Functional characterization of ryanodine receptor (RYR1) sequence variants using a metabolic assay in immortalized B-lymphocytes
Mutations in the RYR1 gene are linked to malignant hyperthermia (MH), central core disease and multi-minicore disease. We screened by DHPLC the RYR1 gene in 24 subjects for mutations, and characterized functional alterations caused by some RYR1 variants. Three novel sequence variants and twenty nove...
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| Veröffentlicht in: | Human mutation 2009-04, Vol.30 (4), p.E575-E590 |
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| creator | Zullo, Alberto Klingler, Werner De Sarno, Claudia Ferrara, Marina Fortunato, Giuliana Perrotta, Giuseppa Gravino, Elvira Di Noto, Rosella Lehmann-Horn, Frank Melzer, Werner Salvatore, Francesco Carsana, Antonella |
| description | Mutations in the RYR1 gene are linked to malignant hyperthermia (MH), central core disease and multi-minicore disease. We screened by DHPLC the RYR1 gene in 24 subjects for mutations, and characterized functional alterations caused by some RYR1 variants. Three novel sequence variants and twenty novel polymorphisms were identified. Immortalized lymphoblastoid cell lines from patients with RYR1 variants and from controls were stimulated with 4-chloro-m-cresol (4-CmC) and the rate of extracellular acidification was recorded. We demonstrate that the increased acidification rate of lymphoblastoid cells in response to 4-CmC is mainly due to RYR1 activation. Cells expressing RYR1 variants in the N-terminal and in the central region of the protein (p.Arg530His, p.Arg2163Pro, p.Asn2342Ser, p.Glu2371Gly and p.Arg2454His) displayed higher activity compared with controls; this could account for the MH-susceptible phenotype. Cell lines harboring RYR1Cys⁴⁶⁶⁴Arg were significantly less activated by 4-CmC. This result indicates that the p.Cys4664Arg variant causes a leaky channel and depletion of intracellular stores. The functional changes detected corroborate the variants analyzed as disease-causing alterations and the acidification rate measurements as a means to monitor Ca²⁺-induced metabolic changes in cells harboring mutant RYR1 channels. |
| doi_str_mv | 10.1002/humu.20991 |
| format | Article |
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We screened by DHPLC the RYR1 gene in 24 subjects for mutations, and characterized functional alterations caused by some RYR1 variants. Three novel sequence variants and twenty novel polymorphisms were identified. Immortalized lymphoblastoid cell lines from patients with RYR1 variants and from controls were stimulated with 4-chloro-m-cresol (4-CmC) and the rate of extracellular acidification was recorded. We demonstrate that the increased acidification rate of lymphoblastoid cells in response to 4-CmC is mainly due to RYR1 activation. Cells expressing RYR1 variants in the N-terminal and in the central region of the protein (p.Arg530His, p.Arg2163Pro, p.Asn2342Ser, p.Glu2371Gly and p.Arg2454His) displayed higher activity compared with controls; this could account for the MH-susceptible phenotype. Cell lines harboring RYR1Cys⁴⁶⁶⁴Arg were significantly less activated by 4-CmC. This result indicates that the p.Cys4664Arg variant causes a leaky channel and depletion of intracellular stores. The functional changes detected corroborate the variants analyzed as disease-causing alterations and the acidification rate measurements as a means to monitor Ca²⁺-induced metabolic changes in cells harboring mutant RYR1 channels.</description><identifier>ISSN: 1059-7794</identifier><identifier>EISSN: 1098-1004</identifier><identifier>DOI: 10.1002/humu.20991</identifier><identifier>PMID: 19191333</identifier><language>eng</language><publisher>Hoboken: Wiley Subscription Services, Inc., A Wiley Company</publisher><subject>B-lymphocytes ; B-Lymphocytes - cytology ; B-Lymphocytes - drug effects ; B-Lymphocytes - metabolism ; calcium channel ; Cell Line, Transformed ; Chromatography, High Pressure Liquid - methods ; Cresols - pharmacology ; DNA Mutational Analysis ; Extracellular Space - chemistry ; Extracellular Space - drug effects ; Family Health ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genetic Testing ; Genetic Variation ; Humans ; Hydrogen-Ion Concentration ; Male ; malignant hyperthermia ; Malignant Hyperthermia - blood ; Malignant Hyperthermia - diagnosis ; Malignant Hyperthermia - genetics ; Mutation ; Myopathies, Structural, Congenital - blood ; Myopathies, Structural, Congenital - diagnosis ; Myopathies, Structural, Congenital - genetics ; Pedigree ; Polymorphism, Genetic ; proton release ; Ryanodine Receptor Calcium Release Channel - genetics ; Ryanodine Receptor Calcium Release Channel - physiology ; RYR1</subject><ispartof>Human mutation, 2009-04, Vol.30 (4), p.E575-E590</ispartof><rights>2009 Wiley‐Liss, Inc.</rights><rights>(c) 2009 Wiley-Liss, Inc.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4891-c3b529b5640107bb2dbc0d830faa7702d1ec201e9dbeb640374de9a027c5896e3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fhumu.20991$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fhumu.20991$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,780,784,1416,27922,27923,45572,45573</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19191333$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Zullo, Alberto</creatorcontrib><creatorcontrib>Klingler, Werner</creatorcontrib><creatorcontrib>De Sarno, Claudia</creatorcontrib><creatorcontrib>Ferrara, Marina</creatorcontrib><creatorcontrib>Fortunato, Giuliana</creatorcontrib><creatorcontrib>Perrotta, Giuseppa</creatorcontrib><creatorcontrib>Gravino, Elvira</creatorcontrib><creatorcontrib>Di Noto, Rosella</creatorcontrib><creatorcontrib>Lehmann-Horn, Frank</creatorcontrib><creatorcontrib>Melzer, Werner</creatorcontrib><creatorcontrib>Salvatore, Francesco</creatorcontrib><creatorcontrib>Carsana, Antonella</creatorcontrib><title>Functional characterization of ryanodine receptor (RYR1) sequence variants using a metabolic assay in immortalized B-lymphocytes</title><title>Human mutation</title><addtitle>Hum. Mutat</addtitle><description>Mutations in the RYR1 gene are linked to malignant hyperthermia (MH), central core disease and multi-minicore disease. We screened by DHPLC the RYR1 gene in 24 subjects for mutations, and characterized functional alterations caused by some RYR1 variants. Three novel sequence variants and twenty novel polymorphisms were identified. Immortalized lymphoblastoid cell lines from patients with RYR1 variants and from controls were stimulated with 4-chloro-m-cresol (4-CmC) and the rate of extracellular acidification was recorded. We demonstrate that the increased acidification rate of lymphoblastoid cells in response to 4-CmC is mainly due to RYR1 activation. Cells expressing RYR1 variants in the N-terminal and in the central region of the protein (p.Arg530His, p.Arg2163Pro, p.Asn2342Ser, p.Glu2371Gly and p.Arg2454His) displayed higher activity compared with controls; this could account for the MH-susceptible phenotype. Cell lines harboring RYR1Cys⁴⁶⁶⁴Arg were significantly less activated by 4-CmC. This result indicates that the p.Cys4664Arg variant causes a leaky channel and depletion of intracellular stores. The functional changes detected corroborate the variants analyzed as disease-causing alterations and the acidification rate measurements as a means to monitor Ca²⁺-induced metabolic changes in cells harboring mutant RYR1 channels.</description><subject>B-lymphocytes</subject><subject>B-Lymphocytes - cytology</subject><subject>B-Lymphocytes - drug effects</subject><subject>B-Lymphocytes - metabolism</subject><subject>calcium channel</subject><subject>Cell Line, Transformed</subject><subject>Chromatography, High Pressure Liquid - methods</subject><subject>Cresols - pharmacology</subject><subject>DNA Mutational Analysis</subject><subject>Extracellular Space - chemistry</subject><subject>Extracellular Space - drug effects</subject><subject>Family Health</subject><subject>Female</subject><subject>Gene Frequency</subject><subject>Genetic Predisposition to Disease</subject><subject>Genetic Testing</subject><subject>Genetic Variation</subject><subject>Humans</subject><subject>Hydrogen-Ion Concentration</subject><subject>Male</subject><subject>malignant hyperthermia</subject><subject>Malignant Hyperthermia - blood</subject><subject>Malignant Hyperthermia - diagnosis</subject><subject>Malignant Hyperthermia - genetics</subject><subject>Mutation</subject><subject>Myopathies, Structural, Congenital - blood</subject><subject>Myopathies, Structural, Congenital - diagnosis</subject><subject>Myopathies, Structural, Congenital - genetics</subject><subject>Pedigree</subject><subject>Polymorphism, Genetic</subject><subject>proton release</subject><subject>Ryanodine Receptor Calcium Release Channel - genetics</subject><subject>Ryanodine Receptor Calcium Release Channel - physiology</subject><subject>RYR1</subject><issn>1059-7794</issn><issn>1098-1004</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc1u1DAUhSMEoj-w4QHAK1SQUvwzjuMlVLRFDEUUBgQb68a503FJ4qmdAOmKR8dhBtgVeWHr6jufbJ8se8DoIaOUP1sN7XDIqdbsVrbLqC7zNJ7dns5S50rp2U62F-MlpbSUUtzNdphOSwixm_08HjrbO99BQ-wKAtgeg7uGaUT8koQROl-7DklAi-veB3Jw_vmcPSERrwbsLJJvEBx0fSRDdN0FAdJiD5VvnCUQI4zEdcS1rQ89NO4aa_Iib8Z2vfJ27DHey-4soYl4f7vvZ4vjlx-OTvP525NXR8_nuZ2VmuVWVJLrShYzyqiqKl5XltaloEsApSivGVpOGeq6wipBQs1q1EC5srLUBYr97PHGuw4-XTz2pnXRYtNAh36IplBUqSn4P5BTWXDBywQe3Aiy5CskFXJCn25QG3yMAZdmHVwLYTSMmqlDM3VofneY4Idb71C1WP9Dt6UlgG2A767B8QaVOV28WfyR5puMiz3--JuB8DW9XChpPp2dGPl6zr-cfVTmXeIfbfgleAMXwUWzeJ8-WFBWMMmVFr8As-PBHQ</recordid><startdate>200904</startdate><enddate>200904</enddate><creator>Zullo, Alberto</creator><creator>Klingler, Werner</creator><creator>De Sarno, Claudia</creator><creator>Ferrara, Marina</creator><creator>Fortunato, Giuliana</creator><creator>Perrotta, Giuseppa</creator><creator>Gravino, Elvira</creator><creator>Di Noto, Rosella</creator><creator>Lehmann-Horn, Frank</creator><creator>Melzer, Werner</creator><creator>Salvatore, Francesco</creator><creator>Carsana, Antonella</creator><general>Wiley Subscription Services, Inc., A Wiley Company</general><scope>FBQ</scope><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QP</scope><scope>7T5</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>200904</creationdate><title>Functional characterization of ryanodine receptor (RYR1) sequence variants using a metabolic assay in immortalized B-lymphocytes</title><author>Zullo, Alberto ; Klingler, Werner ; De Sarno, Claudia ; Ferrara, Marina ; Fortunato, Giuliana ; Perrotta, Giuseppa ; Gravino, Elvira ; Di Noto, Rosella ; Lehmann-Horn, Frank ; Melzer, Werner ; Salvatore, Francesco ; Carsana, Antonella</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4891-c3b529b5640107bb2dbc0d830faa7702d1ec201e9dbeb640374de9a027c5896e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2009</creationdate><topic>B-lymphocytes</topic><topic>B-Lymphocytes - cytology</topic><topic>B-Lymphocytes - drug effects</topic><topic>B-Lymphocytes - metabolism</topic><topic>calcium channel</topic><topic>Cell Line, Transformed</topic><topic>Chromatography, High Pressure Liquid - methods</topic><topic>Cresols - pharmacology</topic><topic>DNA Mutational Analysis</topic><topic>Extracellular Space - chemistry</topic><topic>Extracellular Space - drug effects</topic><topic>Family Health</topic><topic>Female</topic><topic>Gene Frequency</topic><topic>Genetic Predisposition to Disease</topic><topic>Genetic Testing</topic><topic>Genetic Variation</topic><topic>Humans</topic><topic>Hydrogen-Ion Concentration</topic><topic>Male</topic><topic>malignant hyperthermia</topic><topic>Malignant Hyperthermia - blood</topic><topic>Malignant Hyperthermia - diagnosis</topic><topic>Malignant Hyperthermia - genetics</topic><topic>Mutation</topic><topic>Myopathies, Structural, Congenital - blood</topic><topic>Myopathies, Structural, Congenital - diagnosis</topic><topic>Myopathies, Structural, Congenital - genetics</topic><topic>Pedigree</topic><topic>Polymorphism, Genetic</topic><topic>proton release</topic><topic>Ryanodine Receptor Calcium Release Channel - genetics</topic><topic>Ryanodine Receptor Calcium Release Channel - physiology</topic><topic>RYR1</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Zullo, Alberto</creatorcontrib><creatorcontrib>Klingler, Werner</creatorcontrib><creatorcontrib>De Sarno, Claudia</creatorcontrib><creatorcontrib>Ferrara, Marina</creatorcontrib><creatorcontrib>Fortunato, Giuliana</creatorcontrib><creatorcontrib>Perrotta, Giuseppa</creatorcontrib><creatorcontrib>Gravino, Elvira</creatorcontrib><creatorcontrib>Di Noto, Rosella</creatorcontrib><creatorcontrib>Lehmann-Horn, Frank</creatorcontrib><creatorcontrib>Melzer, Werner</creatorcontrib><creatorcontrib>Salvatore, Francesco</creatorcontrib><creatorcontrib>Carsana, Antonella</creatorcontrib><collection>AGRIS</collection><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Immunology Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Human mutation</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Zullo, Alberto</au><au>Klingler, Werner</au><au>De Sarno, Claudia</au><au>Ferrara, Marina</au><au>Fortunato, Giuliana</au><au>Perrotta, Giuseppa</au><au>Gravino, Elvira</au><au>Di Noto, Rosella</au><au>Lehmann-Horn, Frank</au><au>Melzer, Werner</au><au>Salvatore, Francesco</au><au>Carsana, Antonella</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Functional characterization of ryanodine receptor (RYR1) sequence variants using a metabolic assay in immortalized B-lymphocytes</atitle><jtitle>Human mutation</jtitle><addtitle>Hum. Mutat</addtitle><date>2009-04</date><risdate>2009</risdate><volume>30</volume><issue>4</issue><spage>E575</spage><epage>E590</epage><pages>E575-E590</pages><issn>1059-7794</issn><eissn>1098-1004</eissn><abstract>Mutations in the RYR1 gene are linked to malignant hyperthermia (MH), central core disease and multi-minicore disease. We screened by DHPLC the RYR1 gene in 24 subjects for mutations, and characterized functional alterations caused by some RYR1 variants. Three novel sequence variants and twenty novel polymorphisms were identified. Immortalized lymphoblastoid cell lines from patients with RYR1 variants and from controls were stimulated with 4-chloro-m-cresol (4-CmC) and the rate of extracellular acidification was recorded. We demonstrate that the increased acidification rate of lymphoblastoid cells in response to 4-CmC is mainly due to RYR1 activation. Cells expressing RYR1 variants in the N-terminal and in the central region of the protein (p.Arg530His, p.Arg2163Pro, p.Asn2342Ser, p.Glu2371Gly and p.Arg2454His) displayed higher activity compared with controls; this could account for the MH-susceptible phenotype. Cell lines harboring RYR1Cys⁴⁶⁶⁴Arg were significantly less activated by 4-CmC. This result indicates that the p.Cys4664Arg variant causes a leaky channel and depletion of intracellular stores. The functional changes detected corroborate the variants analyzed as disease-causing alterations and the acidification rate measurements as a means to monitor Ca²⁺-induced metabolic changes in cells harboring mutant RYR1 channels.</abstract><cop>Hoboken</cop><pub>Wiley Subscription Services, Inc., A Wiley Company</pub><pmid>19191333</pmid><doi>10.1002/humu.20991</doi><tpages>16</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | B-lymphocytes B-Lymphocytes - cytology B-Lymphocytes - drug effects B-Lymphocytes - metabolism calcium channel Cell Line, Transformed Chromatography, High Pressure Liquid - methods Cresols - pharmacology DNA Mutational Analysis Extracellular Space - chemistry Extracellular Space - drug effects Family Health Female Gene Frequency Genetic Predisposition to Disease Genetic Testing Genetic Variation Humans Hydrogen-Ion Concentration Male malignant hyperthermia Malignant Hyperthermia - blood Malignant Hyperthermia - diagnosis Malignant Hyperthermia - genetics Mutation Myopathies, Structural, Congenital - blood Myopathies, Structural, Congenital - diagnosis Myopathies, Structural, Congenital - genetics Pedigree Polymorphism, Genetic proton release Ryanodine Receptor Calcium Release Channel - genetics Ryanodine Receptor Calcium Release Channel - physiology RYR1 |
| title | Functional characterization of ryanodine receptor (RYR1) sequence variants using a metabolic assay in immortalized B-lymphocytes |
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