Quantifying lymphocyte vacuolization serves as a measure of CLN3 disease severity
Willemijn F. E. Kuper,
Marlies Oostendorp,
Brigitte T. A. van den Broek,
Karin van Veghel,
Lourens J. P. Nonkes,
Edward E. S. Nieuwenhuis,
Sabine A. Fuchs,
Tineke Veenendaal,
Judith Klumperman,
Albert Huisman,
Stefan Nierkens,
Peter M. van Hasselt
Affiliations
Willemijn F. E. Kuper
Department of Metabolic Diseases, Wilhelmina Children's Hospital University Medical Center Utrecht, Utrecht University Utrecht the Netherlands
Marlies Oostendorp
Department of Clinical Chemistry University Medical Center Utrecht Utrecht the Netherlands
Brigitte T. A. van den Broek
Sylvia Toth Center for the Multidisciplinary Follow up of Lysosomal Storage Disorders University Medical Center Utrecht, Utrecht University Utrecht the Netherlands
Karin van Veghel
Laboratory of Translational Immunology University Medical Center Utrecht, Utrecht University Utrecht the Netherlands
Lourens J. P. Nonkes
Department of Clinical Chemistry University Medical Center Utrecht Utrecht the Netherlands
Edward E. S. Nieuwenhuis
Department of Gastroenterology Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht University Utrecht the Netherlands
Sabine A. Fuchs
Department of Metabolic Diseases, Wilhelmina Children's Hospital University Medical Center Utrecht, Utrecht University Utrecht the Netherlands
Tineke Veenendaal
Section Cell Biology, Center for Molecular Medicine University Medical Center Utrecht, Utrecht University Utrecht the Netherlands
Judith Klumperman
Section Cell Biology, Center for Molecular Medicine University Medical Center Utrecht, Utrecht University Utrecht the Netherlands
Albert Huisman
Department of Clinical Chemistry University Medical Center Utrecht Utrecht the Netherlands
Stefan Nierkens
Laboratory of Translational Immunology University Medical Center Utrecht, Utrecht University Utrecht the Netherlands
Peter M. van Hasselt
Department of Metabolic Diseases, Wilhelmina Children's Hospital University Medical Center Utrecht, Utrecht University Utrecht the Netherlands
Abstract Background The CLN3 disease spectrum ranges from a childhood‐onset neurodegenerative disorder to a retina‐only disease. Given the lack of metabolic disease severity markers, it may be difficult to provide adequate counseling, particularly when novel genetic variants are identified. In this study, we assessed whether lymphocyte vacuolization, a well‐known yet poorly explored characteristic of CLN3 disease, could serve as a measure of disease severity. Methods Peripheral blood obtained from healthy controls and CLN3 disease patients was used to assess lymphocyte vacuolization by (a) calculating the degree of vacuolization using light microscopy and (b) quantifying expression of lysosomal‐associated membrane protein 1 (LAMP‐1), using flow cytometry in lymphocyte subsets as well as a qualitative analysis using electron microscopy and ImageStream analysis. Results Quantifying lymphocyte vacuolization allowed to differentiate between CLN3 disease phenotypes (P = .0001). On immunofluorescence, classical CLN3 disease lymphocytes exhibited abundant vacuole‐shaped LAMP‐1 expression, suggesting the use of LAMP‐1 as a proxy for lymphocyte vacuolization. Using flow cytometry in lymphocyte subsets, quantifying intracellular LAMP‐1 expression additionally allowed to differentiate between infection and storage and to differentiate between CLN3 phenotypes even more in‐depth revealing that intracellular LAMP‐1 expression was most pronounced in T‐cells of classical‐protracted CLN3 disease while it was most pronounced in B‐cells of “retina‐only” CLN3 disease. Conclusion Lymphocyte vacuolization serves as a proxy for CLN3 disease severity. Quantifying vacuolization may help interpretation of novel genetic variants and provide an individualized readout for upcoming therapies.
