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Fluorinated chaperone-β-cyclodextrin formulations for neuronopathic Gaucher disease
S50
Abstracts / Molecular Genetics and Metabolism 120 (2016) S17–S145
gene. Initial screening revealed aortic root dilatation, MVP, moderately dilated LV, diminished LV function and LV non-compaction. Also noted were lean habitus, high palate, pectus excavatum, joint laxity, and cutaneous fragility - features suspicious for a connective tissue disorder. As cardiac involvement is not usually associated with the common IVS1(-13) TtoG splice site mutation, targeted sequencing of FBNI, and TGFBR1 and TGFBR2 that was then available was normal. He started on enzyme replacement therapy with good compliance. After 4 years of therapy he had a seizure and brain MRI showed restricted diffusion in the left splenium suggestive of ischemia. A year later, he had palpitations with worsening LV hypokinesis and ejection fraction with inducible self-limiting polymorphic ventricular tachycardia. An ICD was placed. He has since suffered several episodes of ICD shock for ventricular tachyarrhythmia. Interval EKG reveals normal rhythm. Given presentation whole exome sequencing was undertaken. He is heterozygous for a paternally inherited D1937N variant of uncertain significance in MYH6 gene. Sequencing of mitochondrial DNA was normal. Pathogenic variants in MYH6 cause dilated cardiomyopathy type IEE, a late onset disease with mild to moderate disease burden. Synergistic heterozygosity for the genetic changes in MYH6 and GAA is a possibility. Additional studies are planned. His 20-year old sister also affected with LOPD has a normal cardiac exam. His father is asymptomatic. This case illustrates the impact of comorbidities on effective management of Pompe disease and underscores the importance of excluding them when clinical presentation cannot be explained by the diagnosis.
doi:10.1016/j.ymgme.2016.11.105
97 Fluorinated chaperone-β-cyclodextrin formulations for neuronopathic Gaucher disease Jose M. Garcia Fernandeza, Elena M. Sanchez-Fernandezb, Mario de la Matac, Maria I. García Morenob, Juan M. Benitoa, Eiji Nanbad, Yoshiyuki Suzukie, Katsumi Higakid, Jose A. Sanchez-Alcazarc, Carmen Ortiz Melletb, aInstituto de Investigaciones Químicas, CSIC, Sevilla, Spain, bUniversidad de Sevilla, Sevilla, Spain, cCentro Andaluz de Biología del Desarrollo, CSIC-Universidad Pablo de Olavide, Sevilla, Spain, dTottori University, Tottori, Japan, eTokyo Metropolotan Institute of Medical Science, Tokyo, Japan Many of the Gaucher disease-associated mutant glucocerebrosidase (GCase) forms are catalytically competent and able to process the putative substrates, provided the unfolded protein response leading to endoplasmic reticulum-associated degradation is bypassed. The use of pharmacological chaperones (PCs) to rescue the endogenous mutant enzyme by stabilizing the folding conformation and restoring trafficking represent thus an interesting therapeutic option. However, for most PC candidates the chaperoning effect was restricted to mutations located in the catalytic domain of GCase, which do not include the homozygous L444P having the highest prevalence among neuropathic GD patients. Inspection of X-ray structural data revealed that the PCs are distorted upon binding to the enzyme. This inducedfit binding mechanism probably demands a low conformational effort from the protein side. We hypothesized that chaperones encompassing an undistortable moiety, mimicking the glucose portion of glucosylceramide, and substituents providing additional interactions with the enzyme will force the protein to wrap over to form a tightbound PC:GCase complex, thereby propagating the correct folding and stabilizing effects more efficiently beyond the catalytic domain. As a proof of concept, fluoroalkylated nor-tropane (calystegine)-based amphiphilic sp2-iminosugars were developed. To prevent the high tendency
of amphiphilic polyfluorocompounds to form colloidal aggregates, we capitalize on their high avidity towards the cavity of β-cyclodextrin (βCD), a commercially available biocompatible cyclooligosaccharide broadly used in the pharmaceutical industry to improve drug solubility, bioavailability and penetration across the BBB. The design and synthesis of the fluorinated pharmacological chaperone prototypes, the characterization of the corresponding inclusion complexes with βCD and the GCase activity enhancement in GD fibroblasts hosting different mutations associated to neuronopathic variants of the disease have been accomplished. Notably, fluorinated chaperone-β-cyclodextrin formulations exhibiting unprecedented L444P/L444P GCase enhancing capabilities were identified. (The authors acknowledge funds from MINECO (SAF2013-44021-R; CTQ2015-64425-C2-1-R), FIS (PI13/00129) and the Junta de Andalucia ( CTS-5725; FQM-1467)).
doi:10.1016/j.ymgme.2016.11.106
98 In vivo Raman micro-spectroscopy: a new way to screen Fabry disease Roselyne Garnotel, Laboratoire de Biologie et Recherche Pédiatriques, Reims, France Fabry disease is a multisystemic X-linked lysosomal disease caused by a deficiency of α-galactodosidase and resulting in the accumulation of glycosphingolipids in several organs such as skin. Some screening methods are available to determine the activity of this enzyme from a variety of sources such as blood and urine. However, besides to be expensive, these conventional techniques are time-consuming and can be invasive. Thus, our study aims to use Raman micro-spectroscopy, coupled to a confocal micro-probe, as a non-invasive and label-free technique in order to diagnose the pathology directly on the skin of Fabry patients. To do so, 20 healthy volunteers and 21 Fabry patients participated in this study. Stratum corneum thickness was evaluated on protected arm site by using a confocal Raman micro-probe. Principal Component Analysis was used and permitted to separate the healthy volunteers from the Fabry patients. Moreover, the principal components were analyzed and showed the spectral vibrations responsible of this discrimination. Thereafter, a variable selection algorithm, called randfeatures, was used to determine the most spectral discriminant vibrations between two groups of spectra collected respectively from healthy volunteers and Fabry patients. Finally, specific spectroscopic markers associated to the lipid compacity and secondary structure of proteins were assessed. This pilot study allowed to assess the efficiency of the remote confocal Raman micro-probe for the in vivo diagnosis of Fabry disease. In the future, this tool would permit a better detection of the prevalence and the severity of Fabry disease.
doi:10.1016/j.ymgme.2016.11.107
99 Newborn screening and post-screening diagnosis of lysosomal diseases Michael H. Gelb, Univ. of Washington, Seattle, WA, United States Tandem mass spectrometry (MS/MS) for multiplex assay of enzymatic activities in dried blood spots has emerged as the major method being implemented for newborn screening of lysosomal
Abstracts / Molecular Genetics and Metabolism 120 (2016) S17–S145
gene. Initial screening revealed aortic root dilatation, MVP, moderately dilated LV, diminished LV function and LV non-compaction. Also noted were lean habitus, high palate, pectus excavatum, joint laxity, and cutaneous fragility - features suspicious for a connective tissue disorder. As cardiac involvement is not usually associated with the common IVS1(-13) TtoG splice site mutation, targeted sequencing of FBNI, and TGFBR1 and TGFBR2 that was then available was normal. He started on enzyme replacement therapy with good compliance. After 4 years of therapy he had a seizure and brain MRI showed restricted diffusion in the left splenium suggestive of ischemia. A year later, he had palpitations with worsening LV hypokinesis and ejection fraction with inducible self-limiting polymorphic ventricular tachycardia. An ICD was placed. He has since suffered several episodes of ICD shock for ventricular tachyarrhythmia. Interval EKG reveals normal rhythm. Given presentation whole exome sequencing was undertaken. He is heterozygous for a paternally inherited D1937N variant of uncertain significance in MYH6 gene. Sequencing of mitochondrial DNA was normal. Pathogenic variants in MYH6 cause dilated cardiomyopathy type IEE, a late onset disease with mild to moderate disease burden. Synergistic heterozygosity for the genetic changes in MYH6 and GAA is a possibility. Additional studies are planned. His 20-year old sister also affected with LOPD has a normal cardiac exam. His father is asymptomatic. This case illustrates the impact of comorbidities on effective management of Pompe disease and underscores the importance of excluding them when clinical presentation cannot be explained by the diagnosis.
doi:10.1016/j.ymgme.2016.11.105
97 Fluorinated chaperone-β-cyclodextrin formulations for neuronopathic Gaucher disease Jose M. Garcia Fernandeza, Elena M. Sanchez-Fernandezb, Mario de la Matac, Maria I. García Morenob, Juan M. Benitoa, Eiji Nanbad, Yoshiyuki Suzukie, Katsumi Higakid, Jose A. Sanchez-Alcazarc, Carmen Ortiz Melletb, aInstituto de Investigaciones Químicas, CSIC, Sevilla, Spain, bUniversidad de Sevilla, Sevilla, Spain, cCentro Andaluz de Biología del Desarrollo, CSIC-Universidad Pablo de Olavide, Sevilla, Spain, dTottori University, Tottori, Japan, eTokyo Metropolotan Institute of Medical Science, Tokyo, Japan Many of the Gaucher disease-associated mutant glucocerebrosidase (GCase) forms are catalytically competent and able to process the putative substrates, provided the unfolded protein response leading to endoplasmic reticulum-associated degradation is bypassed. The use of pharmacological chaperones (PCs) to rescue the endogenous mutant enzyme by stabilizing the folding conformation and restoring trafficking represent thus an interesting therapeutic option. However, for most PC candidates the chaperoning effect was restricted to mutations located in the catalytic domain of GCase, which do not include the homozygous L444P having the highest prevalence among neuropathic GD patients. Inspection of X-ray structural data revealed that the PCs are distorted upon binding to the enzyme. This inducedfit binding mechanism probably demands a low conformational effort from the protein side. We hypothesized that chaperones encompassing an undistortable moiety, mimicking the glucose portion of glucosylceramide, and substituents providing additional interactions with the enzyme will force the protein to wrap over to form a tightbound PC:GCase complex, thereby propagating the correct folding and stabilizing effects more efficiently beyond the catalytic domain. As a proof of concept, fluoroalkylated nor-tropane (calystegine)-based amphiphilic sp2-iminosugars were developed. To prevent the high tendency
of amphiphilic polyfluorocompounds to form colloidal aggregates, we capitalize on their high avidity towards the cavity of β-cyclodextrin (βCD), a commercially available biocompatible cyclooligosaccharide broadly used in the pharmaceutical industry to improve drug solubility, bioavailability and penetration across the BBB. The design and synthesis of the fluorinated pharmacological chaperone prototypes, the characterization of the corresponding inclusion complexes with βCD and the GCase activity enhancement in GD fibroblasts hosting different mutations associated to neuronopathic variants of the disease have been accomplished. Notably, fluorinated chaperone-β-cyclodextrin formulations exhibiting unprecedented L444P/L444P GCase enhancing capabilities were identified. (The authors acknowledge funds from MINECO (SAF2013-44021-R; CTQ2015-64425-C2-1-R), FIS (PI13/00129) and the Junta de Andalucia ( CTS-5725; FQM-1467)).
doi:10.1016/j.ymgme.2016.11.106
98 In vivo Raman micro-spectroscopy: a new way to screen Fabry disease Roselyne Garnotel, Laboratoire de Biologie et Recherche Pédiatriques, Reims, France Fabry disease is a multisystemic X-linked lysosomal disease caused by a deficiency of α-galactodosidase and resulting in the accumulation of glycosphingolipids in several organs such as skin. Some screening methods are available to determine the activity of this enzyme from a variety of sources such as blood and urine. However, besides to be expensive, these conventional techniques are time-consuming and can be invasive. Thus, our study aims to use Raman micro-spectroscopy, coupled to a confocal micro-probe, as a non-invasive and label-free technique in order to diagnose the pathology directly on the skin of Fabry patients. To do so, 20 healthy volunteers and 21 Fabry patients participated in this study. Stratum corneum thickness was evaluated on protected arm site by using a confocal Raman micro-probe. Principal Component Analysis was used and permitted to separate the healthy volunteers from the Fabry patients. Moreover, the principal components were analyzed and showed the spectral vibrations responsible of this discrimination. Thereafter, a variable selection algorithm, called randfeatures, was used to determine the most spectral discriminant vibrations between two groups of spectra collected respectively from healthy volunteers and Fabry patients. Finally, specific spectroscopic markers associated to the lipid compacity and secondary structure of proteins were assessed. This pilot study allowed to assess the efficiency of the remote confocal Raman micro-probe for the in vivo diagnosis of Fabry disease. In the future, this tool would permit a better detection of the prevalence and the severity of Fabry disease.
doi:10.1016/j.ymgme.2016.11.107
99 Newborn screening and post-screening diagnosis of lysosomal diseases Michael H. Gelb, Univ. of Washington, Seattle, WA, United States Tandem mass spectrometry (MS/MS) for multiplex assay of enzymatic activities in dried blood spots has emerged as the major method being implemented for newborn screening of lysosomal