Characterization of C9orf72 haplotypes to evaluate the effects of normal and pathological variations on its expression and splicing

Author summary Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive neurodegenerative diseases, whose most frequent genetic cause is hexanucleotide repeat (HR) expansion from normal 2 to 20 repeats to pathological hundreds of repeats within a non-coding region of the C9orf72 gene. Haplotype is a specific combination of multiple polymorphic sites along a chromosome that are inherited together in block. We characterized the single nucleotide polymorphism (SNP) signature and HR length of the major C9orf72 haplotypes in Caucasians to identify the allelic origin of C9orf72 transcripts per patient and determine the effects of expanded HR on C9orf72 gene expression and splicing. In C9orf72 ALS patients' cells, the HR expanded allele, compared to non-R allele, was associated with decreased levels of downstream initiated transcript variant, increased levels of upstream initiated transcripts, accumulation of introns 1 and 2, and abnormal splicing at cryptic splice sites along intron 1. The C9orf72 haplotypes DNA signatures described here are valuable for studying C9-ALS/FTD pathogenesis and for developing allele-specific therapeutic strategies. Expansion of the hexanucleotide repeat (HR) in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in Caucasians. All C9orf72-ALS/FTD patients share a common risk (R) haplotype. To study C9orf72 expression and splicing from the mutant R allele compared to the complementary normal allele in ALS/FTD patients, we initially created a detailed molecular map of the single nucleotide polymorphism (SNP) signature and the HR length of the various C9orf72 haplotypes in Caucasians. We leveraged this map to determine the allelic origin of transcripts per patient, and decipher the effects of pathological and normal HR lengths on C9orf72 expression and splicing. In C9orf72 ALS patients' cells, the HR expanded allele, compared to non-R allele, was associated with decreased levels of a downstream initiated transcript variant and increased levels of transcripts initiated upstream of the HR. HR expanded R alleles correlated with high levels of unspliced intron 1 and activation of cryptic donor splice sites along intron 1. Retention of intron 1 was associated with sequential intron 2 retention. The SNP signature of C9orf72 haplotypes described here enables allele-specific analysis of transcriptional products and may pave the way to allele-specific therapeutic strategies.

Automated summary

Expansion of the hexanucleotide repeat (HR) in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in Caucasians. The C9orf72-ALS/FTD patients share a common risk (R) haplotype. Characterizing the SNP signature and HR length of different C9orf72 haplotypes allows for the determination of the effects of expanded HR on C9orf72 gene expression and splicing.