Clinically reported heterozygous mutations in the PINK1 kinase domain exert a gene dosage effect
Identifieur interne : 001175 ( Main/Corpus ); précédent : 001174; suivant : 001176Clinically reported heterozygous mutations in the PINK1 kinase domain exert a gene dosage effect
Auteurs : Eng-King Tan ; F. Shaffra Refai ; Mobin Siddique ; Karen Yap ; Patrick Ho ; Stephanie Fook-Chong ; Yi ZhaoSource :
- Human Mutation [ 1059-7794 ] ; 2009-11.
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Abstract
Mutations in the gene encoding phosphatase and tensin homolog (PTEN)‐induced kinase 1 (PINK1) have been associated with the loss of dopaminergic neurons characteristic of familial and sporadic Parkinson disease. We developed an in vitro system of stable human dopaminergic neuronal cell lines coexpressing an equivalent copy of normal and mutant PINK1 to simulate “heterozygous” and “homozygous” states in patients. Mutants in the N‐terminus, C‐terminus, and kinase domain were generated and cloned into a two‐gene mammalian expression vector to generate stable mammalian expression cell lines producing an equivalent copy number of wild‐type/mutant PINK1. The cell lines were subjected to oxidative stress and the rate of apoptosis and change in mitochondrial membrane potential (ΔΨm) were assessed. Cell lines expressing kinase and C‐terminus mutants exhibited a greater rate of apoptosis and decrease in ΔΨm, and increased time‐dependent cell loss when subjected to oxidative stress compared to the wild‐type. Cell lines expressing two copies of kinase mutants exhibited a greater apoptosis rate and ΔΨm decrease than those expressing one copy of the mutant. In time‐dependent experiments, there was a significant difference between “homozygous,” “heterozygous,” and wild‐type cell lines, with decreasing cell survival in cell lines expressing mutant copies of PINK1 compared to the wild‐type. We provided the first experimental evidence that clinically reported PINK1 heterozygous mutations exert a gene dosage effect, suggesting that haploinsufficiency of PINK1 is the most likely mechanism that increased the susceptibility to dopaminergic cellular loss. Hum Mutat 30:1551–1557, 2009. © 2009 Wiley‐Liss, Inc.
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DOI: 10.1002/humu.21108
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Mutat.</title><idno type="ISSN">1059-7794</idno><idno type="eISSN">1098-1004</idno><imprint><publisher>Wiley Subscription Services, Inc., A Wiley Company</publisher><pubPlace>Hoboken</pubPlace><date type="published" when="2009-11">2009-11</date><biblScope unit="volume">30</biblScope><biblScope unit="issue">11</biblScope><biblScope unit="page" from="1551">1551</biblScope><biblScope unit="page" to="1557">1557</biblScope></imprint><idno type="ISSN">1059-7794</idno></series><idno type="istex">6F1CAD18956AA3A42E02DCC834F55FD8006FA532</idno><idno type="DOI">10.1002/humu.21108</idno><idno type="ArticleID">HUMU21108</idno></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1059-7794</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>PD</term><term>PINK1</term><term>Parkinson</term><term>haploinsufficiency</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Mutations in the gene encoding phosphatase and tensin homolog (PTEN)‐induced kinase 1 (PINK1) have been associated with the loss of dopaminergic neurons characteristic of familial and sporadic Parkinson disease. We developed an in vitro system of stable human dopaminergic neuronal cell lines coexpressing an equivalent copy of normal and mutant PINK1 to simulate “heterozygous” and “homozygous” states in patients. Mutants in the N‐terminus, C‐terminus, and kinase domain were generated and cloned into a two‐gene mammalian expression vector to generate stable mammalian expression cell lines producing an equivalent copy number of wild‐type/mutant PINK1. The cell lines were subjected to oxidative stress and the rate of apoptosis and change in mitochondrial membrane potential (ΔΨm) were assessed. Cell lines expressing kinase and C‐terminus mutants exhibited a greater rate of apoptosis and decrease in ΔΨm, and increased time‐dependent cell loss when subjected to oxidative stress compared to the wild‐type. Cell lines expressing two copies of kinase mutants exhibited a greater apoptosis rate and ΔΨm decrease than those expressing one copy of the mutant. In time‐dependent experiments, there was a significant difference between “homozygous,” “heterozygous,” and wild‐type cell lines, with decreasing cell survival in cell lines expressing mutant copies of PINK1 compared to the wild‐type. We provided the first experimental evidence that clinically reported PINK1 heterozygous mutations exert a gene dosage effect, suggesting that haploinsufficiency of PINK1 is the most likely mechanism that increased the susceptibility to dopaminergic cellular loss. Hum Mutat 30:1551–1557, 2009. © 2009 Wiley‐Liss, Inc.</div></front></TEI><istex><corpusName>wiley</corpusName><author><json:item><name>Eng‐King Tan</name><affiliations><json:string>Department of Neurology, Singapore General Hospital, Singapore</json:string><json:string>Department of Clinical Research, Singapore General Hospital, Singapore</json:string><json:string>Department of Neurology, National Neuroscience Institute, Singapore</json:string><json:string>Neuroscience and Behavioral Disorders (NBD) Program, Duke–National University of Singapore (NUS) Graduate Medical School, Singapore</json:string></affiliations></json:item><json:item><name>F. Shaffra Refai</name><affiliations><json:string>Department of Neurology, Singapore General Hospital, Singapore</json:string></affiliations></json:item><json:item><name>Mobin Siddique</name><affiliations><json:string>Department of Neurology, Singapore General Hospital, Singapore</json:string></affiliations></json:item><json:item><name>Karen Yap</name><affiliations><json:string>Department of Neurology, Singapore General Hospital, Singapore</json:string></affiliations></json:item><json:item><name>Patrick Ho</name><affiliations><json:string>Department of Neurology, Singapore General Hospital, Singapore</json:string></affiliations></json:item><json:item><name>Stephanie Fook‐Chong</name><affiliations><json:string>Department of Clinical Research, Singapore General Hospital, Singapore</json:string></affiliations></json:item><json:item><name>Yi Zhao</name><affiliations><json:string>Department of Clinical Research, Singapore General Hospital, Singapore</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>PINK1</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>haploinsufficiency</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Parkinson</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>PD</value></json:item></subject><articleId><json:string>HUMU21108</json:string></articleId><language><json:string>eng</json:string></language><abstract>Mutations in the gene encoding phosphatase and tensin homolog (PTEN)‐induced kinase 1 (PINK1) have been associated with the loss of dopaminergic neurons characteristic of familial and sporadic Parkinson disease. We developed an in vitro system of stable human dopaminergic neuronal cell lines coexpressing an equivalent copy of normal and mutant PINK1 to simulate “heterozygous” and “homozygous” states in patients. Mutants in the N‐terminus, C‐terminus, and kinase domain were generated and cloned into a two‐gene mammalian expression vector to generate stable mammalian expression cell lines producing an equivalent copy number of wild‐type/mutant PINK1. The cell lines were subjected to oxidative stress and the rate of apoptosis and change in mitochondrial membrane potential (ΔΨm) were assessed. Cell lines expressing kinase and C‐terminus mutants exhibited a greater rate of apoptosis and decrease in ΔΨm, and increased time‐dependent cell loss when subjected to oxidative stress compared to the wild‐type. Cell lines expressing two copies of kinase mutants exhibited a greater apoptosis rate and ΔΨm decrease than those expressing one copy of the mutant. In time‐dependent experiments, there was a significant difference between “homozygous,” “heterozygous,” and wild‐type cell lines, with decreasing cell survival in cell lines expressing mutant copies of PINK1 compared to the wild‐type. We provided the first experimental evidence that clinically reported PINK1 heterozygous mutations exert a gene dosage effect, suggesting that haploinsufficiency of PINK1 is the most likely mechanism that increased the susceptibility to dopaminergic cellular loss. Hum Mutat 30:1551–1557, 2009. © 2009 Wiley‐Liss, Inc.</abstract><qualityIndicators><score>6.968</score><pdfVersion>1.3</pdfVersion><pdfPageSize>595.276 x 793.701 pts</pdfPageSize><refBibsNative>true</refBibsNative><keywordCount>4</keywordCount><abstractCharCount>1714</abstractCharCount><pdfWordCount>4052</pdfWordCount><pdfCharCount>26481</pdfCharCount><pdfPageCount>7</pdfPageCount><abstractWordCount>243</abstractWordCount></qualityIndicators><title>Clinically reported heterozygous mutations in the PINK1 kinase domain exert a gene dosage effect</title><genre><json:string>article</json:string></genre><host><volume>30</volume><publisherId><json:string>HUMU</json:string></publisherId><pages><total>7</total><last>1557</last><first>1551</first></pages><issn><json:string>1059-7794</json:string></issn><issue>11</issue><subject><json:item><value>Research Article</value></json:item></subject><genre><json:string>Journal</json:string></genre><language><json:string>unknown</json:string></language><eissn><json:string>1098-1004</json:string></eissn><title>Human Mutation</title><doi><json:string>10.1002/(ISSN)1098-1004</json:string></doi></host><publicationDate>2009</publicationDate><copyrightDate>2009</copyrightDate><doi><json:string>10.1002/humu.21108</json:string></doi><id>6F1CAD18956AA3A42E02DCC834F55FD8006FA532</id><fulltext><json:item><original>true</original><mimetype>application/pdf</mimetype><extension>pdf</extension><uri>https://api.istex.fr/document/6F1CAD18956AA3A42E02DCC834F55FD8006FA532/fulltext/pdf</uri></json:item><json:item><original>false</original><mimetype>application/zip</mimetype><extension>zip</extension><uri>https://api.istex.fr/document/6F1CAD18956AA3A42E02DCC834F55FD8006FA532/fulltext/zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/document/6F1CAD18956AA3A42E02DCC834F55FD8006FA532/fulltext/tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">Clinically reported heterozygous mutations in the PINK1 kinase domain exert a gene dosage effect</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>Wiley Subscription Services, Inc., A Wiley Company</publisher><pubPlace>Hoboken</pubPlace><availability><p>WILEY</p></availability><date>2009</date></publicationStmt><notesStmt><note type="content">*Communicated by Christine Van Broeckhoven</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Clinically reported heterozygous mutations in the PINK1 kinase domain exert a gene dosage effect</title><author><persName><forename type="first">Eng‐King</forename><surname>Tan</surname></persName><note type="correspondence"><p>Correspondence: Department of Neurology, Singapore General Hospital, Outram Road, Singapore 169608</p></note><affiliation>Department of Neurology, Singapore General Hospital, Singapore</affiliation><affiliation>Department of Clinical Research, Singapore General Hospital, Singapore</affiliation><affiliation>Department of Neurology, National Neuroscience Institute, Singapore</affiliation><affiliation>Neuroscience and Behavioral Disorders (NBD) Program, Duke–National University of Singapore (NUS) Graduate Medical School, Singapore</affiliation></author><author><persName><forename type="first">F. Shaffra</forename><surname>Refai</surname></persName><affiliation>Department of Neurology, Singapore General Hospital, Singapore</affiliation></author><author><persName><forename type="first">Mobin</forename><surname>Siddique</surname></persName><affiliation>Department of Neurology, Singapore General Hospital, Singapore</affiliation></author><author><persName><forename type="first">Karen</forename><surname>Yap</surname></persName><affiliation>Department of Neurology, Singapore General Hospital, Singapore</affiliation></author><author><persName><forename type="first">Patrick</forename><surname>Ho</surname></persName><affiliation>Department of Neurology, Singapore General Hospital, Singapore</affiliation></author><author><persName><forename type="first">Stephanie</forename><surname>Fook‐Chong</surname></persName><affiliation>Department of Clinical Research, Singapore General Hospital, Singapore</affiliation></author><author><persName><forename type="first">Yi</forename><surname>Zhao</surname></persName><affiliation>Department of Clinical Research, Singapore General Hospital, Singapore</affiliation></author></analytic><monogr><title level="j">Human Mutation</title><title level="j" type="abbrev">Hum. Mutat.</title><idno type="pISSN">1059-7794</idno><idno type="eISSN">1098-1004</idno><idno type="DOI">10.1002/(ISSN)1098-1004</idno><imprint><publisher>Wiley Subscription Services, Inc., A Wiley Company</publisher><pubPlace>Hoboken</pubPlace><date type="published" when="2009-11"></date><biblScope unit="volume">30</biblScope><biblScope unit="issue">11</biblScope><biblScope unit="page" from="1551">1551</biblScope><biblScope unit="page" to="1557">1557</biblScope></imprint></monogr><idno type="istex">6F1CAD18956AA3A42E02DCC834F55FD8006FA532</idno><idno type="DOI">10.1002/humu.21108</idno><idno type="ArticleID">HUMU21108</idno></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2009</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Mutations in the gene encoding phosphatase and tensin homolog (PTEN)‐induced kinase 1 (PINK1) have been associated with the loss of dopaminergic neurons characteristic of familial and sporadic Parkinson disease. We developed an in vitro system of stable human dopaminergic neuronal cell lines coexpressing an equivalent copy of normal and mutant PINK1 to simulate “heterozygous” and “homozygous” states in patients. Mutants in the N‐terminus, C‐terminus, and kinase domain were generated and cloned into a two‐gene mammalian expression vector to generate stable mammalian expression cell lines producing an equivalent copy number of wild‐type/mutant PINK1. The cell lines were subjected to oxidative stress and the rate of apoptosis and change in mitochondrial membrane potential (ΔΨm) were assessed. Cell lines expressing kinase and C‐terminus mutants exhibited a greater rate of apoptosis and decrease in ΔΨm, and increased time‐dependent cell loss when subjected to oxidative stress compared to the wild‐type. Cell lines expressing two copies of kinase mutants exhibited a greater apoptosis rate and ΔΨm decrease than those expressing one copy of the mutant. In time‐dependent experiments, there was a significant difference between “homozygous,” “heterozygous,” and wild‐type cell lines, with decreasing cell survival in cell lines expressing mutant copies of PINK1 compared to the wild‐type. We provided the first experimental evidence that clinically reported PINK1 heterozygous mutations exert a gene dosage effect, suggesting that haploinsufficiency of PINK1 is the most likely mechanism that increased the susceptibility to dopaminergic cellular loss. Hum Mutat 30:1551–1557, 2009. © 2009 Wiley‐Liss, Inc.</p></abstract><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>PINK1</term></item><item><term>haploinsufficiency</term></item><item><term>Parkinson</term></item><item><term>PD</term></item></list></keywords></textClass><textClass><keywords scheme="Journal Subject"><list><head>article category</head><item><term>Research Article</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2009-05-24">Received</change><change when="2009-07-30">Registration</change><change when="2009-11">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><original>false</original><mimetype>text/plain</mimetype><extension>txt</extension><uri>https://api.istex.fr/document/6F1CAD18956AA3A42E02DCC834F55FD8006FA532/fulltext/txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Wiley, elements deleted: body"><istex:xmlDeclaration>version="1.0" encoding="UTF-8" standalone="yes"</istex:xmlDeclaration><istex:document><component version="2.0" type="serialArticle" xml:lang="en"><header><publicationMeta level="product"><publisherInfo><publisherName>Wiley Subscription Services, Inc., A Wiley Company</publisherName><publisherLoc>Hoboken</publisherLoc></publisherInfo><doi registered="yes">10.1002/(ISSN)1098-1004</doi><issn type="print">1059-7794</issn><issn type="electronic">1098-1004</issn><idGroup><id type="product" value="HUMU"></id></idGroup><titleGroup><title type="main" xml:lang="en" sort="HUMAN MUTATION">Human Mutation</title><title type="short">Hum. 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Shaffra</givenNames><familyName>Refai</familyName></personName></creator><creator xml:id="au3" creatorRole="author" affiliationRef="#af1"><personName><givenNames>Mobin</givenNames><familyName>Siddique</familyName></personName></creator><creator xml:id="au4" creatorRole="author" affiliationRef="#af1"><personName><givenNames>Karen</givenNames><familyName>Yap</familyName></personName></creator><creator xml:id="au5" creatorRole="author" affiliationRef="#af1"><personName><givenNames>Patrick</givenNames><familyName>Ho</familyName></personName></creator><creator xml:id="au6" creatorRole="author" affiliationRef="#af2"><personName><givenNames>Stephanie</givenNames><familyName>Fook‐Chong</familyName></personName></creator><creator xml:id="au7" creatorRole="author" affiliationRef="#af2"><personName><givenNames>Yi</givenNames><familyName>Zhao</familyName></personName></creator></creators><affiliationGroup><affiliation xml:id="af1" countryCode="SG" type="organization"><unparsedAffiliation>Department of Neurology, Singapore General Hospital, Singapore</unparsedAffiliation></affiliation><affiliation xml:id="af2" countryCode="SG" type="organization"><unparsedAffiliation>Department of Clinical Research, Singapore General Hospital, Singapore</unparsedAffiliation></affiliation><affiliation xml:id="af3" countryCode="SG" type="organization"><unparsedAffiliation>Department of Neurology, National Neuroscience Institute, Singapore</unparsedAffiliation></affiliation><affiliation xml:id="af4" countryCode="SG" type="organization"><unparsedAffiliation>Neuroscience and Behavioral Disorders (NBD) Program, Duke–National University of Singapore (NUS) Graduate Medical School, Singapore</unparsedAffiliation></affiliation></affiliationGroup><keywordGroup xml:lang="en" type="author"><keyword xml:id="kwd1"><i>PINK1</i></keyword><keyword xml:id="kwd2">haploinsufficiency</keyword><keyword xml:id="kwd3">Parkinson</keyword><keyword xml:id="kwd4">PD</keyword></keywordGroup><supportingInformation><p> Additional Supporting Information may be found in the online version of this article. </p><supportingInfoItem><mediaResource alt="supporting information" href="urn-x:wiley:10597794:media:humu21108:humu_21108_sm_SupplInfo"></mediaResource><caption>Supporting Information</caption></supportingInfoItem></supportingInformation><abstractGroup><abstract type="main" xml:lang="en"><title type="main">Abstract</title><p>Mutations in the gene encoding phosphatase and tensin homolog (PTEN)‐induced kinase 1 (PINK1) have been associated with the loss of dopaminergic neurons characteristic of familial and sporadic Parkinson disease. We developed an in vitro system of stable human dopaminergic neuronal cell lines coexpressing an equivalent copy of normal and mutant <i>PINK1</i> to simulate “heterozygous” and “homozygous” states in patients. Mutants in the N‐terminus, C‐terminus, and kinase domain were generated and cloned into a two‐gene mammalian expression vector to generate stable mammalian expression cell lines producing an equivalent copy number of wild‐type/mutant <i>PINK1</i>. The cell lines were subjected to oxidative stress and the rate of apoptosis and change in mitochondrial membrane potential (ΔΨ<sub>m</sub>) were assessed. Cell lines expressing kinase and C‐terminus mutants exhibited a greater rate of apoptosis and decrease in ΔΨ<sub>m</sub>, and increased time‐dependent cell loss when subjected to oxidative stress compared to the wild‐type. Cell lines expressing two copies of kinase mutants exhibited a greater apoptosis rate and ΔΨ<sub>m</sub> decrease than those expressing one copy of the mutant. In time‐dependent experiments, there was a significant difference between “homozygous,” “heterozygous,” and wild‐type cell lines, with decreasing cell survival in cell lines expressing mutant copies of <i>PINK1</i> compared to the wild‐type. We provided the first experimental evidence that clinically reported <i>PINK1</i> heterozygous mutations exert a gene dosage effect, suggesting that haploinsufficiency of <i>PINK1</i> is the most likely mechanism that increased the susceptibility to dopaminergic cellular loss. Hum Mutat 30:1551–1557, 2009. © 2009 Wiley‐Liss, Inc.</p></abstract></abstractGroup></contentMeta><noteGroup><note xml:id="fn1"><p>Communicated by Christine Van Broeckhoven</p></note></noteGroup></header></component></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Clinically reported heterozygous mutations in the PINK1 kinase domain exert a gene dosage effect</title></titleInfo><titleInfo type="alternative" contentType="CDATA" lang="en"><title>Clinically reported heterozygous mutations in the PINK1 kinase domain exert a gene dosage effect</title></titleInfo><name type="personal"><namePart type="given">Eng‐King</namePart><namePart type="family">Tan</namePart><affiliation>Department of Neurology, Singapore General Hospital, Singapore</affiliation><affiliation>Department of Clinical Research, Singapore General Hospital, Singapore</affiliation><affiliation>Department of Neurology, National Neuroscience Institute, Singapore</affiliation><affiliation>Neuroscience and Behavioral Disorders (NBD) Program, Duke–National University of Singapore (NUS) Graduate Medical School, Singapore</affiliation><description>Correspondence: Department of Neurology, Singapore General Hospital, Outram Road, Singapore 169608</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">F. Shaffra</namePart><namePart type="family">Refai</namePart><affiliation>Department of Neurology, Singapore General Hospital, Singapore</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Mobin</namePart><namePart type="family">Siddique</namePart><affiliation>Department of Neurology, Singapore General Hospital, Singapore</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Karen</namePart><namePart type="family">Yap</namePart><affiliation>Department of Neurology, Singapore General Hospital, Singapore</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Patrick</namePart><namePart type="family">Ho</namePart><affiliation>Department of Neurology, Singapore General Hospital, Singapore</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Stephanie</namePart><namePart type="family">Fook‐Chong</namePart><affiliation>Department of Clinical Research, Singapore General Hospital, Singapore</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Yi</namePart><namePart type="family">Zhao</namePart><affiliation>Department of Clinical Research, Singapore General Hospital, Singapore</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="article" displayLabel="article"></genre><originInfo><publisher>Wiley Subscription Services, Inc., A Wiley Company</publisher><place><placeTerm type="text">Hoboken</placeTerm></place><dateIssued encoding="w3cdtf">2009-11</dateIssued><dateCaptured encoding="w3cdtf">2009-05-24</dateCaptured><dateValid encoding="w3cdtf">2009-07-30</dateValid><copyrightDate encoding="w3cdtf">2009</copyrightDate></originInfo><language><languageTerm type="code" authority="rfc3066">en</languageTerm><languageTerm type="code" authority="iso639-2b">eng</languageTerm></language><physicalDescription><internetMediaType>text/html</internetMediaType><extent unit="figures">4</extent><extent unit="tables">1</extent><extent unit="references">20</extent></physicalDescription><abstract lang="en">Mutations in the gene encoding phosphatase and tensin homolog (PTEN)‐induced kinase 1 (PINK1) have been associated with the loss of dopaminergic neurons characteristic of familial and sporadic Parkinson disease. We developed an in vitro system of stable human dopaminergic neuronal cell lines coexpressing an equivalent copy of normal and mutant PINK1 to simulate “heterozygous” and “homozygous” states in patients. Mutants in the N‐terminus, C‐terminus, and kinase domain were generated and cloned into a two‐gene mammalian expression vector to generate stable mammalian expression cell lines producing an equivalent copy number of wild‐type/mutant PINK1. The cell lines were subjected to oxidative stress and the rate of apoptosis and change in mitochondrial membrane potential (ΔΨm) were assessed. Cell lines expressing kinase and C‐terminus mutants exhibited a greater rate of apoptosis and decrease in ΔΨm, and increased time‐dependent cell loss when subjected to oxidative stress compared to the wild‐type. Cell lines expressing two copies of kinase mutants exhibited a greater apoptosis rate and ΔΨm decrease than those expressing one copy of the mutant. In time‐dependent experiments, there was a significant difference between “homozygous,” “heterozygous,” and wild‐type cell lines, with decreasing cell survival in cell lines expressing mutant copies of PINK1 compared to the wild‐type. We provided the first experimental evidence that clinically reported PINK1 heterozygous mutations exert a gene dosage effect, suggesting that haploinsufficiency of PINK1 is the most likely mechanism that increased the susceptibility to dopaminergic cellular loss. Hum Mutat 30:1551–1557, 2009. © 2009 Wiley‐Liss, Inc.</abstract><note type="content">*Communicated by Christine Van Broeckhoven</note><subject lang="en"><genre>Keywords</genre><topic>PINK1</topic><topic>haploinsufficiency</topic><topic>Parkinson</topic><topic>PD</topic></subject><relatedItem type="host"><titleInfo><title>Human Mutation</title></titleInfo><titleInfo type="abbreviated"><title>Hum. Mutat.</title></titleInfo><genre type="Journal">journal</genre><note type="content"> Additional Supporting Information may be found in the online version of this article.Supporting Info Item: Supporting Information - </note><subject><genre>article category</genre><topic>Research Article</topic></subject><identifier type="ISSN">1059-7794</identifier><identifier type="eISSN">1098-1004</identifier><identifier type="DOI">10.1002/(ISSN)1098-1004</identifier><identifier type="PublisherID">HUMU</identifier><part><date>2009</date><detail type="volume"><caption>vol.</caption><number>30</number></detail><detail type="issue"><caption>no.</caption><number>11</number></detail><extent unit="pages"><start>1551</start><end>1557</end><total>7</total></extent></part></relatedItem><identifier type="istex">6F1CAD18956AA3A42E02DCC834F55FD8006FA532</identifier><identifier type="DOI">10.1002/humu.21108</identifier><identifier type="ArticleID">HUMU21108</identifier><accessCondition type="use and reproduction" contentType="copyright">© 2009 Wiley‐Liss, Inc.</accessCondition><recordInfo><recordContentSource>WILEY</recordContentSource><recordOrigin>Wiley Subscription Services, Inc., A Wiley Company</recordOrigin></recordInfo></mods></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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