Abstract
Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation. Funding The European Commission FP7 NeurOmics project; CHDI Foundation; the Medical Research Council UK; the Brain Research Trust; and the Guarantors of Brain.
| Original language | English |
|---|---|
| Pages (from-to) | 701-711 |
| Number of pages | 11 |
| Journal | The Lancet Neurology |
| Volume | 16 |
| Issue number | 9 |
| DOIs | |
| State | Published - Sep 2017 |
Bibliographical note
Publisher Copyright:© 2017 Elsevier Ltd
Funding
The evidence from our study suggests that MSH3 is probably a modifier of disease progression in Huntington's disease. We did an unbiased genetic screen using a novel disease progression measure in the TRACK-HD study, and identified a significant locus on chromosome 5, which encompasses three genes: MTRNR2L2, MSH3 , and DHFR . This locus replicated in an independent group of participants from the European Huntington's disease REGISTRY study using a parallel disease progression measure, and was genome-wide significant in a meta-analysis of the two studies. The lead SNP in TRACK-HD, rs557874766, is a coding variant in MSH3; it is classed of moderate impact, making it genome-wide significant given its annotation. 23 This SNP became clearly genome-wide significant at the more widely used threshold of p=5 × 10 −8 in our meta-analysis of TRACK-HD and REGISTRY. Furthermore, eQTL analyses showed association of lower MSH3 expression with slower disease progression in our analyses. Genetic modifiers of disease in people highlight pathways for therapeutic development; any pathway containing genetic variation that ameliorates or exacerbates disease forms a prevalidated relevant target. However, although the classic case-control design in complex disease has yielded multiple genetic associations highlighting relevant biology for novel treatment design, 24 studies of potential genetic modifiers in genetically simple Mendelian diseases have been difficult. Such diseases are rare and show gene and locus heterogeneity, thus finding genuine modifying associations in such a noisy background is inherently difficult. However, variants that modify disease in the context of a Mendelian causative gene might not be under negative selection pressure in the general population. Modifiers have been identified in specific genetic subtypes of disease 25 and in relatively large samples with consistent clinical data. 8,26 One way to increase the power of genetic studies is to obtain a more accurate measure of phenotype. Prospective multivariate longitudinal measures such as those collected in TRACK-HD are ideal. 27 Our analysis of Huntington's disease progression showed that motor, cognitive, and brain imaging variables typically progress in parallel, and that patterns of loss are not sufficiently distinct to be considered subphenotypes for genetic analysis. Because psychiatric symptoms showed a different trajectory, we developed a single progression measure excluding the psychiatric data ( figure 2 ). Age of onset was correlated with the unified progression measure but did not explain the genetic associations observed with progression. Thus, progression seems to be measuring a different aspect of disease to age of onset, or a similar aspect of disease, but with greater precision. The data available in REGISTRY are less comprehensive than in TRACK-HD; therefore we used a different approach by comparing cross-sectional severity at the most recent visit with that expected based on age and CAG repeat length. The unified progression measures in TRACK-HD and REGISTRY are correlated and again, the genetic associations in REGISTRY are not completely driven by age of onset, showing the usefulness of retrospective composite progression scores in genetic analysis. Prognostic indices for motor onset have been developed, 28 and the development of progression scores for prospective use, for example to stratify patients by predicted rate of progression to empower drug trials, warrants further attention. Our study has limitations. TRACK-HD has the same standardised detailed phenotypic information on nearly all participants, but in only 243 participants carrying the Huntington's disease gene mutations. The REGISTRY study is much larger but the phenotypic data are less complete ( appendix ), often not collected at regular intervals and not on everyone in the study, and the data were collected in multiple centres, which will inevitably lead to variation. Nevertheless, the progression measures show the expected association with change in TMS and TFC in both TRACK-HD and REGISTRY, indicating their clinical relevance. However, future development of the progression statistic and confirmation of the genetic association in participants from ongoing large studies such as ENROLL, 29 with data collected more systematically than in REGISTRY but in less detail than TRACK-HD, would be ideal. The locus we identified by use of the unified TRACK-HD progression measure included three genes, but MSH3 is the best candidate. First, the lead SNP is a coding variant in exon 1 of MSH3 , MSH3 Pro67Ala, with the potential to affect function. 30 Clinically, each copy of the minor allele (G) at this SNP corresponds to a decrease of about 0·4 units per year (95% CI 0·16–0·66) in the rate of change of TMS, and a reduction of about 0·12 units per year (95% CI 0·06–0·18) in the rate of change of TFC ( appendix ). Second, MSH3 has been extensively implicated in the pathogenesis of Huntington's disease in both mouse and in-vitro studies, although this is the first human study to link MSH3 to Huntington's disease. MSH3 is a neuronally expressed member of a family of DNA mismatch repair proteins; 31 it forms a heteromeric complex with MSH2 to form MutSβ, which recognises insertion-deletion loops of up to 13 nucleotides ( figure 4 ). 32 There is, however, a high level of interconnectedness between pathways involved in the DNA damage response, and MutSβ is implicated in other processes. 19 Changes in CAG repeat size occur in terminally differentiated neurons in several Huntington's disease mouse models and in human patient striatum, the brain area most affected in Huntington's disease, and, notably, somatic expansion of the CAG repeat in the brain of patients with Huntington's disease predicts onset. 33 Msh3 is required both for somatic expansion of HTT CAG repeats and for enhancing an early disease phenotype in mouse striatum, 34 Msh3 expression level is associated with repeat instability in mouse brain (whereas Dhfr is not), 32 and expansion of CAG and CTG repeats is prevented by msh3Δ in Saccharomyces cerevisiae . 35 This is a plausible mechanism through which variation in MSH3 could operate in Huntington's disease ( figure 4 ). In patients with myotonic dystrophy type 1, somatic instability of the CTG repeat (CAG on the non-coding strand), is associated with age of onset and an MSH3 variant was recently associated with somatic instability in blood DNA of patients. 36 Variants in DNA repair pathways, including those in MSH3 , contribute to age of onset modification in multiple CAG repeat expansion diseases, 37 implicating the CAG repeat itself as the source of modification. To our knowledge, this is the first study to use a measure of progression to look for modifiers of a neurodegenerative Mendelian disorder. We detected association with a coding variant on chromosome 5, reaching genome-wide significance given its annotation 24 in just 216 participants, which was replicated in a larger independent sample and strengthened on meta-analysis. This indicates that our progression measure developed in TRACK-HD is an excellent reflection of disease pathophysiological progression, or that this is a locus with a large effect size, or, most likely, both. Although there are three genes at the locus, the most significant variant gives a coding change in MSH3, which together with the previous biological evidence makes it the most likely candidate. Somatic expansion of the CAG repeat through alterations in MSH3 is a plausible mechanism for pathogenesis in Huntington's disease, which can be followed up in functional experiments in Huntington's disease models. These data provide additional support for the therapeutic targeting of HTT and the stability of its CAG repeat. Loss of, or variation in, mismatch repair complexes can cause malignancy and thus they are not regarded as ideal drug targets, but MSH3 is not essential because it can tolerate loss-of-function variation 38 and could provide a therapeutic target in Huntington's disease. We note that if MSH3 does operate to alter repeat expansion it might also be a drug target in other repeat expansion disorders. Contributors DJHM collected the data, did the analysis, and wrote the first draft of the manuscript. AFP did the genetic analysis and co-wrote the manuscript. DL did the statistical analysis of the phenotype, and co-wrote the manuscript. KL did the genetic analysis. BRL, RR, and AD the collected data. SM co-supervised the genetic analysis. PH co-supervised the data analyses, did the genetic analysis, and co-wrote the manuscript. LJ helped secure funding, supervised data analyses, and co-wrote the manuscript. SJT conceived the study, secured funding, recruited participants, supervised data analyses, and co-wrote the manuscript. Declarations of interests DL reports grant funding from the CHDI Foundation via UCL, and personal fees from Roche Pharmaceutical, Voyager Pharmaceutical, and Teva Pharmaceuticals. BRL reports grants from the CHDI Foundation via UCL, Teva Pharmaceuticals, and Lifemax Pharmaceuticals, and personal fees from Novartis, Roche, uniQure, Ionis Pharmaceuticals, and Raptor Pharmaceuticals. DJHM, KL, AD, AFP, SM, LJ, RR, PH, and SJT declare no competing interests. Acknowledgments We thank the people who have enabled this work through their participation in the TRACK-HD and REGISTRY studies. We also thank the following organisations for their support of this project: the European Commission 7th Framework Program (FP7/2007–2013; grant agreement number 2012–305121 “Integrated European –omics research project for diagnosis and therapy in rare neuromuscular and neurodegenerative diseases [NeurOmics]”), who provided funding for this project; CHDI Foundation, a non-profit biomedical research organisation exclusively dedicated to developing therapeutics that will substantially improve the lives of Huntington's disease-affected individuals, who funded the TRACK-HD and REGISTRY studies; the Medical Research Council (MRC) for their support of the MRC Centre for Neuropsychiatric Genetics and Genomics, MR/L010305/1; the Brain Research Trust (BRT), the Guarantors of Brain; and the Medical Research Council UK who all supported this project. We thank the people who have enabled this work through their participation in the TRACK-HD and REGISTRY studies. We also thank the following organisations for their support of this project: the European Commission 7th Framework Program (FP7/2007–2013; grant agreement number 2012–305121 “Integrated European –omics research project for diagnosis and therapy in rare neuromuscular and neurodegenerative diseases [NeurOmics]”), who provided funding for this project; CHDI Foundation, a non-profit biomedical research organisation exclusively dedicated to developing therapeutics that will substantially improve the lives of Huntington's disease-affected individuals, who funded the TRACK-HD and REGISTRY studies; the Medical Research Council (MRC) for their support of the MRC Centre for Neuropsychiatric Genetics and Genomics, MR/L010305/1; the Brain Research Trust (BRT), the Guarantors of Brain; and the Medical Research Council UK who all supported this project.
| Funders | Funder number |
|---|---|
| European Commission 7th Framework Program | |
| FP7/2007 | |
| MRC Centre for Neuropsychiatric Genetics and Genomics | |
| Roche Pharmaceutical | |
| TRACK-HD | |
| Teva Pharmaceuticals | |
| Voyager Pharmaceutical | |
| Novartis | |
| Roche | |
| CHDI Foundation | |
| Seventh Framework Programme | 2012–305121 |
| Medical Research Council | MR/P007015/1, MR/L02053X/1, MC_PC_16031, MR/L010305/1, MC_U123160651, MC_PC_14108 |
| Brain Research Trust | |
| Guarantors of Brain | |
| Research Councils UK | |
| UCLH Biomedical Research Centre |