The EryDex System (EDS) is a combination product that is used to load dexamethasone sodium phosphate (DSP) into autologous erythrocytes (EryDex) which is infused into the patient.

In the placebo arm, the subjects will receive autologous erythrocytes prepared with the EDS process using a placebo solution.

Upon completion of all screening assessments for eligibility, subjects meeting all selection criteria at baseline will be randomized in a 1:1 fashion to EryDex or placebo. Approximately 86 subjects 6- to 9-years-old, approximately 43 per group, will be randomized. Approximately 20 subjects 10 years of age and above, 10 per treatment group, may also be enrolled.

Study design: Single arm open-label clinical trial in ataxia-telangiectasia to test the effects of nicotinamide riboside on ataxia scales, immune function, and neurofilament light chain. Study population: 6-10 patients with Ataxia-Telangiectasia. Dose: Nicotinamide riboside 25 mg/kg/day in 3 equal divided doses.

Primary endpoint: Scales for assessment and rating of ataxia (SARA), and International Cooperative Ataxia Rating Scale (ICARS). Improvement of at least ½ standard deviation in key clinical scales which includes either; a) significant improvement in total combined scores from the SARA and ICARS scales, and /or b) significant improvements any aspects of the SARA and ICARS scales individually, especially pertaining to; Postural and gait improvements, Improved syllable speed and articulation, Improved fine motor skills.

Secondary endpoints: Serum analysis of neurofilament light chain (Nfl), Type 1 Interferon (INFs) epigenetic signature

Ataxia Telangiectasia (A-T) is a rare, genetic, progressive, life-limiting, neuro-degenerative condition affecting a variety of body systems resulting in ataxia, immune deficiency, respiratory complications and a predisposition to cancer. Currently there is no cure for A-T.

Over the years, a number of small clinical trials using steroids, antioxidants and anti-inflammatory agents have had little success. The disease natural history is relentless leading to early death. A-T generates a significant disease burden for the individuals, their extended families and on health care resources. With palliative care being the only current option for families, a treatment trial for A-T meets an unmet need. Our group previously demonstrated compelling evidence of reversible mitochondrial dysfunction and preventable cell death in A-T patient cells and the beneficial effects of heptanoate (C7), the primary metabolite of triheptanoin. C7 corrects a defect in endoplasmic reticulum (ER)-mitochondrial signalling in A-T cells and has great potential for application in treating patients. C7 has been utilised with efficacy and safety over the last 15 years for inborn errors of metabolism (IEM) such as long chain fatty acid defects (LC-FAOD).

A-T is due to a genetic defect that results in a defective serine/threonine protein kinase, known as ATM. Normally, ATM, plays a central role in protecting the genome against damage. It is increasingly evident that ATM protects cells against oxidative stress. This protein is also present outside the nucleus, where it is activated by oxidative stress through a separate mechanism from DNA damage, providing an explanation why anti-oxidants have a protective role in A-T cells in culture and in animal models. From these and other studies, it is evident that mitochondrial abnormalities characterise ATM and it has been suggested that A-T should be considered, at least in part, as a mitochondrial disease.

We have added substance to that claim by showing that ATM-deficient (B3) cells are exquisitely sensitive to inhibition of glycolysis by glucose deprivation, compared to controls (HBEC). We have also shown this increased sensitivity to nutrient deprivation for primary epithelial cells from patients and in immortalised patient cells. We demonstrated that this was caused by defective assembly of the VDAC1-GRP75-IP3R1 calcium channel and less ER-mitochondria contact points as determined by transmission electron microscopy. This in turn resulted in reduced calcium release from the ER and less transfer to mitochondria providing further evidence for mitochondrial dysfunction in A-T cells. We have recently completed a Phase 2A/B clinical trial exploring the efficacy and tolerability of C7 in AT patients (https://classic.clinicaltrials.gov/NCT04513002).

Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor for many cellular enzymes, including those involved in mitochondrial biogenesis and maintenance. Nicotinamide adenine dinucleotide exists in two forms, including an oxidized (NAD+) and a reduced (NADH) form, and plays a key role in intermediary metabolism, as obligatory partner in numerous oxidation/reduction reactions. The cellular pool of NAD+ and NADH is tightly regulated through a careful balance between its biosynthesis and its breakdown by NAD+-consuming enzymes. NAD+ deficiency plays a role in disease mechanisms underlying DNA repair disorders. Mitochondrial damage and NAD+ depletion are key features in ataxia telangiectasia.

ATM-deficient mice have neuronal NAD+ deficiency, in particular in the cerebellum.

Fang et al. have demonstrated that mitochondrial dysfunction in ATM deficiency is linked to NAD+/SIRT1 inhibition. NAD+ replenishment significantly extends lifespan and improves health span in both ATM worms and mice through mitophagy and DNA repair. Treatments that replenish intracellular NAD+ reduce the severity of A-T neuropathology, normalize neuromuscular function, delay memory loss, and extend lifespan in both animal models. Mechanistically, treatments that increase intracellular NAD+ also stimulate neuronal DNA repair and improve mitochondrial quality via mitophagy.

Immune deficiency is common in AT, with most patients have humoral and cellular immune defects comprising immunoglobulin-A deficiency, immunoglobulin-G2 and immunoglobulin-G deficiency, and lymphopenia with low numbers of total and naive CD4 T cells. About 10% of patients with classic ataxia-telangiectasia present with hypogammaglobulinaemia with normal or raised immunoglobulin-M levels and follow a severe disease course. Recognition of foreign or misplaced nucleic acids is one of the principal modes by which the immune system detects pathogenic entities. When cytosolic DNA is sensed, a signal is relayed via the cGAS-STING pathway.

ATM deficient cells display elevated levels of INF- induced proteins, a feature also reported in sera of A-T patients. A double knockout of ATM and STING genes in mice attenuated autoinflammatory phenotypes, which was further decreased when the cGAS gene is also deleted in these mice. Inhibition of the cGAS-STING pathway ameliorates the premature senescence phenotype in AT brain organoids. Similar inflammatory manifestations are seen in patients with STING-associated vasculopathy in infancy which is an autosomal dominant type 1 interferonopathy.

Two groups have explored Nicotinamide Riboside (NR) supplementation in small groups of A-T patients via single arm, open label access, proof-of-concept clinical trials. Both have demonstrated improvements in validated ataxia scales. Improvements in immunoglobulin-G (IgG) levels were observed, no alterations were noted in NFlc. Improvements were lost in the wash out period. NR was well tolerated with no reported adverse events.

This is a single arm open-label clinical trial in ataxia-telangiectasia to test the effects of nicotinamide riboside on ataxia scales, immune function, and neurofilament light chain.

Dose will be via oral capsule supplementation at 25mg/kg/day divided into 3 doses (max 300mgs 3 times per day). Dosing will occur via 3 equal doses 3 times a day.

Primary efficacy endpoint: Improvement of at least ½ standard deviation in key clinical scales which includes either; a) significant improvement in total combined scores from the SARA and ICARS scales, and /or b) significant improvements any aspects of the SARA and ICARS scales individually, especially pertaining to; Postural and gait improvements, Improved syllable speed and articulation, Improved fine motor skills.

Secondary endpoints include: Serum analysis of neurofilament light chain (Nfl). Type 1 Interferon (INFs) epigenetic signature specifically the cGAS-STING pathway.

Safety endpoints: Treatment-related adverse events, Routine haematology and biochemical analyses, Paediatric Epilepsy Side Effects Questionnaire (PESQ), Regular clinical assessments.

Ataxia Telangiectasia (AT) is a genetic disease, where patients are born with mutations in the Ataxia- Telangiectasia Mutated (ATM) gene. The gene codes for the ATM kinase, which is required for repair of DNA double-stranded breaks and DNA damage response signalling.

There is no treatment available for the neurological manifestations of AT.

The study investigates the effects of NR (300 mg/day) during 2 years.

Ataxia Telangiectasia (A-T), a life shortening autosomal recessive multisystem disease, is caused by mutations in the ATM gene, and affects 1/300,000 live births. The pathophysiological basis of the disease relates to mutations in the ATM kinase gene that result in a faulty repair of breakages in double-stranded DNA. A-T causes a progressive ataxia, dystonia, and movement disorder, and is complicated by immunodeficiency, lung disease, faltering growth, limb and spinal deformities, and increased susceptibility to cancers. The disease first manifests in early childhood with balance problems and is slowly progressive, with loss of control of limb and eye movements, and speech, through the school years, resulting in severe disability by adulthood. Patients with classical A-T rarely live beyond their 20s, and there is no effective treatment. Because it is so rare, little has been published on the natural history of the condition.

The Department of Health funds the national multidisciplinary clinic for children with A-T in Nottingham, with follow-up of adults at the Royal Papworth Hospital, Cambridgeshire. Clinical and laboratory data have been collected systematically from over 170 patients, most on more than one occasion, from 2001 to date. This unique clinical collection will allow us to elucidate the natural history of A-T using a mixture of cross-sectional and longitudinal analyses, from presentation in the pre-school years to adulthood.

We will discover more about the presentation and diagnosis of A-T, and the detailed evolution of the disease as children grow to adulthood. Preliminary analyses of the neurological phenotype in 134 patients has shown important genotype-phenotype relationships and a more variable deterioration over time than anticipated, which will be investigated further. In parallel, investigation of the morbidity and mortality due to deterioration in lung function, the immune system, infections, cancers, malnutrition, and skeletal deformity will be undertaken. We will learn about the interactions between the various disease manifestations, e.g. lung disease and nutritional status, immunological impairment and neurology and risk of cancers. These discoveries will inform patient care, and help to generate hypotheses for further research projects. From the analysis of the cross-sectional data we will establish an initial Core Outcome Set, which will be further developed at Patient and Public Involvement (PPI) meetings with young people with A-T and their parents/carers.

This study is needed to accurately map the course of A-T from childhood to adulthood, to help us recognise different patterns of disease in individual patients, improve early diagnosis by understanding the reasons for diagnostic delay, anticipate complications more accurately and elucidate the best ages to intervene before rapid deterioration.

The expected clinical course involves deterioration in gross and fine motor skills, oculomotorpraxia, choreoform movements, and difficulty with chewing and swallowing. By the end of their first decade children are usually wheelchair dependent as cerebellar destruction progresses and further neurodisability ensues with development of incapacitating movement disorder and peripheral neuropathy. Sinopulmonary infections and recurrent lower respiratory tract infections with bronchiectasis are common in A-T, and current management aims to avoid and treat lung infection. The treatment for malignancy must take into consideration the fact that many oncological therapies have adverse-effects that will impact on individual function in other domains for example drugs that cause muscle weakness will exacerbate motor impairments. Also the DNA of patients with A-T is particularly susceptible to double strand breakage, which can itself cause secondary malignancies.

Understanding the natural history and its variability is not only vital to planning effective patient-centred services, and counselling patients and their families, but will also inform the design of future clinical research, particularly clinical trials. The choice of age group to include in a trial, duration of treatment, and choice of core outcome measures will depend on the results of this natural history study.

This study will centre on data analysis and will not involve any therapeutic interventions, although it is intended that the results will enable future research to determine optimum therapeutic strategies. The benefits of this study thus relate to the compilation of vast amounts of data that have been gathered on A-T over the last 15 years and extracting meaningful information that will inform future care. There is no identifiable risk from inclusion in the study as it involves patient data that are gathered through standard clinical interaction and does not involve introducing or changing treatments or any increased patient interaction apart from voluntary participation of a sample of parents / guardians and young people with A-T in the PPI focus group meetings, for which informed consent will be sought.

There are no established treatments for A-T. Symptomatic treatments are used in patients depending on their symptoms, e.g. gastrostomy feeds are used when eating becomes too slow, or too difficult because of swallowing difficulties. Antibiotics and chest physiotherapy are used to prevent and treat chest infections. Immunodeficiency can be treated in selected cases, as can the various complications of A-T as and when they arise, including scoliosis, cancers, granulomas, movement disorders and spasticity.

Current therapy in A-T is aimed at mitigating neurological deterioration, prevention and treatment of respiratory complications, early identification and treatment of malignancy whilst minimising adverse-effects and treating immunodeficiency with immunoglobulin, in other words, managing the downstream complications of the gene mutation. Each aspect and complication requires specialist assessment and input and monitoring of progression and response to treatment. A-T is managed from a multi-speciality and multi-disciplinary perspective and in Nottingham this happens in the setting of the National A-T Clinic that children attend with their families every two years. Neurological progression is determined using the A-T Index and AT-NEST scoring systems. Assessment by a clinician with paediatric respiratory expertise also occurs alongside input from a neurologist, immunologist, geneticist, dietician, occupational and physiotherapist, clinical psychologist and speech and language therapist. One of the aims of this natural history study is to avoid complications developing by elucidating the optimal timing of treatment interventions.

Respiratory disease patterns in A-T include sinopulmonary disease and bronchiectasis, interstitial lung disease and lung disease associated with neuromuscular, especially bulbar, dysfunction. At present treatment depends on the pattern of lung disease and can range from antibiotics, immunoglobulin replacement, systemic steroids, chest physiotherapy, and steps to limit aspiration. Assessment of response to treatment includes assessment of resolution or persistence of symptoms, spirometry measurements, and imaging such as chest x-rays and where indicated, CT scans, although limiting exposure to radiation is always a factor to consider in patients with A-T.

There have been several small scale pilot trials of medication for the neurological impairments with mixed success, including the use of amantidine5, betamethasone6,7, and dexamethasone loaded autologous redcells8.

Potential but as yet untested treatments include anti-oxidants which have the potential to slow down progression of neurodegeneration by protecting Purkinje cells in the cerebellum from oxidative stress caused by free radicals. ATM has a role in the inhibition of oxidative stress and absence or reduced function of ATM could result in increased oxidative stress and accelerated neuronal loss.11

However, all these suggestions in fact impact the downstream consequences of the mutations. In cystic fibrosis, another multisystem genetic disease which largely impacts the respiratory system, the treatment paradigm has shifted from downstream treatment of the consequences of gene dysfunction, to gene therapy and small molecule treatments addressing the basic defect. It is likely that in the near future A-T will follow this paradigm, e.g. with the development of drugs to allow read-through of premature termination codons, or the skipping of mutations, or the incorporation of functioning genetic sequences into the genome. These and other, as yet unknown, therapeutic approaches will require carefully designed clinical trials, and this natural history study will inform the design of such future trials.

The study will allow a systematic approach to A-T and an understanding of the progression of respiratory, neurological, immunological and oncological disease that underline this multi-systemic disorder, as well as the impact of growth impairment. It will enable evaluation of procedures and medical treatments as well as surgical therapies. There will be insight into the psychological impact of the illness of children and their families and whether there is a particular period where increased or additional support needs to be offered.

As discussed earlier, a critical outcome will be determining the timing of appropriate therapeutic interventions to prevent complications and delay progression of disease, as well as the potential to inform the design of future clinical trials involving therapies for A-T. As a natural history study there will be scope to extract information about A-T within all domains and identify any important variables in treatment, identify whether timing of treatment needs adjustment for maximal impact, for example, by earlier introduction of therapy there may be slower progression of disease or fewer complications.

A possible negative consequence is that the information gleaned might be of little immediate clinical use, however we do not think that will be the case. We think that the data will be valuable in highlighting evidence gaps and informing new studies. We may find that there are inconsistencies in treatments offered to patients and which will encourage us to try to rationalise and streamline our clinical assessments and advice.

Study design: Parallel group, placebo-controlled, dose-escalation each 2 months for 12 months. Dose based on percent (%) of calculated caloric intake. Thirty participants will be randomised in blocks on a 1:1:1 ratio into one of three groups stratified by age (< 5 years, 5-10 years, > 10 years of age). Group 1: 10%, 20%, 35%, 35%, 35% (no placebo). Group 2: placebo, 10%, 20%, 35%, 35% Group 3: placebo, placebo, 10%, 20%, 35%.

Primary endpoint: The percent cell death induced by glucose deprivation in cell culture. Secondary endpoints include: Scales for assessment and rating of ataxia, International Cooperative Ataxia Rating Scale, Ataxia Telangiectasia Neurological Examination Scale Toolkit, speech and language assessment, EyeSeeCam assessment, MRI lung imaging, Lung function, Upper respiratory microbiome, Faecal microbiome, Survival and inflammatory phenotype of airway epithelial cells, macrophages and in serum, Metabolomic biomarker discovery in serum and measurement of neuroflament light chain.

Ataxia Telangiectasia (A-T) is a rare, genetic, progressive, life-limiting, neuro-degenerative condition affecting a variety of body systems resulting in ataxia, immune deficiency, respiratory complications and a predisposition to cancer. Currently there is no cure for A-T. Over the years, a number of small clinical trials using steroids, antioxidants and anti-inflammatory agents have had little success. The disease natural history is relentless leading to early death. A-T generates a significant disease burden for the individuals, their extended families and on health care resources. With palliative care being the only current option for families, a treatment trial for A-T meets an unmet need. The investigators preliminary data provide compelling evidence of reversible mitochondrial dysfunction and preventable cell death in A-T patient cells and the beneficial effects of heptanoate (C7), the primary metabolite of triheptanoin. C7 corrects a defect in endoplasmic reticulum (ER)-mitochondrial signalling in A-T cells and has great potential for application in treating participants. C7 has been utilised with efficacy and safety over the last 15 years for inborn errors of metabolism (IEM) such as long chain fatty acid defects (LC-FAOD).

A-T is due to a genetic defect that results in a defective serine/threonine protein kinase, known as ATM. Normally, ATM, plays a central role in protecting the genome against damage. It is increasingly evident that ATM protects cells against oxidative stress. This protein is also present outside the nucleus, where it is activated by oxidative stress through a separate mechanism from DNA damage, providing an explanation why anti-oxidants have a protective role in A-T cells in culture and in animal models. From these and other studies, it is evident that mitochondrial abnormalities characterise ATM and it has been suggested that A-T should be considered, at least in part, as a mitochondrial disease. The Investigators have added substance to that claim by showing that ATM-deficient (B3) cells are exquisitely sensitive to inhibition of glycolysis by glucose deprivation, compared to controls (HBEC). The investigators have also shown this increased sensitivity to nutrient deprivation for primary epithelial cells from patients and in immortalised patient cells. Together these data point to a reduced capacity of A-T mitochondria to support energy metabolism and provide additional evidence for a mitochondrial defect in A-T cells. The investigators have recently demonstrated that this hypersensitivity to glucose deprivation can be explained by a novel mechanism involving defective signalling between the ER and the mitochondrion. The investigators demonstrated that this was caused by defective assembly of the VDAC1-GRP75-IP3R1 calcium channel and less ER-mitochondria contact points as determined by transmission electron microscopy. This in turn resulted in reduced calcium release from the ER and less transfer to mitochondria providing further evidence for mitochondrial dysfunction in A-T cells.

The investigators selected triheptanoin, a highly purified, synthetic medium odd-chain triglyceride that is catabolized to heptanoate and can traverse the mitochondrial membrane without the carnitine carrier. Free heptanoate is then metabolized by the medium chain fatty acid oxidation enzymes to yield both acetyl CoA and propionyl CoA that act as anaplerotics to replenish the TCA cycle and enhance energy metabolism by providing NADPH and generating ATP. The investigators demonstrated that heptanoate partially corrects the extreme sensitivity to glycolysis inhibition in both the ATM-deficient cell line as well as in primary epithelial cells from a patient with A-T. Excitingly, heptanoate also corrected all of the defects in ER-mitochondrial signalling including calcium uptake into mitochondria. Based on the importance of mitochondrial dysfunction in the A-T phenotype and our results revealing correction of mitochondrial function by heptanoate, the investigators consider that triheptanoin has excellent potential in correcting many aspects of the A-T phenotype including the progressive neurodegenerative phenotype.

Triheptanoin has been used for over 15 years to treat LC-FAOD, with demonstrated improvements in cardiac function and reductions in rhabdomyolysis episodes. Triheptanoin and heptanoate are known to protect against cell death in experimental conditions largely characterised by oxidative stress, such as stroke and motor neurone disease, adult polyglucosan body disease, alternating hemiplegia of childhood, Glucose-1 transporter deficiency, and mouse models and humans with epilepsy. Heptanoate protects cultured neurons against H2O2-induced cell death. Collectively these studies demonstrate that triheptanoin is well tolerated and is effective in treating a range of neurological conditions associated with neuronal energy deficiency.

Seamless Phase II to Phase III go/no-go criteria Interim monitoring for the intervention program in the A-T2020/01 trial will occur at the times of the two interim analyses (first, when the study cohort has completed the initial 2 months treatment, and second, after 6 months treatment when Group One has completed 2 months of the 35% dose). A blinded report will be presented to the iDSMB containing pertinent descriptive statistics of the groups, a standard between-group comparison for the primary and secondary outcomes, and a Bayesian estimation of the (posterior) probability that each of the three intervention groups is superior for the primary outcomes. The information to be presented to the iDSMB will be agreed with the iDSMB prior to the first iDSMB meeting, and will be updated at the time of the iDSMB meetings. Data will be presented to the iDSMB in a blind fashion, but the iDSMB can request unblinded data to confirm or ratify any reported interim results. The iDSMB may however make a recommendation about stopping current interventions if they show poor promise or futility.

The primary endpoints for interim iDSMB reports are the percent cell death induced by glucose deprivation in cell culture, and reversal/correction of their abnormal mitochondrial profile in primary epithelial cells resulting in cell death over the treatment period.

Secondary scales clinical neurological assessments assist formulating the go/no-go criteria and will include: SARA and ICARS. SARA is a validated cerebellar ataxia tool, measuring gait, stance, sitting, speech, finger-chase test, finger nose-test, fast alternating movements and heel-shin test. It has eight categories with accumulative score ranging from 0 (no ataxia) to 40 (most severe ataxia); Gait (0-8 points), Stance (0-6 points), Sitting (0-4 points), Speech disturbance (0-6 points), Finger chase (0-4 points), Nose-finger test (0-4 points), Fast alternating hand movement (0-4 points), Heel-shin slide (0-4 points). ICARS is a scale recorded out of 100 with 19 items and 4 subscales and has been used in A-T. Disorders rated as subscales within the ICARS are: Postural and gait disturbances, (7 items, 0-34 points) Limb Ataxia (7 items, 0-52 points), Dysarthria (2 items, 0-8 points), and Oculomotor disorders (3 items, 0-6 points). Minimum Score: 0 Maximum score: 100.

go/no-go triggers

go triggers Seamless progress from Phase II to Phase III will be triggered under the following pre-set parameters;

• If clinically or statistically significant improvement in the primary study outcome is observed in combination with a measured improvement of at least ½ standard deviation in key clinical scales which includes either;
• significant improvement in total combined scores from the SARA and ICARS scales.
• And/or significant improvements any aspects of the SARA and ICARS scales individually, especially pertaining to; Postural and gait improvements, Speech disturbance, Improved fine motor skills, Fine motor disturbance, Kinetic functions

No-go triggers Seamless progress from Phase II to Phase III will not occur under the following pre-set parameters;

• Adverse events
• If no clinically significant improvement in the primary study outcome is observed
• If a clinically significant improvement in the primary study outcome occurs without any improvements in key secondary scales specifically the SARA and ICARS.

Ataxia Telangiectasia (A-T) is an inherited disorder characterised by cerebellar neurodegeneration, immunodeficiency and respiratory disease. People with A-T have abnormal DNA repair and consequently have an increased risk of cancer. Despite this, current guidelines for management of children and young people with A-T do not include cancer surveillance.

Improvements in MRI technology have allowed whole-body MRI (WB-MRI) scanning with relatively short acquisition times. Currently, WB-MRI protocols are used for diagnosing and monitoring some primary and secondary cancers, including cancer surveillance in people with the Li-Fraumeni syndrome, which is another genetic cancer predisposition syndrome. Therefore, the research team believe that whole-body MRI provides a safe method for cancer surveillance in children and young people with A-T. However, the investigators do not know whether cancer surveillance in children and young people with A-T using whole-body MRI is feasible and desirable.

The research team proposes a feasibility study of MRI-based cancer surveillance with qualitative evaluation of participant experience with the primary aim to establish:

• feasibility of whole-body MRI for cancer surveillance in children and young people with A-T
• views of, and psychological impact on, participants and families / carers participating in whole-body MRI for cancer surveillance.
• feasibility of conducting a formal screening trial in terms of statistical design, sample size, screening interval, comparator arms and international collaboration Completion of this study will provide us with evidence of technical feasibility, very strong evidence of child / family views, a viable formal screening trial design and an engaged international research community, allowing us to proceed to a formal trial establishing the efficacy of a cancer surveillance programme for children and young people with A-T.

Ataxia Telangiectasia (A-T) is an autosomal recessive disorder characterised with cerebellar neurodegeneration, immunodeficiency, respiratory disease and cancer susceptibility (22% by age 20). A-T is a complex disorder caused by mutations in the Ataxia Telangiectasia Mutated (ATM) gene which creates highly unstable ATM protein fragments. In the classical clinical presentation of A-T there is no kinase activity due to the absence of ATM protein. One consequence of this is that cellular DNA-repair pathways are compromised which allows breaks in DNA strands leading to genomic instability and/or cell death. As a result, there is an increase sensitivity to ionizing radiation and elevated cancer risk. Children less than 16 years with classical A-T demonstrate a predisposition to the development of different types of lymphoid cancers (lymphoma and leukaemia). However non-lymphoid tumours can also occur.

The current practice at the United Kingdom Paediatric A-T clinic for cancer surveillance is to perform blood testing, including full blood account, liver function tests and measurement of circulating tumour marker Alpha-fetoprotein. Full blood account can detect an increase white cell count and abnormal white cell populations and therefore, is effective in detecting most types of leukaemia. Elevation of Alpha-Fetoprotein can be a marker of different types of tumours, for example hepatocellular carcinoma and liver metastases, however is also often elevated in people with A-T. Therefore, its use for detecting cancer in A-T is uncertain and not currently evidence-based. The lack of evidence-based guidelines regarding the optimal cancer surveillance strategy in children with A-T could delay the diagnosis and consequently the treatment plan.

Magnetic Resonance Imaging (MRI) technology has progress rapidly since its discovery, making it possible currently to perform whole-body MRI scans with relatively short acquisition times. Presently, whole-body MRI protocols that include diffusion-weighted imaging and structural (T1/Dixon) acquisitions are used for diagnosing and monitoring cancers (sarcomas, metastases and haematological tumours like myeloma). A further advantage of this technique compared to computed tomography is that does not involve radiation, which is important given the elevated radiosensitivity of people with A-T.

The value of whole-body MRI in people with cancer predisposition has been shown in Li-Fraumeni syndrome, syndrome that, like A-T, is associated with increased risk of haematological and solid tumours. Despite the successful results of using a multimodality protocol for cancer surveillance in Li-Fraumeni syndrome, it cannot be assumed that the results will be the same in people with A-T as the also have respiratory and neurological dysfunction that could influence the tolerability and image quality of the MRI scans. Also, the profile of the tumours differs between these two syndromes. To date, the value of whole-body MRI for cancer surveillance in children and young people with A-T has not been demonstrated. It is unknown whether is possible to obtain diagnostic images, and the spectrum of findings (both cancer and non-cancer) that can be detected has not been reported.

In addition to technical considerations, it is vital to understand the perspectives of people affected by A-T regarding cancer surveillance. Participation in a cancer surveillance programme could increase anxiety for both the participant and family, and it is possible that negative psychological aspects could outweigh the perceived benefits or earlier cancer detection. Compared to other screening programmes, it should be noted that these families are already having to deal with a family member with complex progressive chronic disease, and so emotional and psychological capacity to deal with added anxiety may be limited. Hence, it is important to know, before conducting a full scale trial that will inform the current lack of evidence-based guidelines, whether cancer surveillance in children and young people with A-T using whole-body MRI is feasible and desirable.

This study aims to investigate the link between the Ataxia Telangiectasia Mutated (ATM) gene and metformin response. This link has been identified from large studies of the human genome, and this study aims to confirm this link in a clinical study. The ATM gene is involved in DNA repair - if a person inherits a "faulty" copy of this gene from both their parents, they have a genetic condition called Ataxia-telangiectasia (A-T).

A-T is associated with, among other things, a resistance to insulin, which causes fatty liver and diabetes. This study will recruit people who have A-T, but have not developed diabetes, and compare this group to "healthy" controls, i.e. people who do not have A-T or diabetes. The study will compare how the groups respond to two drugs used to treat diabetes (metformin and pioglitazone), with the intention that this will guide the management of diabetes in A-T.

This is an, open label unblinded study recruiting 15 people with A-T and 15 age and gender matched controls. Each participant will have three study visits to the Clinical Research Centre at Ninewells hospital in Dundee - one at baseline, a second after 8 weeks of metformin and the final visit after eight weeks of pioglitazone. During each visit we will carry out a number of investigations to study the insulin resistance of A-T and how it responds to metformin and pioglitazone.

Metformin is a commonly-prescribed drug, used as first-line medical management of type 2 diabetes mellitus (T2DM), but also off-license in non-diabetics for Polycystic Ovary Syndrome (PCOS). Over 120 million people worldwide are prescribed metformin. Despite this, its mechanism of action is not fully understood. Both tolerance of and response to metformin varies greatly from patient to patient, and highlights the need for further research into the pharmacokinetics and dynamics of the drug.

As a team of diabetes researchers, our group have been interested in the genetics of drug response in diabetes. A Genome Wide Association Study carried out by members of our group highlighted the locus carrying the ATM gene as a potential link to metformin response. We have designed this study to investigate this link clinically. In doing so we hope to guide the management of diabetes in a condition called Ataxia Telangiectasia.

ATM (Ataxia Telangiectasia Mutated) is a gene involved in DNA repair - homozygous recessive mutations in this gene cause Ataxia Telangiectasia (A-T), which is associated with cerebellar ataxia, ocular telangiectasia and lymphoproliferative cancers. The incidence of A-T is between 1 in 40,000 to 1 in 100,000 live births, though this increases dramatically with consanguineous parents. Interestingly, A-T has also been associated with insulin resistance. Both "fatty liver" and diabetes have been documented in this patient group, but there is little research into the link between A-T and these conditions. Approximately 1 in 100 people are carriers of a loss of function mutation in the ATM gene, which is associated with an increased risk of ischaemic heart disease and certain cancers.

Several studies of ATM deficiency in a mouse model have been carried out. Atm -/- mice display an early defect in glucose-stimulated insulin release and later develop hyperglycaemia, and insulin resistance[1]. Unpublished data from the McCrimmon Group at University of Dundee suggest that mice heterozygous for ATM deficiency have impaired fasting glucose, but demonstrated a marked improvement in fasting glucose with metformin. A Cell Reports paper, detailing a mouse model of ATM deficiency, demonstrates insulin resistance in ATM deficient mice, with a lipodystrophic phenotype[2]. This phenotype (paucity of subcutaneous fat, and increased visceral fat) was attenuated by metformin and thiazolidinedione (TZD) use.

A small study published by the Pearson group from University of Dundee, which compared data from oral glucose tolerance tests (OGTTs) in A-T patients versus healthy individuals, confirmed increased insulin resistance in A-T patients[3]. As mentioned above, a genome wide association study, also by the Pearson group, highlights the ATM locus as a potential genetic link to metformin response[4, 5]. With the mouse models and information from genetic studies, this study now aims to assess insulin resistance in this A-T patient group, and to understand how they respond to drugs commonly used to treat insulin resistance / diabetes.

The potential genetic link between ATM and metformin response will be investigated. Thus far it is unclear from the genetic studies how ATM deficiency affects an individual's metformin response - is their response greater or less than that of the general population? Similarly, magnitude of this response has not been quantified. There are anecdotal reports of patients with A-T who have had a marked improvement in glycaemic control with metformin use. Mouse model studies have indicated an increased response to metformin in heterozygous ATM deficient mice.

The study will also investigate the response of individuals with A-T to TZDs. Studies of the mouse model of A-T has demonstrated response to the TZD pioglitazone,. Of note, pioglitazone is now used off-licence in non-diabetics for the treatment of Non-Alcoholic Fatty Liver Disease (NAFLD) [6, 7], therefore we know it is safe for use in a non-diabetic cohort.

This study will assess the effect of ATM deficiency on metformin and pioglitazone response in humans, by studying people with A-T, and comparing their response to that of a matched control group.

This study will recruit non-diabetic individuals - 15 cases (people with A-T) vs 15 age and gender matched controls - in a crossover design. Participants will be 18 - 30 years of age. This age group is most realistic for recruiting patients with "classic", as opposed to "mild variant" A-T, as people with classic A-T rarely survive to their 30s. Exclusion of individuals with other milder forms of A-T will provide a more detectable difference between the two cohorts. Participants will be of white European descent, as this will narrow the genetic differences between individuals. Ethnic origin also has an effect on an individual's insulin resistance, therefore all the participants should be of the same ethnicity.

The study is made up of two treatment periods each lasting eight weeks, and separated by a one week washout period. Initial treatment shall be with metformin, titrated to 1000mg twice daily. The second treatment will be pioglitazone titrated to 30mg once daily.

The study will last a total of approximately 17 weeks, and involves three visits to the Clinical Research Centre at Ninewells hospital in Dundee. The first visit will be the longest, lasting 1.5 days, and the other two visits last one full day with a short preparatory visit for 30 minutes the day before.

Multiple methods will be used to investigate the relationship between ATM, diabetes and drug response:

• Dual tracer mixed meal tests with indirect calorimetry (see Tracer Studies SOP)
• MRI (see MRI SOP)
• Blood and urine sampling (see Sample Collection SOP)
• Fat biopsy (see Fat Biopsy SOP)

In summary, each individual will have three tracer studies: an initial study at baseline; a second after eight weeks of metformin; and a final tracer study after eight weeks of the TZD, pioglitazone. Tracer studies involve a standardised meal the night before the study, and fasting from midnight in preparation. No alcohol should be consumed for 24 hours before the study. On the day of the tracer study two cannulae will be inserted, one in each forearm. One cannula will be used to infuse a stable glucose tracer [6,6-2H2] for the duration of the eight hour study. After two hours of this infusion, the participant will be fed a mixed meal containing [U-13C] glucose (stable). The participant is observed for a further six hours, while the [6,6-2H2] glucose infusion continues. Throughout the tracer study, blood will be taken from the second cannula at multiple time-points, to allow for measurement of glucose (including the tracers), insulin, C-peptide, glucagon, glucagon-like peptide 1 (GLP-1) and nonesterified fatty acids (NEFAs). A total of 150ml of blood will be taken during the tracer study. Urine is collected in two phases (before and after the meal) during the tracer study, for the measurement of glucose excretion. Indirect calorimetry will be used for twenty minute episodes at several time-points to measure substrate utilisation during the study. This involves the participant wearing a "hood" which is lightweight, and has a see-through visor. All of these measurements will enable us to model glucose fluxes in the participant and calculate indices of insulin sensitivity. Repetition of the tracer studies on two anti-hyperglycaemic agents will provide comparison of these indices on and off treatment.

At the baseline visit an MRI, blood sampling and fat biopsy will take place during the half day visit before the tracer study. This will allow us to assess fat distribution using MRI and obtain adipose tissue to carry out lab-based studies to assess the adipocyte function and response to metformin and pioglitazone. Blood samples are taken for "safety bloods" (i.e. to ensure normal renal function and HbA1c <48mmol/mol) but also for future DNA analysis, to confirm the diagnosis in the A-T group, and to check for carrier status of the controls.

Visits two and three involve a full day at the clinical research centre (CRC) for a tracer study. On the day before the tracer, a short half-hour visit to obtain "safety bloods" (in this case, to check renal function) and provide the standardised meal is necessary to prepare for the tracer study the following day.

If this study can clinically confirm the hypothesis that individuals with ATM-deficiency respond well to either metformin or pioglitazone, and individuals show marked improvement in insulin resistance while taking either study drug, this study could direct clinical decision-making in the care of patients with A-T and fatty liver / insulin resistance.

In conjunction with the clinical studies cell experiment studies will be carried out on induced pluripotent stem cell (IPSC) derived hepatocytes from individuals with A-T, to assess drug response at a cellular level. This will be a collaboration with the Sanger Institute in Cambridge, where they have already developed IPSC-derived hepatocytes from the blood of A-T patients. These cells will be used to create a drug response model at a cellular level. These cell lines are not from our recruited patients directly, but serve as a cellular model of A-T. However, we will offer the A-T group the chance to donate blood to the INSIGNIA study, run by the Sanger Institute, which is a study focused on the investigation of patterns of mutations (signatures) in inherited and other progressive genetic diseases (please see INSIGNIA PIS and consent forms). This is optional and taking part in RAMP does not commit those with A-T to contributing to INSIGNIA.