Limits...
Co-inherited mutations of Fas and caspase-10 in development of the autoimmune lymphoproliferative syndrome.

Cerutti E, Campagnoli MF, Ferretti M, Garelli E, Crescenzio N, Rosolen A, Chiocchetti A, Lenardo MJ, Ramenghi U, Dianzani U - BMC Immunol. (2007)

Bottom Line: However, other mutations, namely of the FasL gene (ALPS-Ib) and the caspase-10 gene (ALPS-II) are occasionally detected, whereas some patients do not present any known mutations (ALPS-III).Fas expression was reduced and caspase-10 activity was decreased in both patients.In both patients, the mutations were inherited from distinct healthy parents.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interdisciplinary Research Center of Autoimmune Diseases (IRCAD) and Department of Medical Science, A, Avogadro University of Eastern Piedmont, Novara, Italy. cerutti@med.unipmn.it

ABSTRACT

Background: Autoimmune lymphoproliferative syndrome (ALPS) is a rare inherited disorder characterized by defective function of Fas, autoimmune manifestations that predominantly involve blood cells, polyclonal accumulation of lymphocytes in the spleen and lymph nodes with lymphoadenomegaly and/or splenomegaly, and expansion of TCRalphabeta+ CD4/CD8 double-negative (DN) T cells in the peripheral blood. Most frequently, it is due to Fas gene mutations, causing ALPS type Ia (ALPS-Ia). However, other mutations, namely of the FasL gene (ALPS-Ib) and the caspase-10 gene (ALPS-II) are occasionally detected, whereas some patients do not present any known mutations (ALPS-III). Recently, mutations of the NRAS gene have been suggested to cause ALPS-IV.

Results: This work reports two patients that are combined heterozygous for single nucleotide substitutions in the Fas and caspase-10 genes. The first patient carried a splice site defect suppressing allele expression in the Fas gene and the P501L substitution in caspase-10. The second had a mutation causing a premature stop codon (Q47X) in the Fas gene and the Y446C substitution in caspase-10. Fas expression was reduced and caspase-10 activity was decreased in both patients. In both patients, the mutations were inherited from distinct healthy parents.

Conclusion: These data strongly suggest that co-transmission of these mutation was responsible for ALPS.

Show MeSH

Related in: MedlinePlus

Caspase-10 activity in 293T cells transfected with P501L-caspase-10. Analysis of 293T cells transiently transfected with the mock, WTFLAG, P501LHA, or P501LHA+WTFLAG plasmids, as indicated in the panel. a) Western blot analysis of cell transfectant lysates performed with anti-caspase-10 antibody; expression of the transfected molecules was confirmed using anti-HA and -FLAG antibodies (data not shown). The white arrow shows the pro-caspase-10; black arrows indicate the cleaved forms. b) Fluorimetric enzyme assay of caspase-10 activity evaluated in the cell transfectant lysates 24 h after transfection; data are relative to those displayed by mock-transfected cells (indicated as 100%) and are the means ± SE of data from 6 independent experiments. The asterisks mark the data significantly different from those obtained with P501LHA-transfected cells (p < 0.01, Mann Whitney test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2211507&req=5

Figure 3: Caspase-10 activity in 293T cells transfected with P501L-caspase-10. Analysis of 293T cells transiently transfected with the mock, WTFLAG, P501LHA, or P501LHA+WTFLAG plasmids, as indicated in the panel. a) Western blot analysis of cell transfectant lysates performed with anti-caspase-10 antibody; expression of the transfected molecules was confirmed using anti-HA and -FLAG antibodies (data not shown). The white arrow shows the pro-caspase-10; black arrows indicate the cleaved forms. b) Fluorimetric enzyme assay of caspase-10 activity evaluated in the cell transfectant lysates 24 h after transfection; data are relative to those displayed by mock-transfected cells (indicated as 100%) and are the means ± SE of data from 6 independent experiments. The asterisks mark the data significantly different from those obtained with P501LHA-transfected cells (p < 0.01, Mann Whitney test).

Mentions: To further assess the activity of the novel P501L-caspase-10, the cDNAs coding for it or the wild-type protein (isoform d) were cloned into the pcDNA3.1 Myc-His vector, fused to HA- or FLAG-tag sequences respectively (P501LHA and WTFLAG plasmids). Both were transiently transfected into 293T cells, expressing minimal levels of endogenous caspase-10. Moreover, 293T cells were cotrasfected with the P501LHA and WTFLAG plasmids to determine whether the mutated form exterted a dominant negative activity on the wild type form. Western blot analysis showed that both constructs were expressed at comparable levels in all trasfectants and both proteins were spontaneously cleaved, the mutated form even more efficiently than the wild type form (Fig. 3). However, analysis of the caspase-10 enzyme activity on the lysates by a fluorimetric assay showed that the P501L-caspase-10 displayed about 50% of the activity displayed by the wild type. Cells cotransfected with the P501LHA and WTFLAG plasmids showed levels of caspase-10 activity intermediate between those of cells transfected with each plasmid alone, which indicates that the mutated form does not exert a dominant negative activity on the wild type.


Co-inherited mutations of Fas and caspase-10 in development of the autoimmune lymphoproliferative syndrome.

Cerutti E, Campagnoli MF, Ferretti M, Garelli E, Crescenzio N, Rosolen A, Chiocchetti A, Lenardo MJ, Ramenghi U, Dianzani U - BMC Immunol. (2007)

Caspase-10 activity in 293T cells transfected with P501L-caspase-10. Analysis of 293T cells transiently transfected with the mock, WTFLAG, P501LHA, or P501LHA+WTFLAG plasmids, as indicated in the panel. a) Western blot analysis of cell transfectant lysates performed with anti-caspase-10 antibody; expression of the transfected molecules was confirmed using anti-HA and -FLAG antibodies (data not shown). The white arrow shows the pro-caspase-10; black arrows indicate the cleaved forms. b) Fluorimetric enzyme assay of caspase-10 activity evaluated in the cell transfectant lysates 24 h after transfection; data are relative to those displayed by mock-transfected cells (indicated as 100%) and are the means ± SE of data from 6 independent experiments. The asterisks mark the data significantly different from those obtained with P501LHA-transfected cells (p < 0.01, Mann Whitney test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211507&req=5

Figure 3: Caspase-10 activity in 293T cells transfected with P501L-caspase-10. Analysis of 293T cells transiently transfected with the mock, WTFLAG, P501LHA, or P501LHA+WTFLAG plasmids, as indicated in the panel. a) Western blot analysis of cell transfectant lysates performed with anti-caspase-10 antibody; expression of the transfected molecules was confirmed using anti-HA and -FLAG antibodies (data not shown). The white arrow shows the pro-caspase-10; black arrows indicate the cleaved forms. b) Fluorimetric enzyme assay of caspase-10 activity evaluated in the cell transfectant lysates 24 h after transfection; data are relative to those displayed by mock-transfected cells (indicated as 100%) and are the means ± SE of data from 6 independent experiments. The asterisks mark the data significantly different from those obtained with P501LHA-transfected cells (p < 0.01, Mann Whitney test).
Mentions: To further assess the activity of the novel P501L-caspase-10, the cDNAs coding for it or the wild-type protein (isoform d) were cloned into the pcDNA3.1 Myc-His vector, fused to HA- or FLAG-tag sequences respectively (P501LHA and WTFLAG plasmids). Both were transiently transfected into 293T cells, expressing minimal levels of endogenous caspase-10. Moreover, 293T cells were cotrasfected with the P501LHA and WTFLAG plasmids to determine whether the mutated form exterted a dominant negative activity on the wild type form. Western blot analysis showed that both constructs were expressed at comparable levels in all trasfectants and both proteins were spontaneously cleaved, the mutated form even more efficiently than the wild type form (Fig. 3). However, analysis of the caspase-10 enzyme activity on the lysates by a fluorimetric assay showed that the P501L-caspase-10 displayed about 50% of the activity displayed by the wild type. Cells cotransfected with the P501LHA and WTFLAG plasmids showed levels of caspase-10 activity intermediate between those of cells transfected with each plasmid alone, which indicates that the mutated form does not exert a dominant negative activity on the wild type.

Bottom Line: However, other mutations, namely of the FasL gene (ALPS-Ib) and the caspase-10 gene (ALPS-II) are occasionally detected, whereas some patients do not present any known mutations (ALPS-III).Fas expression was reduced and caspase-10 activity was decreased in both patients.In both patients, the mutations were inherited from distinct healthy parents.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interdisciplinary Research Center of Autoimmune Diseases (IRCAD) and Department of Medical Science, A, Avogadro University of Eastern Piedmont, Novara, Italy. cerutti@med.unipmn.it

ABSTRACT

Background: Autoimmune lymphoproliferative syndrome (ALPS) is a rare inherited disorder characterized by defective function of Fas, autoimmune manifestations that predominantly involve blood cells, polyclonal accumulation of lymphocytes in the spleen and lymph nodes with lymphoadenomegaly and/or splenomegaly, and expansion of TCRalphabeta+ CD4/CD8 double-negative (DN) T cells in the peripheral blood. Most frequently, it is due to Fas gene mutations, causing ALPS type Ia (ALPS-Ia). However, other mutations, namely of the FasL gene (ALPS-Ib) and the caspase-10 gene (ALPS-II) are occasionally detected, whereas some patients do not present any known mutations (ALPS-III). Recently, mutations of the NRAS gene have been suggested to cause ALPS-IV.

Results: This work reports two patients that are combined heterozygous for single nucleotide substitutions in the Fas and caspase-10 genes. The first patient carried a splice site defect suppressing allele expression in the Fas gene and the P501L substitution in caspase-10. The second had a mutation causing a premature stop codon (Q47X) in the Fas gene and the Y446C substitution in caspase-10. Fas expression was reduced and caspase-10 activity was decreased in both patients. In both patients, the mutations were inherited from distinct healthy parents.

Conclusion: These data strongly suggest that co-transmission of these mutation was responsible for ALPS.

Show MeSH
Related in: MedlinePlus