Specifications of the ACMG/AMP variant curation guidelines for the analysis of germline ATM sequence variants.

The ClinGen Hereditary Breast, Ovarian, and Pancreatic Cancer (HBOP) Variant Curation Expert Panel (VCEP) is composed of internationally recognized experts in clinical genetics, molecular biology, and variant interpretation. This VCEP made specifications for the American College of Medical Genetics and Association for Molecular Pathology (ACMG/AMP) guidelines for the ataxia telangiectasia mutated (ATM) gene according to the ClinGen protocol. These gene-specific rules for ATM were modified from the ACMG/AMP guidelines and were tested against 33 ATM variants of various types and classifications in a pilot curation phase. The pilot revealed a majority agreement between the HBOP VCEP classifications and the ClinVar-deposited classifications. Six pilot variants had conflicting interpretations in ClinVar, and re-evaluation with the VCEP's ATM-specific rules resulted in four that were classified as benign, one as likely pathogenic, and one as a variant of uncertain significance (VUS) by the VCEP, improving the certainty of interpretations in the public domain. Overall, 28 of the 33 pilot variants were not VUS, leading to an 85% classification rate. The ClinGen-approved, modified rules demonstrated value for improved interpretation of variants in ATM.

Reduced levels of MRE11 cause disease phenotypes distinct from ataxia telangiectasia-like disorder.

The MRE11/RAD50/NBS1 (MRN) complex plays critical roles in cellular responses to DNA double-strand breaks. MRN is involved in end binding and processing, and it also induces cell cycle checkpoints by activating the ataxia-telangiectasia mutated (ATM) protein kinase. Hypomorphic pathogenic variants in the MRE11, RAD50, or NBS1 genes cause autosomal recessive genome instability syndromes featuring variable degrees of dwarfism, neurological defects, anemia, and cancer predisposition. Disease-associated MRN alleles include missense and nonsense variants, and many cause reduced protein levels of the entire MRN complex. However, the dramatic variability in the disease manifestation of MRN pathogenic variants is not understood. We sought to determine if low protein levels are a significant contributor to disease sequelae and therefore generated a transgenic murine model expressing MRE11 at low levels. These mice display dramatic phenotypes including small body size, severe anemia, and impaired DNA repair. We demonstrate that, distinct from ataxia telangiectasia-like disorder caused by MRE11 pathogenic missense or nonsense variants, mice and cultured cells expressing low MRE11 levels do not display the anticipated defects in ATM activation. Our findings indicate that ATM signaling can be supported by very low levels of the MRN complex and imply that defective ATM activation results from perturbation of MRN function caused by specific hypomorphic disease mutations. These distinct phenotypic outcomes underline the importance of understanding the impact of specific pathogenic MRE11 variants, which may help direct appropriate early surveillance for patients with these complicated disorders in a clinical setting.

Contribution and therapeutic implications of retroelement insertions in ataxia telangiectasia.

Certain classes of genetic variation still escape detection in clinical sequencing analysis. One such class is retroelement insertion, which has been reported as a cause of Mendelian diseases and may offer unique therapeutic implications. Here, we conducted retroelement profiling on whole-genome sequencing data from a cohort of 237 individuals with ataxia telangiectasia (A-T). We found 15 individuals carrying retroelement insertions in ATM, all but one of which integrated in noncoding regions. Systematic functional characterization via RNA sequencing, RT-PCR, and/or minigene splicing assays showed that 12 out of 14 intronic insertions led or contributed to ATM loss of function by exon skipping or activating cryptic splice sites. We also present proof-of-concept antisense oligonucleotides that suppress cryptic exonization caused by a deep intronic retroelement insertion. These results provide an initial systematic estimate of the contribution of retroelements to the genetic architecture of recessive Mendelian disorders as ∼2.1%-5.5%. Our study highlights the importance of retroelement insertions as causal variants and therapeutic targets in genetic diseases.

Histone Ubiquitination by the DNA Damage Response Is Required for Efficient DNA Replication in Unperturbed S Phase.

Chromatin ubiquitination by the ubiquitin ligase RNF168 is critical to regulate the DNA damage response (DDR). DDR deficiencies lead to cancer-prone syndromes, but whether this reflects DNA repair defects is still elusive. We identified key factors of the RNF168 pathway as essential mediators of efficient DNA replication in unperturbed S phase. We found that loss of RNF168 leads to reduced replication fork progression and to reversed fork accumulation, particularly evident at repetitive sequences stalling replication. Slow fork progression depends on MRE11-dependent degradation of reversed forks, implicating RNF168 in reversed fork protection and restart. Consistent with regular nucleosomal organization of reversed forks, the replication function of RNF168 requires H2A ubiquitination. As this novel function is shared with the key DDR players ATM, γH2A.X, RNF8, and 53BP1, we propose that double-stranded ends at reversed forks engage classical DDR factors, suggesting an alternative function of this pathway in preventing genome instability and human disease.

Phosphoproteomics reveals novel modes of function and inter-relationships among PIKKs in response to genotoxic stress.

The DNA damage response (DDR) is a complex signaling network that relies on cascades of protein phosphorylation, which are initiated by three protein kinases of the family of PI3-kinase-related protein kinases (PIKKs): ATM, ATR, and DNA-PK. ATM is missing or inactivated in the genome instability syndrome, ataxia-telangiectasia (A-T). The relative shares of these PIKKs in the response to genotoxic stress and the functional relationships among them are central questions in the genome stability field. We conducted a comprehensive phosphoproteomic analysis in human wild-type and A-T cells treated with the double-strand break-inducing chemical, neocarzinostatin, and validated the results with the targeted proteomic technique, selected reaction monitoring. We also matched our results with 34 published screens for DDR factors, creating a valuable resource for identifying strong candidates for novel DDR players. We uncovered fine-tuned dynamics between the PIKKs following genotoxic stress, such as DNA-PK-dependent attenuation of ATM. In A-T cells, partial compensation for ATM absence was provided by ATR and DNA-PK, with distinct roles and kinetics. The results highlight intricate relationships between these PIKKs in the DDR.

Inhibition of ATM kinase rescues planarian regeneration after lethal radiation.

As stem cells divide, they acquire mutations that can be passed on to daughter cells. To mitigate potentially deleterious outcomes, cells activate the DNA damage response (DDR) network, which governs several cellular outcomes following DNA damage, including repairing DNA or undergoing apoptosis. At the helm of the DDR are three PI3-like kinases including Ataxia-Telangiectasia Mutated (ATM). We report here that knockdown of ATM in planarian flatworms enables stem cells to withstand lethal doses of radiation which would otherwise induce cell death. In this context, stem cells circumvent apoptosis, replicate their DNA, and recover function using homologous recombination-mediated DNA repair. Despite radiation exposure, atm knockdown animals survive long-term and regenerate new tissues. These effects occur independently of ATM's canonical downstream effector p53. Together, our results demonstrate that in planarians, ATM promotes radiation-induced apoptosis. This acute, ATM-dependent apoptosis is a key determinant of long-term animal survival. Our results suggest that inhibition of ATM in these organisms could, therefore, potentially favor cell survival after radiation without obvious effects on stem cell behavior.

Causative mechanisms and clinical impact of immunoglobulin deficiencies in ataxia telangiectasia.

BACKGROUND: Ataxia telangiectasia (AT) is characterized by cerebellar ataxia, telangiectasia, immunodeficiency, and increased cancer susceptibility and is caused by mutations in the ataxia telangiectasia mutated (ATM) gene. The immunodeficiency comprises predominantly immunoglobulin deficiency, mainly IgA and IgG2, with a variable severity. So far, the exact mechanisms underlying the immunoglobulin deficiency, especially the variable severity, remain unelucidated. OBJECTIVE: We characterized the clinical impact of immunoglobulin deficiencies in AT and elucidated their mechanisms in AT. METHODS: We analyzed long-term immunoglobulin levels, immunophenotyping, and survival time in our cohort (n = 87, median age 16 years; maximum 64 years). Somatic hypermutation and class-switch junctions in B cells were analyzed by next-generation sequencing. Furthermore, an in vitro class-switching induction assay was performed, followed by RNA sequencing, to assess the effect of ATM inhibition. RESULTS: Only the hyper-IgM AT phenotype significantly worsened survival time, while IgA or IgG2 deficiencies did not. The immunoglobulin levels showed predominantly decreased IgG2 and IgA. Moreover, flow cytometric analysis demonstrated reduced naive B and T lymphocytes and a deficiency of class-switched IgG2 and IgA memory B cells. Somatic hypermutation frequencies were lowered in IgA- and IgG2-deficient patients, indicating hampered germinal center reaction. In addition, the microhomology of switch junctions was elongated, suggesting alternative end joining during class-switch DNA repair. The in vitro class switching and proliferation were negatively affected by ATM inhibition. RNA sequencing analysis showed that ATM inhibitor influenced expression of germinal center reaction genes. CONCLUSION: Immunoglobulin deficiency in AT is caused by disturbed development of class-switched memory B cells. ATM deficiency affects both germinal center reaction and choice of DNA-repair pathway in class switching.

Ataxia Telangiectasia patient-derived neuronal and brain organoid models reveal mitochondrial dysfunction and oxidative stress.

Ataxia Telangiectasia (AT) is a rare disorder caused by mutations in the ATM gene and results in progressive neurodegeneration for reasons that remain poorly understood. In addition to its central role in nuclear DNA repair, ATM operates outside the nucleus to regulate metabolism, redox homeostasis and mitochondrial function. However, a systematic investigation into how and when loss of ATM affects these parameters in relevant human neuronal models of AT was lacking. We therefore used cortical neurons and brain organoids from AT-patient iPSC and gene corrected isogenic controls to reveal levels of mitochondrial dysfunction, oxidative stress, and senescence that vary with developmental maturity. Transcriptome analyses identified disruptions in regulatory networks related to mitochondrial function and maintenance, including alterations in the PARP/SIRT signalling axis and dysregulation of key mitophagy and mitochondrial fission-fusion processes. We further show that antioxidants reduce ROS and restore neurite branching in AT neuronal cultures, and ameliorate impaired neuronal activity in AT brain organoids. We conclude that progressive mitochondrial dysfunction and aberrant ROS production are important contributors to neurodegeneration in AT and are strongly linked to ATM's role in mitochondrial homeostasis regulation.

Absence of ATM leads to altered NK cell function in mice.

Ataxia-telangiectasia (A-T) is a rare disorder caused by genetic defects of A-T mutated (ATM) kinase, a key regulator of stress response, and characterized by neurodegeneration, immunodeficiency, and high incidence of cancer. Here we investigated NK cells in a mouse model of A-T (Atm-/-) showing that they are strongly impaired at killing tumor cells due to a block of early signaling events. On the other hand, in Atm-/- littermates with thymic lymphoma NK cell cytotoxicity is enhanced as compared with ATM-proficient mice, possibly via tumor-produced TNF-α. Results also suggest that expansion of exhausted NKG2D+ NK cells in Atm-/- mice is driven by low-level expression of stress-inducible NKG2D ligands, whereas development of thymoma expressing the high-affinity MULT1 ligand is associated with NKG2D down-regulation on NK cells. These results expand our understanding of immunodeficiency in A-T and encourage exploring NK cell biology in A-T patients in the attempt to identify cancer predictive biomarkers and novel therapeutic targets.

Patient-Reported Outcomes and Medical Provider Satisfaction Among Adult and Pediatric Ataxia-Telangiectasia Patients.

PURPOSE: Ataxia-telangiectasia (A-T) is a rare genetic condition with malfunctioning DNA repair processes resulting in significant clinical findings, including progressive neurologic decline, elevated malignancy risk, immunodeficiency, oculocutaneous telangiectasias, and severe pulmonary disease. Research has been limited into the quality of life of such patients and yet to be completed are studies quantitatively analyzing psychosocial, physical, and cognitive patient-reported outcomes (PROs) within the A-T population. METHODS: PRO evaluations of 90 international adult and pediatric A-T patients and their caregivers were completed via secure online administration of Patient-Reported Outcomes Measurement Information System (PROMIS) short forms evaluating anger, cognition, mood, social health, fatigue, pain, anxiety, and upper extremity function. The impact of age, gender, race/ethnicity, prior malignancy diagnosis, and current supportive treatment interventions on such PROs was additionally assessed. Finally, given the importance of medical providers in the care of A-T patients and the impact of patient satisfaction on healthcare outcomes, we further analyzed, via a novel survey, how patients and caregivers perceived their primary A-T healthcare provider's A-T expertise, trustworthiness, accessibility, and level of compassion. RESULTS/CONCLUSION: It was found that a diagnosis of A-T complexly impacts patient PROs, but such data offers the potential for preventative and therapeutic interventions to improve the care of such patients. While most A-T patients and their caregivers feel their primary A-T medical provider has expertise and compassion in addition to being accessible and trustworthy, a significant percentage of study subjects did not agree that their provider was an expert in A-T or overall trustworthy.

53BP1 regulates the self-renewal ability of neural stem/progenitor cells through modulating mitochondrial homeostasis.

The regulation of intracellular reactive oxygen species (ROS) levels is important for maintaining the self-renewal ability of neural stem/progenitor cells (NSCs). In this study, we demonstrate that 53BP1, a DNA damage response factor known to facilitate the repair of DNA double-strand breaks, supports the maintenance of NSC stemness. ReNcell VM human NSCs with depleted 53BP1 exhibited reduced self-renewal ability compared with control NSCs, as revealed by a decrease in neurosphere size and an increase in differentiation into neural or glial cells within an NSC culture. Furthermore, 53BP1 depletion elevated cellular ROS levels, accompanied by mitochondrial abnormalities. The reduced self-renewal ability and elevated ROS levels in 53BP1-deficient NSCs were restored with the treatment of a radical scavenger, N-acetyl-l-cysteine. In addition, we investigated the functional relationship in the NSC self-renewal ability between 53BP1 and ataxia-telangiectasia mutated (ATM) or forkhead box O3a (FOXO3a), factors required for mitochondrial homeostasis, and the maintenance of NSC stemness. We found that ATM inhibition or FOXO3a deficiency, in addition to 53BP1 deficiency, did not induce further NSC stemness impairment. Collectively, our findings show that 53BP1, by cooperatively functioning with ATM and FOXO3a, supports the maintenance of NSC stemness by modulating mitochondrial homeostasis.

Meeting report: AT workshop 2023-A platform for discussing cutting-edge science in DNA damage signaling, repair, and human disorders.

Ataxia-telangiectasia (A-T) is a rare devastating hereditary condition, which affects multiple organ systems including cerebellar motor function as well as DNA repair, resulting in a higher incidence of cancer and immunodeficiency. The genetic defect in A-T lies in ATM kinase, which is activated by DNA damage and regulates a plethora of substrates including the p53 tumor suppressor. We have organized an international meeting "The 19th Ataxia-Telangiectasia Workshop 2023 (ATW2023)" with support from the Molecular Biology Society of Japan (MBSJ) and other funders. Here, we report that ATW2023 was successfully held in Kyoto from March 2nd to 5th, 2023 with more than 150 participants traveling from all over the world, despite the still smoldering COVID-19 pandemic. In this meeting report, we will briefly describe the highlights of the meeting and would like to express our gratitude to the MBSJ for the financial support.

Long-Term Nicotinamide Riboside Use Improves Coordination and Eye Movements in Ataxia Telangiectasia.

BACKGROUND: Supplementation of nicotinamide riboside (NR) ameliorates neuropathology in animal models of ataxia telangiectasia (A-T). In humans, short-term NR supplementation showed benefits in neurological outcome. OBJECTIVES: The study aimed to investigate the safety and benefits of long-term NR supplementation in individuals with A-T. METHODS: A single-arm, open-label clinical trial was performed in individuals with A-T, receiving NR over a period of 2 years. Biomarkers and clinical examinations were used to assess safety parameters. Standardized and validated neuromotor tests were used to monitor changes in neurological symptoms. Using generalized mixed models, test results were compared to expected disease progression based on historical data. RESULTS: NAD+ concentrations increased rapidly in peripheral blood and stabilized at a higher level than baseline. NR supplementation was well tolerated for most participants. The total scores in the neuromotor test panels, as evaluated at the 18-month time point, improved for all but one participant, primarily driven by improvements in coordination subscores and eye movements. A comparison with historical data revealed that the progression of certain neuromotor symptoms was slower than anticipated. CONCLUSIONS: Long-term use of NR appears to be safe and well tolerated, and it improves motor coordination and eye movements in patients with A-T of all ages. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

ATM: Translating the DNA Damage Response to Adaptive Immunity.

ATM is often dubbed the master regulator of the DNA double stranded break (DSB) response. Since proper induction and repair of DNA DSBs forms the core of immunological diversity, it is surprising that patients with ataxia telangiectasia generally have a mild immunodeficiency in contrast to other DSB repair syndromes. In this review, we address this discrepancy by delving into the functions of ATM in DSB repair and cell cycle control and translate these to adaptive immunity. We conclude that ATM, despite its myriad functions, is not an absolute requirement for acquiring sufficient levels of immunological diversity to prevent severe viral and opportunistic infections. There is, however, a more clinically pronounced antibody deficiency in ataxia telangiectasia due to disturbed class switch recombination.

Cerebellar Cognitive Affective Syndrome in Mexican Pediatric Patients with Ataxia-Telangiectasia.

Ataxia-telangiectasia (A-T) is a disease caused by mutations in the ATM gene (11q22.3-23.1) that induce neurodegeneration Sasihuseyinoglu AS et al.  Pediatr Allergy Immunol Pulmonol 31(1):9-14, 2018, Teive HAG et al. Parkinsonism Relat Disord 46:3-8, 2018. Clinically, A-T is characterized by ataxia, mucocutaneous telangiectasia, immunodeficiency, and malignancy. Movement disorders have been the most described and well-studied symptoms of A-T. Other studies have reported visuospatial processing disorders, executive function disorders and emotional regulation disorders, which are clinical manifestations that characterize cerebellar cognitive affective syndrome (CCAS) Choy KR et al. Dev Dyn 247(1):33-46, 2018. To describe the neurocognitive and emotional state of pediatric patients with ataxia-telangiectasia and to discuss whether they have cerebellar cognitive affective syndrome. This observational, cross-sectional, and descriptive study included 9 patients with A-T from May 2019 to May 2021. A complete medical history was retrieved, and tests were applied to assess executive functions, visual-motor integration and abilities, language, psychological disorders, and ataxia. Six girls and 3 boys agreed to participate. The age range was 6 to 14 years. The participants included five schoolchildren and four teenagers. Eight patients presented impaired executive functioning. All patients showed some type of error in copying and tracing (distortion) in the performance of visual perceptual abilities. Emotional disorders such as anxiety and depression were observed in six patients. Eight patients presented with dyslalia and impairments in word articulation, all patients presented with ataxia, and seven patients used a wheelchair. All patients presented symptoms consistent with CCAS and had variable cognitive performance.

Disproportionate Expression of ATM in Cerebellar Cortex During Human Neurodevelopment.

Cerebellar neurodegeneration is a classical feature of ataxia telangiectasia (A-T), an autosomal recessive condition caused by loss-of-function mutation of the ATM gene, a gene with multiple regulatory functions. The increased vulnerability of cerebellar neurones to degeneration compared to cerebral neuronal populations in individuals with ataxia telangiectasia implies a specific importance of intact ATM function in the cerebellum. We hypothesised that there would be elevated transcription of ATM in the cerebellar cortex relative to ATM expression in other grey matter regions during neurodevelopment in individuals without A-T. Using ATM transcription data from the BrainSpan Atlas of the Developing Human Brain, we demonstrate a rapid increase in cerebellar ATM expression relative to expression in other brain regions during gestation and remaining elevated during early childhood, a period corresponding to the emergence of cerebellar neurodegeneration in ataxia telangiectasia patients. We then used gene ontology analysis to identify the biological processes represented in the genes correlated with cerebellar ATM expression. This analysis demonstrated that multiple processes are associated with expression of ATM in the cerebellum, including cellular respiration, mitochondrial function, histone methylation, and cell-cycle regulation, alongside its canonical role in DNA double-strand break repair. Thus, the enhanced expression of ATM in the cerebellum during early development may be related to the specific energetic demands of the cerebellum and its role as a regulator of these processes.

Correlation Between the SARA and A-T NEST Clinical Severity Scores in Adults with Ataxia-Telangiectasia.

Ataxia-Telangiectasia (A-T) is an autosomal recessive neurodegenerative disease associated with cerebellar ataxia and extrapyramidal features. A-T has a complex and diverse phenotype with varying rates of disease progression. The development of robust natural history studies and therapeutic trials relies on the accurate recording of phenotype using relevant and validated severity of illness indexes. We compared the commonly used Scale for the Assessment and Rating of Ataxia (SARA) and the disease-specific A-T Neurological Examination Scale Toolkit (A-T NEST), in our adult A-T cohort. We found a strong correlation between A-T NEST and the established SARA score, validating the use of A-T NEST and SARA in capturing the natural history of A-T patients.

The Care and Management of Children and Young People with Ataxia Telangiectasia Provided by Nurses and Allied Health Professionals: a Scoping Review.

Ataxia telangiectasia (A-T) is a rare, multisystem progressive condition that typically presents in early childhood. In the absence of cure, people with A-T require coordinated multidisciplinary care to manage their complex array of needs and to minimize the disease burden. Although symptom management has proven benefits for this population, including improved quality of life and reduced complications, there is a need for guidance specific to the nursing and allied healthcare teams who provide care within the community. A scoping review, adopting the Joanna Briggs Institute methodology, was undertaken. It aimed to identify and map the available expertise from nursing and allied healthcare and management of children and young people with A-T ≤ 18 years of age. A rigorous search strategy was employed which generated a total of 21,118 sources of evidence, of which 50 were selected for review following screening by experts. A range of interventions were identified that reported a positive impact on A-T-related impairments, together with quality of life, indicating that outcomes can be improved for this population. Most notable interventions specific to A-T include therapeutic exercise, inspiratory muscle training, and early nutritional assessment and intervention. Further research will be required to determine the full potential of the identified interventions, including translatability to the A-T setting for evidence related to other forms of ataxia. Large gaps exist in the nursing and allied health evidence-base, highlighting a need for robust research that includes children and young people with A-T and their families to better inform and optimize management strategies.

The Latest Developments for the Treatment of Ataxia Telangiectasia: A Narrative Review.

Ataxia telangiectasia (AT), Louis-Bar syndrome, is a rare neurodegenerative disorder caused by autosomal recessive biallelic mutations within the ataxia telangiectasia mutated (ATM) gene. Currently, there are no curative therapies available for this disorder. This review provides an overview of the latest advances in treatment methods including 1- Acetyl-DL-leucine, 2- Bone Marrow Transplantation, 3- Gene Therapy, 4- Dexamethasone, and finally 5- Red Blood Cells (RBCs) as a carrier for dexamethasone (encapsulation of dexamethasone sodium phosphate into autologous erythrocytes, known as EryDex). Most of the treatments under investigation are in the early stages, except for the EryDex System. It appears that the EryDex system and N-Acetyl-DL-Leucine may hold promise as potential treatment options.

Videoocular assessment of eye movement activity in an ataxia-telangiectasia: a case study.

PURPOSE: Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by progressive neurological deficits, including prominent oculomotor dysfunction. We report 5 cases of eye movement assessment in children 9-15 years old with A-T. METHODS: Three different oculomotor tasks (gaze holding, visually guided saccades and visual search) were used, and video-oculography was performed. Additionally, the scale for the assessment and rating of ataxia (SARA) score was used to assess severity of the cerebellar ataxia. RESULTS: Unstable gaze holding, nystagmus and saccadic intrusions were found. In addition to psychophysiological assessment results, we provide quantitative analysis of oculomotor activity, revealing a specific abnormal oculomotor pattern, consisting of (i) marked saccade hypermetria, (ii) unstable gaze holding, and (iii) gaze-evoked nystagmus. CONCLUSION: Our study opens the prospect to evaluate efficacy and safety of alternative methods for supporting the patient and improving his/her life quality.