Name: in vitro enzyme measurement to test pharmacological chaperone responsiveness in fabry and pompe disease
Uploaded: 2017-12-20T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease at JoVE.com

The authors would like to acknowledge Mandy Loebert and Tina Czajka for excellent technical support. We thank Flora Luo (Harvard Medical School, Boston, MA, USA) for language editing help.

1. Preparation of Mutant pcDNA3.1/GLA and pcDNA3.1/GAA Constructs
NOTE: The cloning strategies for the GLA and GAA coding sequences (cds) have been reported earlier15,18.
<ol>
	<li>Site-directed Mutagenesis Using Site-Directed Mutagenesis
	<ol>
		<li>Use the reference sequences NM_000169.2 and NM_000152.4 as templates for the mutagenesis of GLA and GAA genes, respectively. Have a s...

<ol>
	<li>Ishii, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+alpha-galactosidase+A+enzymes+identified+in+Fabry+disease+patients+with+residual+enzyme+activity:+biochemical+characterization+and+restoration+of+normal+intracellular+processing+by+1-deoxygalactonojirimycin.">Mutant alpha-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin.</a> Biochem J. 406, 285-295 (2007).</li><li>Tajima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+biochemical+studies+on+Pompe+disease+and+a+&amp;#34;pseudodeficiency+of+acid+alpha-glucosidase&amp;#34.">Structural and biochemical studies on Pompe disease and a &amp;#34;pseudodeficiency of acid alpha-glucosidase&amp;#34.</a> J Hum Genet. 52, 898-906 (2007).</li><li>Lukas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+Clinical+Consequences+of+Novel+&#945;-Galactosidase+A+Mutations+in+Fabry+Disease.">Functional and Clinical Consequences of Novel &#945;-Galactosidase A Mutations in Fabry Disease.</a> Hum Mutat. 37, 43-51 (2016).</li><li>Parenti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treating+lysosomal+storage+diseases+with+pharmacological+chaperones:+from+concept+to+clinics.">Treating lysosomal storage diseases with pharmacological chaperones: from concept to clinics.</a> EMBO Mol Med. 1, 268-279 (2009).</li><li>Yasuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabry+disease:+characterization+of+alpha-galactosidase+A+double+mutations+and+the+D313Y+plasma+enzyme+pseudodeficiency+allele.">Fabry disease: characterization of alpha-galactosidase A double mutations and the D313Y plasma enzyme pseudodeficiency allele.</a> Hum Mutat. 22, 486-492 (2003).</li><li>Shimotori, M., Maruyama, H., Nakamura, G., Suyama, T., Sakamoto, F., Itoh, M., Miyabayashi, S., Ohnishi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+mutations+of+the+GLA+gene+in+Japanese+patients+with+Fabry+disease+and+their+functional+characterization+by+active+site+specific+chaperone.">Novel mutations of the GLA gene in Japanese patients with Fabry disease and their functional characterization by active site specific chaperone.</a> Hum Mutat. 29, 331 (2008).</li><li>Andreotti, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapy+of+Fabry+disease+with+pharmacological+chaperones:+from+in+silico+predictions+to+in+vitro+tests.">Therapy of Fabry disease with pharmacological chaperones: from in silico predictions to in vitro tests.</a> Orphanet J Rare Dis. 6, 66 (2011).</li><li>Khanna, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pharmacological+chaperone+AT2220+increases+the+specific+activity+and+lysosomal+delivery+of+mutant+acid+alpha-glucosidase,+and+promotes+glycogen+reduction+in+a+transgenic+mouse+model+of+Pompe+disease.">The pharmacological chaperone AT2220 increases the specific activity and lysosomal delivery of mutant acid alpha-glucosidase, and promotes glycogen reduction in a transgenic mouse model of Pompe disease.</a> PLoS One. 9, e102092 (2014).</li><li>Siekierska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;-Galactosidase+aggregation+is+a+determinant+of+pharmacological+chaperone+efficacy+on+Fabry+disease+mutants.">&#945;-Galactosidase aggregation is a determinant of pharmacological chaperone efficacy on Fabry disease mutants.</a> J Biol Chem. 287, 28386-28397 (2012).</li><li>Parenti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+enhancement+of+mutated+alpha-glucosidase+activity+in+fibroblasts+from+patients+with+Pompe+disease.">Pharmacological enhancement of mutated alpha-glucosidase activity in fibroblasts from patients with Pompe disease.</a> Mol Ther. 15, 508-514 (2007).</li><li>Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pharmacogenetic+approach+to+identify+mutant+forms+of+&#945;-galactosidase+A+that+respond+to+a+pharmacological+chaperone+for+Fabry+disease.">A pharmacogenetic approach to identify mutant forms of &#945;-galactosidase A that respond to a pharmacological chaperone for Fabry disease.</a> Hum Mutat. 32, 965-977 (2011).</li><li>Lukas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterisation+of+alpha-galactosidase+a+mutations+as+a+basis+for+a+new+classification+system+in+fabry+disease.">Functional characterisation of alpha-galactosidase a mutations as a basis for a new classification system in fabry disease.</a> PLoS Genet. 9, e1003632 (2013).</li><li>Andreotti, G., Citro, V., Correra, A., Cubellis, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thermodynamic+assay+to+test+pharmacological+chaperones+for+Fabry+disease.">A thermodynamic assay to test pharmacological chaperones for Fabry disease.</a> Biochim Biophys Acta. 1840, 1214-1224 (2014).</li><li>. Available from: <a target="_blank" href="https://www.google.ch/patents/US9095584">https://www.google.ch/patents/US9095584</a> (2017)</li><li>Lukas, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+enhancers+for+the+treatment+of+Fabry+and+Pompe+disease.">Enzyme enhancers for the treatment of Fabry and Pompe disease.</a> Mol Ther. 23, 456-4564 (2015).</li><li>Yam, G. H., Bosshard, N., Zuber, C., Steinmann, B., Roth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+chaperone+corrects+lysosomal+storage+in+Fabry+disease+caused+by+trafficking-incompetent+variants.">Pharmacological chaperone corrects lysosomal storage in Fabry disease caused by trafficking-incompetent variants.</a> Am J Physiol Cell Physiol. 290, C1076-C1082 (2006).</li><li>Shin, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+pharmacological+chaperones+in+Fabry+disease.">Screening for pharmacological chaperones in Fabry disease.</a> Biochem Biophys Res Commun. 359, 168-173 (2007).</li><li>Shin, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+response+of+mutated+alpha-galactosidase+A+to+a+pharmacological+chaperone.">Prediction of response of mutated alpha-galactosidase A to a pharmacological chaperone.</a> Pharmacogenet Genomics. 18, 773-7780 (2008).</li><li>Filoni, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+studies+of+new+GLA+gene+mutations+leading+to+conformational+Fabry+disease.">Functional studies of new GLA gene mutations leading to conformational Fabry disease.</a> Biochim Biophys Acta. 1802, 247-252 (2010).</li><li>Citro, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Large+Phenotypic+Spectrum+of+Fabry+Disease+Requires+Graduated+Diagnosis+and+Personalized+Therapy:+A+Meta-Analysis+Can+Help+to+Differentiate+Missense+Mutations.">The Large Phenotypic Spectrum of Fabry Disease Requires Graduated Diagnosis and Personalized Therapy: A Meta-Analysis Can Help to Differentiate Missense Mutations.</a> Int J Mol Sci. 17, (2016).</li><li>Cammisa, M., Correra, A., Andreotti, G., Cubellis, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabry_CEP:+a+tool+to+identify+Fabry+mutations+responsive+to+pharmacological+chaperones.">Fabry_CEP: a tool to identify Fabry mutations responsive to pharmacological chaperones.</a> Orphanet J Rare Dis. 8, 111 (2013).</li><li>Leinekugel, P., Michel, S., Conzelmann, E., Sandhoff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+correlation+between+the+residual+activity+of+beta-hexosaminidase+A+and+arylsulfatase+A+and+the+severity+of+the+resulting+lysosomal+storage+disease.">Quantitative correlation between the residual activity of beta-hexosaminidase A and arylsulfatase A and the severity of the resulting lysosomal storage disease.</a> Hum Genet. 88, 513-523 (1992).</li><li>Germain, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+pharmacodynamic+effects+of+a+pharmacological+chaperone+on+&#945;-galactosidase+A+activity+and+globotriaosylceramide+clearance+in+Fabry+disease:+report+from+two+phase+2+clinical+studies.">Safety and pharmacodynamic effects of a pharmacological chaperone on &#945;-galactosidase A activity and globotriaosylceramide clearance in Fabry disease: report from two phase 2 clinical studies.</a> Orphanet J Rare Dis. 7, 91 (2012).</li><li>Hughes, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+pharmacological+chaperone+migalastat+compared+with+enzyme+replacement+therapy+in+Fabry+disease:+18-month+results+from+the+randomised+phase+III+ATTRACT+study.">Oral pharmacological chaperone migalastat compared with enzyme replacement therapy in Fabry disease: 18-month results from the randomised phase III ATTRACT study.</a> J Med Genet. 54, 288-296 (2017).</li><li>Parenti, G., Andria, G., Valenzano, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Chaperone+Therapy:+Preclinical+Development,+Clinical+Translation,+and+Prospects+for+the+Treatment+of+Lysosomal+Storage+Disorders.">Pharmacological Chaperone Therapy: Preclinical Development, Clinical Translation, and Prospects for the Treatment of Lysosomal Storage Disorders.</a> Mol Ther. 23, 1138-1148 (2015).</li><li>Parenti, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Chaperone+Enhances+Blood+&#945;-Glucosidase+Activity+in+Pompe+Disease+Patients+Treated+With+Enzyme+Replacement+Therapy.">A Chaperone Enhances Blood &#945;-Glucosidase Activity in Pompe Disease Patients Treated With Enzyme Replacement Therapy.</a> Mol Ther. 22, 2004-2012 (2014).</li></ol>

The protocol described herein delivers robust results for enzyme damage assessment in hereditary lysosomal diseases of metabolism. This manuscript is an amendment to the protocol published earlier15. The most crucial modifications involve stringency (i.e., in the process of mutant vector construct preparation), standardization of the cell culture protocol (i.e., HEK293H cell maintenance and transfection conditions), and the high number of experimental repetitions (at least 5), wh...

There are over a dozen lysosomal storage disorders (LSDs) related to glycosidase dysfunction as a result of primary gene mutations. In Fabry (OMIM #301500) and Pompe (OMIM #232300) disease, more than 500 and 200 missense mutations1,2,3 have been reported, respectively, which corresponds to about 60% of the total mutation count. Numerous new gene variants are still being identified, many of which have unknown significance. Extensive biochemical studies revealed that certain genotypes do not lead to a complete loss-of-function of the GLA gene (OMIM *300644) in Fabry disease, but cause the corresponding enzyme to fail to reach a thermodynamically favored folding state4. This results in ER retention and premature degradation of the otherwise functional enzyme. Similar conclusions have been drawn in other LSDs including Pompe disease5. Moreover, molecular characterization of enzyme variants can facilitate clinical interpretation of the mutations at the time of diagnosis6, suggesting that LSD progression is an individual process based on the nature of the mutation. Therefore, the conventional classification into typically 2 to 3 different clinical types should be reassessed in order to streamline clinical counselling and therapeutic decisions.
Enzyme Replacement Therapy (ERT) is available for both diseases. ERT, however, has limited efficacy in affected tissues/organs such as the brain and skeletal muscle. Furthermore, ERT can elicit an immunogenic response that jeopardizes its therapeutic benefits. Pharmacological Chaperones (PCs) are an attractive treatment alternative for patients with so-called responsive mutations. PCs serve as a molecular scaffold for correct protein folding and stabilization which in turn prevents endoplasmic reticulum (ER) retention and ER-associated degradation of the enzyme. Moreover, PCs can be administered orally and are potentially able to cross the blood brain barrier. Therefore, PCT might be a more viable option for treating patients with certain genotypes. For an extensive review on PC application in LSDs, refer to the excellent review by Parenti7.
The discovery of hundreds of disease causing mutant alleles challenges pre-clinical drug testing and necessitates a simple, fast, and highly standardized assessment of amenable patients for a personalized medicine approach. In order to assess the detrimental effects of LSD gene mutations and to test candidate mutations to predict amenable patients for PCT, a highly standardized over-expression system in HEK293H cells that allows for fast and reliable enzyme activity measurement was developed. Similar over-expression systems have been previously described for Fabry and Pompe disease using either COS-78,9,10,11, HeLa cells12, or HEK29313,14,15,16 cells for the glycosidase gene.
A very similar method has even been patented as a &#34;Method to predict response to Pharmacological Chaperone treatment of diseases&#34;17 indicating the relevance of a cell culture system capable of being integrated into clinical practice.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuikChange II XL Site-Directed Mutagenesis Kit&#160;</td>
			<td>Stratagene, La Jolla, CA, USA</td>
			<td>#200522</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[R301Q]-frw: 5&#180;- CTA ATG ACC TCC AAC ACA TCA GCC C-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[R301Q]-rev: 5&#180;- GGG CTG ATG TGT TGG AGG TCA TTA G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A156V]-frw: 5&#180;-CTA CGA CAT TGA TGT CCA GAC CTT TGC TG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A156V]-rev: 5&#180;-CAG CAA AGG TCT GGA CAT CAA TGT CGT AG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A156V]-frw: 5&#180;-GGA AAT AAA ACC TGC ACA GGC TTC CCT GGG A-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GLA[A143T]-rev: 5&#180;-TCC CAG GGA AGC CTG TGC AGG TTT TAT TTC C-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[F455Y]-frw: 5&#180;-CTG CCG GGA GCT TCA GGC CCT ACG A-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[F455Y]-rev: 5&#180;-TCG TAG GGC CTG AAG CTC CCG GCA G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[P545L]-frw: 5&#180;-CAC CCT ACG TGC TTG GGG TGG TTG G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[P545L]-rev: 5&#180;-CCA ACC ACC CCA AGC ACG TAG GGT G-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[L552P]-frw: 5&#180;-TTG GGG GGA CCC CCC AGG CGG CCA C-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer GAA[P545L]-rev: 5&#180;-GTG GCC GCC TGG GGG GTC CCC CCA A-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer T7: 5&#180;-TAA TAC GAC TCA CTA TAG GG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer BGHrev: 5&#180;-TAG AAG GCA CAG TCG AGG-3&#180;</td>
			<td>MWG Eurofins Operon, Ebersberg, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>8952.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>103,753</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,064,041,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potasium chloride</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,049,360,500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 x 6H2O</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,058,330,250</td>
			<td></td>
		</tr>
		<tr>
			<td>D (+)-Glucose monohydrate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,083,422,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,064,980,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>3908.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>X863.3</td>
			<td></td>
		</tr>
		<tr>
			<td>disodium hydrogen phosphate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,065,860,500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1,062,640,050</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>X969.2</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Medium</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>X968.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Zyppy Plasmid Miniprep Kit</td>
			<td>ZymoResearch, Freiburg, Germany</td>
			<td>D4020</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAfilter Plasmid Midi Kit</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>12245</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293H cell line</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>11631-017&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s Modified Eagle Medium&#160;</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>31966-047</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone fetal bovine serum&#160;</td>
			<td>GE Healthcare, South Logan, Utah, USA</td>
			<td>SV30160.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR Standard Cell Culture Flasks, Greiner Bio One</td>
			<td>VWR International GmbH, Hannover, Germany</td>
			<td>82050-854</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates (24 well)</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>831,836</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellstar 96 well plate</td>
			<td>Greiner bio one, Frickenhausen, Germany</td>
			<td>655185</td>
			<td></td>
		</tr>
		<tr>
			<td>SafeSeal reaction tubes, 1,5mL&#160;</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>72,706</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Deoxygalactonojirimycin Hydrochloride&#160;</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>D9641</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Deoxygalactonojirimycin Hydrochloride&#160;</td>
			<td>Toronto Research Chemicals, Toronto, Canada</td>
			<td>D236500</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Deoxynojirimycin&#160;</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>D9305</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Dulbecco w/o Calcium, w/o Magnesium</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>L 1825</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Thermo Scientific, Braunschweig, Germany</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific, Braunschweig, Germany</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Methylumbelliferone</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>M1381</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Methylumbelliferyl &#945;-D-galactopyranoside</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>M7633</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Methylumbelliferyl &#945;-D-glucopyranoside</td>
			<td>Sigma Aldrich, Munich, Germany</td>
			<td>M9766</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>incubator type T6</td>
			<td>Heraeus Instruments, Hanau, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminous Plate Gr. 2 E</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>P265.1</td>
			<td></td>
		</tr>
		<tr>
			<td>3130 xl Genetic Analyzer&#160;</td>
			<td>Applied Biosystems, Darmstadt, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>GFL-3032 bacterial shaker</td>
			<td>GFL, Burgwedel, Germany&#160;</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-25 centrifuge</td>
			<td>Beckman/Coulter, Krefeld, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraspec 3100 pro Spectrophotometer</td>
			<td>Amersham Biosciences, Buckinghamshire, United Kingdom</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>water-jacket incubator&#160;</td>
			<td>Binder, Tuttlingen, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genie 1 touch mixer</td>
			<td>Scientific Industries, Bohemia, NY, USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Z 233 MK-2 refrigerated microtube centrifuge</td>
			<td>Hermle, Tuttlingen, Germany&#160;</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>LaboStar ultrapure water device</td>
			<td>Siemens, Berlin, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Shaker PST-60HL-4 orbital shaker</td>
			<td>Biosan, Riga, Latvia</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Tecan GENios Reader</td>
			<td>Tecan, M&#228;nnedorf, Switzerland</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>HI 223 Microprocessor pH Meter</td>
			<td>HANNA Instruments, Arvore, Portugal</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>CASY 
			Cell Counter + Analyser System 
			Model TT</td>
			<td>Innovatis AG, Cham/Zug, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magellan data analysis software v6.6</td>
			<td>Tecan, M&#228;nnedorf, Switzerland</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 5.04</td>
			<td>GraphPad Software, Inc., La Jolla, CA; USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Office 2010</td>
			<td>Microsoft Corporation, Redmond, WA, USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>BioEdit Alignment Editor, V7.0.1</td>
			<td>http://www.mbio.ncsu.edu/bioedit/bioedit.html</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Abbreviations</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>frw = forward; rev = reverse</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>n.a. = not applicable</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro enzyme measurement to test pharmacological chaperone responsiveness in fabry and pompe disease

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, high costs, and low patient numbers. Hundreds of mutant alleles are implicated in most of the LSDs. The diseases are typically classified into 2 to 3 different clinical types according to severity. Moreover, molecular characterization of the genotype can help predict clinical outcomes and inform patient care. Therefore, we developed a simple cell culture assay based on HEK293H cells heterologously over-expressing the mutations identified in Fabry and Pompe disease. A similar assay has recently been introduced as a preclinical test to identify amenable mutations for Pharmacological Chaperone Therapy (PCT) in Fabry disease. This manuscript describes an amended cell culture assay which enables rapid phenotypic assessment of allelic variants in Fabry and Pompe disease to identify eligible patients for PCT and may aid in the development of novel pharmacochaperones.

The mutagenesis procedure 
To assess the efficiency of GLA gene mutagenesis, the mutations were classified into one of the following categories. This approach to generate mutations revealed that about 66.5% of the GLA mutations were obtained in the first attempt. A further 25% could be obtained after a slightly modified second PCR.
Category 1: The mutagenesis PCR was effective at first att...

Watch this Scientific Journal Video about In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease at JoVE.com

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

There is a demand to make pre-clinical testing for a novel class of "orphan" drugs called pharmacological chaperones reproducible, fast, and efficient. We developed a simple, highly standardized, and versatile cell culture-based assay to screen for eligible patients as well as novel pharmacological chaperone drugs.

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, ...

The overall goal of this amended cell culture protocol is to supply a rapid phenotypic assessment of allelic variance in Fabry and Pompe disease. This method can help to answer key questions in the field of lysosomal storage diseases, such as prognostic outcomes and therapeutic decisions. The main advantage of this technique is that it is highly reproducible and that it can aid in the development of novel drugs, such as pharmaco-chaperones.To carry out site-directed mutagenesis, begin by using the reference sequences NM 00169.2 and NM 00152.4 as templates for the mutagenesis of GLA and GAA genes, respectively. Use the free Primer Design tool to support primer design. Then, have a set of high-purity, salt-free primers synthesized by a commercial provider, with sense and antisense primers carrying one of the respective sequence modifications, central to their length, to individually introduce the mutation.Prepare the reaction mixture in a 50 microliter volume and run the PCR program using standard conditions provided by the manufacturer and as listed in the text protocol. Following PCR, add one microliter of Dpn1 restriction enzyme and continue to incubate the reaction vials at 37 degrees Celsius for one hour. After transforming the plasma DNA into competent E.coli cells and preparing plasmids of the desired transformance according to the text protocol, use a suitable molecular biology tool to analyze the sequence.When the desired mutation is detected and no further sequence abnormality compared to NM 000169.2 for alpha-galactosidase A or NM 000152.4 for acid alpha-galactosidase is seen, select the clone for transfection-grade plasmid purification. Determine the purity of the DNA by measuring the absorbance in a spectrophotometer. Following the cultivation of HEK293H cells in a T75 culture flask according to the text protocol, 24 hours prior to the transfection, use PBS without calcium or magnesium to wash the cells once.With 0.05%Trypsin-EDTA, harvest the cells and in the cavities of a 24-well culture plate use 500 microliters of DMEM supplemented with 10%FBS to seed 1.5 times 10 to the five cells. Carry out cell transfection according to the manufacturer's manual. Using a mixture of one microgram of plasmid DNA dissolved in 100 microliters of serum-free DMEM and 2.5 microliters of transfection reagent.Incubate the solution for 20 minutes at room temperature, then add it to the cells in a dropwise manner. After a period of four hours at 37 degrees Celsius and 5%CO2, remove the medium containing the transfection reagent and add 500 microliters of fresh DMEM with 10%FBS and 1%penicillin-streptomycin. As an option during this step, add DGJ or DNJ to the culture medium where intended.On the day of the harvest, remove the cells from the incubator. Then aspirate the medium and use PBS with calcium and magnesium to carefully wash the cells two times. This step is critical because DGJ and DNJ are inhibitors of both enzymes, and therefore any left over would invalidate the test.Following the wash, add 200 microliters of deionized water directly on top of the cells. Then rinse the cells from the plate and transfer them to a 1.5 milliliter reaction tube. Place the samples in an appropriate foam rack and vortex them for five seconds to make the lysis more efficient.Then alternate the samples between liquid nitrogen for 10 seconds and a room-temperature water bath until the thawing is complete. After repeating the homogenization procedure five times, spin the samples for five minutes at 10, 000g. Then transfer the supernatant into a new reaction tube.To carry out the BCA assay, prepare a fresh tube for each sample containing 40 microliters of deionized H20 and add 10 microliters of sample. Mix each solution by briefly vortexing and transfer 10 microliters of each sample in triplicate into the cavities of a 96-well plate. To prepare a standard curve, dilute a 2 milligram per milliliter BSA stock solution and deionized H2O according to the text protocol.After combining reagent A and reagent B, start the reaction by adding 200 microliters of the BCA reagent solution and incubate the samples in the dark at 37 degrees Celsius and 300 rpm on an orbital shaker for one hour. Then measure the absorbance at 560 nanometers in a plate reader. The samples typically contain between one and 1.5 micrograms of protein per microliter.Dilute the calculated amount of each sample and pipette it into fresh 1.5 milliliter reaction tubes to obtain 0.05 micrograms of alpha-galactosidase A or 0.5 micrograms of acid alpha-glucosidase per microliter of solution. Vortex the samples for five seconds again and pipette 10 microliters of this dilution in duplicate into a 96-well plate. Start the reactions by adding 20 microliters of the respective substrate solution.For alpha-galactosidase A, add two millimolar 4-methylumbelliferyl alpha-D galactopyranoside, or 4-MU-gal, in 0.06 molar phosphate-citrate buffer, pH 4.7. For acid alpha-glucosidase, add 2 millimolar 4-methylumbelliferyl alpha-D glucopyranoside, or 4-MU-glu, in 0.025 molar sodium acetate, pH 4.0. Incubate the enzyme reactions for one hour in the dark at 37 degrees Celsius and 300 rpm on an orbital shaker.Then terminate the reaction by adding 200 microliters of 1.0 molar pH 10.5-adjusted glycine sodium-hydroxide buffer. Prepare a standard curve of 4-MU from a 0.01 milligram per milliliter stock according to the text protocol and pipette 10 microliters of the solutions in duplicate into a 96-well plate. Add 200 microliters of the 1.0 molar glycine sodium-hydroxide buffer to each well in order to adjust the volume and pH.Finally, measure the enzyme activity in a florescence reader equipped with the appropriate filter set. And analyze the data using the appropriate software for the fluorescence-reader device. To assess the efficiency of GLA gene mutagenesis, the mutations were classified into three categories and revealed that about 66.5%were obtained in the first attempt.A further 25%could be obtained after a slightly modified second PCR. In category three, more effort, such as primer redesign, had to be undertaken to yield the desired clone. This table refers to the enzyme-activity measurement results for three alpha-galactosidase A and three acid alpha-glucosidase mutations either left untreated or treated with DGJ and DNJ.The data are displayed as absolute values for the substrate turnover and has relative values normalized to the wild-type enzyme. The absolute enzyme activity data was corrected for endogenous enzyme activity of the HEK293H cells using cells transfected with an empty pcDNA 3.1 vector. Enzyme activity values were normalized to wild-type enzyme from corresponding experiments, which explains the deviation of relative values between the different mutants.Once mastered, the post-cell culture fraction of this experiment, which represents most of the hands-on time, can be done within four hours. After its development, this technique paved the way for researchers in the field of lysosomal storage diseases to explore genotype-phenotype correlations in Fabry and Pompe disease. This protocol can aid in the development of novel drugs in lysosomal storage diseases.

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