Residual association at C9orf72 suggests an alternative amyotrophic lateral sclerosis-causing hexanucleotide repeat

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motor neurons. Single-nucleotide polymorphism rs3849942 is associated with ALS, tagging a hexanucleotide repeat mutation in the C9orf72 gene. It is possible that there is more than 1 disease-causing genetic variation at this locus, in which case association might remain after removal of cases carrying the mutation. DNA from patients with ALS was therefore tested for the mutation. Genome-wide association testing was performed first using all samples, and then restricting the analysis to samples not carrying the mutation. rs3849942 and rs903603 were strongly associated with ALS when all samples were included (rs3849942, p = [3 × 2] × 10(-6), rank 7/442,057; rs903603, p = [7 × 6] × 10(-8), rank 2/442,057). Removal of the mutation-carrying cases resulted in loss of association for rs3849942 (p = [2 × 6] × 10(-3), rank 1225/442,068), but had little effect on rs903603 (p = [1 × 9] × 10(-5), rank 8/442,068). Those with a risk allele of rs903603 had an excess of apparent homozygosity for wild type repeat alleles, consistent with polymerase chain reaction failure of 1 allele because of massive repeat expansion. These results indicate residual association at the C9orf72 locus suggesting a second disease-causing repeat mutation.

Supplementary Fig. 1

Genome-wide association study of all case (599) versus control (4136) samples. The red line is the genome correction threshold (5 × 10⁻⁸) and blue line is the Bonferroni threshold (1.13 × 10⁻⁷).

Supplementary Fig. 2

Genome-wide association study of mutation cases removed (560) versus control (4136) samples. The red line is the genome correction threshold (5 × 10⁻⁸) and the blue line is the Bonferroni threshold (1.13 × 10⁻⁷).

Supplementary Fig. 3

Q-Q plot for genome-wide association study comparing nonrepeat mutation case (560) versus control (4136) samples.

Supplementary Fig. 4

Manhattan plot of genome-wide association study comparing repeat mutation case (39) versus control (4136) samples. The red line is the genome correction threshold (5 × 10⁻⁸) and the blue line is the Bonferroni threshold (1.13 × 10⁻⁷).

Supplementary Fig. 5

Q-Q plot for genome-wide association study comparing repeat mutation case (39) versus control (4136) samples (λ_GCof 1.02): 79 single-nucleotide polymorphism mutation-specific haplotype association analysis of mutation case (39) versus control (1182) samples: χ² = 64.62; p = 9.08 × 10⁻¹⁴ and 11 single-nucleotide polymorphism residual haplotype association analysis of nonmutation case (560) versus control (1182) samples: χ² = 15.58; p = 7.90 × 10⁻⁵.

Supplementary Fig. 6a

A seventy-nine single-nucleotide polymorphism haplotype for cases with the pathological expansion (A) and an 11 single-nucleotide polymorphism haplotype for those without (B).

Supplementary Fig. 6b

Supplementary Fig. 9

Amplified fragment length polymorphism of an example case showing homozygosity for 5 repeats.

Fig. 1

Relationship between rs3849942 alleles and repeat length in nonmutation cases. The risk allele is strongly associated with longer repeat lengths, suggesting it might lie on a haplotype promoting repeat length instability.

Fig. 2

The relationship between hexanucleotide allele repeat length and single-nucleotide polymorphisms (SNPs) showing residual association at the C9orf72 locus SNP rs903603 (A), rs10967976 (B), and rs10812611 (C). Just as for the relationship for rs3849942 shown in Fig. 1, the risk allele for SNPs on the alternative risk haplotype is overwhelmingly likely to be associated with repeat sizes greater than 2.

Fig. 3

Twelve single-nucleotide polymorphism haplotypes for case samples with repeat length greater than size 2.

Fig. 4

Repeat-primed polymerase chain reaction results. Repeat-primed polymerase chain reaction of example case samples showing a small expansion in the nonpathological size range (A), and a sample known to have a pathological (GGGGCC)_n expansion mutation (B).