Investigational Assay for Haplotype Phasing of the Huntingtin Gene

Abstract

Novel treatments for Huntington’s disease (HD), a progressive neurodegenerative disorder, include selective targeting of the mutant allele of the huntingtin gene (m HTT ) carrying the abnormally expanded disease-causing cytosine-adenine-guanine (CAG) repeat. WVE-120101 and WVE-120102 are investigational stereopure antisense oligonucleotides that enable selective suppression of m HTT by targeting single-nucleotide polymorphisms (SNPs) that are in haplotype phase with the CAG repeat expansion. Recently developed long-read sequencing technologies can capture CAG expansions and distant SNPs of interest and potentially facilitate haplotype-based identification of patients for clinical trials of oligonucleotide therapies. However, improved methods are needed to phase SNPs with CAG repeat expansions directly and reliably without need for familial genotype/haplotype data. Our haplotype phasing method uses single-molecule real-time sequencing and a custom algorithm to determine with confidence bases at SNPs on mutant alleles, even without familial data. Herein, we summarize this methodology and validate the approach using patient-derived samples with known phasing results. Comparison of experimentally measured CAG repeat lengths, heterozygosity, and phasing with previously determined results showed improved performance. Our methodology enables the haplotype phasing of SNPs of interest and the disease-causing, expanded CAG repeat of the huntingtin gene, enabling accurate identification of patients with HD eligible for allele-selective clinical studies.

An update on the neurological short tandem repeat expansion disorders and the emergence of long-read sequencing diagnostics

“…An advantage of the long and highly accurate reads generated by PacBio SMRT sequencing, is the ability to resolve the STR length and sequence, as well as detecting and phasing possible variants in the surrounding regions. For example, a recent study developed a haplotype phasing protocol for the HTT gene using PacBio SMRT sequencing, enabling detection of relevant SNPs and ‘CAG’ expansions in HTT on the same amplicon [ 153 ]. Several new bioinformatics tools, such as IsoPhase [ 163 ], SHAPEIT4 [ 33 ] and NanoCaller [ 1 ], use long reads to accurately phase SNV, insertions and deletions.…”

Chemical engineering of therapeutic siRNAs for allele-specific gene silencingin vivoin CNS

“…Up to 40% of patients of European descent are heterozygous at rs362273 36 and the A isoform is more common on HD chromosomes 65 , we have successfully targeted the A isoform, but to treat 40% of patients both isoforms would need to be targeted. If the allele selective silencing of HTT becomes essential for clinical disease modification then, the linkage between the CAG repeats and the SNP site heterozygosity would need to be established individually for eligible patients using long read sequencing 72 . For screening purposes, siRNAs are prepared in OptiMEM at 3M concentration.…”

Uncovering Essential Tremor Genetics: The Promise of Long-Read Sequencing

“…For this purpose, the No-Amp targeted sequencing has been used to overcome the difficulties due to PCR stutter and to the high GC content, thus permitting a detailed sequence information and the assessment of somatic variability of repeated elements without the confounding errors related to the PCR stutter ( 7 ). In addition, LRS demonstrated the feasibility of rapidly genotyping and haplotype phasing to differentiate the genetic variability inherited either from paternal or maternal origins without familial genotype/haplotype data ( 31 ). In HD, this has led to determine the allele bearing both the pathogenic CAG expansion and SNPs 165 kb downstream, essential for personalized therapies.…”