Huntingtin Haplotypes Provide Prioritized Target Panels for Allele-specific Silencing in Huntington Disease Patients of European Ancestry

Abstract

Huntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in the Huntingtin gene (HTT). Heterozygous polymorphisms in cis with the mutation allow for allele-specific suppression of the pathogenic HTT transcript as a therapeutic strategy. To prioritize target selection, precise heterozygosity estimates are needed across diverse HD patient populations. Here we present the first comprehensive investigation of all common target alleles across the HTT gene, using 738 reference haplotypes from the 1000 Genomes Project and 2364 haplotypes from HD patients and relatives in Canada, Sweden, France, and Italy. The most common HD haplotypes (A1, A2, and A3a) define mutually exclusive sets of polymorphisms for allele-specific therapy in the greatest number of patients. Across all four populations, a maximum of 80% are treatable using these three target haplotypes. We identify a novel deletion found exclusively on the A1 haplotype, enabling potent and selective silencing of mutant HTT in approximately 40% of the patients. Antisense oligonucleotides complementary to the deletion reduce mutant A1 HTT mRNA by 78% in patient cells while sparing wild-type HTT expression. By suppressing specific haplotypes on which expanded CAG occurs, we demonstrate a rational approach to the development of allele-specific therapy for a monogenic disorder.

Figure 1

SNPs across HTT represent specific gene-spanning haplotypes. (a) Single-nucleotide polymorphisms (SNPs) across HTT belong to gene-spanning haplotypes representing three major haplogroups A, B, and C. Transcribed (intragenic) SNPs are shaded gray. The primary Huntington disease (HD) haplotype, A1, is defined by rs362307. The secondary HD haplotype A2 is defined by rs2798235 and rs363080. One SNP (rs2298969) distinguishes the A haplogroup from the B and C haplogroups (black box). In our panel, 8 of 51 intragenic SNPs exclusively distinguish the A and B haplogroups from the C haplogroup (bold). (b) Pairwise linkage disequilibrium (LD) of SNP genotypes (r²) reveals a complex haplotype structure across the HTT gene region (black, r² = 1; shades of gray, 1 > r² > 0; white, r² = 0). Alleles present on similar haplotypes are in high pairwise LD across HTT, such as A2-defining rs2798235 and rs363080. Haplotype A1-defining SNP rs362307 is not in LD with any other variant in our initial 63-SNP panel. Positions correspond to GRCh37. Representative extragenic crossover sequences are colored according to the most likely originating haplotype.

Figure 2

A1, A2, and A3a are the most common gene-spanning HD haplotypes. (a) In the UBC HD BioBank, the expanded CAG repeat (CAG > 35) is found on the A1 haplotype in 48.1% of phased, unrelated Canadian Huntington disease (HD) chromosomes. Intragenic recombinant haplotypes (X) are rare (3.0% of controls) whereas >95% of HTT haplotypes show no evidence of recombination within the transcribed gene sequence. C1 is the most common intragenic haplotype among Canadian control chromosomes, representing 29.8%. (b) The most common HD haplotype, A1, is uniquely defined by three transcribed polymorphisms in high pairwise linkage disequilibrium (LD) across HTT. The 4 bp indel rs72239206 represents a novel polymorphism associated with the CAG expansion (bold). The second most common HD haplotype, A2, is defined by five intragenic single-nucleotide polymorphisms (SNPs), three of which are novel (bold). HD-associated A3a, the third most common HD haplotype, is specifically marked by the novel SNP rs113407847. (c) Pairwise LD plot (r²) of A1 and A2 haplotype-defining polymorphisms as calculated from 700 phased haplotypes of European Caucasians.

Figure 3

In four European Huntington disease (HD) cohorts, distinct distributions of HD haplotypes are observed. As in Canada, A1 is the most frequent HD haplotype in Finland (60%) and Sweden (51%), and France (45%). In contrast, A2 is the most frequent HD haplotype in Italy (58%).

Figure 4

Cumulative treatment coverage of Huntington disease (HD) patients by A1, A2, and A3a allele-specific HTT silencing targets in four patient populations of European ancestry. Targeting one A1 allele (among three) and one A2 allele (among five) would permit selective HTT silencing treatment in 68% of HD patients in the Canadian cohort. In combination, A1 + A2 + A3a targets would permit selective silencing of an average of 80% of HD patients across all four populations.

Figure 5

ASOs targeting ΔACTT (rs72239206) potently and selectively silence A1 HTT mRNA and protein in human cells. (a) Design of antisense oligonucleotide (ASO) gapmers selectively targeting mutant HTT A1 mRNA at the ΔACTT sequence. (b) Transfection of patient-derived lymphoblasts (44/18 CAG) with ΔACTT-complementary ASOs selectively reduces mutant HTT mRNA. Patient lymphoblasts transfected with 5-9-5, 5-7-5, and 4-7-4 LNA gapmers show dose-dependent reduction of mutant HTT mRNA relative to untreated controls, falling to 21.5% mutant HTT mRNA at the highest 4-7-4 dose. Wild-type HTT mRNA levels do not fall below untreated levels at any tested dose of 5-7-5 or 4-7-4 LNA gapmer. (c) Dose-dependent reduction of mutant HTT protein relative to untreated controls, sparing wtHTT at all tested 5-7-5 and 4-7-4 LNA gapmer doses. ** and *** represent P = 0.01 and P = 0.001 by analysis of variance with Bonferroni post hoc. LNA, locked nucleic acid.