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Polycystic kidney disease is an inherited degenerative disease in which the uriniferous tubules are replaced by expanding fluid-filled cysts that ultimately destroy organ function. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form, afflicting approximately 1 in 1,000 people. It primarily is caused by mutations in the transmembrane proteins polycystin-1 (Pkd1) and polycystin-2 (Pkd2). The most proximal effects of Pkd mutations leading to cyst formation are not known, but pro-proliferative signaling must be involved for the tubule epithelial cells to increase in number over time. The c-Jun N-terminal kinase (JNK) pathway promotes proliferation and is activated in acute and chronic kidney diseases. Using a mouse model of cystic kidney disease caused by Pkd2 loss, we observe JNK activation in cystic kidneys and observe increased nuclear phospho c-Jun in cystic epithelium. Genetic removal of Jnk1 and Jnk2 suppresses the nuclear accumulation of phospho c-Jun, reduces proliferation and reduces the severity of cystic disease. While Jnk1 and Jnk2 are thought to have largely overlapping functions, we find that Jnk1 loss is nearly as effective as the double loss of Jnk1 and Jnk2. Jnk pathway inhibitors are in development for neurodegeneration, cancer, and fibrotic diseases. Our work suggests that the JNK pathway should be explored as a therapeutic target for ADPKD., Author summary Autosomal dominant polycystic kidney disease is a leading cause of end stage renal disease requiring dialysis or kidney transplant. During disease development, the cells lining the kidney tubules proliferate. This proliferation transforms normally small diameter tubules into fluid-filled cysts that enlarge with time, eventually destroying all kidney function. Despite decades of research, polycystic kidney disease remains incurable. Furthermore, the precise signaling events involved in cyst initiation and growth remain unclear. The c-Jun N-terminal kinase (JNK), is a major pathway regulating cellular proliferation and differentiation but its importance to polycystic kidney disease was not known. We show that JNK activity is elevated in cystic kidneys and that reducing JNK activity decreases cyst growth pointing to JNK inhibition as a therapeutic strategy for treating polycystic kidney disease.

Disease-associated trinucleotide repeats form secondary DNA structures that interfere with replication and repair. Replication has been implicated as a mechanism that can cause repeat expansions and contractions. However, because structure-forming repeats are also replication barriers, it has been unclear whether the instability occurs due to slippage during normal replication progression through the repeat, slippage or misalignment at a replication stall caused by the repeat, or during subsequent replication of the repeat by a restarted fork that has altered properties. In this study, we have specifically addressed the fidelity of a restarted fork as it replicates through a CAG/CTG repeat tract and its effect on repeat instability. To do this, we used a well-characterized site-specific replication fork barrier (RFB) system in fission yeast that creates an inducible and highly efficient stall that is known to restart by recombination-dependent replication (RDR), in combination with long CAG repeat tracts inserted at various distances and orientations with respect to the RFB. We find that replication by the restarted fork exhibits low fidelity through repeat sequences placed 2–7 kb from the RFB, exhibiting elevated levels of Rad52- and Rad8ScRad5/HsHLTF-dependent instability. CAG expansions and contractions are not elevated to the same degree when the tract is just in front or behind the barrier, suggesting that the long-traveling Polδ-Polδ restarted fork, rather than fork reversal or initial D-loop synthesis through the repeat during stalling and restart, is the greatest source of repeat instability. The switch in replication direction that occurs due to replication from a converging fork while the stalled fork is held at the barrier is also a significant contributor to the repeat instability profile. Our results shed light on a long-standing question of how fork stalling and RDR contribute to expansions and contractions of structure-forming trinucleotide repeats, and reveal that tolerance to replication stress by fork restart comes at the cost of increased instability of repetitive sequences., Author summary Trinucleotide repeat expansions are the cause of several muscular- and neuro-degenerative diseases, and further expansions during intergenerational inheritance often leads to an earlier age-of-onset in the offspring. Some intergenerational expansions appear to occur during germ cell replication, but how those replication-associated expansions arise is incompletely understood. Because there are many replication barriers in genomes, including the structure-forming repeats themselves, we investigated whether a repeat placed after such a barrier had altered instability. We discovered that a restarted replication fork traversing a CAG repeat tract is highly error prone and repeat expansions and contractions are more prevalent in this case compared to normal replication. These results reveal a mechanism for replication-associated repeat instability relevant to disease-associated trinucleotide repeat expansions.

The clinical evaluation of a genetic syndrome relies upon recognition of a characteristic pattern of signs or symptoms to guide targeted genetic testing for confirmation of the diagnosis. However, individuals displaying a single phenotype of a complex syndrome may not meet criteria for clinical diagnosis or genetic testing. Here, we present a phenome-wide association study (PheWAS) approach to systematically explore the phenotypic expressivity of common and rare alleles in genes associated with four well-described syndromic diseases (Alagille (AS), Marfan (MS), DiGeorge (DS), and Noonan (NS) syndromes) in the general population. Using human phenotype ontology (HPO) terms, we systematically mapped 60 phenotypes related to AS, MS, DS and NS in 337,198 unrelated white British from the UK Biobank (UKBB) based on their hospital admission records, self-administrated questionnaires, and physiological measurements. We performed logistic regression adjusting for age, sex, and the first 5 genetic principal components, for each phenotype and each variant in the target genes (JAG1, NOTCH2 FBN1, PTPN1 and RAS-opathy genes, and genes in the 22q11.2 locus) and performed a gene burden test. Overall, we observed multiple phenotype-genotype correlations, such as the association between variation in JAG1, FBN1, PTPN11 and SOS2 with diastolic and systolic blood pressure; and pleiotropy among multiple variants in syndromic genes. For example, rs11066309 in PTPN11 was significantly associated with a lower body mass index, an increased risk of hypothyroidism and a smaller size for gestational age, all in concordance with NS-related phenotypes. Similarly, rs589668 in FBN1 was associated with an increase in body height and blood pressure, and a reduced body fat percentage as observed in Marfan syndrome. Our findings suggest that the spectrum of associations of common and rare variants in genes involved in syndromic diseases can be extended to individual phenotypes within the general population., Author summary Standard medical evaluation of genetic syndromes relies upon recognizing a characteristic pattern of signs or symptoms to guide targeted genetic testing for confirmation of the diagnosis. This may lead to missing diagnoses in patients with silent or a low expressed form of the syndrome. Here we take advantage of a rich electronic health record, various phenotypic measurements, and genetic information in 337,198 unrelated white British from the UKBB, to study the relation between single syndromic disease phenotypes and genes related to syndromic disease. We show multiple phenotype-genotype associations in concordance with phenotypes variations found in syndromic diseases. For example, we show that a commonly found variant in FBN1 was associated with high standing/sitting height ratio and reduced body fat percentage as observed in individuals with Marfan syndrome. Our findings suggest that common and rare alleles in syndromic disease genes are causative of individual component phenotypes present in a general population; further research is needed to characterize the pleiotropic effect of alleles in syndromic genes in persons without the syndromic disease.

Neurofibromatosis type 1 is a monogenetic disorder that predisposes individuals to tumor formation and cognitive and behavioral symptoms. The neuronal circuitry and developmental events underlying these neurological symptoms are unknown. To better understand how mutations of the underlying gene (NF1) drive behavioral alterations, we have examined grooming in the Drosophila neurofibromatosis 1 model. Mutations of the fly NF1 ortholog drive excessive grooming, and increased grooming was observed in adults when Nf1 was knocked down during development. Furthermore, intact Nf1 Ras GAP-related domain signaling was required to maintain normal grooming. The requirement for Nf1 was distributed across neuronal circuits, which were additive when targeted in parallel, rather than mapping to discrete microcircuits. Overall, these data suggest that broadly-distributed alterations in neuronal function during development, requiring intact Ras signaling, drive key Nf1-mediated behavioral alterations. Thus, global developmental alterations in brain circuits/systems function may contribute to behavioral phenotypes in neurofibromatosis type 1., Author summary Neurofibromatosis type 1 results in increased susceptibility to cognitive and behavioral symptoms, such as attention-deficit/hyperactivity disorder. This suggests that the underlying genetic mutations affect neuronal function. Where and when these effects occur in the brain is not understood. We approached these questions in the genetically-powerful fly model, examining how loss of the NF1 gene affects a complex motor behavior, grooming. Loss of NF1 during brain development caused excessive grooming in adult animals. This suggests that the gene plays a key role in brain development, which may be important for treatment of the disorder. Further, we found that a specific signaling pathway, Ras, is involved in regulating this grooming behavior. Finally, we mapped where in the brain the neurofibromin (Nf1) protein is required to maintain normal levels of grooming. The data suggested that it is required across many neurons, rather than in small subsets. When Nf1 was knocked down in smaller subsets, there was no effect, but if two subsets were added together, an effect was produced. This means that Nf1 regulates the function of many neurons and brain regions to influence complex behaviors.

Neurofibromatosis type-1 (NF1) patients suffer from cutaneous and subcutaneous neurofibromas (CNF) and large plexiform neurofibromas (PNF). Whole gene deletions of the NF1 gene can cause a more severe phenotype compared to smaller intragenic changes. Two distinct groups of NF1 whole gene deletions are type-1 deletions and atypical deletions. Our aim was to assess volumes and averaged annual growth-rates of CNF and PNF in patients with NF1 whole gene deletions and to compare these with NF1 patients without large deletions of the NF1 gene. We retrospectively evaluated 140 whole-body MR examinations of 38 patients with NF1 whole gene deletions (type-1 group: n = 27/atypical group n = 11) and an age- and sex matched collective of 38 NF1-patients. Age-dependent subgroups were created (0–18 vs >18 years). Sixty-four patients received follow-up MRI examinations (NF1whole gene deletion n = 32/control group n = 32). Whole-body tumor-volumes were semi-automatically assessed (MedX, V3.42). Tumor volumes and averaged annual growth-rates were compared. Median tumor-burden was significantly higher in the type-1 group (418ml; IQR 77 – 950ml, p = 0.012) but not in the atypical group (356ml;IQR 140–1190ml, p = 0.099) when compared to the controls (49ml; IQR 11–691ml). Averaged annual growth rates were significantly higher in both the type-1 group (14%/year; IQR 45–36%/year, p = 0.004) and atypical group (11%/year; IQR 5–23%/year, p = 0.014) compared to the controls (4%/year; IQR1–8%/year). Averaged annual growth rates were significantly higher in pediatric patients with type-1 deletions (21%/year) compared with adult patients (8%/year, p = 0.014) and also compared with pediatric patients without large deletions of the NF1 gene (3.3%/year, p = 0.0015). NF1 whole gene deletions cause a more severe phenotype of NF1 with higher tumor burden and higher growth-rates compared to NF1 patients without large deletions of the NF1 gene. In particular, pediatric patients with type-1 deletions display a pronounced tumor growth., Author summary Neurofibromatosis type-1 (NF1) is an autosomal dominantly inherited tumor predisposition syndrome and is caused by a variety of mutations of the NF1 gene. Large deletions encompassing the entire NF1 gene are known to be associated with a more severe clinical phenotype. A hallmark feature of NF1 is the development of different benign tumors: cutaneous or subcutaneous neurofibromas (CNF) and the potentially large plexiform neurofibromas (PNF). Importantly, PNF can undergo transformation into malignant peripheral nerve sheath tumors, which contribute to the increased mortality of NF1 patients. Identified risk factors for malignant transformation are NF1 whole gene deletions, large whole-body tumor burden and a high number of subcutaneous neurofibromas. We performed semi-automated volumetry of these tumors to determine volumes and growth rates of CNF and PNF in patients with whole gene deletions and a control group of NF1 patients without large deletions of the NF1 gene. Our results indicate that NF1 whole gene deletions lead to higher tumor burden and higher growth rates. In particular, pediatric NF1 patients with a specific type of whole gene deletion (type-1 deletion) exhibit rapid growth of PNF. We therefore suggest that these patients should be monitored in close intervals to evaluate tumor growth and risk of malignant transformation.

Loss of the XPF-ERCC1 endonuclease causes a dramatic phenotype that results in progeroid features associated with liver, kidney and bone marrow dysfunction. As this nuclease is involved in multiple DNA repair transactions, it is plausible that this severe phenotype results from the simultaneous inactivation of both branches of nucleotide excision repair (GG- and TC-NER) and Fanconi anaemia (FA) inter-strand crosslink (ICL) repair. Here we use genetics in human cells and mice to investigate the interaction between the canonical NER and ICL repair pathways and, subsequently, how their joint inactivation phenotypically overlaps with XPF-ERCC1 deficiency. We find that cells lacking TC-NER are sensitive to crosslinking agents and that there is a genetic interaction between NER and FA in the repair of certain endogenous crosslinking agents. However, joint inactivation of GG-NER, TC-NER and FA crosslink repair cannot account for the hypersensitivity of XPF-deficient cells to classical crosslinking agents nor is it sufficient to explain the extreme phenotype of Ercc1-/- mice. These analyses indicate that XPF-ERCC1 has important functions outside of its central role in NER and FA crosslink repair which are required to prevent endogenous DNA damage. Failure to resolve such damage leads to loss of tissue homeostasis in mice and humans., Author summary The integrity of DNA is essential for life so even the most primitive life forms have evolved a DNA repair ‘toolkit’ to detect and fix different types of DNA damage. XPF-ERCC1 is an enzyme that can cut DNA and a key player in many of these DNA repair transactions. Consistent with this, inactivating mutations of XPF-ERCC1 in humans and mice lead to a dramatic premature ageing phenotype associated with liver, kidney and bone marrow dysfunction. Here, we ask which of the many functions of XPF-ERCC1 are required to protect tissues from endogenous DNA damage. To do this, we generated cells and mice lacking two of the best characterised functions of XPF-ERCC1: nucleotide excision repair and inter-strand crosslink repair. Surprisingly, neither the cells nor mice lacking these two repair pathways behave like the XPF-ERCC1 mutants, in fact the mice are remarkable in their lack of phenotype. Our work suggests that there are functions of XPF-ERCC1 outside of the canonical repair pathways which are important for DNA repair and the homeostasis of multiple organs.

Disrupted circadian rhythms is a prominent and early feature of neurodegenerative diseases including Huntington’s disease (HD). In HD patients and animal models, striatal and hypothalamic neurons expressing molecular circadian clocks are targets of mutant Huntingtin (mHtt) pathogenicity. Yet how mHtt disrupts circadian rhythms remains unclear. In a genetic screen for modifiers of mHtt effects on circadian behavior in Drosophila, we discovered a role for the neurodegenerative disease gene Ataxin2 (Atx2). Genetic manipulations of Atx2 modify the impact of mHtt on circadian behavior as well as mHtt aggregation and demonstrate a role for Atx2 in promoting mHtt aggregation as well as mHtt-mediated neuronal dysfunction. RNAi knockdown of the Fragile X mental retardation gene, dfmr1, an Atx2 partner, also partially suppresses mHtt effects and Atx2 effects depend on dfmr1. Atx2 knockdown reduces the cAMP response binding protein A (CrebA) transcript at dawn. CrebA transcript level shows a prominent diurnal regulation in clock neurons. Loss of CrebA also partially suppresses mHtt effects on behavior and cell loss and restoration of CrebA can suppress Atx2 effects. Our results indicate a prominent role of Atx2 in mediating mHtt pathology, specifically via its regulation of CrebA, defining a novel molecular pathway in HD pathogenesis., Author summary Circadian clocks evolved to anticipate 24 h environmental rhythms driven by the earth’s daily rotation and regulate nearly all aspects of behavior, physiology and the genome. Disruptions of the circadian clock have been associated with a wide range of human diseases, including neurodegenerative diseases such as Huntington’s disease (HD). Using an HD animal model in which a mutant Huntingtin (mHtt) protein is expressed, we identify a role for the RNA binding protein and neurodegenerative disease gene Ataxin-2 (Atx2) in mediating mHtt effects on circadian behavioral rhythms. Using transcriptomics, we identify the transcription factor CrebA as a potential target of both Atx2 and the circadian clock. Finally, we demonstrate a role for CrebA in mediating mHtt effects on circadian behavior, defining a novel Atx2-CrebA pathway in a neurodegenerative disease model. These studies define the molecular mechanisms by which mHtt can disrupt circadian rhythms identifying potential novel therapeutic targets for this uniformly fatal disease.

CFTR modulators have revolutionized the treatment of individuals with cystic fibrosis (CF) by improving the function of existing protein. Unfortunately, almost half of the disease-causing variants in CFTR are predicted to introduce premature termination codons (PTC) thereby causing absence of full-length CFTR protein. We hypothesized that a subset of nonsense and frameshift variants in CFTR allow expression of truncated protein that might respond to FDA-approved CFTR modulators. To address this concept, we selected 26 PTC-generating variants from four regions of CFTR and determined their consequences on CFTR mRNA, protein and function using intron-containing minigenes expressed in 3 cell lines (HEK293, MDCK and CFBE41o-) and patient-derived conditionally reprogrammed primary nasal epithelial cells. The PTC-generating variants fell into five groups based on RNA and protein effects. Group A (reduced mRNA, immature (core glycosylated) protein, function 1% (n = 5)), Group D (reduced mRNA, mature protein, function >1% (n = 5)) and Group E (aberrant RNA splicing, mature protein, function > 1% (n = 1)) variants responded to modulators. Increasing mRNA level by inhibition of NMD led to a significant amplification of modulator effect upon a Group D variant while response of a Group A variant was unaltered. Our work shows that PTC-generating variants should not be generalized as genetic ‘nulls’ as some may allow generation of protein that can be targeted to achieve clinical benefit., Author summary The development of variant specific modulators that correct dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) protein is an excellent example of precision medicine. Currently there is no molecular treatment available for individuals with cystic fibrosis (CF) carrying nonsense or frameshift variants because such variants introduce a premature termination codon (PTC), and are not expected to produce CFTR protein. We have performed a systematic study of nonsense and frameshift variants located in four regions of CFTR that we postulated should have varying effects on mRNA stability, protein production, and/or function. Using primary nasal cells and three different cell line models stably expressing CFTR expression mini-genes (EMGs), we report molecular consequences of 26 PTC-generating variants in CFTR, and identify which variants allow generation of CFTR protein that is responsive to currently available modulator therapies and which require alternative therapeutic approaches.

Pseudomonas aeruginosa colonizes the airways of cystic fibrosis (CF) patients, causing infections that can last for decades. During the course of these infections, P. aeruginosa undergoes a number of genetic adaptations. One such adaptation is the loss of swimming motility functions. Another involves the formation of the rugose small colony variant (RSCV) phenotype, which is characterized by overproduction of the exopolysaccharides Pel and Psl. Here, we provide evidence that the two adaptations are linked. Using random transposon mutagenesis, we discovered that flagellar mutations are linked to the RSCV phenotype. We found that flagellar mutants overexpressed Pel and Psl in a surface-contact dependent manner. Genetic analyses revealed that flagellar mutants were selected for at high frequencies in biofilms, and that Pel and Psl expression provided the primary fitness benefit in this environment. Suppressor mutagenesis of flagellar RSCVs indicated that Psl overexpression required the mot genes, suggesting that the flagellum stator proteins function in a surface-dependent regulatory pathway for exopolysaccharide biosynthesis. Finally, we identified flagellar mutant RSCVs among CF isolates. The CF environment has long been known to select for flagellar mutants, with the classic interpretation being that the fitness benefit gained relates to an impairment of the host immune system to target a bacterium lacking a flagellum. Our new findings lead us to propose that exopolysaccharide production is a key gain-of-function phenotype that offers a new way to interpret the fitness benefits of these mutations., Author summary Microbiologists have known for decades that Pseudomonas aeruginosa mutates during chronic respiratory infection of cystic fibrosis (CF) patients. One of the most reported functions lost during these infections is flagellar motility. A long-standing interpretation of this observation is that the flagellum is disadvantageous for the bacterium in the CF environment. We report the surprising finding that mutation of a wide range of flagellar genes results in the overproduction of the biofilm matrix polysaccharides Psl and Pel. We propose, therefore, that flagellar mutations represent a gain-of-function that would help the bacterium to form biofilms and persist in the CF airways.

FSHD is characterized by the misexpression of DUX4 in skeletal muscle. Although DUX4 upregulation is thought to be the pathogenic cause of FSHD, DUX4 is lowly expressed in patient samples, and analysis of the consequences of DUX4 expression has largely relied on artificial overexpression. To better understand the native expression profile of DUX4 and its targets, we performed bulk RNA-seq on a 6-day differentiation time-course in primary FSHD2 patient myoblasts. We identify a set of 54 genes upregulated in FSHD2 cells, termed FSHD-induced genes. Using single-cell and single-nucleus RNA-seq on myoblasts and differentiated myotubes, respectively, we captured, for the first time, DUX4 expressed at the single-nucleus level in a native state. We identified two populations of FSHD myotube nuclei based on low or high enrichment of DUX4 and FSHD-induced genes (“FSHD-Lo” and “FSHD Hi”, respectively). FSHD-Hi myotube nuclei coexpress multiple DUX4 target genes including DUXA, LEUTX and ZSCAN4, and also upregulate cell cycle-related genes with significant enrichment of E2F target genes and p53 signaling activation. We found more FSHD-Hi nuclei than DUX4-positive nuclei, and confirmed with in situ RNA/protein detection that DUX4 transcribed in only one or two nuclei is sufficient for DUX4 protein to activate target genes across multiple nuclei within the same myotube. DUXA (the DUX4 paralog) is more widely expressed than DUX4, and depletion of DUXA suppressed the expression of LEUTX and ZSCAN4 in late, but not early, differentiation. The results suggest that the DUXA can take over the role of DUX4 to maintain target gene expression. These results provide a possible explanation as to why it is easier to detect DUX4 target genes than DUX4 itself in patient cells and raise the possibility of a self-sustaining network of gene dysregulation triggered by the limited DUX4 expression., Author summary Although misexpression of DUX4 has been known as the major cause in FSHD, it is lowly expressed in patient samples and analysis of the consequences of DUX4 expression has largely relied on artificial overexpression. Here, we took advantage of recent methodological advances to observe native DUX4 expression at the single-nucleus level in FSHD2 patient-derived myotubes. Using single-nucleus RNA-seq (snRNA-seq), we were able to detect endogenous DUX4-expressing nuclei and the extent of spreading of DUX4-target gene expression across many nuclei. Our highly sensitive snRNA-seq method further allowed us to identify two populations of FSHD myotube nuclei with distinct transcriptional profiles. One is highly enriched with DUX4 and target genes (FSHD-Hi) while the other has sparser DUX4 and FSHD-induced genes expressed (FSHD-Lo), reflecting two potentially different pathological states of patient myotubes. We observed a set of transcription factors specifically upregulated in FSHD-Hi myotube nuclei associated with the cell cycle, and significant upregulation of DUX4 paralog DUXA that contributes to further upregulation of DUX4 target genes. We propose that transcription factors downstream of DUX4 may amplify DUX4 signal and thus act to perpetuate FSHD.

Modifiers of Mendelian disorders can provide insights into disease mechanisms and guide therapeutic strategies. A recent genome-wide association (GWA) study discovered genetic modifiers of Huntington's disease (HD) onset in Europeans. Here, we performed whole genome sequencing and GWA analysis of a Venezuelan HD cluster whose families were crucial for the original mapping of the HD gene defect. The Venezuelan HD subjects develop motor symptoms earlier than their European counterparts, implying the potential for population-specific modifiers. The main Venezuelan HD family inherits HTT haplotype hap.03, which differs subtly at the sequence level from European HD hap.03, suggesting a different ancestral origin but not explaining the earlier age at onset in these Venezuelans. GWA analysis of the Venezuelan HD cluster suggests both population-specific and population-shared genetic modifiers. Genome-wide significant signals at 7p21.2–21.1 and suggestive association signals at 4p14 and 17q21.2 are evident only in Venezuelan HD, but genome-wide significant association signals at the established European chromosome 15 modifier locus are improved when Venezuelan HD data are included in the meta-analysis. Venezuelan-specific association signals on chromosome 7 center on SOSTDC1, which encodes a bone morphogenetic protein antagonist. The corresponding SNPs are associated with reduced expression of SOSTDC1 in non-Venezuelan tissue samples, suggesting that interaction of reduced SOSTDC1 expression with a population-specific genetic or environmental factor may be responsible for modification of HD onset in Venezuela. Detection of population-specific modification in Venezuelan HD supports the value of distinct disease populations in revealing novel aspects of a disease and population-relevant therapeutic strategies., Author summary Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in HTT, which encodes huntingtin. HD involves various neurological manifestations such as involuntary movements, cognitive decline, and personality change; age at onset of diagnostic signs is partly determined by the length of the CAG repeat. However, genome-wide analysis of European HD subjects revealed that age at onset corrected for individual CAG repeat size is modified by other genetic factors, revealing potential disease-delaying pathways that form targets for therapeutic strategies. In this study, we set out to identify genetic modifiers in a Venezuelan HD cluster whose families were crucial for discovering the cause of HD. Through genome-wide association analysis using age at onset corrected for CAG repeat size as the phenotype, we found genome-wide significant (chromosome 7) and suggestive significant association signals (chromosomes 4 and 17). Significant modification signals in Venezuelan HD appear to be population-specific because genetic analysis of European HD subjects did not reveal modification signals at those locations. Nevertheless, established modification signals in European HD at a chromosome 15 locus were also augmented by Venezuelan HD data, supporting the existence of both shared and population-specific genetic modifiers in HD. Together with the full sequence of HTT shared by the majority of these Venezuelan HD subjects, our genetic analysis provides insights into the geographical origin of the founder mutation, meaningful allele-specific sites for gene targeting approaches, and potential targets for disease-modifying intervention in this population.

Considering that mutations in known prostate cancer (PrCa) predisposition genes, including those responsible for hereditary breast/ovarian cancer and Lynch syndromes, explain less than 5% of early-onset/familial PrCa, we have sequenced 94 genes associated with cancer predisposition using next generation sequencing (NGS) in a series of 121 PrCa patients. We found monoallelic truncating/functionally deleterious mutations in seven genes, including ATM and CHEK2, which have previously been associated with PrCa predisposition, and five new candidate PrCa associated genes involved in cancer predisposing recessive disorders, namely RAD51C, FANCD2, FANCI, CEP57 and RECQL4. Furthermore, using in silico pathogenicity prediction of missense variants among 18 genes associated with breast/ovarian cancer and/or Lynch syndrome, followed by KASP genotyping in 710 healthy controls, we identified “likely pathogenic” missense variants in ATM, BRIP1, CHEK2 and TP53. In conclusion, this study has identified putative PrCa predisposing germline mutations in 14.9% of early-onset/familial PrCa patients. Further data will be necessary to confirm the genetic heterogeneity of inherited PrCa predisposition hinted in this study., Author summary Prostate cancer (PrCa) is the most frequent cancer diagnosed in men worldwide, estimated to be responsible for the death of 27,540 men in the United States in 2015. Contrarily to other cancer types, the genetic contribution to the 10–20% of PrCa cases occurring in families with aggregation of the disease is largely unknown. Germline mutations in the BRCA2 and the MSH2 breast and colon cancer predisposing genes, respectively, explain only about 1.5% of our early-onset/familial PrCa cases. Taking advantage of recent deep sequencing technologies and an analysis pipeline established in our group, we have screened 121 PrCa patients with strong evidence of an hereditary component for mutations in 94 genes involved in several cancer predisposing syndromes. We found truncating/functionally deleterious mutations in seven genes and “likely pathogenic” missense variants in four genes, of which five and one, respectively, have not been previously associated with PrCa predisposition. We believe this study significantly contributes to the understanding of the genetic heterogeneity behind early-onset/familial PrCa.

Substitutions that disrupt pre-mRNA splicing are a common cause of genetic disease. On average, 13.4% of all hereditary disease alleles are classified as splicing mutations mapping to the canonical 5′ and 3′ splice sites. However, splicing mutations present in exons and deeper intronic positions are vastly underreported. A recent re-analysis of coding mutations in exon 10 of the Lynch Syndrome gene, MLH1, revealed an extremely high rate (77%) of mutations that lead to defective splicing. This finding is confirmed by extending the sampling to five other exons in the MLH1 gene. Further analysis suggests a more general phenomenon of defective splicing driving Lynch Syndrome. Of the 36 mutations tested, 11 disrupted splicing. Furthermore, analyzing past reports suggest that MLH1 mutations in canonical splice sites also occupy a much higher fraction (36%) of total mutations than expected. When performing a comprehensive analysis of splicing mutations in human disease genes, we found that three main causal genes of Lynch Syndrome, MLH1, MSH2, and PMS2, belonged to a class of 86 disease genes which are enriched for splicing mutations. Other cancer genes were also enriched in the 86 susceptible genes. The enrichment of splicing mutations in hereditary cancers strongly argues for additional priority in interpreting clinical sequencing data in relation to cancer and splicing., Author summary To understand the extent to which disrupted pre-mRNA splicing causes human disease, we re-analyzed coding mutations in MLH1, one of the causal genes of Lynch Syndrome. We found that a high fraction of the MLH1 coding mutations resulted in disrupted splicing. To further investigate a more general role of defective splicing across human disease genes, simulation strategies were used to identify 86 disease genes prone to splice site mutations. In these 86 genes, there was an enrichment of cancer genes including the three main casual genes of Lynch Syndrome (MLH1, MSH2, and PMS2). Thus, it appears defective splicing may be the main driver of Lynch Syndrome and other cancers. Genes prone to splicing mutations have certain features that allow for the comprehensive prediction of splicing-prone diseases genes in the human genome. Our findings strongly argue for additional clinical sequencing prioritization in both cancer genes and genes prone to splice site mutations.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF) and are associated with congenital bilateral absence of the vas deferens (CBAVD), which is the major cause of infertility in male patients with CF. However, most Taiwanese patients with CBAVD do not carry major CFTR mutations. Some patients have a single copy deletion of the solute carrier family 9 isoform 3 (SLC9A3) gene. SLC9A3 is a Na+/H+ exchanger, and depleted Slc9a3 in male mice causes infertility due to the abnormal dilated lumen of the rete testis and efferent ductules. Furthermore, SLC9A3 interacts with CFTR in the pancreatic duct and functions as a genetic modifier of CF. However, SLC9A3 function and its relation to CFTR expression in the male reproductive tract in vivo remain elusive. In the present study, we found that CFTR expression was dramatically decreased in the epididymis and vas deferens of Slc9a3 knockout mice. Adult Slc9a3-/- mice showed not only significantly decreased epididymis and vas deferens weight but also increased testis weight. Furthermore, Slc9a3-/- mice developed obstructive azoospermia because of abnormal abundant secretions and calcification in the lumen of the reproductive tract. Ultrastructural analysis of the epithelium in Slc9a3–/–epididymis and vas deferens displayed disorganized and reduced number of stereocilia and numerous secretory apparatuses. Our data revealed that interdependence between SLC9A3 and CFTR is critical for maintaining a precise microenvironment in the epithelial cytoarchitecture of the male reproductive tract. The Slc9a3-deficient mice with impaired male excurrent ducts in this study provide proof for our clinical findings that some Taiwanese of CBAVD carry SLC9A3 deletion but without major CFTR mutations., Author summary Cystic fibrosis (CF) is the most common inherited life-threatening disease in Caucasians. The most well-known cause of CF is a genetic defect in CFTR, an apical membrane chloride and bicarbonate channel. The symptoms of CF include defects in the respiratory, digestive, and male reproductive systems. Most male patients with CF are infertile due to congenital bilateral absence of the vas deferens (CBAVD), which leads to obstructive azoospermia. Nevertheless, Taiwanese patients with CBAVD do not carry the common mutations of CFTR found in Caucasians. We have identified a potential candidate, SLC9A3, of which a single copy is lost in Taiwanese patients with CBAVD. In addition to the previously reported role of SLC9A3 in the digestive system and efferent ductules, we now report that the SLC9A3 deficiency causes obstructive azoospermia and impairs the epithelial structure of the reproductive tract. Loss of SLC9A3 also leads to dramatic reduced expression of CFTR in the reproductive tract. We suggest that the interplay between SLC9A3 and CFTR is responsible for CF-related infertility. Thus, we have characterized a potential critical player in the pathogenesis of CBAVD and provide a new diagnostic candidate for Asian patients with CBAVD.

Huntington’s Disease (HD) is an autosomal dominant progressive neurodegenerative disorder characterized by cognitive, behavioral and motor dysfunctions. HD is caused by a CAG repeat expansion in exon 1 of the HD gene that is translated into an expanded polyglutamine tract in the encoded protein, huntingtin (HTT). While the most significant neuropathology of HD occurs in the striatum, other brain regions are also affected and play an important role in HD pathology. To date there is no cure for HD, and recently strategies aiming at silencing HTT expression have been initiated as possible therapeutics for HD. However, the essential functions of HTT in the adult brain are currently unknown and hence the consequence of sustained suppression of HTT expression is unpredictable and can potentially be deleterious. Using the Cre-loxP system of recombination, we conditionally inactivated the mouse HD gene homologue at 3, 6 and 9 months of age. Here we show that elimination of Htt expression in the adult mouse results in behavioral deficits, progressive neuropathological changes including bilateral thalamic calcification, and altered brain iron homeostasis., Author summary Huntington’s Disease is a genetic disorder characterized by progressive cognitive, behavioral and motor dysfunctions. Usually the first symptoms appear around 40 years of age, and lead to death within 15–20 years after the onset of symptoms. To date there is no cure for Huntington’s Disease, and current therapeutic strategies are only palliative, and far from optimal. Gene silencing currently appears as the most attractive approach for the treatment of Huntington’s Disease. However, since normal and mutant huntingtin (the protein product of the Huntington’s disease gene) differ only on the polyglutamine length, unless allele-specific silencing is planned, normal huntingtin (that is neuroprotective) will also be inactivated with unknown implications. To address these questions, we investigated the consequences of elimination of normal huntingtin function in adulthood. In summary, our studies show that huntingtin plays a role in brain iron homeostasis, and that elimination of huntingtin in the adult mouse results in behavioral deficits and progressive neuropathological changes including bilateral thalamic calcification.

The Huntington’s disease (HD) protein, huntingtin (HTT), is a large protein consisting of 3144 amino acids and has conserved N-terminal sequences that are followed by a polyglutamine (polyQ) repeat. Loss of Htt is known to cause embryonic lethality in mice, whereas polyQ expansion leads to adult neuronal degeneration. Whether N-terminal HTT is essential for neuronal development or contributes only to late-onset neurodegeneration remains unknown. We established HTT knock-in mice (N160Q-KI) expressing the first 208 amino acids of HTT with 160Q, and they show age-dependent HTT aggregates in the brain and neurological phenotypes. Importantly, the N-terminal mutant HTT also preferentially accumulates in the striatum, the brain region most affected in HD, indicating the importance of N-terminal HTT in selective neuropathology. That said, homozygous N160Q-KI mice are also embryonic lethal, suggesting that N-terminal HTT alone is unable to support embryonic development. Using Htt knockout neurons, we found that loss of Htt selectively affects the survival of developing neuronal cells, but not astrocytes, in culture. This neuronal degeneration could be rescued by a truncated HTT lacking the first 237 amino acids, but not by N-terminal HTT (1–208 amino acids). Also, the rescue effect depends on the region in HTT known to be involved in intracellular trafficking. Thus, the N-terminal HTT region may not be essential for the survival of developing neurons, but when carrying a large polyQ repeat, can cause selective neuropathology. These findings imply a possible therapeutic benefit of removing the N-terminal region of HTT containing the polyQ repeat to treat the neurodegeneration in HD., Author Summary The 17 amino acids in the N-terminal region of huntingtin (HTT) are conserved in a wide range of species and are followed by a polyglutamine repeat whose expansion causes selective neurodegeneration in Huntington’s disease (HD). Loss of Htt can affect developing neurons and early embryonic development in mice. Whether N-terminal HTT is important for the survival of developing neurons or contributes mainly to a gain of toxic function in HD remains unknown. In the current study, we generated N-terminal mutant HTT knock-in mice and found that N-terminal HTT with an expanded polyQ repeat is unable to support the early development of mice, but can cause age-dependent neurological phenotypes. Further, we show that a truncated HTT without the N-terminal region can rescue the Htt loss-mediated degeneration of developing neurons. Our studies suggest that removal of the N-terminal region of mutant HTT could be a strategy to abolish the neuronal toxicity of mutant HTT.

Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of peripheral neuropathies with diverse genetic causes. In this study, we identified p.I43N mutation in PMP2 from a family exhibiting autosomal dominant demyelinating CMT neuropathy by whole exome sequencing and characterized the clinical features. The age at onset was the first to second decades and muscle atrophy started in the distal portion of the leg. Predominant fatty replacement in the anterior and lateral compartment was similar to that in CMT1A caused by PMP22 duplication. Sural nerve biopsy showed onion bulbs and degenerating fibers with various myelin abnormalities. The relevance of PMP2 mutation as a genetic cause of dominant CMT1 was assessed using transgenic mouse models. Transgenic mice expressing wild type or mutant (p.I43N) PMP2 exhibited abnormal motor function. Electrophysiological data revealed that both mice had reduced motor nerve conduction velocities (MNCV). Electron microscopy revealed that demyelinating fibers and internodal lengths were shortened in both transgenic mice. These data imply that overexpression of wild type as well as mutant PMP2 also causes the CMT1 phenotype, which has been documented in the PMP22. This report might expand the genetic and clinical features of CMT and a further mechanism study will enhance our understanding of PMP2-associated peripheral neuropathy., Author Summary Isolation of causative mutation is still challenging in genetic diseases with a variety of genetic causes. We discovered a mutation in a novel gene from a family exhibiting a peripheral neuropathy by virtue of next-generation sequencing. Although the family shows characteristic clinical features of hereditary motor and sensory neuropathy, we could not find a mutation from well-known genes. To demonstrate the clinical relevance of the novel gene, we generated transgenic mice, which carry the patients’ mutation within their chromosome. The transgenic mice exhibited the same phenotype as the patients including peripheral neuropathic symptoms and reduced locomotor function. We also observed the affected peripheral nervous system through electron microscopy. It seems that the expression of the mutant protein impairs the myelin of peripheral nervous system. These data might expand the genetic, clinical, and pathophysiological features of the peripheral neuropathy and a further investigation will enhance our understanding of disease in the peripheral nervous system.

Among hereditary colorectal cancer predisposing syndromes, Lynch syndrome (LS) caused by mutations in DNA mismatch repair genes MLH1, MSH2, MSH6 or PMS2 is the most common. Patients with LS have an increased risk of early onset colon and endometrial cancer, but also other tumors that generally have an earlier onset compared to the general population. However, age at first primary cancer varies within families and genetic anticipation, i.e. decreasing age at onset in successive generations, has been suggested in LS. Anticipation is a well-known phenomenon in e.g neurodegenerative diseases and several reports have studied anticipation in heritable cancer. The purpose of this study is to determine whether anticipation can be shown in a nationwide cohort of Swedish LS families referred to the regional departments of clinical genetics in Lund, Stockholm, Linköping, Uppsala and Umeå between the years 1990–2013. We analyzed a homogenous group of mutation carriers, utilizing information from both affected and non-affected family members. In total, 239 families with a mismatch repair gene mutation (96 MLH1 families, 90 MSH2 families including one family with an EPCAM–MSH2 deletion, 39 MSH6 families, 12 PMS2 families, and 2 MLH1+PMS2 families) comprising 1028 at-risk carriers were identified among the Swedish LS families, of which 1003 mutation carriers had available follow-up information and could be included in the study. Using a normal random effects model (NREM) we estimate a 2.1 year decrease in age of diagnosis per generation. An alternative analysis using a mixed-effects Cox proportional hazards model (COX-R) estimates a hazard ratio of exp(0.171), or about 1.19, for age of diagnosis between consecutive generations. LS-associated gene-specific anticipation effects are evident for MSH2 (2.6 years/generation for NREM and hazard ratio of 1.33 for COX-R) and PMS2 (7.3 years/generation and hazard ratio of 1.86). The estimated anticipation effects for MLH1 and MSH6 are smaller., Author summary Genetic anticipation is a phenomenon where symptoms of a hereditary disease appear at an earlier age and/or are more severe in successive generations. In genetic disorders such as Fragile X syndrome, Myotonic dystrophy type 1 and Huntington disease, anticipation is caused by the expansion of unstable trinucleotide repeats during meiosis. Anticipation is also reported to occur in some hereditary cancers though the underlying mechanism behind this observation is unknown. Several studies have investigated anticipation in Lynch syndrome, the most common hereditary colorectal cancer syndrome, yet there is a debate concerning whether anticipation occurs and what underlying mechanism there is. The objective of this project is to study if anticipation is part of the clinical picture in Swedish families with LS, with the long term goal to enable better prediction of age at onset in family members. Our results suggest that anticipation occurs in families with mutation in MSH2 and PMS2, while the evidence is equivocal for MLH1 and MSH6.

Inherited hypertrichoses are rare syndromes characterized by excessive hair growth that does not result from androgen stimulation, and are often associated with additional congenital abnormalities. In this study, we investigated the genetic defect in a case of autosomal recessive congenital generalized hypertrichosis terminalis (CGHT) (OMIM135400) using whole-exome sequencing. We identified a single base pair substitution in the 5′ donor splice site of intron 32 in the ABC lipid transporter gene ABCA5 that leads to aberrant splicing of the transcript and a decrease in protein levels throughout patient hair follicles. The homozygous recessive disruption of ABCA5 leads to reduced lysosome function, which results in an accumulation of autophagosomes, autophagosomal cargos as well as increased endolysosomal cholesterol in CGHT keratinocytes. In an unrelated sporadic case of CGHT, we identified a 1.3 Mb cryptic deletion of chr17q24.2-q24.3 encompassing ABCA5 and found that ABCA5 levels are dramatically reduced throughout patient hair follicles. Collectively, our findings support ABCA5 as a gene underlying the CGHT phenotype and suggest a novel, previously unrecognized role for this gene in regulating hair growth., Author Summary Inherited hypertrichoses represent a group of hair overgrowth syndromes that are extremely rare in humans and have remained an area of great interest to evolutionary geneticists since they are considered to be recurrences of an ancestral phenotype. These syndromes often present with additional congenital abnormalities including bone, heart and dental defects; thus, it is crucial to identify the mechanisms and genes underlying the pathology. Copy number variants (CNVs) have previously been reported in several cases of congenital generalized hypertrichosis terminalis (CGHT) with a minimal overlapping region of 555 kb encompassing four genes. However, no point mutations in these or any other single genes have been described to underlie the CGHT phenotype. In this study, we report the first loss-of-function mutation in an ABC transporter, ABCA5 and identified an additional copy number variant in a separate case that lies within the minimal common region. We found high levels of ABCA5 expression in both epithelial and mesenchymal compartments of human and mouse hair follicles, and in CGHT patients, this expression is significantly reduced or completely lost. ABCA5 is a lysosomal protein, and its loss-of-function compromises the integrity of lysosomes and leads to an intra-endolysosomal accumulation of cholesterol. Importantly, our findings support a novel role for ABCA5 in regulating hair growth.

It is common practice in genome-wide association studies (GWAS) to focus on the relationship between disease risk and genetic variants one marker at a time. When relevant genes are identified it is often possible to implicate biological intermediates and pathways likely to be involved in disease aetiology. However, single genetic variants typically explain small amounts of disease risk. Our idea is to construct allelic scores that explain greater proportions of the variance in biological intermediates, and subsequently use these scores to data mine GWAS. To investigate the approach's properties, we indexed three biological intermediates where the results of large GWAS meta-analyses were available: body mass index, C-reactive protein and low density lipoprotein levels. We generated allelic scores in the Avon Longitudinal Study of Parents and Children, and in publicly available data from the first Wellcome Trust Case Control Consortium. We compared the explanatory ability of allelic scores in terms of their capacity to proxy for the intermediate of interest, and the extent to which they associated with disease. We found that allelic scores derived from known variants and allelic scores derived from hundreds of thousands of genetic markers explained significant portions of the variance in biological intermediates of interest, and many of these scores showed expected correlations with disease. Genome-wide allelic scores however tended to lack specificity suggesting that they should be used with caution and perhaps only to proxy biological intermediates for which there are no known individual variants. Power calculations confirm the feasibility of extending our strategy to the analysis of tens of thousands of molecular phenotypes in large genome-wide meta-analyses. We conclude that our method represents a simple way in which potentially tens of thousands of molecular phenotypes could be screened for causal relationships with disease without having to expensively measure these variables in individual disease collections.

With the advent of whole exome sequencing, cases where no pathogenic coding mutations can be found are increasingly being observed in many diseases. In two large, distantly-related families that mapped to the Charcot-Marie-Tooth neuropathy CMTX3 locus at chromosome Xq26.3-q27.3, all coding mutations were excluded. Using whole genome sequencing we found a large DNA interchromosomal insertion within the CMTX3 locus. The 78 kb insertion originates from chromosome 8q24.3, segregates fully with the disease in the two families, and is absent from the general population as well as 627 neurologically normal chromosomes from in-house controls. Large insertions into chromosome Xq27.1 are known to cause a range of diseases and this is the first neuropathy phenotype caused by an interchromosomal insertion at this locus. The CMTX3 insertion represents an understudied pathogenic structural variation mechanism for inherited peripheral neuropathies. Our finding highlights the importance of considering all structural variation types when studying unsolved inherited peripheral neuropathy cases with no pathogenic coding mutations., Author Summary Next generation sequencing technologies have greatly advanced disease gene discovery for Charcot-Marie-Tooth (CMT) disease and related inherited peripheral neuropathies. However, many families with CMT remain unsolved after all protein-coding sequences have been interrogated through whole exome sequencing. The pathogenic mutations in these unsolved families may be non-coding point mutations, small indels or large structural variations involving thousands to millions of base pairs. In two large, distantly related families with X-linked CMT, all known protein-coding sequence variants were tested and no causal variant was found. Using whole genome sequencing we identified a 78 kb 8q24.3 insertion at chromosome Xq27.1 as the likely underlying cause of neuropathy in these two families. This is the first report of a large insertion causing CMT and highlights an understudied disease mechanism for inherited peripheral neuropathy.

Recent systems-based analyses have demonstrated that sleep and stress traits emerge from shared genetic and transcriptional networks, and clinical work has elucidated the emergence of sleep dysfunction and stress susceptibility as early symptoms of Huntington's disease. Understanding the biological bases of these early non-motor symptoms may reveal therapeutic targets that prevent disease onset or slow disease progression, but the molecular mechanisms underlying this complex clinical presentation remain largely unknown. In the present work, we specifically examine the relationship between these psychiatric traits and Huntington's disease (HD) by identifying striatal transcriptional networks shared by HD, stress, and sleep phenotypes. First, we utilize a systems-based approach to examine a large publicly available human transcriptomic dataset for HD (GSE3790 from GEO) in a novel way. We use weighted gene coexpression network analysis and differential connectivity analyses to identify transcriptional networks dysregulated in HD, and we use an unbiased ranking scheme that leverages both gene- and network-level information to identify a novel astrocyte-specific network as most relevant to HD caudate. We validate this result in an independent HD cohort. Next, we computationally predict FOXO3 as a regulator of this network, and use multiple publicly available in vitro and in vivo experimental datasets to validate that this astrocyte HD network is downstream of a signaling pathway important in adult neurogenesis (TGFβ-FOXO3). We also map this HD-relevant caudate subnetwork to striatal transcriptional networks in a large (n = 100) chronically stressed (B6xA/J)F2 mouse population that has been extensively phenotyped (328 stress- and sleep-related measurements), and we show that this striatal astrocyte network is correlated to sleep and stress traits, many of which are known to be altered in HD cohorts. We identify causal regulators of this network through Bayesian network analysis, and we highlight their relevance to motor, mood, and sleep traits through multiple in silico approaches, including an examination of their protein binding partners. Finally, we show that these causal regulators may be therapeutically viable for HD because their downstream network was partially modulated by deep brain stimulation of the subthalamic nucleus, a medical intervention thought to confer some therapeutic benefit to HD patients. In conclusion, we show that an astrocyte transcriptional network is primarily associated to HD in the caudate and provide evidence for its relationship to molecular mechanisms of neural stem cell homeostasis. Furthermore, we present a unified systems-based framework for identifying gene networks that are associated with complex non-motor traits that manifest in the earliest phases of HD. By analyzing and integrating multiple independent datasets, we identify a point of molecular convergence between sleep, stress, and HD that reflects their phenotypic comorbidity and reveals a molecular pathway involved in HD progression., Author Summary Huntington’s disease is a complex neurodegenerative disorder caused by CAG expansion in the huntingtin gene. Huntington’s disease leads to abnormal involuntary movements in patients, but recent studies have shown that many non-motor psychiatric and sleep changes also manifest in the earliest phases of the disease, often before gross changes in movement. This study identifies gene networks that are affected by Huntington’s disease and associated with these non-motor traits, revealing biological pathways and the cell-types likely involved in the earliest stages of Huntington’s disease.

Cardiac remodelling and contractile dysfunction occur during both acute and chronic disease processes including the accumulation of insoluble aggregates of misfolded amyloid proteins that are typical features of Alzheimer's, Parkinson's and Huntington's disease (HD). While HD has been described mainly as a neurological disease, multiple epidemiological studies have shown that HD patients exhibit a high incidence of cardiovascular events leading to heart failure, and that this is the second highest cause of death. Given that huntingtin is ubiquitously expressed, cardiomyocytes may be at risk of an HD-related dysfunction. In mice, the forced expression of an expanded polyQ repeat under the control of a cardiac specific promoter led to severe heart failure followed by reduced lifespan. However the mechanism leading to cardiac dysfunction in the clinical and pre-clinical HD settings remains unknown. To unravel this mechanism, we employed the R6/2 transgenic and HdhQ150 knock-in mouse models of HD. We found that pre-symptomatic animals developed connexin-43 relocation and a significant deregulation of hypertrophic markers and Bdnf transcripts. In the symptomatic animals, pronounced functional changes were visualised by cardiac MRI revealing a contractile dysfunction, which might be a part of dilatated cardiomyopathy (DCM). This was accompanied by the re-expression of foetal genes, apoptotic cardiomyocyte loss and a moderate degree of interstitial fibrosis. To our surprise, we could identify neither mutant HTT aggregates in cardiac tissue nor a HD-specific transcriptional dysregulation, even at the end stage of disease. We postulate that the HD-related cardiomyopathy is caused by altered central autonomic pathways although the pathogenic effects of mutant HTT acting intrinsically in the heart may also be a contributing factor., Author Summary Huntington's disease (HD) is a neurodegenerative disorder for which the mutation results in an extra-long tract of glutamines that causes the huntingtin protein to aggregate. It is characterized by neurological symptoms and brain pathology that is associated with nuclear and cytoplasmic aggregates and with transcriptional dysregulation. Despite the fact that HD has been recognized principally as a neurological disease, there are multiple epidemiological studies showing that HD patients exhibit a high rate of cardiovascular events leading to heart failure. To unravel the cause of cardiac dysfunction in HD models, we employed a wide range of molecular and physiological methods using two well established genetic mouse models of this disease. We found that pre-symptomatic animals developed aberrant gap junction channel expression and a significant deregulation of hypertrophic markers that may predispose them to arrhythmia and an overall change in cardiac function. These changes were accompanied by the re-expression of foetal genes, apoptotic cardiomyocyte loss and a moderate degree of interstitial fibrosis in the symptomatic animals. Surprisingly, we could identify neither mutant HTT aggregates in cardiac tissue nor a HD-specific transcriptional dysregulation. Therefore, we conclude that the HD-related cardiomyopathy could be driven by altered central autonomic pathways.