This project is being done in collaboration with Dr. Jordi Diaz-Manera and Dr. Ana Topf and their project “Latin-Seq for Neutrophil Assay Validation”.
With the advent of Next Generation Sequencing, an increasing number of neuromuscular diseases can be diagnosed directly by DNA test but despite the rapidly evolving technologies, the analysis is still time consuming and the classification of genetic variants as pathogenic presents considerable challenges. Absence or significant reduction of dysferlin as measured by Western blot of muscle or CD14+ monocytes is a reliable diagnostic indicator of disease. CD14+ immunoblot is generally preferred over the muscle biopsy as being a less invasive and more cost-effective assay. However, its technical complexity has limited the application to specialized diagnostic laboratories. Dysferlin is also expressed in primary granules and secretory vesicles of neutrophils. Our lab has developed a simplified diagnostic assay that involves the use of immunohistochemistry on peripheral blood films (PBF) to detect dysferlin expression in these cells. Preliminary analysis of accuracy has been carried out on samples obtained from healthy controls, patients with genetic diagnosis of dysferlinopathy and individuals with alternative genetic diagnoses. Further studies including participants in whom the disease status is unknown are required to predict how the test is likely to perform in clinical practice as a pre-screening for mutations in the DYSF gene and to validate the assay as a functional assessment for variants of uncertain significance (VUS).
We will undertake a large-scale prospective study in an undiagnosed Latin American cohort, in combination with a training program for sample collection. This study will have access to 200 samples from individuals recruited in the LATIN-SEQ project. LATIN-SEQ is an international research collaboration coordinated by the John Walton Muscular Dystrophy Research Centre in Newcastle upon Tyne, UK, that applies targeted whole exome sequencing to >1,000 patients with undiagnosed neuromuscular conditions from Latin America. On completion of on-site training sessions for sample collection and quality check, PBF will be collected from adult participants who had onset of muscle weakness after age 15. Protein data will be compared to DYSF genetic status of participants when WES result interpretation takes place through LATIN-SEQ. The outcome of the study will demonstrate whether this assay is suitable to large population screening and as a test for VUS’ pathogenicity. The study will also be useful to assess the requirements for a broader training program to ensure the reproducibility of the assay in diagnostic centers worldwide.