20-4 Gene Correction of Epidermolysis Bullosa Simplex Mutations using CRISPR/Cas9 Technology
Epidermolysis bullosa simplex (EBS) is a rare genetic disease characterized by fragility of the skin resulting in blistering and erosion caused by minor mechanical trauma. To date, there is no cure, and current therapies are palliative, aimed at treating infections and trauma and maintaining an acceptable quality of life. The goal of this project is to develop an effective and permanent therapy for this disease. The researchers are proposing a treatment based on a patient's own cells, corrected in the laboratory for the disease causing mutation. They will use a recently developed technology: CRISPR/Cas9, a model very similar to “find and replace” often used in word processing programs. The first step will be to produce pluripotent stem cells, i.e. which can differentiate into any type of cells in the body, from skin cells isolated from cutaneous biopsies of patients. Then, in these pluripotent cells, they will induce DNA cuts close to the known mutation sites using the CRISPR/Cas9 genome editing system (“find”). These DNA cuts will be repaired by homologous recombination, a process that replaces the portion of the DNA with the mutation (genetic variation) by exogenous synthesized similar DNA molecules without any mutation (“replace”). The repaired cells will then be differentiated into specialized cells of the skin: keratinocytes. Finally, a skin model will be made with these corrected keratinocytes and will be used for autografts in order to treat the patient. In principle, these autografts back onto the fragile blisters will persist longer. The advantages of this approach would be absence of the severe potential side effects associated with the problems of rejection since the patient own cells will be used. It is hoped that this will alleviate blistering apparent and that the researchers can realize the promise of their project, which is treating this genetic disease and improve survival of EBS patients.
20-7 Elemental Diet for Chronic Granulomatous Disorder-Associated Colitis
Chronic granulomatous disorder (CGD) is a rare primary immune deficiency in which the patient’s white blood cells do not work properly. Patients suffer severe infections but also often develop a form of inflammatory bowel disease (‘colitis’) which causes diarrhea, blood loss and anemia. Current treatment includes steroids, which have many side effects (such as weight gain, weakening of the bones and development of diabetes) or immune-suppressing medicines which further increase the risk of infection. Ultimately many patients end up requiring major surgery such as removal of diseased bowel, often needing a long-term stoma bag. We plan to give an ‘elemental diet’ to one or two patients with CGD-associated colitis. This is a simple liquid diet of nutrients, known to be safe and effective in Crohn’s disease. We will see whether this therapy improves symptoms, reduces bowel inflammation and changes the abundance or pattern of bacteria in the stool. We will recruit patient(s) from an existing study comparing MRI scans with colonoscopy to assess CGD colitis: these investigations will give us accurate baseline appearances of the bowel. Once the patient(s) starts the elemental diet they will be asked to keep a daily symptom diary and to provide weekly stool samples. We will use these samples to measure inflammation and to look at changes in the stool bacteria (‘microbiome’) during the period of therapy. If there has been an effect, we may repeat the MRI scan or endoscopy at the end of the period of diet. If successful, this has the potential to significantly improve care of patients with CGD colitis, by substituting a safe treatment for potentially hazardous ones. There may be a particular role for this therapy around the time of stem cell transplant, where it is important to control inflammation but also to avoid risk of infection.
Results - One patient with severe Chronic Granulomatous Disorder (CGD)-associated colitis received elemental diet. This resulted in prompt cessation of diarrhoea but paradoxically his symptoms progressed to severe constipation with abdominal pain, an increase in blood inflammatory markers and a likely new peri-anal fistula. We therefore discontinued therapy and have subsequently tried the novel inflammatory bowel disease therapy vedolizumab in this patient and two others. Interestingly, all patients also experienced some constipation and abdominal pain initially, with a paradoxical rise in stool markers of inflammation. However, in the two patients receiving therapy for longest, symptoms and inflammation markers have subsequently improved. The initial patient has had a repeat endoscopy which reveals much improved appearances of the bowel. There have not been any serious infections. This suggests that vedolizumab may be a useful treatment for this condition but can lead to troublesome side effects initially. We are currently planning to analyse changes in the bacteria in the stool (‘microbiome’) during the therapy.
20-14 Development of a Pre-Operative Risk Stratification Score for Pediatric Thyroid Carcinoma
Thyroid nodules (lumps) are found in up to 10% of children. These nodules are often detected by the patient or caregiver, or found incidentally when neck imaging is performed for other reasons. While most nodules are benign, up to 25% of them are cancerous, this rate is roughly five times higher in children than in adults. It can be challenging to distinguish benign from malignant nodules using our existing tools. While thyroid cancer is a common malignancy in women (and is on-track to become the second most common women’s cancer within the next 5 years), it is rare in children. Pediatric cases of thyroid cancer account for less than 2% of all thyroid cancers. As a result very little research on thyroid cancer reflects pediatric data. Despite this, it has become clear that thyroid cancer behaves differently in children than it does in adults. As a result, physicians treating children with thyroid cancer, do so based on limited information. The current approach to tell if a nodule is benign or cancerous includes a review of the medical history, physical examination, blood work, ultrasound and biopsy. Unfortunately, none of these tests – on their own - are reliable enough to distinguish between benign and cancerous nodules. Ultimately, this distinction can only be made definitively after surgery. As a result many children undergo surgery for what ultimately proves to be a benign (noncancerous) nodule. This exposes them to the inherent risks and complications of surgery. The researchers on this project are developing a score that combines information gathered in the pre-surgical period (clinical evaluation, bloodwork and ultrasound) to better predict the likelihood that a nodule is cancerous, and thus to inform the decision-making process that patients and their physicians undertake, when deciding about surgery. Ultimately, it is hoped that this will reduce the rates of surgery for non-cancerous nodules and thereby limit children’s exposure to surgical risks.
20-1 Investigating a Recommender System Approach to Tangible Support for People with Rare Diseases
Online communities are valuable sources of peer support for people with rare diseases, especially for informational and emotional support. Unfortunately, there are barriers to facilitating more tangible forms of support within these communities. The low prevalence of specific rare diseases makes in person tangible support challenging to coordinate (i.e., if there is no one with the same condition in the same geographic area). Additionally, some of the tasks patients have described needing help with are things that would be challenging for someone with the same condition to provide; if a person requires help with groceries because their condition makes lifting the grocery bags challenging, it is likely that someone with the same disease might have similar support needs, rather than being able to help. We are developing an algorithm that matches a given patient with people with DIFFERENT rare diseases in their local area based on abilities, skills, and expertise to facilitate this exchange of tangible support. We hypothesize that the experience of living with a rare disease may serve as common ground, while coordinating support among DIFFERENT rare diseases may help overcome the symptomatic and geographic limitations of trying to coordinate tangible support between people with the same condition. Although it might be possible to facilitate one-on-one exchanges of tangible support, this may be more effectively achieved using a pay-it-forward mentality. We propose that a patient would earn “time dollars” for help they provide to others that they could redeem for help with their own needs. In this project, we intend to conduct a web-based experiment to collect the necessary data to build the algorithm that matches a given patient with other people with rare diseases in their local area based on skills and expertise for purposes of giving/receiving help on a given task. This experiment allows us to learn about the design of such a platform and assess its feasibility.
Results - People with rare diseases often describe receiving frequent and high-quality support from other people with rare diseases through online health communities or Facebook groups. Although these existing rare disease support groups excel at providing emotional and informational forms of support there are barriers to facilitating more tangible forms of support (e.g., help with physical tasks around the home), within these communities; tangible support often requires people to connect in-person and this is challenging when diseases are rare and patients are geographically distributed. In this project, I investigated the possibility of extending these groups to create a helping network for people with different rare diseases to facilitate exchanges of social, and especially tangible, support. I conducted a two-part survey to identify the tasks that may be best suited for digitally facilitated support exchanges, the most important characteristics for matching people with different rare diseases, and people’s information sharing. I found that many people with rare diseases were searching for help with the same tasks even when they have different rare diseases. I also found that people with rare diseases prefer to receive help from someone whose expertise complements theirs and who has had similar health experiences. Where people with rare diseases self-reported that the demographic characteristics of the helper are not important, their behavior when asked to select from several example helpers suggests that they prefer to receive help from someone who is demographically similar to them. Finally, I found that people with rare diseases are more willing than people with common chronic illnesses to share private information about themselves on social media sites.
20-2 Utilization of CRISPR/Cas9-Based Gene Editing for Correction of Deletion Mutations in DMD
Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disease, affecting approximately 1 in 5000 boys. The disease is caused by the lack of dystrophin protein expression, leading to progressive muscle deterioration, loss of ambulation, cardiac and respiratory complications, and limited life expectancy. Currently, there is no cure with only symptomatic management available. The lack of dystrophin is caused by mutations in the DMD gene, where deletion mutations account for 70% of all DMD cases. The researchers working on this project propose that deletion mutations causing DMD can be corrected using a genome engineering technology called clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to produce full length dystrophin. As a proof of concept study, we will be using immortalized muscle cells from the novel DMD mouse model containing the deletion of exons 52-54 (Dmd del52-54). The cDNA coding for the missing exons will be re-introduced (knocked-in) using CRISPR/Cas9 to restore the full length dystrophin. Successful restoration of full length dystrophin offers a tremendous therapeutic and possible curative potential for DMD patients. The optimized knock-in strategy can also be adapted to other diseases affected by deletion mutations.
20-3 Assessing Variants of Uncertain Significance in Schwannomatosis Disease
Schwannomatosis is a rare disease in which people develop tumours on their nerves. These tumours cause severe pain that is not helped by pain killers. There are currently two genes that are known to cause the disease when they are damaged, called SMARCB1 and LZTR1. Damage to one of these two genes can be found in around half of all people with schwannomatosis. It is sometimes difficult to tell the difference between schwannomatosis and another disease, called neurofibromatosis type 2 (NF2), which is caused by a different gene. Genetic testing can be important to be able to tell these diseases apart in some cases. Recently, some rare changes have been found in LZTR1 in people with nerve tumours, which are not clearly causing the disease, but are also not clearly unrelated. This project will study these changes to find out whether they are damaging to the gene product. The study will help understand which changes in LZTR1 are disease-causing and which are unrelated to the disease. If it cn be can proved that a gene change is disease-causing, then the person who has that change can be told that they definitely have schwannomatosis. If the change is not disease-causing, then that person can be referred for further testing. Knowing that a gene change is disease-causing would also mean that their family members could be tested for the same change. This would be useful for testing whether their relatives are at risk of having the same disease. The results of this project will also help doctors and genetic counsellors to understand how the disease is likely to develop in each case, which will help them to plan appropriate medical care.
Results - Schwannomatosis is a rare disease in which people develop tumours on their nerves. These tumours cause severe pain that is not helped by pain killers. There are currently two genes that are known to cause the disease when they are damaged, called SMARCB1 and LZTR1. Damage to one of these two genes can be found in around half of all people with schwannomatosis. Recently, some rare changes have been found in LZTR1 in people with nerve tumours, but it is not clear whether they are causing the disease, or not. We have studied some of these changes to find out whether they have a damaging effect on the gene.
Using the funding from the Rare Disease Foundation, we have been able to show that the structure of the LZTR1 gene product has been damaged in three unrelated people with different gene changes. These results can now be tested on a clinical basis and used to give a clear diagnosis to the patients. These gene changes can also be used to test relatives of the patients to check whether they are also at risk of developing tumours.
For the remaining cases where no structural changes have been found in the gene transcript, we are using other methods to test for changes to the function of the LZTR1 protein. We have recently submitted a grant application to the Wellcome Trust to extend these studies.
20-5 MRI versus Colonoscopy for the Assessment of Chronic Granulomatous Disorder-Associated Colitis
Chronic granulomatous disorder (CGD) is a rare genetic immune deficiency in which the affected person’s white blood cells do not work properly. Patients suffer recurrent infections, but also often develop a form of inflammatory bowel disease of the colon (‘colitis’) which causes diarrhea, blood loss and anemia. CGD-associated colitis is currently assessed with colonoscopy, but this is invasive and carries risks such as perforation (‘puncturing’ the bowel). The researchers on this project aim to see whether MRI scans, which do not involve radiation, can give them the same information as a colonoscopy, and to assess which test patients prefer. They will also look at whether simple stool tests for inflammation or a clinical score can predict disease activity. Consenting adult participants will be examined so that the reserchers can calculate the clinical score. The patients will then submit stool samples to exclude infection and for measurement of inflammation. The following day they will have the MRI scan (after drinking a special ‘contrast’ drink) and in the afternoon will have a colonoscopy. Patients will be given questionnaires after each investigation and again a week later to assess satisfaction and preference. The colonoscopy appearance, the MRI scan and any biopsies taken will all be carefully scored by two specialist doctors (gastroenterologists, radiologists and histopathologists) and an average of the clinical scores will be used. The primary outcome measure will be the correlation between the score awarded to the MRI appearance and the score for the colonoscopy appearance. If the MRI can provide as much information as the colonoscopy, and if patients prefer MRI or have no specific preference, then the researchers hope to be able to substitute this safe monitoring test in place of a potentially hazardous one.
Results - This project was successfully completed with 10 patients undergoing sequential MRI and colonoscopy on the same day to assess Chronic Granulomatous Disorder (CGD)-associated colitis. MRI was able to accurately identify the extent of colitis. It was also generally better tolerated and preferred by patients, took less time and was completed in all participants (colonoscopy was not completed in 3 patients, and in one patient less than half the colon was visualised). We were also able to identify additional unanticipated pathology with MRI such as an undescended testis. A stool test for inflammation (calprotectin) accurately discriminated between those with and without colitis, with the level broadly correlating with the severity of disease. However, a clinical scoring system adapted from Crohn’s disease performed poorly – mainly because the patients with CGD have little pain. Overall this research suggests that colitis in CGD can be monitored using non-invasive methods and practice should be changed accordingly.
20-6 Predicting NF1 Severity using CADD Scores
Neurofibromatosis 1 (NF1) is an inherited, progressive condition with highly variable severity. Its lack of predictability is a major burden to families: nobody can determine at the birth of an affected child whether he or she will have just a few benign skin tumours (neurofibromas) or will have significant disability, which can include severe disfigurement, life-threatening tumours, cardiovascular or bony abnormalities, or learning disability. It is known that there are links between a person’s genetics and their NF1 manifestations, but except in rare cases, these links are not useful for severity prediction. The researchers plan to use a new method, called CADD scoring, which looks at a number of different genetic factors to determine the effect of a mutation on the severity of a condition. Families with NF1 have told us that having some sort of a future “road map” would be invaluable for their peace of mind, and would help to direct screening and early interventions.
20-8 Designing Drugs to Treat ATP-Sensitive Potassium (KATP) Channel Mutations that Cause Hyperinsulinism (HI)
Potassium ion channels are essential for a healthy pancreas, and are formed from two different proteins: (1) the Kir6.2 protein that forms the channel pore and (2) the SUR1 protein that controls pore opening and closing. Changes in the SUR1 gene that result in defective channels cause hyperinsulinism (HI). HI is rare (1/50,000 live births) and severe disease that results in excessive insulin in the blood, and is treated by partial or total removal of the pancreas leading to severe health consequences. If left untreated HI causes brain damage. The researchers on this project will determine, at a molecular level, how changes in SUR1 (called mutations) cause the potassium channel to stop working, which will allow us to design specialized drugs to treat HI caused by specific mutations. The study will focus on regions of SUR1 called the nucleotide binding domains (NBDs). The NBDs use the energy of ATP (the energy currency of cells) to control pore opening and closing, and contain HI-causing mutations. Our previous work showed that mutations alter the 3-D arrangement of atoms in the NBDs and impair their binding to ATP. Mutations may also impair how SUR1 physically associates with other parts of the channel, which would disrupt transport of the channel from where it is made inside the cell to the cell surface where it functions. Using a technique similar to medical MRI, the researchers will provide a 3-D picture describing which parts of the NBDs bind candidate drugs. They will also test if drugs can correct defects in the 3-D architecture of mutant NBDs and rescue any impairment of mutant NBDs to bind other parts of the channel. The ability of drugs to correct defects in the SUR1 mutants will be addressed through studies that examine the transport of newly-made channels to the cell surface and with experiments that measure opening and closing of the channel pore. The molecular-level knowledge will address how well potential drugs work and promote the design of new drugs to treat HI.
Results - Potassium ion channels, which are essential for a healthy pancreas, are formed from two different proteins: (1) the Kir6.2 protein that forms the channel pore and (2) the SUR1 protein that controls pore opening and closing. Changes in the SUR1 gene that result in defective channels cause hyperinsulinism (HI), a rare (1/50,000 live births) and severe disease that results in excessive insulin in the blood, and is treated by partial or total removal of the pancreas leading to severe health consequences. If left untreated HI causes brain damage.
Using a technique similar to medical MRI, we have determined which residues in SUR1 bind drugs that open the potassium ion channel or that assist the potassium channel reach the cell surface. Using the MRI-based information, we have also calculated structures of the protein/drug complexes. We have also shown that changes SUR1 (called mutations) affect how different regions of the protein interact with one another, which is one of the molecular mechanisms underlying disease. This work has formed the basis of an M.Sc. thesis (September 2017) and a Ph.D. thesis (March 2018) in the laboratory. The corresponding manuscripts are currently being revised. Note that there is variability in where different drugs bind SUR1, in the defects caused by different SUR1 mutations, and in the types of drugs that can target and correct different SUR1 mutations. Thus, our data underscores the need for these studies in characterization of different HI disease-causing mutations and their treatment.
20-9 Understanding the Molecular Basis of Unexplained X-linked Intellectual Disability in a Large Kindred with Multiple Affected Individuals
Intellectual disability (ID) is one of the most common causes of disabilities in the population and is one of the most common birth defects. Understanding genetic factors which underlie ID has been a major challenge. While major advances have been made in identifying genetic aetiologies of some IDs, for many forms the genetics is complex and poorly understood, hence many children with ID do not have an accurate diagnosis. To date the research has focused predominantly on protein coding variation, with the non-coding portions of the genome (98% of the genome) unexplored. Recently, due to the utilisation of whole-genome sequencing (WGS) we can reliably identify all non-coding variations within the genome and subsequently study their consequences in human diseases. This project is based around an extended family with 7 males affected by X-linked ID, physical disability, speech disorder and variable obesity. In these males, no genetic cause has been discovered after extensive genetic investigations have been performed, including whole exome sequencing (WES) and microarray analysis. The researchers in this project will perform WGS to sequence the entire genome and look for genomic variants located on the X chromosome. Fortunately, from previous studies using a powerful tool known as linkage analysis they have localized a possible location of the disease gene. This will facilitate discovering the elusive mutation within the significant volume of data which will be generated. Identifying genes responsible for this unknown form of hereditary ID will benefit this unfortunate family as it will provide a molecular diagnosis for the patients. In addition, it will allow for genetic testing for carriers and prenatal testing, as well as improve our understanding of the mechanisms leading to ID in this family. Ultimately, the findings from this study will aid in the improvement of genetic testing available for other patients without a molecular diagnosis for ID.
20-10 Immunophenotyping in DYRK1A-deficiency
The researchers on this project have diagnosed a teenage girl with DYRK1A-related intellectual disability syndrome, caused by mutation in DYRK1A gene. She has short stature, microcephaly, seizures, and recurrent ear infections. This syndrome affects approximately 0.1%-0.5% of individuals with intellectual disability and/or autism. DYRK1A is believed to be a major regulator of the immune system. Healthy people need to be able to turn on their immune response when they run into dangerous bacteria, fungi or viruses, and to turn it back off when they have fought off the infection. However, if the immune system stays turned off even when pathogens invade the body, a child will have impaired immunity and will get sick more often. Inflammation is one of the ways the immune system helps the body to fight foreign pathogens; the Th17 and T regulatory (Treg) cells help turn this on and off. The researchers believe that their patient’s DYRK1A mutation is weakening her immune system by reducing Tregs, and allowing too many Th17 cells. Her repeated ear infections have led to dangerously high fevers on many occasions, requiring treatment in the Emergency Department and placement of tubes in both ears to drain ear fluid. Thus, they want to measure the proportions of her T regulatory (Treg) and Th17 cells. Once they know this, they will aim to find out the cause of her weak immune system and will be able to select the right antibiotics and antifungals to treat her.
20-11 A Retrospective Study to Understand the Late Stage of Intellectual and Cognitive Outcomes in Myotonic Dystrophy Type 1
Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder with multisystemic involvement, including the muscular, cardiac and central nervous system. It is the most common form of adult-onset muscular dystrophy. Among the 5 phenotypes of DM1, the adult-onset is the most prevalent. Its cognitive profile is generally well characterized in patients between 40 to 60 years old. Moreover, our team has recently demonstrated an early decline in cognitive functions, in a cohort of 152 patients with this phenotype followed within a longitudinal study over 9 years (an additional assessment after 14 years has been recently conducted). However, the patients that were suspected of presenting dementia have been excluded due to their inability to perform the tests. Thus, the present exploratory study aims to enhance the understanding of the late evolution of intellectual and cognitive abilities by answering two questions: 1) May cognitive impairment observed in DM1 lead to a specific dementia profile?; and 2) Is there any factor allowing to predict the onset of dementia in DM1? This is crucial for clinical practice, in order to better anticipate and facilitate home cares or the transition towards specialized institutions.
20-12 Development and Evaluation of a Patient Resource for Psychiatric Risk Involved in 22q11.2DS
Individuals with 22qDS are at an increased risk of developing psychiatric disorders. Parents of individuals with 22qDS have reported receiving little information about psychiatric manifestations of the syndrome from healthcare professionals. Parents have reported gathering information regarding psychiatric risks from non-medical sources like the internet. Having a reliable source of information can help reduce parents’ anxiety whilst enabling them to be aware of early signs of mental illness in their child. As early detection and treatment of mental health problems can lead to better long-term outcomes, this could improve outcomes for people with the syndrome too. The purpose of this study is to develop and evaluate a patient resource in the form of a booklet that focuses on psychiatric disorders in the context of 22qDS. Interviews with parents of individuals with 22qDS who have previously received psychiatric genetic counselling will help identify important elements to include in the resource. The booklet will then be presented to individuals from 22qDS support groups and family organizations and evaluated using online surveys. Links to these surveys will be accessed on support group websites. Information from these surveys will guide further improvements to the resource by understanding what participants liked and what they thought could be improved.
20-13 Polyphosphate: a Novel Universal Regulator in Hermansky-Pudlak Syndrome
AN OVERLOOKED SYNDROME WITH MULTIPLE SUBTYPES: The treatment regimen of multi-system and multi-causative syndromes is often difficult to design due to extreme variability within and between patients. One example is Hermansky-Pudlak Syndrome (HPS) – a rare, hereditary genetic disorder affecting ~ 1 in 750,000 people worldwide. Patients present with albinism, visual impairment, and prolonged bleeding due to platelet dysfunction. There are at least 10 different subtypes of HPS, characterized by which gene is mutated and the associated symptoms. Due to its rarity, HPS receives little attention in biomedical research; patients are counselled to manage their symptoms and have little hope of actual treatments. A COMMON DEFECT IN HPS: Despite the variability seen in HPS, the common overall cause between all subtypes is the production of defective protein(s) that normally function to promote protein trafficking to the lysosome-related organelle (LRO). These specialized organelles are found in platelets, skin cells, and lung cells and are involved in specific functions, like blood clotting. The molecular underpinnings of the disease remain a mystery. AN ANCIENT MOLECULE PRESENTS A NEW OPPORTUNITY: Recently, a molecule called polyphosphate (polyP) has garnered a lot of attention. This molecule is mostly stored in LROs and found in all organisms, from yeast to humans. In humans, polyP accumulates to high concentrations in platelets and perpetuates the blood coagulation cascade to stop bleeding. The novel idea on which this project is based is that defects in polyP biology may be a common variable in HPS subtypes. In this study, the researchers will exploit innovative approaches to polyP biology to uncover novel links to HPS. Since polyP biology is already being targeted for multiple therapeutic purposes, it is hoped that this project will pave the way for the first treatments for HPS patient.