To determine whether the lack of segregation was the result of single allele amplification due to the presence of

an unamplifiable repeat expansion, we used a repeat-primed PCR method specifically designed to the observed GGGGCC hexanucleotide repeat. This method suggested the presence of repeat expansions in all affected members of family VSM-20, but not in unaffected relatives ( Figure 2C). Subsequent analysis www.selleckchem.com/products/OSI-906.html of 909 healthy controls by fluorescent fragment-length analysis identified 315 who were homozygous, however no repeat expansions were observed by repeat-primed PCR. The maximum size of the repeat in controls was 23 units. These findings suggested the presence of a unique repeat expansion in family VSM-20 and prompted us to perform Southern blot analysis

on DNA from four different affected and one unaffected member of VSM-20. In addition to the expected normal allele, we detected a variably sized expanded allele, too large to be amplified by PCR, which was found only in the affected individuals ( Figure 2D). In all but one patient, the expanded alleles appeared as single discrete bands; however, in patient 20-17 ( Figure 2D, lane 5) two discrete high molecular weight bands were observed, suggesting somatic instability of the repeat. Based on this small number of patients, selleck chemicals llc we estimated the number of GGGGCC repeat units to range from approximately 700 to 1600. The proband of family VSM-20 (20-6) is part of a highly selected series of 26 probands ascertained at UBC, Vancouver, Canada, with a confirmed pathological diagnosis of FTLD-TDP and a positive family history of FTD and/or ALS. We previously identified

GRN mutations in seven probands (26.9%) from this series, all from families with a clinically pure FTD phenotype; however, the genetic basis for the disease in the other families remained unknown. Using a combination of fluorescent fragment-length and repeat-primed PCR analyses, we then found that 16 of the 26 FTLD-TDP families in this series (61.5%) carried expanded alleles of the GGGGCC hexanucleotide repeat; nine with a combined FTD/ALS phenotype and seven with clinically pure FTD. In five of these families, DNA was available from multiple GPX6 affected members and in all cases, the repeat expansion was found to segregate with disease ( Figure 2 and see Figure S1 available online). These findings suggest that GGGGCC expansions in C9ORF72 are the most common cause of familial FTLD-TDP. To further determine the frequency of GGGGCC hexanucleotide expansions in C9ORF72 in patients with FTLD-TDP pathology and to assess the importance of this genetic defect in the etiology of patients clinically diagnosed with FTD and ALS, we analyzed 696 patients (93 pathologically diagnosed FTLD-TDP, 374 clinical FTD, and 229 clinical ALS) derived from three well-characterized patient series ascertained at the Mayo Clinic Florida (MCF) and MCR ( Table S1).