The G4C2 repeat expansion in C9orf72 is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We tested the hypothesis that the repeat expansion causes aberrant CpG methylation near the G4C2 repeat, which could be responsible for the downregulation of gene expression. We investigated the CpG methylation profile by two methods using genomic DNA from the blood of individuals with ALS (37 expansion carriers and 64 noncarriers), normal controls (n = 76), and family members of 7 ALS probands with the expansion. We report that hypermethylation of the CpG island 5' of the G4C2 repeat is associated with the presence of the expansion (p < 0.0001). A higher degree of methylation was significantly correlated with a shorter disease duration (p < 0.01), associated with familial ALS (p = 0.009) and segregated with the expansion in 7 investigated families. Notably, we did not detect methylation for either normal or intermediate alleles (up to 43 repeats), bringing to question the current cutoff of 30 repeats for pathological alleles. Our study raises several important questions for the future investigation of large data sets, such as whether the degree of methylation corresponds to clinical presentation (ALS versus FTLD).

Importance Common single nucleotide polymorphisms in the SORL1 gene have been associated with late onset Alzheimer’s disease (LOAD) but causal variants have not been fully characterized nor has the mechanism been established. Objective To identify functional SORL1 mutations in patients with LOAD. Design and Participants This was a family- and cohort-based genetic association study. Caribbean Hispanics with familial and sporadic LOAD and similarly aged controls recruited from the United States and the Dominican Republic, and patients with sporadic disease of Northern European origin recruited from Canada. Main Outcome Measure(s) Prioritized coding variants in SORL1 detected by targeted re-sequencing and validated by genotyping in additional family members and unrelated healthy controls. Variants transfected into human embryonic kidney 293 (HEK) cell lines were tested for Aβ40 and Aβ42 secretion and the amount of the amyloid precursor protein (APP) secreted at the cell surface was determined. Results 17 coding exonic variants were significantly associated with disease. Two rare variants (rs117260922-E270K and rs143571823-T947M) with MAF<1% and one common variant (rs2298813-A528T) with MAF=14.9% segregated within families and were deemed deleterious to the coding protein. Transfected cell lines showed increased Aβ40 and Aβ42 secretion for the rare variants (E270K and T947M) and increased Aβ42 secretion for the common variant (A528T). All mutants increased the amount of APP at the cell surface, though in slightly different ways, thereby failing to direct full-length APP into the retromer-recycling endosome pathway. Conclusions and Relevance Common and rare variants in SORL1 elevate the risk of LOAD by directly affecting APP processing which, in turn can result in increased Aβ40 and Aβ42 secretion.

ObjectiveTo detect rare coding variants underlying loci detected by genome‐wide association studies (GWAS) of late onset Alzheimer disease (LOAD).MethodsWe conducted targeted sequencing of ABCA7, BIN1, CD2AP, CLU, CR1, EPHA1, MS4A4A/MS4A6A, and PICALM in 3 independent LOAD cohorts: 176 patients from 124 Caribbean Hispanics families, 120 patients and 33 unaffected individuals from the 129 National Institute on Aging LOAD Family Study; and 263 unrelated Canadian individuals of European ancestry (210 sporadic patients and 53 controls). Rare coding variants found in at least 2 data sets were genotyped in independent groups of ancestry‐matched controls. Additionally, the Exome Aggregation Consortium was used as a reference data set for population‐based allele frequencies.ResultsOverall we detected a statistically significant 3.1‐fold enrichment of the nonsynonymous mutations in the Caucasian LOAD cases compared with controls (p = 0.002) and no difference in synonymous variants. A stop‐gain mutation in ABCA7 (E1679X) and missense mutation in CD2AP (K633R) were highly significant in Caucasian LOAD cases, and mutations in EPHA1 (P460L) and BIN1 (K358R) were significant in Caribbean Hispanic families with LOAD. The EPHA1 variant segregated completely in an extended Caribbean Hispanic family and was also nominally significant in the Caucasians. Additionally, BIN1 (K358R) segregated in 2 of the 6 Caribbean Hispanic families where the mutations were discovered.InterpretationTargeted sequencing of confirmed GWAS loci revealed an excess burden of deleterious coding mutations in LOAD, with the greatest burden observed in ABCA7 and BIN1. Identifying coding variants in LOAD will facilitate the creation of tractable models for investigation of disease‐related mechanisms and potential therapies. Ann Neurol 2015;78:487–498