Figure 1

(A) Diagram of the TP-PCR principle. Grey squares represent tandem GCC repeats at the AFF2 gene 5′ UTR. Black squares represent annealing of "CGG primer” within the GCC tract and in a second priming site overriding the rs1333167094 “T” SNP (when GCT is located exactly in the middle of the (CGG)5C primer sequence). Above is an electropherogram illustration after capillary electrophoresis on automatic sequencer, reflecting the total repeat length (higher rightmost peak), that corresponds to the distance between the forward primer and the sole GCC priming site 52 bp downstream of the end of the repeat. The image is not at scale; (B) Partial AFF2 sequence (GRCh37/hg19, NM_002025.3) harbouring the primer binding sites. Annealing sites within the GCC repeats (light blue), generating stutters, and at (GCC)2GCT(GCC)2G sequence located 52 nucleotides downstream (dark blue). “Forward primer” and corresponding binding site (yellow); “CGG primer” (blue arrow and dashed purple line) (CGG)5C; Tail primer (green arrow) and SNPs c.-413T > C rs868949662 (*) (highest population MAF: 0.02) and c.-355T > C rs1333167094 (#) (highest population MAF: < 0.01) are also indicated. In case of rs868949662 “C”, the nucleotide following the 3rd GCC triplet is a “C” hampering its recognition, as a result instead of the three additional GCC triplets, only two are recognized (region underlined); (C) Examples of TP-PCR results. (i) Female heterozygous sample with normal and intermediate alleles; (ii) Female heterozygous sample with normal and expanded alleles; superimposed close-up electropherogram shows the continuous stuttering after the prominent peak with 188 bp, an indication of the presence of an expanded allele that is identified but not correctly sized by this assay; (iii) Female heterozygous sample with two normal alleles differing by one repeat (14 and 15 GCCs).