Danon disease – lysosomal dyfunction disease - molecular pathology of LAMP2 protein - implications for clinical diagnostics

Konference: 2011 7. Sympozium a workshop molekulární patologie a histo-cyto-chemie

Kategorie: Onkologická diagnostika

Téma: Keynote lectures of invited speakers II

Číslo abstraktu: 010

Autoři: J. Sikora; F. Majer; M. Kubánek; H. Vlášková; L. Stolnaya; l. Kró; MUDr. Tomáš Kalina, Ph.D.; RNDr. Lenka Dvořáková; Prof. MUDr. Milan Elleder (1938 - 2011), DrSc.

Danon disease (DD) is a monogenic X-linked disorder, which is clinically characterized by combination of cardiomyopathy, skeletal myopathy and variable degree of mental retardation. DD develops due to mutations in the gene coding lysosomal associated membrane protein 2 (LAMP2). The ultrastructural changes in DD reflect a defect in autophagosomal (AP) clearance due to inefficient AP/lysosomal membrane interaction and fusion impaired by defects in LAMP2.

We present a DD family with a clinical phenotype of hypertrophic cardiomyopathy associated with pre-excitation, myopia and mild myopathy in affected males. The diagnosis of DD in the family members was based on the assessment of their clinical status and evaluation of the skeletal or cardiac muscle biopsies documenting typical histological and ultrastructural DD changes including the absence of LAMP2 protein by immuno staining. Direct sequence analysis of LAMP2 gene and its mRNA revealed a novel protein truncation mutation in all affected family members.

Our major aim is to present the results of the studies on the cellular pathology in two explanted DD hearts, comment on the utility of peripheral white blood cells (WBCs) for clinical DD diagnostics, and discuss the functional studies focusing on the cellular processing of the novel pathologic LAMP2 protein variant.

Histological findings corresponding to AP accumulation were present in all segments of the male DD hemizygote’s heart. By ultrastructural analysis, we have documented dilatations of T-tubular cardiomyocyte conductance system. Such changes, possibly affecting cardiomyocyte excitability and contractility, have never been, to our best knowledge, reported neither in endomyocardial DD biopsies or DD KO mice. Due to X chromosome inactivation, myocardium from a female DD heterozygote showed minute fraction of LAMP2 immuno positive cardiomyocytes.

To test the qualitative utility of WBCs in DD clinical diagnostics, we performed LAMP2 immuno detection in peripheral blood smears of all three patients. Samples from male hemizygotes (patient 1 and 3) were devoid of LAMP2, whereas small fraction of myeloid lineage cells was LAMP2 positive in the female DD heterozygote. Electron microscopy of WBC pellets demonstrated scattered APs in neutrophilic granulocytes and monocytes, comparable to autophagic changes found in cardiomyocytes. LAMP2 expression was further quantitatively evaluated in specific WBC sub-populations (granulocytes, CD4+ and CD8+ T-lymphocytes, CD20+ B-lymphocytes, CD14+ monocytes and CD56+ natural killer cells) by polychromatic flow cytometry analysis. This approach delineated granulocytes and CD14+ monocytes as the dominant LAMP2 expressing WBC types in healthy human controls. Whereas male DD patients lacked LAMP2 in all WBC subtypes, female DD heterozygote expressed LAMP2 in 14% of her peripheral granulocytes and monocytes. Comparative plotting of ratio values of LAMP2 expression in CD14+ monocytes over LAMP2 expression in CD20+ B lymphocytes provided a mean to clearly discriminate DD patients from healthy subjects. Summarized, we propose to consider peripheral WBCs as optimal ‘first choice’ cellular population for LAMP2 expression testing when DD is a clinically relevant differential diagnostic option. Flow cytometry analysis, in contrast to peripheral blood smear LAMP2 labeling, represents a quantitative way to disclose a skewed LAMP2 expression in DD female heterozygotes.

As the novel mutation results in a putative truncated 334 amino acids long LAMP2 protein, which lacks the transmembrane and C-terminal cytosolic domain, we hypothesized that this mutated LAMP2 variant may attain a relatively stable folding state and while not targeted to late endosomes/lysosomes, may become exocytosed. Therefore we performed LAMP2 Western blotting of cardiac muscle, WBCs and cultured skin fibroblasts as well as corresponding extra cellular media (plasma and tissue culture medium) but found no intra- or extra cellular truncated LAMP2. Additionally, we compared the expression and intracellular targeting of the three physiological LAMP2 splicing variants (A, B and C) C-terminally tagged by GFP with a mutated lAMP2 fusion protein in cultured skin fibroblasts. Contrary to normal LAMP2 variants, which became expressed and were targeted to late endosomes/lysosomes as evaluated by GFP fluorescence, the mutated LAMP2 variant has not even been expressed (demonstrated by the lack of mature GFP fluorescence) suggesting folding instability and possibly its co- or early post-translational degradation.

Acknowledgements: Malušková J., Honsová E. (Clinical and Transplantation Pathology Department – Institute for Clinical and Experimental Medicine, Prague, Czech Republic)

Datum přednesení příspěvku: 29. 4. 2011