Disease Collaborative Research Group Research Articles

Identification of genetic modifiers of Huntington's disease somatic CAG repeat instability by in vivo CRISPR-Cas9 genome editing.

Huntington's disease (HD), one of >50 inherited repeat expansion disorders (Depienne and Mandel, 2021), is a dominantly-inherited neurodegenerative disease caused by a CAG expansion in HTT (The Huntington's Disease Collaborative Research Group, 1993). Inherited CAG repeat length is the primary determinant of age of onset, with human genetic studies underscoring that the property driving disease is the CAG length-dependent propensity of the repeat to further expand in brain (Swami et al ., 2009; GeM-HD, 2015; Hensman Moss et al ., 2017; Ciosi et al ., 2019; GeM-HD, 2019; Hong et al ., 2021). Routes to slowing somatic CAG expansion therefore hold great promise for disease-modifying therapies. Several DNA repair genes, notably in the mismatch repair (MMR) pathway, modify somatic expansion in HD mouse models (Wheeler and Dion, 2021). To identify novel modifiers of somatic expansion, we have used CRISPR-Cas9 editing in HD knock-in mice to enable in vivo screening of expansion-modifier candidates at scale. This has included testing of HD onset modifier genes emerging from human genome-wide association studies (GWAS), as well as interactions between modifier genes, thereby providing new insight into pathways underlying CAG expansion and potential therapeutic targets.

Huntington's Disease Gene Hunters: An Expanding Tale.

Huntington's Disease Gene Hunters: An Expanding Tale.

Issue Highlights

Issue Highlights

Biologics Delay Progression of Crohn’s Disease, but Not Early Surgery, in Children

Up to 30% of patients with Crohn's disease (CD) require surgery within the first 5 years from diagnosis. We investigated the recent risk of bowel surgery in an inception cohort of pediatric patients with CD and whether early use of biologics (tumor necrosis factor antagonists) alters later disease course. We collected data from the Pediatric Inflammatory Bowel Disease Collaborative Research Group registry on 1442 children (age, ≤16 y) diagnosed with CD from January 2002 throughDecember 2014. Data were collected at diagnosis, 30 days following diagnosis, and then quarterly and during hospitalizations for up to 12 years. Our primary aim was to determine the 10-year risk for surgery in children with CD. Our secondary aim was todetermine whether early use of biologics (<3 mo of diagnosis) affected risk of disease progression. The 10-year risk of first bowel surgery was 26%. The 5-year risk of bowel surgery did not change from 2002 through 2014, and remained between 13% and 14%. Most surgeries occurred within 3 years from diagnosis. The only predictor of surgery was disease behavior at diagnosis. CD with inflammatory behavior had the lowest risk of surgery compared to stricturing disease, penetrating disease, or both. We associated slowing of disease progression to stricturing or penetrating disease (but not surgery) with early use of biologics, but this effect only became evident after 5 years of disease. Our results indicate that biologics slow disease progression over time (hazard ratio, 0.85; 95% CI, 0.76-0.95). In an analysis of data from a registry of pediatric patients with CD, we found that amongthose with significant and progressing disease at or shortly after presentation, earlysurgery is difficult to prevent, even with early use of biologics. Early use of biologics (<3mo of diagnosis) can delay later disease progression to stricturing and/or penetrating disease, but this affect could become evident only years after initial management decisions are made.

Journey from theory to practice: Setting up a Huntington Disease service in Romania

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder that has benefited from considerable research during the last two decades and the progress made is remarkable.Clinical diagnosis is based on symptoms and signs that include motor signs (i.e. chorea, hypokinesia, dystonia, tics), psychiatric aspects (e.g. depression, anxiety, irritability, mania, apathy, psychosis) and cognitive decline (e.g. executive functioning). The motor signs, mainly unwanted movements, remain the most characteristic and specific for a clinical diagnosis (Roos, 2014). Although the motor symptoms are immediately evident, psychiatric and cognitive symptoms contribute significantly to patients' impaired functioning and have great impact on their daily lives (Craufurd & Snowden, 2014; Tabrizi et al., 2013).Genetic testing by direct mutation analysis has been available for more than two decades (The Huntington's Disease Collaborative Research Group, 1993) and can be used either to confirm a clinical diagnosis of HD in symptomatic people or to predict whether or not an at-risk person will go on to develop HD.Currently there are a number of guidelines and standards of care available that take into account the complexity of HD and suggest best practice models for the assessment, diagnosis, management and counseling of this condition that have been shown to be effective and acceptable for patients and families (Craufurd et al., 2015; Semaka & Hayden, 2014; Losekoot et al., 2013). In fact, multidisciplinary teams in numerous centers have contributed to the significant advances we are continuously seeing in HD research and every day life of HD families. The aim of this paper is to discuss our progress in developing the first HD multidisciplinary clinic in Romania.Journey from theory to practiceProfessionals familiar with HD are very much aware of the complexity of this condition, its clinical features and the main implications for patients and families. Therefore, for most professionals it is rather reassuring to be able to rely on international guidelines when managing patients' care, to discuss challenging cases with more experienced colleagues, or to adapt existing services that are in place for similar conditions. Not being able to do either can bring one very close to helplessness.In Romania, most often, HD families see a neurologist or a psychiatrist that have seen very few HD patients their whole career. The family history is rarely available or helpful. Patients are in most cases not aware of any previous HD in the family and seem to vaguely identify relatives with dementia-like, schizophrenia-like or Parkinson's like symptoms. DNA analysis would probably be the most reasonable step at this stage; unfortunately that it is not available. The healthcare system has yet to concentrate on so many other priorities and genetic centers are not entirely functional yet. The Romanian Health Ministry has set up in 2014 a national network for genetic medicine that includes six regional centers (Ministerial order nr. 1358/2014). The centers struggle to become fully functional because of insufficient funding, equipment and expertise. Specialist outpatient clinics are another problematic aspect as they are not available in each center, which clearly makes patients' access to adequate healthcare difficult. Having said that, it is no surprise that there is no national or regional registry for most genetic conditions and very few specialist genetic services are in place (none for HD).In our country, generally, a patient with HD symptoms is referred to a neurology or psychiatry service, depending on their symptoms, and is treated as a neurological or psychiatric patient. Diagnosis is most often confirmed with genetic testing which is not funded and is only available in private practice (the cost for genetic testing for HD is around 100 Eur). Once the diagnosis is confirmed, most patients are treated under the care of a psychiatrist or a neurologist. …

Huntington's Disease: The Most Curable Incurable Brain Disorder?

Huntington's Disease: The Most Curable Incurable Brain Disorder?

Huntington's Disease as Neurodevelopmental Disorder: Altered Chromatin Regulation, Coding, and Non-Coding RNA Transcription.

Huntington's disease (HD) is a monogenic autosomal dominant, fatal disorder due to CAG trinucleotide expansion in exon 1 of the HD gene (HTT) (The Huntington's Disease Collaborative Research Group, 1993; Figure ​Figure1A).1A). Nowadays, there is no cure or effective treatment for the disease which presents with motor, cognitive and psychiatric dysfunction. Although typically conceived as a “neurodegenerative disease,” mainly affecting the GABAergic medium-sized spiny neurons (MSN) of the striatum and deep layers of the cortex (Rosas et al., 2003), an increasing body of evidence has surfaced indicating that abnormal neurodevelopment might also have a crucial role in HD (Mehler and Gokhan, 2000, 2001; Humbert, 2010). Neurodegenerative diseases have been classically defined as chronic and progressive disorders of the nervous system affecting neurologic and behavioral functions, which start with specific biochemical changes that ultimately lead to distinct histopathologic and clinical syndromes. On the contrary, neurodevelopmental disorders result from an anomaly of brain maturation, during fetal or early postnatal life, which is postulated to alter the structure and/or function of neuronal and synaptic populations (Harrison, 1995).

Concomitant Use of Immunomodulators Affects the Durability of Infliximab Therapy in Children With Crohn’s Disease

It is important to determine the effects of immunomodulators on the ability of children to remain on infliximab therapy for Crohn's disease (durability of therapy), given the potential benefits and risks of concomitant therapy-especially with thiopurines in male patients. We investigated how immunomodulatory treatment affects the durability of infliximab therapy. We collected data from the Pediatric Inflammatory Bowel Disease Collaborative Research Group Registry, from January 2002 through August 2014, on 502 children with Crohn's disease who participated in a prospective multicenter study. Data were collected from patients who received at least a 3-dose induction regimen of infliximab, and their concomitant use of immunomodulators: no thiopurine or methotrexate treatment, treatment for 6 months or less during infliximab therapy, or treatment for more than 6 months during infliximab therapy. The probabilities (± standard error) that children remained on infliximab therapy for 1 year, 3 years, and 5 years after the treatment began were 0.84 ± 0.02, 0.69 ± 0.03, and 0.60 ± 0.03, respectively. Age, sex, and disease extent or location did not affect the durability of infliximab therapy. Greater length of concomitant use of immunomodulators was associated with increased time of infliximab therapy. The probability that patients with more than 6 months of immunomodulator use remained on infliximab therapy for 5 years was 0.70 ± 0.04, compared with 0.48 ± 0.08 for patients who did not receive immunomodulators and 0.55 ± 0.06 for patients who received immunomodulators for 6 months or less (P < .001). In boys who received immunomodulators for 6 months or more after starting infliximab, the overall durability of infliximab therapy was greater among patients receiving methotrexate than thiopurine (P < .01); the probabilities that they remained on infliximab therapy for 5 years were 0.97 ± 0.03 vs 0.58 ± 0.08, respectively. In children with Crohn's disease, concomitant treatment with an immunomodulator for more than 6 months after starting infliximab therapy increases the chances that patients will remain on infliximab. In boys, methotrexate appears to increase the durability of infliximab therapy compared with thiopurine.

Stuck in the Middle: Huntington’s Disease or not Huntington’s Disease?

Stuck in the Middle: Huntington’s Disease or not Huntington’s Disease?

Liver enzyme elevations within 3 months of diagnosis of inflammatory bowel disease and likelihood of liver disease.

Inflammatory bowel disease-associated liver diseases (IBD-LDs) include autoimmune hepatitis (AIH), primary sclerosing cholangitis (PSC), and an overlap syndrome. Prospective unbiased multicenter data regarding the frequency of IBD-LD in patients with pediatric inflammatory bowel disease (IBD) are lacking. We examined early alanine aminotransferase (ALT) and γ-glutamyl transpeptidase (GGT) elevations in children diagnosed as having IBD and assessed the likelihood of IBD-LD. Data collected from the prospective observational Pediatric Inflammatory Bowel Disease Collaborative Research Group Registry enrolling children of age <16 years within 30 days of diagnosis. AIH, PSC, and overlap syndrome were diagnosed using local institutional criteria. A total of 1569 subjects had liver enzymes available. Of the total, 757 had both ALT and GGT, 800 had ALT only (no GGT), and 12 had GGT only (no ALT). Overall, 29 of 1569 patients (1.8%) had IBD-LD. IBD-LD was diagnosed in 1 of 661 (0.15%) of patients with both ALT and GGT ≤ 50 IU/L compared with 21 of 42 (50%) of patients with both ALT and GGT > 50 (odds ratio 660, P < 0.0001). Of the 29 patients with IBD-LD, 21 had PSC, 2 had AIH, and 6 had overlap syndrome. IBD-LD was more common in patients with ulcerative colitis and IBD-unclassified (indeterminate colitis) than in those with Crohn disease (4% vs 0.8%, respectively, P < 0.001). Elevation of both ALT and GGT within 90 days after the diagnosis of IBD is associated with a markedly increased likelihood of IBD-LD. Both ALT and GGT levels should be measured in all of the pediatric patients newly diagnosed as having IBD.

An N-terminal Nuclear Export Signal Regulates Trafficking and Aggregation of Huntingtin (Htt) Protein Exon 1*

Huntington disease is a dominantly inherited neurodegenerative condition caused by polyglutamine expansion in the N terminus of the huntingtin protein (Htt). The first 17 amino acids (N17) of Htt play a key role in regulating its toxicity and aggregation. Both nuclear export and cytoplasm retention functions have been ascribed to N17. We have determined that N17 acts as a nuclear export sequence (NES) within Htt exon and when fused to yellow fluorescent protein. We have defined amino acids within N17 that constitute the nuclear export sequence (NES). Mutation of any of the conserved residues increases nuclear accumulation of Htt exon 1. Nuclear export of Htt is sensitive to leptomycin B and is reduced by knockdown of exportin 1. In HEK293 cells, NES mutations decrease overall Htt aggregation but increase the fraction of cells with nuclear inclusions. In primary cultured neurons, NES mutations increase nuclear accumulation and increase overall aggregation. This work defines a bona fide nuclear export sequence within N17 and links it to effects on protein aggregation. This may help explain the important role of N17 in controlling Htt toxicity.

Outcome Following Aminosalicylate Therapy in Children Newly Diagnosed As Having Ulcerative Colitis

Despite a paucity of published supporting data, 5-aminosalicylate (5-ASA) use in pediatric ulcerative colitis (UC) is common. The present study describes the use and outcome of a large multicenter inception cohort of children with UC treated with 5-ASA. Data were obtained from the Pediatric Inflammatory Bowel Disease Collaborative Research Group Registry, a prospective North American observational study of children newly diagnosed as having inflammatory bowel disease ages 16 years or younger. Patient data are recorded at diagnosis, 30 days, and then quarterly. Patients are managed by physician dictate, not protocol. Disease activity is classified by physician global assessment. The primary outcome examined was corticosteroid (CS) free, inactive UC at 1 year following initiation of 5-ASA within 30 days of diagnosis (with or without concomitant CS use) without the need for rescue therapy (immunomodulators, biologics, or colectomy). Study subjects included 213 patients newly diagnosed as having UC who received oral 5-ASA compounds (115 of whom also received CS) during the first 30 days after diagnosis, and no other oral therapies for the treatment of UC. Of these 213 patients, 86 (40%) were CS free and physician global assessment inactive at 1 year without rescue. Outcome was not associated with disease severity at diagnosis, demographic or laboratory factors examined, or initial dose of 5-ASA used. Forty percent of children taking 5-ASA as primary maintenance therapy at diagnosis are in CS-free remission after 1 year of treatment. Further pediatric studies will be needed to address whether increased adherence and/or higher dosing schedules will improve outcomes.

Huntington's disease: fighting on many fronts

The discovery in 1993 of the genetic abnormality that causes Huntington's disease triggered an explosion in our understanding of this inherited neurodegenerative disorder (The Huntington's Disease Collaborative Research Group, 1993). Since the Huntington's disease mutation is fully penetrant, everyone with the same underlying CAG triplet repeat expansion in the huntingtin ( HTT ) gene will develop a devastating combination of motor, cognitive and psychiatric disturbances. The monogenic nature of Huntington's disease suggests that disease modifying therapies should be within reach. Currently, however, no such treatments exist (Mestre et al. , 2009). Nevertheless, much has been done to characterize the huntingtin (Htt) protein and the effects of its toxic twin, mutant huntingtin. We know that Htt is a large and ubiquitously expressed protein with a range of possible functions, and that Huntington's disease is predominantly caused by the damaging effects of mutant Htt on neurons. A range of animal models recapitulate the molecular, cellular and clinical phenotypes of Huntington's disease, enhancing our understanding and facilitating the search for treatments (Ross and Tabrizi, 2011). Numerous potential pathogenic mechanisms have been identified, leading to promising therapeutic approaches in transgenic Huntington's disease mice and other models. Although none has yet been found effective in human patients, there is optimism in the field that Huntington's disease is a disease for which the goal of effective therapy might be achieved (Munoz-Sanjuan and Bates, 2011). Two articles in this issue of Brain highlight the Huntington's disease research community's multi-faceted approach to developing new treatments for Huntington's disease. Grondin and colleagues report success in a 6-month study of HTT gene silencing using RNA interference in the adult non-human primate brain (Grondin et al. , 2012); while Labbadia et al. (2012) describe the means by which a molecular chaperone can suppress aggregation of disease-associated Htt complexes. Gene silencing is an …

Exome and whole-genome sequencing for gene discovery: The future is now!

Exome and whole-genome sequencing for gene discovery: The future is now!

Huntington's Disease: Advocacy Driving Science

My mother, Leonore, was diagnosed with Huntington's disease (HD) in 1968 at age 53. I was 23, my sister Alice 26, and our father, Milton Wexler, 60 years old. The same year, our father created the Hereditary Disease Foundation (HDF), dedicated to finding treatments and cures for HD. HD is an autosomal dominant, neurodegenerative disorder. Alice and I each have a 50% chance of inheriting and dying from the disorder. Over the past 43 years, we have been proud to change the face of science. Through Milton Wexler Interdisciplinary Workshops, judicious funding, and focusing on innovation and creativity, the HDF is an integral partner in key discoveries. The HDF recruited and supported >100 scientists worldwide who worked together as the Huntington's Disease Collaborative Research Group in a successful ten-year search for the HD gene. We found a DNA marker for the HD gene in 1983-the first marker to be found when the chromosomal location was unknown. We isolated the HD gene itself a decade later. These breakthroughs helped launch the Human Genome Project. We supported creating the first mouse model of HD and many other model systems. Currently, we focus on gene silencing, among other approaches, to create new treatments and cures.

Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Associates with Huntingtin Protein and Promotes Its Atypical Ubiquitination to Enhance Aggregate Formation

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys6, Lys27, and Lys29 linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys6, Lys27, and Lys29 ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis.

Interaction with Polyglutamine-expanded Huntingtin Alters Cellular Distribution and RNA Processing of Huntingtin Yeast Two-hybrid Protein A (HYPA)

Huntington disease (HD) is an autosomal inherited disorder that causes the deterioration of brain cells. The polyglutamine (polyQ) expansion of huntingtin (Htt) is implicated in the pathogenesis of HD via interaction with an RNA splicing factor, Htt yeast two-hybrid protein A/forming-binding protein 11 (HYPA/FBP11). Besides the pathogenic polyQ expansion, Htt also contains a proline-rich region (PRR) located exactly in the C terminus to the polyQ tract. However, how the polyQ expansion influences the PRR-mediated protein interaction and how this abnormal interaction leads to the biological consequence remain elusive. Our NMR structural analysis indicates that the PRR motif of Htt cooperatively interacts with the tandem WW domains of HYPA through domain chaperoning effect of WW1 on WW2. The polyQ-expanded Htt sequesters HYPA to the cytosolic location and then significantly reduces the efficiency of pre-mRNA splicing. We propose that the toxic gain-of-function of the polyQ-expanded Htt that causes dysfunction of cellular RNA processing contributes to the pathogenesis of HD.

Factors That Determine Risk for Surgery in Pediatric Patients With Crohn's Disease

We examined the incidence of Crohn's disease (CD)-related surgery in a multi-center, inception cohort of pediatric patients with CD. We also examined the effect of starting immunomodulator therapy within 30 days of diagnosis. Data from 854 children with CD from the Pediatric Inflammatory Bowel Disease Collaborative Research Group who were diagnosed with CD between 2002 and 2008 were analyzed. Overall, 76 (9%) underwent a first CD-related surgery, 57 (7%) underwent a first bowel surgery (bowel resection, ostomy, strictureplasty, or appendectomy), and 19 (2%) underwent a first non-bowel surgery (abscess drainage or fistulotomy). The cumulative risks for bowel surgery, non-bowel surgery, and all CD-related surgeries were 3.4%, 1.4%, and 4.8%, respectively, at 1 year after diagnosis and 13.8%, 4.5%, and 17.7%, respectively, at 5 years after diagnosis. Older age at diagnosis, greater disease severity, and stricturing or penetrating disease increased the risk of bowel surgery. Disease between the transverse colon and rectum decreased the risk. Initiation of immunomodulator therapy within 30 days of diagnosis, sex, race, and family history of inflammatory bowel disease did not influence the risk of bowel surgery. In an analysis of pediatric patients with CD, the 5-year cumulative risk of bowel surgery was lower than that reported in recent studies of adult and pediatric patients but similar to that of a recent retrospective pediatric study. Initiation of immunomodulator therapy at diagnosis did not alter the risk of surgery within 5 years of diagnosis.

An ovine transgenic Huntington's disease model.

Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disorder caused by an expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene [Huntington's Disease Collaborative Research Group (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The Huntington's Disease Collaborative Research Group. Cell, 72, 971-983]. Despite identification of the gene in 1993, the underlying life-long disease process and effective treatments to prevent or delay it remain elusive. In an effort to fast-track treatment strategies for HD into clinical trials, we have developed a new large-animal HD transgenic ovine model. Sheep, Ovis aries L., were selected because the developmental pattern of the ovine basal ganglia and cortex (the regions primarily affected in HD) is similar to the analogous regions of the human brain. Microinjection of a full-length human HTT cDNA containing 73 polyglutamine repeats under the control of the human promotor resulted in six transgenic founders varying in copy number of the transgene. Analysis of offspring (at 1 and 7 months of age) from one of the founders showed robust expression of the full-length human HTT protein in both CNS and non-CNS tissue. Further, preliminary immunohistochemical analysis demonstrated the organization of the caudate nucleus and putamen and revealed decreased expression of medium size spiny neuron marker DARPP-32 at 7 months of age. It is anticipated that this novel transgenic animal will represent a practical model for drug/clinical trials and surgical interventions especially aimed at delaying or preventing HD initiation. New sequence accession number for ovine HTT mRNA: FJ457100.

Outcome Following Infliximab Therapy in Children With Ulcerative Colitis

Infliximab is effective in treating moderate/severe ulcerative colitis (UC) in adults. The aim of this study was to determine the outcome after treatment with infliximab in pediatric UC. We performed a multicenter cohort study of 332 pediatric patients with UC enrolled in the Pediatric Inflammatory Bowel Disease Collaborative Research Group Registry. Children<or=16 years of age and newly diagnosed with UC are enrolled in the registry. Disease and medication information are collected prospectively from the treating physician at diagnosis, 30 days, and quarterly thereafter. No interventions were specified, per protocol. Of 332 patients, 52 (16%) received infliximab (23%<3 months from diagnosis, 38% 3-12 months, 38% >12 months). Mean age at infliximab initiation was 13.3+/-2.6 (range 6-17) years; 87% of patients had pancolitis. Median follow-up was 30 months. Continuous maintenance (CM) therapy was given in 65%, episodic in 21%, episodic converted to CM in 6%, and insufficient data in 8% of patients. Sixty-three percent of patients were corticosteroid refractory, and 35% were corticosteroid dependent. Concomitant medications at first infliximab infusion included corticosteroids (87%), thiopurines (63%), and 5-aminosalicylates (51%). Corticosteroid-free inactive disease by physician global assessment was noted in 12/44 (27%), 15/39 (38%), and 6/28 (21%) patients at 6, 12, and 24 months, respectively. Kaplan-Meier analysis showed that the likelihood of remaining colectomy free after treatment with infliximab was 75% at 6 months, 72% at 12 months, and 61% at 2 years. In this cohort of children with UC receiving infliximab, corticosteroid-free inactive disease was observed in 38 and 21% of patients at 12 and 24 months, respectively. By 24 months, 61% of patients had avoided colectomy.