Figure 2. GGGGCC hexanucleotide repeat expansion in the first intron and promoter of C9ORF72.
(A) Physical map of the chromosome 9p21 ALS/FTD locus showing the p-values for SNPs genotyped in the previous GWAs (Laaksovirta et al., 2010), the location of the GWAs association signal within a 232kb block of linkage disequilibrium, the MOBKL2B, IFNK and C9ORF72 genes within this region, and the position of the GGGGCC hexanucleotide repeat expansion within the two main transcripts of C9ORF72 (RefSeq accession numbers NM_018325.2 and NM_145005.4, see online www.ncbi.nlm.nih.gov/RefSeq/ for further details; GenBank accession numbers GI:209863035 and GI:209863036, see online www.ncbi.nlm.nih.gov/genbank/ for further details); (B) A graphical representation of primer binding for repeat-primed PCR analysis is shown in the upper panel. In the lower panel, capillary-based sequence traces of the repeat-primed PCR are shown. Orange lines indicate the size markers, and the vertical axis represents fluorescence intensity. A typical saw tooth tail pattern that extends beyond the 300 bp marker with a 6 bp periodicity is observed in the case carrying the GGGGCC repeat expansion; (C) Detection of the repeat expansion in the lymphoblastoid cell line from the affected proband of the GWENT#1 kindred (ND06769) by FISH using Alexa Fluor 488 - labeled oligonucleotide probe seen as a green fluorescence signal on one of the homologues of chromosome 9p (i) consistent with a repeat expansion size of more than 1.5kb. DAPI-inverted image (ii & iv). No hybridization signal was detected on metaphase cells or interphase nuclei from the lymphoblastoid cell line of control individual ND 11463 (iii) and 5 other normal control individuals (data not shown). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, red color), x60 objective.