Periodic Reporting for period 1 - OST-CDG-omics (Untangling the pathophysiology of congenital disorders of glycosylation affecting the OST complex) Reporting period: 2020-07-01 to 2022-06-30 Summary of the context and overall objectives of the project The OST-CDG-omics project was conceived in order to further investigate the cause of disease in patients with Congenital Disorders of Glycosylation (CDG). These are inborn errors of metabolism (IEM) that disrupt glycosylation. This is the process of attaching sugar chains, or glycans, to lipids and proteins as they are being made by the cell. Glycans are important for the eventual function of these proteins or lipids. For example, they may be required to fold a protein correctly, or to traffic it to the correct place within the cell. Over 150 genetically distinct types of CDG have been discovered, this number will likely grow; 2% of the human genome encodes proteins responsible for glycan processing or recognition. CDG cause an extraordinarily wide range of often multisystemic clinical presentations. This makes diagnosis of CDG very difficult; it is still an under-diagnosed disorder, even in countries where screening for CDG is common. We have used state-of-the-art technology that allows us to thoroughly investigate the genetic code of affected patients who are known to have a CDG, but are yet to receive a genetic diagnosis. This, alongside expert cell biological analysis of the resulting glycosylation defect, has provided us with a number of breakthroughs involving the identification of new forms of CDG, or clarifying the disease mechanisms of existing CDG. These have helped undiagnosed patients to finally come to an end of their diagnostic odyssey, as well as provided fruitful further avenues of research for scientists in their quest to fully understand the process of glycosylation and its biological impacts. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far A genomic analysis workflow was developed for the diagnosis of patients with a suspected CDG that were unable to receive a diagnosis after CDG gene panel analysis. This included a total of 36 individuals. 12 of these were patients, the rest family members. Five patients received a diagnosis due to whole genome sequencing (WGS) analysis. Two novel CDG were identified (CAMLG-CDG, and another (gene X) as yet unpublished) and a dominant form of an existing recessive CDG was identified (STT3A-CDG). Still undiagnosed patients are still undergoing analysis and/or being taken forward for RNAseq (transcriptomic) analysis, this is ongoing work.In addition, previously collected whole exome sequencing (WES) data from 12 other undiagnosed CDG patients was reanalysed using state-of-the-art pipelines. This led to two additional diagnoses in these individuals. Each main aspect of the project is listed below:i) Identification of a dominant form of STT3A-CDGAlongside collaborators, a total of 16 patients from 9 families were identified with dominant mutations in STT3A leading to a broad phenotype including neurological and skeletal abnormalities. Molecular and cell biological techniques were used to characterise the glycosylation abnormalities in patient cells and to prove the dominant pathomechanism in a yeast model. This was a particularly novel aspect of this part of the project and to our knowledge, this type of analysis to prove a dominant effect had not previously been performed. This work was published in 2021.ii) Identification and characterisation of a novel CDG and trafficking disorder, CAMLG-CDGIn a single patient included in our WGS cohort, a homozygous splice site variant was identified in CAMLG. Firstly, it was proven in patient fibroblasts that this led to splice changes that caused depletion of the resulting protein product, GET2. Further models using a HeLa cell line were then used to prove the cause of disease. This finding was entirely novel and sheds light on the role of CAML within the transmembrane domain recognition complex (TRC) pathway. This work was published in early 2022.iii) The second patient with SLC37A4-CDGWe identified the c.1267C>T (p.R423*) variant in SLC37A4 in a historical patient in our centre. This variant had previously been shown to cause a dominant CDG via an unusual and unexpected mechanism in a single patient. We were therefore able to confirm this novel dominant CDG in a second patient. After characterisation work, this was published in early 2021.iv) Further elucidation of the pathomechanism of MAGT1-CDGThrough the study of individuals with mutations in the MAGT1 gene, the link between MAGT1-CDG (the resulting disorder) and NKG2D hypoglycosylation was elucidated and a direct link to pathogenesis was found. This sheds light on the function of different aspects of the OST, as MAGT1 is a subunit specific to the post-translational OST-B complex. This work was published in early 2022.v) Identification of a novel CDG caused by mutations in gene XPerhaps the most exciting outcome of this project is the identification of pathogenic variants in a gene, unnamed here, that we will term gene X. We believe that gene X leads to the disruption of dolichol metabolism. Dolichol is a precursor required for the glycosylation process. We believe that by revealing the role of gene X, we can not only identify a novel (severe) disorder leading to a CDG involving deafness and psychomotor delay, but shed significant light on novel aspects of dolichol metabolism. Our group has launched a doctorate student project investigating this, and we are close to being able to publish the characterisation of Gene X. This will potentially be the biggest outcome of this project.Additionally, pathogenic variants in three other patients with a CDG were identified and characterised in our lab. These are unpublished but were shared with treating clinicians, providing diagnoses. Specifically, a variant causing the deletion of exon 16 of ATP6V0A2 was identified in a longstanding undiagnosed patient with cutis laxa. In another patient, a missense variant in the CAD gene was shown to lead to pathogenicity. This allowed continued treatment with uridine, a rare case of an effective treatment for a CDG. Finally, in a patient with developmental delay, epilepsy and hypotonia, a previously overlooked mutation in ATP6AP2, a known CDG gene, was shown to cause disease.This work was communicated at a total of 6 symposia and resulted in a total of 5 publications. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) OST-CDG-omics has had several important impacts in the study of CDG which will provide insight to future researchers and clinicians. These include the new disorders described above. In addition, a review article detailing the state-of-the-art of CDG genetics was published during the project. In addition, the further study of Gene X, also discussed above, will likely lead to the most impactful outcome of this research project, once published.Users of the project results include three main groups. Firstly, clinicians treating patients with CDG will be able to diagnose according to our findings in this study. One example of this would be a previously uncharacterised variant in STT3A could now conclusively be categorised as pathogenic due to our work. Secondly, patients themselves who have received a diagnosis are clearly directly impacted by the research goals of this project. Finally, other researchers will be able to use our results, particularly those based on the study of CAMLG (and more broadly the TRC pathway) and Gene X, to inform their own work. Outside of the CDG field, these aspects of the project in particularly will have impact in the fields of membrane trafficking and insertion, or lipid metabolism.