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X-Chromosome tissue inactivation in Fabry disease
S42
Abstracts
Fabry disease patients show a deficiency in the activity of the lysosomal enzyme alpha-galactosidase (alpha-GAL A). One proposed treatment for Fabry disease is pharmacological chaperone therapy, where a small molecule stabilizes the alpha-GAL A protein, leading to increased enzymatic activity. Using enzyme kinetics, tryptophan fluorescence, circular dichroism, and proteolysis assays, we show that the pharmacological chaperones 1-deoxygalactonojirimycin (DGJ) and galactose stabilize the human alpha-GAL A glycoprotein. Crystal structures of complexes of alpha-GAL A and chaperones explain the molecular basis for the higher potency of DGJ over galactose. Using site-directed mutagenesis, we show the higher potency of DGJ results from an ionic interaction with D170. We propose that protonation of D170 in acidic conditions leads to weaker binding of DGJ. The results establish a biochemical basis for pharmacological chaperone therapy applicable to other protein misfolding diseases. doi:10.1016/j.ymgme.2012.11.091
78 Clinical event status of patients with Fabry disease after long-term treatment with agalsidase beta and follow-up from The Fabry Registry Dominique P. Germaina, Gabor E. Linthorstb, William R. Wilcoxc,d, Frank Weidemanne, Marta Cizmarikf, David G. Warnockg, aUniversité de Versailles - Saint Quentin en Yvelines and Hôpital Raymond Poincaré, Garches, France, bAcademic Medical Center, Amsterdam, The Netherlands, cMedical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA, dDepartment of Pediatrics, UCLA School of Medicine, Los Angeles, CA, USA, eUniversity of Würzburg, Würzburg, Germany, f Genzyme, a Sanofi company, Biomedical Data Sciences and Informatics, Cambridge, MA, USA, gUniversity of Alabama at Birmingham, Birmingham, AL, USA Results from a phase 3 placebo-controlled, double-blind study demonstrated safety and efficacy of agalsidase beta as enzyme replacement therapy for Fabry disease (1 mg/kg/2wks for 20 weeks, NCT00074971); a subsequent open-label extension study demonstrated long-term renal stabilization (4.5 to 5 years). Of the 58 patients who participated in these studies, 50 men and 2 women were subsequently followed observationally through the Fabry Registry (NCT00196742). The objective was to evaluate the types and incidence of major clinical events that occurred since the time agalsidase beta treatment was initiated. Analyses included data from the clinical study and its extension study while patients received agalsidase beta at a dose of 1 mg/kg/2wks, as well as observational data subsequently reported to the Fabry Registry while patients reported receiving a dose of 0.9 to 1.1 mg/kg/2wks. Mean follow-up time from patients' first infusion in the clinical study to their most recent Fabry Registry assessment was 10.9–2.0 years (mean SD, min 4.9, max 12.9). At the time of their most recent follow-up, 42 of 52 patients (81%) were alive and had not experienced a major renal event, cardiovascular event, or stroke during the overall treatment period. Eight patients experienced 1 or more major clinical events during the 5-year clinical trial period, including 5 with strokes, 3 with renal dialysis, 1 with myocardial infarction, and 2 who died. During the subsequent follow-up period, it was reported that 1 patient received renal dialysis and 2 patients died. Thus, most patients remained alive and event-free with long-term agalsidase beta treatment. doi:10.1016/j.ymgme.2012.11.092
79 X-Chromosome tissue inactivation in Fabry disease Javier Gervas-Arrugaa, Jorge Javier Cebollac, Pilar Giraldoa,b,d, Miguel Pocovia,b,c, aIIS Aragón, Zaragoza, Zaragoza, Spain, bCentro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), cDepartamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, Zaragoza, Spain, dHaematology Department, Hospital Universitario Miguel Servet, Zaragoza, Spain Anderson-Fabry disease (FD) is an X-linked lysosomal storage disorder caused by deficiency or absence of the lysosomal hydrolase fabry-galactosidase. Heterozygous females can either be asymptomatic or develop the classic phenotype due to random X-chromosomal inactivation. One of the two X-chromosomes in each somatic cell of healthy human females becomes inactivated very early in embryonic development. Studies on normal females demonstrated that X-chromosome inactivation patterns appeared to be tissue-specific. DNA methylation is an important mechanism of X-chromosome inactivation. The aim of this study was to identify the methylation patterns and genotyping of the maternally and paternally derived X-chromosomes inactivation in heterozygous GLA mutated females to perform a correct diagnosis and subsequent genetic counseling of affected families. X-chromosome inactivation was analyzed in peripheral blood mononuclear cells in 8 FD female 3 carriers of G183V mutation, 5 of a complex intronic haplotype (CIH), a female control, and in skin fibroblasts of 2 female carriers of G183V and a female control by using short tandem repeat (STR) fluorescence labeling PCR amplification with bisulfite DNA treated and combined with capillary electrophoresis. Our results showed that the skin fibroblast methylation profile was related to the enzyme activity pattern. The enzymatic activity was higher with the increase in the activation of non-mutated chromosome. We did not find correlations between percentage of activation of X-chromosome and enzyme activity in skin fibroblast and in peripheral blood mononuclear cells. These findings support that the methylation patterns may play a role in the FD phenotype depending on X-chromosome activation in tissues. doi:10.1016/j.ymgme.2012.11.093
80 Activation of PPAR-a:RXRa pathway upregulates tripeptidyl peptidase 1 in brain cells: Implications for late infantile neuronal ceroid lipofuscinosis therapy Arunava Ghosha, Grant Corbetta, Frank Gonzalezb, Kalipada Pahana, a Rush University, Chicago, IL, USA, bNational Institutes of Health, Bethesda, MD, USA Background: Late infantile neuronal ceroid lipofuscinosis (LINCL) is an autosomal recessive disease caused due to mutation of Cln2 gene resulting in absence or dysfunctional tripeptidyl peptidase I (TPP1). This causes accumulation of autofluorescent storage materials in neurons and other cell types. Currently there is no established treatment for this fatal disease. Study: In this study, we investigated the effects of FDA approved fibrate drugs like gemfibrozil and fenofibrate in conjunction with alltrans retinoic acid, on the expression pattern of TPP1. We also delineate a possible molecular mechanism of upregulation of TPP1 by the fibrate drug treatments. Results: We discovered that both fenofibrate and gemfibrozil along with all trans retinoic acid were able to upregulate the expression of TPP1 both in brain cells and in vivo in the brain. Since gemfibrozil and fenofibrate are known to activate peroxisome proliferator-activated receptor (PPAR), the role of PPAR-alpha in fibrate-mediated
Abstracts
Fabry disease patients show a deficiency in the activity of the lysosomal enzyme alpha-galactosidase (alpha-GAL A). One proposed treatment for Fabry disease is pharmacological chaperone therapy, where a small molecule stabilizes the alpha-GAL A protein, leading to increased enzymatic activity. Using enzyme kinetics, tryptophan fluorescence, circular dichroism, and proteolysis assays, we show that the pharmacological chaperones 1-deoxygalactonojirimycin (DGJ) and galactose stabilize the human alpha-GAL A glycoprotein. Crystal structures of complexes of alpha-GAL A and chaperones explain the molecular basis for the higher potency of DGJ over galactose. Using site-directed mutagenesis, we show the higher potency of DGJ results from an ionic interaction with D170. We propose that protonation of D170 in acidic conditions leads to weaker binding of DGJ. The results establish a biochemical basis for pharmacological chaperone therapy applicable to other protein misfolding diseases. doi:10.1016/j.ymgme.2012.11.091
78 Clinical event status of patients with Fabry disease after long-term treatment with agalsidase beta and follow-up from The Fabry Registry Dominique P. Germaina, Gabor E. Linthorstb, William R. Wilcoxc,d, Frank Weidemanne, Marta Cizmarikf, David G. Warnockg, aUniversité de Versailles - Saint Quentin en Yvelines and Hôpital Raymond Poincaré, Garches, France, bAcademic Medical Center, Amsterdam, The Netherlands, cMedical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA, dDepartment of Pediatrics, UCLA School of Medicine, Los Angeles, CA, USA, eUniversity of Würzburg, Würzburg, Germany, f Genzyme, a Sanofi company, Biomedical Data Sciences and Informatics, Cambridge, MA, USA, gUniversity of Alabama at Birmingham, Birmingham, AL, USA Results from a phase 3 placebo-controlled, double-blind study demonstrated safety and efficacy of agalsidase beta as enzyme replacement therapy for Fabry disease (1 mg/kg/2wks for 20 weeks, NCT00074971); a subsequent open-label extension study demonstrated long-term renal stabilization (4.5 to 5 years). Of the 58 patients who participated in these studies, 50 men and 2 women were subsequently followed observationally through the Fabry Registry (NCT00196742). The objective was to evaluate the types and incidence of major clinical events that occurred since the time agalsidase beta treatment was initiated. Analyses included data from the clinical study and its extension study while patients received agalsidase beta at a dose of 1 mg/kg/2wks, as well as observational data subsequently reported to the Fabry Registry while patients reported receiving a dose of 0.9 to 1.1 mg/kg/2wks. Mean follow-up time from patients' first infusion in the clinical study to their most recent Fabry Registry assessment was 10.9–2.0 years (mean SD, min 4.9, max 12.9). At the time of their most recent follow-up, 42 of 52 patients (81%) were alive and had not experienced a major renal event, cardiovascular event, or stroke during the overall treatment period. Eight patients experienced 1 or more major clinical events during the 5-year clinical trial period, including 5 with strokes, 3 with renal dialysis, 1 with myocardial infarction, and 2 who died. During the subsequent follow-up period, it was reported that 1 patient received renal dialysis and 2 patients died. Thus, most patients remained alive and event-free with long-term agalsidase beta treatment. doi:10.1016/j.ymgme.2012.11.092
79 X-Chromosome tissue inactivation in Fabry disease Javier Gervas-Arrugaa, Jorge Javier Cebollac, Pilar Giraldoa,b,d, Miguel Pocovia,b,c, aIIS Aragón, Zaragoza, Zaragoza, Spain, bCentro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), cDepartamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, Zaragoza, Spain, dHaematology Department, Hospital Universitario Miguel Servet, Zaragoza, Spain Anderson-Fabry disease (FD) is an X-linked lysosomal storage disorder caused by deficiency or absence of the lysosomal hydrolase fabry-galactosidase. Heterozygous females can either be asymptomatic or develop the classic phenotype due to random X-chromosomal inactivation. One of the two X-chromosomes in each somatic cell of healthy human females becomes inactivated very early in embryonic development. Studies on normal females demonstrated that X-chromosome inactivation patterns appeared to be tissue-specific. DNA methylation is an important mechanism of X-chromosome inactivation. The aim of this study was to identify the methylation patterns and genotyping of the maternally and paternally derived X-chromosomes inactivation in heterozygous GLA mutated females to perform a correct diagnosis and subsequent genetic counseling of affected families. X-chromosome inactivation was analyzed in peripheral blood mononuclear cells in 8 FD female 3 carriers of G183V mutation, 5 of a complex intronic haplotype (CIH), a female control, and in skin fibroblasts of 2 female carriers of G183V and a female control by using short tandem repeat (STR) fluorescence labeling PCR amplification with bisulfite DNA treated and combined with capillary electrophoresis. Our results showed that the skin fibroblast methylation profile was related to the enzyme activity pattern. The enzymatic activity was higher with the increase in the activation of non-mutated chromosome. We did not find correlations between percentage of activation of X-chromosome and enzyme activity in skin fibroblast and in peripheral blood mononuclear cells. These findings support that the methylation patterns may play a role in the FD phenotype depending on X-chromosome activation in tissues. doi:10.1016/j.ymgme.2012.11.093
80 Activation of PPAR-a:RXRa pathway upregulates tripeptidyl peptidase 1 in brain cells: Implications for late infantile neuronal ceroid lipofuscinosis therapy Arunava Ghosha, Grant Corbetta, Frank Gonzalezb, Kalipada Pahana, a Rush University, Chicago, IL, USA, bNational Institutes of Health, Bethesda, MD, USA Background: Late infantile neuronal ceroid lipofuscinosis (LINCL) is an autosomal recessive disease caused due to mutation of Cln2 gene resulting in absence or dysfunctional tripeptidyl peptidase I (TPP1). This causes accumulation of autofluorescent storage materials in neurons and other cell types. Currently there is no established treatment for this fatal disease. Study: In this study, we investigated the effects of FDA approved fibrate drugs like gemfibrozil and fenofibrate in conjunction with alltrans retinoic acid, on the expression pattern of TPP1. We also delineate a possible molecular mechanism of upregulation of TPP1 by the fibrate drug treatments. Results: We discovered that both fenofibrate and gemfibrozil along with all trans retinoic acid were able to upregulate the expression of TPP1 both in brain cells and in vivo in the brain. Since gemfibrozil and fenofibrate are known to activate peroxisome proliferator-activated receptor (PPAR), the role of PPAR-alpha in fibrate-mediated