Sequence-Level Analysis of the Major European Huntington Disease Haplotype

Abstract

Huntington disease (HD) reflects the dominant consequences of a CAG-repeat expansion in HTT. Analysis of common SNP-based haplotypes has revealed that most European HD subjects have distinguishable HTT haplotypes on their normal and disease chromosomes and that ∼50% of the latter share the same major HD haplotype. We reasoned that sequence-level investigation of this founder haplotype could provide significant insights into the history of HD and valuable information for gene-targeting approaches. Consequently, we performed whole-genome sequencing of HD and control subjects from four independent families in whom the major European HD haplotype segregates with the disease. Analysis of the full-sequence-based HTT haplotype indicated that these four families share a common ancestor sufficiently distant to have permitted the accumulation of family-specific variants. Confirmation of new CAG-expansion mutations on this haplotype suggests that unlike most founders of human disease, the common ancestor of HD-affected families with the major haplotype most likely did not have HD. Further, availability of the full sequence data validated the use of SNP imputation to predict the optimal variants for capturing heterozygosity in personalized allele-specific gene-silencing approaches. As few as ten SNPs are capable of revealing heterozygosity in more than 97% of European HD subjects. Extension of allele-specific silencing strategies to the few remaining homozygous individuals is likely to be achievable through additional known SNPs and discovery of private variants by complete sequencing of HTT. These data suggest that the current development of gene-based targeting for HD could be extended to personalized allele-specific approaches in essentially all HD individuals of European ancestry.

Figure 1

Pedigree Structures of Four Families Used for Haplotype Analysis Four independent families in whom the hap.01 haplotype segregates with disease were analyzed by whole-genome sequencing followed by haplotype phasing. Of the 38 individuals sequenced, 29 are shown here, permitting the haplotype-phasing analysis of 16 father-mother-child trios (numbered by family and child; filled and open symbols represent subjects with an expanded HTT CAG repeat and normal individuals, respectively). CAG repeats of study subjects are provided under pedigree symbols. Small CAG differences between parent and child pairs (e.g., ±2) are within the margin of error of the CAG genotyping assay, which takes the highest peak among neighbor peaks as the repeat size of the sample.

Figure 2

Sequence-Based Haplotype hap.01 Is Compared to the Human Reference Sequence Site variant alleles shared by the independently established consensus hap.01 disease chromosomes of all four families were compared to the reference genome, identifying 27 differential sites, all of which are reported in dbSNP. The HTT structure is based on GenBank: NM_002111 and is depicted from left (5′) to right (3′) on the line (representing the genomic region); narrow and thickened segments represent non-coding and coding portions of the mRNA, respectively. The locations of variant sites are based on the hg19 genome assembly. “D” indicates a single-nucleotide deletion. The site noted in green is a synonymous coding variant, and a codon loss (three consecutive “D” sites) is shown in red. All other variants are non-coding.

Figure 3

Locations and Control-Population Frequencies of Family-Specific Variants (A) The final hap.01 sequences revealed family-specific differences at seven sites, whose locations, genomic reference alleles, and disease-chromosome alleles for each family are shown. (B) Kaviar3 data were used for obtaining allele frequencies for the seven family-specific variants, shown as pie charts. In three cases, alleles for variations not seen previously are shown at 0% frequency in the pie chart.

Figure 4

Recombination Events on hap.01 HD Chromosomes (A) RefSeq genes are indicated in blue arrows above the orange traces, which represent the HapMap recombination rates (chr4: 2,500,000–4,000,000 bp region). Recombination rates are very low around HTT, and recombination peaks are located centromerically. The red arrow indicates the highest recombination peak in this region, where two recombination events were detected. (B) In an expanded view of the highest recombination peak, the reference genomic coordinates with recombination rates are aligned with actual recombination events in (C) and (D). Both recombination events were located within the recombination peak at chr4: 3,725,000 bp. (C) A recombination event in family 1 is shown. Five transmitted HD chromosomes were compared for chr4: 22,500,000–4,000,000, revealing a recombination event in the maternal transmission in trio 2. The mother’s disease chromosome (red) and normal chromosome (green) are compared to the transmitted disease chromosomes (red and green). Only bases at sites of heterozygosity are shown. (D) A similar analysis to that in (C) was performed for family 4. One recombination event was detected in the paternal transmission of trio 4.