P298 Controlled storage conditions improve specificity and sensitivity of a blood-based assay for dysferlinopathy: a pilot study in an Indian cohort

• Published in: Neuromuscular disorders : NMD 2023-10, Vol.33, p.S118-S118
• Main Authors: Barresi, R., Cox, D., Henderson, M., Emmons, S., Gaitonde, P., Dastur, R.
• Format: Article
• Language: English
• ISSN: 0960-8966; 1873-2364
• DOI: 10.1016/j.nmd.2023.07.208

Mutations the gene encoding for dysferlin (DYSF) are responsible for dysferlinopathy, a rare autosomal recessive muscular dystrophy with either proximal or distal onset. The absence of dysferlin expression assessed by immunoblot of muscle biopsies or CD14+ peripheral blood monocytes is a very accurate diagnostic predictor of disease. These techniques, however, are specialized and laborious. Since dysferlin is also expressed in primary granules and secretory vesicles of neutrophils, we have developed a simplified immunohistochemical assay to detect dysferlin expression in neutrophils on peripheral blood films (PBF). We validated the assay on a cohort of individuals of Indian background attending the Dysferlin Registry meeting “Strength in Numbers” in Mumbai, coordinated by Jain Foundation in November 2019. 32 patients with genetic diagnosis of dysferlinopathy and 18 healthy individuals volunteered for the study. PBF immunolabeled for dysferlin were assessed for quality and blind-scored. Results were then correlated with the genetic diagnosis. Labelling for dysferlin was absent or very reduced in all patients. Negative labelling was also seen in control PBF that were of poor quality or stored at extreme environmental and climate conditions for a prolonged time (>4 months). In contrast, storage at 4°C for up to 16 weeks did not affect dysferlin labelling, indicating that controlled temperature conditions are key to the specificity of the assay. Further studies are planned, which include participants in whom the disease status is unknown, to validate the assay diagnostically as a prospective pre-screening for mutations in the DYSF gene and as a functional assessment for variants’ pathogenicity.