Hexanucleotide repeat expansionswithin the C9orf72geneare themostimportant genetic cause ofamyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The difficulty of developing a precise method to determine the expansion size has hampered the study of possible correlations between the hexanucleotide repeat numberandclinical phenotype. Herewecharacterize, throughanewnon-radioactive Southern blot protocol, the expansion size range in a series of 38 ALS and 22 FTD heterozygous carriers of >30 copies of the repeat. Maximum, median and modal hexanucleotide repeat number were higher in ALS patients than in FTD patients (P < 0.05 in all comparisons). A higher median number of repeats correlated with a bigger range of repeat sizes (Spearman's p = 0.743, P = 1.05 × 10 -11 ).Wedid not find any correlation between age of onset or disease duration with the repeat size in neither ALS nor FTD mutation carriers. Clinical presentation (bulbar or spinal) in ALS patients did not correlate either with the repeat length. We finally analyzed two families with affected and unaffected repeat expansion carriers, compared the size of the repeat expansion between two monozygotic (MZ) twins (one affected of ALS and the other unaffected), and examined the expansion size in two different tissues (cerebellumandperipheral blood) belonging to thesameFTDpatient.Theresults suggested that the length. © The Author 2013. Published by Oxford University Press. All rights reserved.