amplifiable product on flanking PCR Presence of a saw-tooth decremental pattern on repeat-primed PCR (RP-PCR) for the pathogenic AAGGG pentanucleotide expansion Optional: Absence of a saw-tooth decremental pattern on repeat-primed PCR for non-pathogenic repeat expansions of (e.g.,) AAAAG, AAAGG, and other possible configurations [Akçimen et al 2019] These expansions can be large enough to prevent amplification of a PCR product on standard flanking PCR conditions. Given the large size of the AAGGG pentanucleotide repeat expansions, sizing can be obtained only by Southern blotting, which is the only available method to confirm the presence of biallelic expansions, showing either 2 discreet bands or 1 band corresponding to 2 expanded alleles of similar size.
Somatic instability	Data not available
Germline instability	Data not available

Technical Issue

Comment [Reference]

Sequence of repeat

AAAAG (normal) & AAGGG (expanded pathogenic). However, expansions may be AAAAG, AAAGG, AAGAG, AGAGG, ACAGG or AAGGG; imperfect repeats w/interruptions are also possible.

Methods to detect
expanded allele

Conventional PCR, repeat-primed PCR (RP-PCR) [Cortese et al 2019, Akçimen et al 2019], & Southern blotting [Cortese et al 2019] have been described. The presence of biallelic AAGGG pentanucleotide expansions is suggested by the following:

Absence of PCR amplifiable product on flanking PCR
Presence of a saw-tooth decremental pattern on repeat-primed PCR (RP-PCR) for the pathogenic AAGGG pentanucleotide expansion
Optional: Absence of a saw-tooth decremental pattern on repeat-primed PCR for non-pathogenic repeat expansions of (e.g.,) AAAAG, AAAGG, and other possible configurations [Akçimen et al 2019]
These expansions can be large enough to prevent amplification of a PCR product on standard flanking PCR conditions.

Given the large size of the AAGGG pentanucleotide repeat expansions, sizing can be obtained only by Southern blotting, which is the only available method to confirm the presence of biallelic expansions, showing either 2 discreet bands or 1 band corresponding to 2 expanded alleles of similar size.

Somatic instability

Data not available

Germline instability

Data not available

From: RFC1 CANVAS / Spectrum Disorder

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