Splice modulators target PMS1 to reduce somatic expansion of the Huntington's disease-associated CAG repeat.

Huntington's disease (HD) is a dominant neurological disorder caused by an expanded HTT exon 1 CAG repeat that lengthens huntingtin's polyglutamine tract. Lowering mutant huntingtin has been proposed for treating HD, but genetic modifiers implicate somatic CAG repeat expansion as the driver of onset. We find that branaplam and risdiplam, small molecule splice modulators that lower huntingtin by promoting HTT pseudoexon inclusion, also decrease expansion of an unstable HTT exon 1 CAG repeat in an engineered cell model. Targeted CRISPR-Cas9 editing shows this effect is not due to huntingtin lowering, pointing instead to pseudoexon inclusion in PMS1. Homozygous but not heterozygous inactivation of PMS1 also reduces CAG repeat expansion, supporting PMS1 as a genetic modifier of HD and a potential target for therapeutic intervention. Although splice modulation provides one strategy, genome-wide transcriptomics also emphasize consideration of cell-type specific effects and polymorphic variation at both target and off-target sites.

Modification of Huntington's disease by short tandem repeats.

Expansions of glutamine-coding CAG trinucleotide repeats cause a number of neurodegenerative diseases, including Huntington's disease and several of spinocerebellar ataxias. In general, age-at-onset of the polyglutamine diseases is inversely correlated with the size of the respective inherited expanded CAG repeat. Expanded CAG repeats are also somatically unstable in certain tissues, and age-at-onset of Huntington's disease corrected for individual HTT CAG repeat length (i.e. residual age-at-onset), is modified by repeat instability-related DNA maintenance/repair genes as demonstrated by recent genome-wide association studies. Modification of one polyglutamine disease (e.g. Huntington's disease) by the repeat length of another (e.g. ATXN3, CAG expansions in which cause spinocerebellar ataxia 3) has also been hypothesized. Consequently, we determined whether age-at-onset in Huntington's disease is modified by the CAG repeats of other polyglutamine disease genes. We found that the CAG measured repeat sizes of other polyglutamine disease genes that were polymorphic in Huntington's disease participants but did not influence Huntington's disease age-at-onset. Additional analysis focusing specifically on ATXN3 in a larger sample set (n = 1388) confirmed the lack of association between Huntington's disease residual age-at-onset and ATXN3 CAG repeat length. Additionally, neither our Huntington's disease onset modifier genome-wide association studies single nucleotide polymorphism data nor imputed short tandem repeat data supported the involvement of other polyglutamine disease genes in modifying Huntington's disease. By contrast, our genome-wide association studies based on imputed short tandem repeats revealed significant modification signals for other genomic regions. Together, our short tandem repeat genome-wide association studies show that modification of Huntington's disease is associated with short tandem repeats that do not involve other polyglutamine disease-causing genes, refining the landscape of Huntington's disease modification and highlighting the importance of rigorous data analysis, especially in genetic studies testing candidate modifiers.

Somatic CAG Repeat Stability in a Transgenic Sheep Model of Huntington's Disease.

Somatic instability of the huntingtin (HTT) CAG repeat mutation modifies age-at-onset of Huntington's disease (HD). Understanding the mechanism and pathogenic consequences of instability may reveal therapeutic targets. Using small-pool PCR we analyzed CAG instability in the OVT73 sheep model which expresses a full-length human cDNA HTT transgene. Analyses of five- and ten-year old sheep revealed the transgene (CAG)69 repeat was remarkably stable in liver, striatum, and other brain tissues. As OVT73 sheep at ten years old have minimal cell death and behavioral changes, our findings support instability of the HTT expanded-CAG repeat as being required for the progression of HD.

PMS1 as a target for splice modulation to prevent somatic CAG repeat expansion in Huntington's disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder whose motor, cognitive, and behavioral manifestations are caused by an expanded, somatically unstable CAG repeat in the first exon of HTT that lengthens a polyglutamine tract in huntingtin. Genome-wide association studies (GWAS) have revealed DNA repair genes that influence the age-at-onset of HD and implicate somatic CAG repeat expansion as the primary driver of disease timing. To prevent the consequent neuronal damage, small molecule splice modulators (e.g., branaplam) that target HTT to reduce the levels of huntingtin are being investigated as potential HD therapeutics. We found that the effectiveness of the splice modulators can be influenced by genetic variants, both at HTT and other genes where they promote pseudoexon inclusion. Surprisingly, in a novel hTERT-immortalized retinal pigment epithelial cell (RPE1) model for assessing CAG repeat instability, these drugs also reduced the rate of HTT CAG expansion. We determined that the splice modulators also affect the expression of the mismatch repair gene PMS1, a known modifier of HD age-at-onset. Genome editing at specific HTT and PMS1 sequences using CRISPR-Cas9 nuclease confirmed that branaplam suppresses CAG expansion by promoting the inclusion of a pseudoexon in PMS1, making splice modulation of PMS1 a potential strategy for delaying HD onset. Comparison with another splice modulator, risdiplam, suggests that other genes affected by these splice modulators also influence CAG instability and might provide additional therapeutic targets.

Tissue-specific and repeat length-dependent somatic instability of the X-linked dystonia parkinsonism-associated CCCTCT repeat.

X-linked dystonia-parkinsonism (XDP) is a progressive adult-onset neurodegenerative disorder caused by insertion of a SINE-VNTR-Alu (SVA) retrotransposon in the TAF1 gene. The SVA retrotransposon contains a CCCTCT hexameric repeat tract of variable length, whose length is inversely correlated with age at onset. This places XDP in a broader class of repeat expansion diseases, characterized by the instability of their causative repeat mutations. Here, we observe similar inverse correlations between CCCTCT repeat length with age at onset and age at death and no obvious correlation with disease duration. To gain insight into repeat instability in XDP we performed comprehensive quantitative analyses of somatic instability of the XDP CCCTCT repeat in blood and in seventeen brain regions from affected males. Our findings reveal repeat length-dependent and expansion-based instability of the XDP CCCTCT repeat, with greater levels of expansion in brain than in blood. The brain exhibits regional-specific patterns of instability that are broadly similar across individuals, with cerebellum exhibiting low instability and cortical regions exhibiting relatively high instability. The spectrum of somatic instability in the brain includes a high proportion of moderate repeat length changes of up to 5 repeats, as well as expansions of ~ 20- > 100 repeats and contractions of ~ 20-40 repeats at lower frequencies. Comparison with HTT CAG repeat instability in postmortem Huntington's disease brains reveals similar brain region-specific profiles, indicating common trans-acting factors that contribute to the instability of both repeats. Analyses in XDP brains of expansion of a different SVA-associated CCCTCT located in the LIPG gene, and not known to be disease-associated, reveals repeat length-dependent expansion at overall lower levels relative to the XDP CCCTCT repeat, suggesting that expansion propensity may be modified by local chromatin structure. Together, the data support a role for repeat length-dependent somatic expansion in the process(es) driving the onset of XDP and prompt further investigation into repeat dynamics and the relationship to disease.

Genetic modifiers of Huntington disease differentially influence motor and cognitive domains.

Genome-wide association studies (GWASs) of Huntington disease (HD) have identified six DNA maintenance gene loci (among others) as modifiers and implicated a two step-mechanism of pathogenesis: somatic instability of the causative HTT CAG repeat with subsequent triggering of neuronal damage. The largest studies have been limited to HD individuals with a rater-estimated age at motor onset. To capitalize on the wealth of phenotypic data in several large HD natural history studies, we have performed algorithmic prediction by using common motor and cognitive measures to predict age at other disease landmarks as additional phenotypes for GWASs. Combined with imputation with the Trans-Omics for Precision Medicine reference panel, predictions using integrated measures provided objective landmark phenotypes with greater power to detect most modifier loci. Importantly, substantial differences in the relative modifier signal across loci, highlighted by comparing common modifiers at MSH3 and FAN1, revealed that individual modifier effects can act preferentially in the motor or cognitive domains. Individual components of the DNA maintenance modifier mechanisms may therefore act differentially on the neuronal circuits underlying the corresponding clinical measures. In addition, we identified additional modifier effects at the PMS1 and PMS2 loci and implicated a potential second locus on chromosome 7. These findings indicate that broadened discovery and characterization of HD genetic modifiers based on additional quantitative or qualitative phenotypes offers not only the promise of in-human validated therapeutic targets but also a route to dissecting the mechanisms and cell types involved in both the somatic instability and toxicity components of HD pathogenesis.

Mutations causing Lopes-Maciel-Rodan syndrome are huntingtin hypomorphs.

Huntington's disease pathogenesis involves a genetic gain-of-function toxicity mechanism triggered by the expanded HTT CAG repeat. Current therapeutic efforts aim to suppress expression of total or mutant huntingtin, though the relationship of huntingtin's normal activities to the gain-of-function mechanism and what the effects of huntingtin-lowering might be are unclear. Here, we have re-investigated a rare family segregating two presumed HTT loss-of-function (LoF) variants associated with the developmental disorder, Lopes-Maciel-Rodan syndrome (LOMARS), using whole-genome sequencing of DNA from cell lines, in conjunction with analysis of mRNA and protein expression. Our findings correct the muddled annotation of these HTT variants, reaffirm they are the genetic cause of the LOMARS phenotype and demonstrate that each variant is a huntingtin hypomorphic mutation. The NM_002111.8: c.4469+1G>A splice donor variant results in aberrant (exon 34) splicing and severely reduced mRNA, whereas, surprisingly, the NM_002111.8: c.8157T>A NP_002102.4: Phe2719Leu missense variant results in abnormally rapid turnover of the Leu2719 huntingtin protein. Thus, although rare and subject to an as yet unknown LoF intolerance at the population level, bona fide HTT LoF variants can be transmitted by normal individuals leading to severe consequences in compound heterozygotes due to huntingtin deficiency.

Promotion of somatic CAG repeat expansion by Fan1 knock-out in Huntington's disease knock-in mice is blocked by Mlh1 knock-out.

Recent genome-wide association studies of age-at-onset in Huntington's disease (HD) point to distinct modes of potential disease modification: altering the rate of somatic expansion of the HTT CAG repeat or altering the resulting CAG threshold length-triggered toxicity process. Here, we evaluated the mouse orthologs of two HD age-at-onset modifier genes, FAN1 and RRM2B, for an influence on somatic instability of the expanded CAG repeat in Htt CAG knock-in mice. Fan1 knock-out increased somatic expansion of Htt CAG repeats, in the juvenile- and the adult-onset HD ranges, whereas knock-out of Rrm2b did not greatly alter somatic Htt CAG repeat instability. Simultaneous knock-out of Mlh1, the ortholog of a third HD age-at-onset modifier gene (MLH1), which suppresses somatic expansion of the Htt knock-in CAG repeat, blocked the Fan1 knock-out-induced acceleration of somatic CAG expansion. This genetic interaction indicates that functional MLH1 is required for the CAG repeat destabilizing effect of FAN1 loss. Thus, in HD, it is uncertain whether the RRM2B modifier effect on timing of onset may be due to a DNA instability mechanism. In contrast, the FAN1 modifier effects reveal that functional FAN1 acts to suppress somatic CAG repeat expansion, likely in genetic interaction with other DNA instability modifiers whose combined effects can hasten or delay onset and other CAG repeat length-driven phenotypes.

Patterns of CAG repeat instability in the central nervous system and periphery in Huntington's disease and in spinocerebellar ataxia type 1.

The expanded HTT CAG repeat causing Huntington's disease (HD) exhibits somatic expansion proposed to drive the rate of disease onset by eliciting a pathological process that ultimately claims vulnerable cells. To gain insight into somatic expansion in humans, we performed comprehensive quantitative analyses of CAG expansion in ~50 central nervous system (CNS) and peripheral postmortem tissues from seven adult-onset and one juvenile-onset HD individual. We also assessed ATXN1 CAG repeat expansion in brain regions of an individual with a neurologically and pathologically distinct repeat expansion disorder, spinocerebellar ataxia type 1 (SCA1). Our findings reveal similar profiles of tissue instability in all HD individuals, which, notably, were also apparent in the SCA1 individual. CAG expansion was observed in all tissues, but to different degrees, with multiple cortical regions and neostriatum tending to have the greatest instability in the CNS, and liver in the periphery. These patterns indicate different propensities for CAG expansion contributed by disease locus-independent trans-factors and demonstrate that expansion per se is not sufficient to cause cell type or disease-specific pathology. Rather, pathology may reflect distinct toxic processes triggered by different repeat lengths across cell types and diseases. We also find that the HTT CAG length-dependent expansion propensity of an individual is reflected in all tissues and in cerebrospinal fluid. Our data indicate that peripheral cells may be a useful source to measure CAG expansion in biomarker assays for therapeutic efforts, prompting efforts to dissect underlying mechanisms of expansion that may differ between the brain and periphery.

Genetic and Functional Analyses Point to FAN1 as the Source of Multiple Huntington Disease Modifier Effects.

A recent genome-wide association study of Huntington disease (HD) implicated genes involved in DNA maintenance processes as modifiers of onset, including multiple genome-wide significant signals in a chr15 region containing the DNA repair gene Fanconi-Associated Nuclease 1 (FAN1). Here, we have carried out detailed genetic, molecular, and cellular investigation of the modifiers at this locus. We find that missense changes within or near the DNA-binding domain (p.Arg507His and p.Arg377Trp) reduce FAN1's DNA-binding activity and its capacity to rescue mitomycin C-induced cytotoxicity, accounting for two infrequent onset-hastening modifier signals. We also idenified a third onset-hastening modifier signal whose mechanism of action remains uncertain but does not involve an amino acid change in FAN1. We present additional evidence that a frequent onset-delaying modifier signal does not alter FAN1 coding sequence but is associated with increased FAN1 mRNA expression in the cerebral cortex. Consistent with these findings and other cellular overexpression and/or suppression studies, knockout of FAN1 increased CAG repeat expansion in HD-induced pluripotent stem cells. Together, these studies support the process of somatic CAG repeat expansion as a therapeutic target in HD, and they clearly indicate that multiple genetic variations act by different means through FAN1 to influence HD onset in a manner that is largely additive, except in the rare circumstance that two onset-hastening alleles are present. Thus, an individual's particular combination of FAN1 haplotypes may influence their suitability for HD clinical trials, particularly if the therapeutic agent aims to reduce CAG repeat instability.

Genetic Risk Underlying Psychiatric and Cognitive Symptoms in Huntington's Disease.

BACKGROUND: Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the HTT gene. It is diagnosed following a standardized examination of motor control and often presents with cognitive decline and psychiatric symptoms. Recent studies have detected genetic loci modifying the age at onset of motor symptoms in HD, but genetic factors influencing cognitive and psychiatric presentations are unknown. METHODS: We tested the hypothesis that psychiatric and cognitive symptoms in HD are influenced by the same common genetic variation as in the general population by 1) constructing polygenic risk scores from large genome-wide association studies of psychiatric and neurodegenerative disorders and of intelligence and 2) testing for correlation with the presence of psychiatric and cognitive symptoms in a large sample (n = 5160) of patients with HD. RESULTS: Polygenic risk score for major depression was associated specifically with increased risk of depression in HD, as was schizophrenia risk score with psychosis and irritability. Cognitive impairment and apathy were associated with reduced polygenic risk score for intelligence. CONCLUSIONS: Polygenic risk scores for psychiatric disorders, particularly depression and schizophrenia, are associated with increased risk of the corresponding psychiatric symptoms in HD, suggesting a common genetic liability. However, the genetic liability to cognitive impairment and apathy appears to be distinct from other psychiatric symptoms in HD. No associations were observed between HD symptoms and risk scores for other neurodegenerative disorders. These data provide a rationale for treatments effective in depression and schizophrenia to be used to treat depression and psychotic symptoms in HD.

A patient-derived cellular model for Huntington's disease reveals phenotypes at clinically relevant CAG lengths.

The huntingtin protein participates in several cellular processes that are disrupted when the polyglutamine tract is expanded beyond a threshold of 37 CAG DNA repeats in Huntington's disease (HD). Cellular biology approaches to understand these functional disruptions in HD have primarily focused on cell lines with synthetically long CAG length alleles that clinically represent outliers in this disease and a more severe form of HD that lacks age onset. Patient-derived fibroblasts are limited to a finite number of passages before succumbing to cellular senescence. We used human telomerase reverse transcriptase (hTERT) to immortalize fibroblasts taken from individuals of varying age, sex, disease onset, and CAG repeat length, which we have termed TruHD cells. TruHD cells display classic HD phenotypes of altered morphology, size and growth rate, increased sensitivity to oxidative stress, aberrant adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratios, and hypophosphorylated huntingtin protein. We additionally observed dysregulated reactive oxygen species (ROS)-dependent huntingtin localization to nuclear speckles in HD cells. We report the generation and characterization of a human, clinically relevant cellular model for investigating disease mechanisms in HD at the single-cell level, which, unlike transformed cell lines, maintains functions critical for huntingtin transcriptional regulation and genomic integrity.

Genetic Modification of Huntington Disease Acts Early in the Prediagnosis Phase.

Age at onset of Huntington disease, an inherited neurodegenerative disorder, is influenced by the size of the disease-causing CAG trinucleotide repeat expansion in HTT and by genetic modifier loci on chromosomes 8 and 15. Stratifying by modifier genotype, we have examined putamen volume, total motor score (TMS), and symbol digit modalities test (SDMT) scores, both at study entry and longitudinally, in normal controls and CAG-expansion carriers who were enrolled prior to the emergence of manifest HD in the PREDICT-HD study. The modifiers, which included onset-hastening and onset-delaying alleles on chromosome 15 and an onset-hastening allele on chromosome 8, revealed no major effect in controls but distinct patterns of modification in prediagnosis HD subjects. Putamen volume at study entry showed evidence of reciprocal modification by the chromosome 15 alleles, but the rate of loss of putamen volume was modified only by the deleterious chromosome 15 allele. By contrast, both alleles modified the rate of change of the SDMT score, but neither had an effect on the TMS. The influence of the chromosome 8 modifier was evident only in the rate of TMS increase. The data indicate that (1) modification of pathogenesis can occur early in the prediagnosis phase, (2) the modifier loci act in genetic interaction with the HD mutation rather than through independent additive effects, and (3) HD subclinical phenotypes are differentially influenced by each modifier, implying distinct effects in different cells or tissues. Together, these findings indicate the potential benefit of using genetic modifier strategies for dissecting the prediagnosis pathogenic process in HD.

HttQ111/+ Huntington's Disease Knock-in Mice Exhibit Brain Region-Specific Morphological Changes and Synaptic Dysfunction.

BACKGROUND: Successful disease-modifying therapy for Huntington's disease (HD) will require therapeutic intervention early in the pathogenic process. Achieving this goal requires identifying phenotypes that are proximal to the HTT CAG repeat expansion. OBJECTIVE: To use Htt CAG knock-in mice, precise genetic replicas of the HTT mutation in patients, as models to study proximal disease events. METHODS: Using cohorts of B6J.HttQ111/+ mice from 2 to 18 months of age, we analyzed pathological markers, including immunohistochemistry, brain regional volumes and cortical thickness, CAG instability, electron microscopy of striatal synapses, and acute slice electrophysiology to record glutamatergic transmission at striatal synapses. We also incorporated a diet perturbation paradigm for some of these analyses. RESULTS: B6J.HttQ111/+ mice did not exhibit significant neurodegeneration or gliosis but revealed decreased striatal DARPP-32 as well as subtle but regional-specific changes in brain volumes and cortical thickness that parallel those in HD patients. Ultrastructural analyses of the striatum showed reduced synapse density, increased postsynaptic density thickness and increased synaptic cleft width. Acute slice electrophysiology showed alterations in spontaneous AMPA receptor-mediated postsynaptic currents, evoked NMDA receptor-mediated excitatory postsynaptic currents, and elevated extrasynaptic NMDA currents. Diet influenced cortical thickness, but did not impact somatic CAG expansion, nor did it show any significant interaction with genotype on immunohistochemical, brain volume or cortical thickness measures. CONCLUSIONS: These data show that a single HttQ111 allele is sufficient to elicit brain region-specific morphological changes and early neuronal dysfunction, highlighting an insidious disease process already apparent in the first few months of life.

A modifier of Huntington's disease onset at the MLH1 locus.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by an expanded CAG repeat in HTT. Many clinical characteristics of HD such as age at motor onset are determined largely by the size of HTT CAG repeat. However, emerging evidence strongly supports a role for other genetic factors in modifying the disease pathogenesis driven by mutant huntingtin. A recent genome-wide association analysis to discover genetic modifiers of HD onset age provided initial evidence for modifier loci on chromosomes 8 and 15 and suggestive evidence for a locus on chromosome 3. Here, genotyping of candidate single nucleotide polymorphisms in a cohort of 3,314 additional HD subjects yields independent confirmation of the former two loci and moves the third to genome-wide significance at MLH1, a locus whose mouse orthologue modifies CAG length-dependent phenotypes in a Htt-knock-in mouse model of HD. Both quantitative and dichotomous association analyses implicate a functional variant on ∼32% of chromosomes with the beneficial modifier effect that delays HD motor onset by 0.7 years/allele. Genomic DNA capture and sequencing of a modifier haplotype localize the functional variation to a 78 kb region spanning the 3'end of MLH1 and the 5'end of the neighboring LRRFIP2, and marked by an isoleucine-valine missense variant in MLH1. Analysis of expression Quantitative Trait Loci (eQTLs) provides modest support for altered regulation of MLH1 and LRRFIP2, raising the possibility that the modifier affects regulation of both genes. Finally, polygenic modification score and heritability analyses suggest the existence of additional genetic modifiers, supporting expanded, comprehensive genetic analysis of larger HD datasets.

Haplotype-based stratification of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by expansion of a CAG trinucleotide repeat in HTT, resulting in an extended polyglutamine tract in huntingtin. We and others have previously determined that the HD-causing expansion occurs on multiple different haplotype backbones, reflecting more than one ancestral origin of the same type of mutation. In view of the therapeutic potential of mutant allele-specific gene silencing, we have compared and integrated two major systems of HTT haplotype definition, combining data from 74 sequence variants to identify the most frequent disease-associated and control chromosome backbones and revealing that there is potential for additional resolution of HD haplotypes. We have used the large collection of 4078 heterozygous HD subjects analyzed in our recent genome-wide association study of HD age at onset to estimate the frequency of these haplotypes in European subjects, finding that common genetic variation at HTT can distinguish the normal and CAG-expanded chromosomes for more than 95% of European HD individuals. As a resource for the HD research community, we have also determined the haplotypes present in a series of publicly available HD subject-derived fibroblasts, induced pluripotent cells, and embryonic stem cells in order to facilitate efforts to develop inclusive methods of allele-specific HTT silencing applicable to most HD patients. Our data providing genetic guidance for therapeutic gene-based targeting will significantly contribute to the developments of rational treatments and implementation of precision medicine in HD.

High resolution time-course mapping of early transcriptomic, molecular and cellular phenotypes in Huntington's disease CAG knock-in mice across multiple genetic backgrounds.

Huntington's disease is a dominantly inherited neurodegenerative disease caused by the expansion of a CAG repeat in the HTT gene. In addition to the length of the CAG expansion, factors such as genetic background have been shown to contribute to the age at onset of neurological symptoms. A central challenge in understanding the disease progression that leads from the HD mutation to massive cell death in the striatum is the ability to characterize the subtle and early functional consequences of the CAG expansion longitudinally. We used dense time course sampling between 4 and 20 postnatal weeks to characterize early transcriptomic, molecular and cellular phenotypes in the striatum of six distinct knock-in mouse models of the HD mutation. We studied the effects of the HttQ111 allele on the C57BL/6J, CD-1, FVB/NCr1, and 129S2/SvPasCrl genetic backgrounds, and of two additional alleles, HttQ92 and HttQ50, on the C57BL/6J background. We describe the emergence of a transcriptomic signature in HttQ111/+ mice involving hundreds of differentially expressed genes and changes in diverse molecular pathways. We also show that this time course spanned the onset of mutant huntingtin nuclear localization phenotypes and somatic CAG-length instability in the striatum. Genetic background strongly influenced the magnitude and age at onset of these effects. This work provides a foundation for understanding the earliest transcriptional and molecular changes contributing to HD pathogenesis.

Novel allele-specific quantification methods reveal no effects of adult onset CAG repeats on HTT mRNA and protein levels.

Huntington's disease (HD) reflects dominant consequences of a CAG repeat expansion mutation in HTT. Expanded CAG repeat size is the primary determinant of age at onset and age at death in HD. Although HD pathogenesis is driven by the expanded CAG repeat, whether the mutation influences the expression levels of mRNA and protein from the disease allele is not clear due to the lack of sensitive allele-specific quantification methods and the presence of confounding factors. To determine the impact of CAG expansion at the molecular level, we have developed novel allele-specific HTT mRNA and protein quantification methods based on principles of multiplex ligation-dependent probe amplification and targeted MS/MS parallel reaction monitoring, respectively. These assays, exhibiting high levels of specificity and sensitivity, were designed to distinguish allelic products based upon expressed polymorphic variants in HTT, including rs149 109 767. To control for other cis-haplotype variations, we applied allele-specific quantification assays to a panel of HD lymphoblastoid cell lines, each carrying the major European disease haplotype (i.e. hap.01) on the mutant chromosome. We found that steady state levels of HTT mRNA and protein were not associated with expanded CAG repeat length. Rather, the products of mutant and normal alleles, both mRNA and protein, were balanced, thereby arguing that a cis-regulatory effect of the expanded CAG repeat is not a critical component of the underlying mechanism of HD. These robust allele-specific assays could prove valuable for monitoring the impact of allele-specific gene silencing strategies currently being explored as therapeutic interventions in HD.

Genetic Contributors to Intergenerational CAG Repeat Instability in Huntington's Disease Knock-In Mice.

Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat in exon 1 of the HTT gene. Longer repeat sizes are associated with increased disease penetrance and earlier ages of onset. Intergenerationally unstable transmissions are common in HD families, partly underlying the genetic anticipation seen in this disorder. HD CAG knock-in mouse models also exhibit a propensity for intergenerational repeat size changes. In this work, we examine intergenerational instability of the CAG repeat in over 20,000 transmissions in the largest HD knock-in mouse model breeding datasets reported to date. We confirmed previous observations that parental sex drives the relative ratio of expansions and contractions. The large datasets further allowed us to distinguish effects of paternal CAG repeat length on the magnitude and frequency of expansions and contractions, as well as the identification of large repeat size jumps in the knock-in models. Distinct degrees of intergenerational instability were observed between knock-in mice of six background strains, indicating the occurrence of trans-acting genetic modifiers. We also found that lines harboring a neomycin resistance cassette upstream of Htt showed reduced expansion frequency, indicative of a contributing role for sequences in cis, with the expanded repeat as modifiers of intergenerational instability. These results provide a basis for further understanding of the mechanisms underlying intergenerational repeat instability.