Trimodal pattern of C9ORF72 GGGGCC normal allele repeat number in sporadic amyotrophic lateral sclerosis and lack of association with disease risk and age at onset

BACKGROUND: Recent research has discovered a non-coding hexanucleotide repeat expansion mutation (HREM) of the chromosome 9 open reading frame 72 (C9ORF72) gene, which produces hundreds of pathological GGGGCC repeats on one allele in up to 40% of familial and 10% of patients with sporadic amyotrophic lateral sclerosis (ALS). We studied patients with sporadic ALS who had a normal number of repeats (up to 23) on both alleles (non-HREM cases), patients with the expanded repeat on one allele (HREM cases), and healthy controls, to determine whether a higher normal number of GGGGCC repeats increases the risk of developing sporadic ALS, and whether it is associated with an earlier age at symptom onset. METHODS: We used PCR techniques and amplified fragment length polymorphism analysis to characterise repeat numbers on each allele in 397 white UK patients with sporadic ALS and 235 white UK controls. This method identified 357 non-HREM cases and 40 HREM cases. We then performed logistic regressions of repeat number against disease status (cases vs controls) and linear regressions of repeat number against age at onset of symptoms (cases), all adjusted for sex. All four models of allelic effect (recessive, dominant, additive, and multiplicative) were evaluated for each analysis. FINDINGS: Normal repeat numbers on each allele were not significantly associated with disease risk or age at onset of symptoms (p>0·05 for all analyses). These factors may therefore be determined by other environmental or genetic influences in patients with sporadic ALS. We found a trimodal pattern of repeat number expression, with two, five, or eight GGGGCC repeats on each allele in most of the cases and controls. This pattern may relate to the normal structure or function of this region or of C9ORF72 mRNA transcripts. INTERPRETATION: Determination of the effects of repeat number pattern on C9ORF72 gene expression and function, and an understanding of what increases the risk of developing the expansion mutation, will be vital in elucidating the pathological mechanisms of the C9ORF72 mutation in ALS. FUNDING: National Institute for Health Research.