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Danon disease : A focus on processing of the novel LAMP2 mutation and comments on the beneficial use of peripheral white blood cells in the diagnosis of LAMP2 deficiency

Majer, F. ; Vlaskova, H. ; Krol, L. LU orcid ; Kalina, T. ; Kubanek, M. ; Stolnaya, L. ; Dvorakova, L. ; Elleder, M. and Sikora, J. (2012) In Gene 498(2). p.183-195
Abstract

Danon disease (DD) is a monogenic X-linked disorder characterized by cardiomyopathy, skeletal myopathy and variable degrees of intellectual disability. DD develops due to mutations in the gene encoding lysosomal-associated membrane protein 2 (LAMP2). We report on a family exhibiting the clinical phenotype comprising of hypertrophic cardiomyopathy and ventricular pre-excitation, myopia and mild myopathy in two male patients and cardiomyopathy and myopia in a female patient. The diagnosis of DD in this family was based on the assessment of the clinical phenotypes and the absence of LAMP2 in skeletal and/or cardiac muscle biopsy specimens. Sequence analysis of the LAMP2 gene and its mRNA revealed a novel LAMP2 mutation (c.940delG) in all... (More)

Danon disease (DD) is a monogenic X-linked disorder characterized by cardiomyopathy, skeletal myopathy and variable degrees of intellectual disability. DD develops due to mutations in the gene encoding lysosomal-associated membrane protein 2 (LAMP2). We report on a family exhibiting the clinical phenotype comprising of hypertrophic cardiomyopathy and ventricular pre-excitation, myopia and mild myopathy in two male patients and cardiomyopathy and myopia in a female patient. The diagnosis of DD in this family was based on the assessment of the clinical phenotypes and the absence of LAMP2 in skeletal and/or cardiac muscle biopsy specimens. Sequence analysis of the LAMP2 gene and its mRNA revealed a novel LAMP2 mutation (c.940delG) in all three patients.Approximately 25% of the female patient's cardiomyocytes were LAMP2 positive apparently due to the unfavorable skewing of X chromosome inactivation. We further performed qualitative LAMP2 immunohistochemistry on peripheral white blood cells using the smear technique and revealed the absence of LAMP2 in the male patients. LAMP2 expression was further assessed in granulocytes, CD4. + and CD8. + T lymphocytes, CD20. + B lymphocytes, CD14. + monocytes and CD56. + natural killer cells by quantitative polychromatic flow cytometry. Whereas the male DD patients lacked LAMP2 in all WBC populations, the female patient expressed LAMP2 in 15.1% and 12.8% of monocytes and granulocytes, respectively. LAMP2 expression ratiometrics of highly vs. weakly expressing WBC populations discriminated the DD patients from the healthy controls. WBCs are thus suitable for initial LAMP2 expression testing when DD is a differential diagnostic option. Moreover, flow cytometry represents a quantitative method to assess the skewing of LAMP2 expression in female heterozygotes. Because LAMP2 is a major protein constituent of the membranes of a number of lysosome-related organelles, we also tested the exocytic capacity of the lytic granules from CD8. + T lymphocytes in the patient samples. The degranulation triggered by a specific stimulus (anti-CD3 antibody) was normal. Therefore, this process can be considered LAMP2 independent in human T cells.The c.940delG mutation results in a putatively truncated protein (p.A314QfsX32), which lacks the transmembrane domain and the cytosolic tail of the wild-type LAMP2. We tested whether this variant becomes exocytosed because of a failure in targeting to late endosomes/lysosomes. Western blotting of cardiac muscle, WBCs and cultured skin fibroblasts (and their culture media) showed no intra- or extracellular truncated LAMP2. By comparing the expression pattern and intracellular targeting in cultured skin fibroblasts of normal LAMP2 isoforms (A, B and C) tagged with green fluorescent protein (GFP) and the A314Qfs32-GFP fusion, we found that the A314Qfs32-GFP protein is not even expressed. These observations suggest that the truncated protein is unstable and is co-translationally or early post-translationally degraded.

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publishing date
type
Contribution to journal
publication status
published
subject
keywords
Cardiomyopathy, Danon disease, Lysosomal-associated membrane protein 2, Polychromatic flow cytometry, White blood cells, X-chromosome inactivation
in
Gene
volume
498
issue
2
pages
183 - 195
publisher
Elsevier
external identifiers
  • pmid:22365987
  • scopus:84858751035
ISSN
0378-1119
DOI
10.1016/j.gene.2012.02.004
language
English
LU publication?
no
additional info
Funding Information: This work was supported by the Research Project no. 0021620806 and partly by the grant IGA MZ NS/10342-3/2009 . T.K. and L.K. were supported by the grant IGA MZ NS/9996-4 and T.K. is supported as “ISAC Scholar” by the International Society of Advancement of Cytometry . The authors would further like to acknowledge the following colleagues — Dr. Zamecnik (Department of Pathology and Molecular Medicine, 2nd Medical School, Charles University in Prague), Dr. Snorek (Department of Cardiology, Hospital Ceske Budejovice); Dr. Ridzon (Neurological Clinic, Thomayer University Hospital); Drs. Maluskova and Honsova (Department of Clinical and Transplantation Pathology); Drs. Kotrc and Malek (Department of Cardiology); and Dr. Pirk (Department of Cardiovascular Surgery). All the latter colleagues are from the Institute for Clinical and Experimental Medicine, Prague, Czech Republic. Copyright: Copyright 2012 Elsevier B.V., All rights reserved.
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@article{ff719ce6-6f23-448b-9ed5-34173461b892,
  abstract     = {{<p>Danon disease (DD) is a monogenic X-linked disorder characterized by cardiomyopathy, skeletal myopathy and variable degrees of intellectual disability. DD develops due to mutations in the gene encoding lysosomal-associated membrane protein 2 (LAMP2). We report on a family exhibiting the clinical phenotype comprising of hypertrophic cardiomyopathy and ventricular pre-excitation, myopia and mild myopathy in two male patients and cardiomyopathy and myopia in a female patient. The diagnosis of DD in this family was based on the assessment of the clinical phenotypes and the absence of LAMP2 in skeletal and/or cardiac muscle biopsy specimens. Sequence analysis of the LAMP2 gene and its mRNA revealed a novel LAMP2 mutation (c.940delG) in all three patients.Approximately 25% of the female patient's cardiomyocytes were LAMP2 positive apparently due to the unfavorable skewing of X chromosome inactivation. We further performed qualitative LAMP2 immunohistochemistry on peripheral white blood cells using the smear technique and revealed the absence of LAMP2 in the male patients. LAMP2 expression was further assessed in granulocytes, CD4. + and CD8. + T lymphocytes, CD20. + B lymphocytes, CD14. + monocytes and CD56. + natural killer cells by quantitative polychromatic flow cytometry. Whereas the male DD patients lacked LAMP2 in all WBC populations, the female patient expressed LAMP2 in 15.1% and 12.8% of monocytes and granulocytes, respectively. LAMP2 expression ratiometrics of highly vs. weakly expressing WBC populations discriminated the DD patients from the healthy controls. WBCs are thus suitable for initial LAMP2 expression testing when DD is a differential diagnostic option. Moreover, flow cytometry represents a quantitative method to assess the skewing of LAMP2 expression in female heterozygotes. Because LAMP2 is a major protein constituent of the membranes of a number of lysosome-related organelles, we also tested the exocytic capacity of the lytic granules from CD8. + T lymphocytes in the patient samples. The degranulation triggered by a specific stimulus (anti-CD3 antibody) was normal. Therefore, this process can be considered LAMP2 independent in human T cells.The c.940delG mutation results in a putatively truncated protein (p.A314QfsX32), which lacks the transmembrane domain and the cytosolic tail of the wild-type LAMP2. We tested whether this variant becomes exocytosed because of a failure in targeting to late endosomes/lysosomes. Western blotting of cardiac muscle, WBCs and cultured skin fibroblasts (and their culture media) showed no intra- or extracellular truncated LAMP2. By comparing the expression pattern and intracellular targeting in cultured skin fibroblasts of normal LAMP2 isoforms (A, B and C) tagged with green fluorescent protein (GFP) and the A314Qfs32-GFP fusion, we found that the A314Qfs32-GFP protein is not even expressed. These observations suggest that the truncated protein is unstable and is co-translationally or early post-translationally degraded.</p>}},
  author       = {{Majer, F. and Vlaskova, H. and Krol, L. and Kalina, T. and Kubanek, M. and Stolnaya, L. and Dvorakova, L. and Elleder, M. and Sikora, J.}},
  issn         = {{0378-1119}},
  keywords     = {{Cardiomyopathy; Danon disease; Lysosomal-associated membrane protein 2; Polychromatic flow cytometry; White blood cells; X-chromosome inactivation}},
  language     = {{eng}},
  month        = {{05}},
  number       = {{2}},
  pages        = {{183--195}},
  publisher    = {{Elsevier}},
  series       = {{Gene}},
  title        = {{Danon disease : A focus on processing of the novel LAMP2 mutation and comments on the beneficial use of peripheral white blood cells in the diagnosis of LAMP2 deficiency}},
  url          = {{http://dx.doi.org/10.1016/j.gene.2012.02.004}},
  doi          = {{10.1016/j.gene.2012.02.004}},
  volume       = {{498}},
  year         = {{2012}},
}