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

Israel Ben-Dor,Yuval Nevo,Crystal Pacut,Benjamin E Reubinoff,Eva L Feldman,Giorgio Sirugo

doi:10.1371/journal.pgen.1009445

Abstract

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.

Highlights

Amyotrophic lateral sclerosis (ALS [OMIM: 612069]) and frontotemporal dementia (FTD [OMIM: 600274]) are two fatal neurodegenerative diseases
Haplotype is a specific combination of multiple polymorphic sites along a chromosome that are inherited together in block
We show that pathological hexanucleotide repeat (HR) expansion lowers chromosome 9 open reading frame 72 (C9orf72) first- and second-intron splicing efficiency and increases aberrant splicing at first-intron cryptic donor splice sites

Summary

Introduction

Amyotrophic lateral sclerosis (ALS [OMIM: 612069]) and frontotemporal dementia (FTD [OMIM: 600274]) are two fatal neurodegenerative diseases. GGGGCC hexanucleotide repeat (HR) expansion in chromosome 9 open reading frame 72 (C9orf72) gene is the most common genetic cause of ALS and FTD in Caucasians [3,4]. It accounts for around 39% of familial and 7% of sporadic ALS cases and 25% of familial and 6% of sporadic FTD cases in the Caucasians [5]. HR expansion is bidirectionally transcribed to sense and antisense transcripts that sequester crucial RNA binding proteins [9] In the cytoplasm, these transcripts undergo repeat-associated non-ATG (RAN) translation, resulting in the production and accumulation of pathogenic dipeptide repeat proteins [10]. Hypermethylation occurs in 36% and 17% of C9orf72-ALS and FTD patients, respectively [15], but not in normal and intermediate repeat lengths [14,16]

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Conclusion