The GGGGCC repeat length in healthy individuals ranged from 2–23 hexanucleotide units, whereas we estimated the repeat length to be 700–1600 units in FTD/ALS patients based on DNA from lymphoblast cell lines. Accurate sizing of the repeat is challenging, especially in DNA extracted from peripheral blood and brain tissue samples, where a smear of high

molecular weight bands suggested somatic repeat instability (Figure S1). Notably, the large number of repeats observed in our patients is similar to other noncoding repeat expansion disorders where more than 1000 repeat copies are common (Liquori et al., 2001, Mahadevan et al., 1992, Moseley et al., 2006, Sato et al., 2009 and Timchenko et al., 1996). However, Pifithrin �� Decitabine molecular weight the minimal repeat size needed to cause FTD/ALS remains to be determined and may be significantly smaller. Importantly, anticipation was not apparent in most of our families, although occasionally a significantly earlier onset was observed in the youngest generation. This could simply reflect heightened awareness by family members or caregivers; however, it remains possible that repeat length is correlated with the age of disease onset or clinical presentation. Future studies are needed to fully resolve this question. In previous studies, we and others suggested that a single ∼140 kb “risk” haplotype, broadly defined by

SNP rs3849942 allele “A,” was shared by all affected family members of chromosome 9p-linked families and that this same haplotype was responsible for the ALS and FTLD-TDP GWAS hits at chromosome 9p (Mok et al., 2011). The presence of the “risk”

haplotype in all 75 unrelated expanded repeat carriers in our study further confirms the strong association of this haplotype with disease. While these findings are consistent with the previously proposed hypothesis of a single founder mutation, the identification of an expanded hexanucleotide repeat as the basis for disease in these patients now suggests the possibility that the abnormal repeat may occur on a predisposing haplotypic background that is prone to expansion. This alternative hypothesis is supported by our finding of significantly longer repeats on P-type ATPase the “risk” haplotype (defined by rs3849942 allele “A”) compared to the wild-type haplotype (defined as rs3849942 allele “G”) in the normal population. The somewhat unusual observation that the GGGGCC repeat was uninterrupted in control individuals carrying a range of normal allele sizes further supports this alternative hypothesis. De novo expansions of uninterrupted GGGGCC sequences at the long end of the normal spectrum could potentially explain the sporadic nature of the disease in a subset of our patients. In summary, we identified a noncoding expanded GGGGCC hexanucleotide repeat in C9ORF72 as the cause of chromosome 9p-linked FTD/ALS and showed that this genetic defect is the most common cause of ALS and FTD identified to date.