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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Loss of Function Mutation in the Palmitoyl-Transferase HHAT Leads to Syndromic 46,XY Disorder of Sex Development by Impeding Hedgehog Protein Palmitoylation and Signaling</title><author><name sortKey="Callier, Patrick" sort="Callier, Patrick" uniqKey="Callier P" first="Patrick" last="Callier">Patrick Callier</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><addr-line>FHU-TRANSLAD, Département de Génétique, Hôpital Le Bocage, CHU, Dijon, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"><addr-line>EA 4271 GAD Génétique des Anomalies du Développement, Université de Bourgogne, Dijon, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Calvel, Pierre" sort="Calvel, Pierre" uniqKey="Calvel P" first="Pierre" last="Calvel">Pierre Calvel</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Matevossian, Armine" sort="Matevossian, Armine" uniqKey="Matevossian A" first="Armine" last="Matevossian">Armine Matevossian</name><affiliation><nlm:aff id="aff4"><addr-line>Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff5"><addr-line>Gerstner Sloan-Kettering Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff6"><addr-line>Graduate Program in Pharmacology, Weill Graduate School of Medical Sciences of Cornell University, New York, New York, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Makrythanasis, Periklis" sort="Makrythanasis, Periklis" uniqKey="Makrythanasis P" first="Periklis" last="Makrythanasis">Periklis Makrythanasis</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Bernard, Pascal" sort="Bernard, Pascal" uniqKey="Bernard P" first="Pascal" last="Bernard">Pascal Bernard</name><affiliation><nlm:aff id="aff7"><addr-line>Department of Anatomy and Developmental Biology, Monash University, Clayton, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Kurosaka, Hiroshi" sort="Kurosaka, Hiroshi" uniqKey="Kurosaka H" first="Hiroshi" last="Kurosaka">Hiroshi Kurosaka</name><affiliation><nlm:aff id="aff8"><addr-line>Stowers Institute for Medical Research, Kansas City, Missouri, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Vannier, Anne" sort="Vannier, Anne" uniqKey="Vannier A" first="Anne" last="Vannier">Anne Vannier</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Thauvin Robinet, Christel" sort="Thauvin Robinet, Christel" uniqKey="Thauvin Robinet C" first="Christel" last="Thauvin-Robinet">Christel Thauvin-Robinet</name><affiliation><nlm:aff id="aff2"><addr-line>FHU-TRANSLAD, Département de Génétique, Hôpital Le Bocage, CHU, Dijon, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"><addr-line>EA 4271 GAD Génétique des Anomalies du Développement, Université de Bourgogne, Dijon, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Borel, Christelle" sort="Borel, Christelle" uniqKey="Borel C" first="Christelle" last="Borel">Christelle Borel</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Mazaud Guittot, Severine" sort="Mazaud Guittot, Severine" uniqKey="Mazaud Guittot S" first="Séverine" last="Mazaud-Guittot">Séverine Mazaud-Guittot</name><affiliation><nlm:aff id="aff9"><addr-line>Institut National de la Santé et de la Recherche Médicale (Inserm) U1085-IRSET, Université de Rennes 1, Structure Fédérative Recherche Biosit, Campus de Beaulieu, Rennes, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Rolland, Antoine" sort="Rolland, Antoine" uniqKey="Rolland A" first="Antoine" last="Rolland">Antoine Rolland</name><affiliation><nlm:aff id="aff9"><addr-line>Institut National de la Santé et de la Recherche Médicale (Inserm) U1085-IRSET, Université de Rennes 1, Structure Fédérative Recherche Biosit, Campus de Beaulieu, Rennes, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Desdoits Lethimonier, Christele" sort="Desdoits Lethimonier, Christele" uniqKey="Desdoits Lethimonier C" first="Christèle" last="Desdoits-Lethimonier">Christèle Desdoits-Lethimonier</name><affiliation><nlm:aff id="aff9"><addr-line>Institut National de la Santé et de la Recherche Médicale (Inserm) U1085-IRSET, Université de Rennes 1, Structure Fédérative Recherche Biosit, Campus de Beaulieu, Rennes, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Guipponi, Michel" sort="Guipponi, Michel" uniqKey="Guipponi M" first="Michel" last="Guipponi">Michel Guipponi</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Zimmermann, Celine" sort="Zimmermann, Celine" uniqKey="Zimmermann C" first="Céline" last="Zimmermann">Céline Zimmermann</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Stevant, Isabelle" sort="Stevant, Isabelle" uniqKey="Stevant I" first="Isabelle" last="Stévant">Isabelle Stévant</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Kuhne, Francoise" sort="Kuhne, Francoise" uniqKey="Kuhne F" first="Françoise" last="Kuhne">Françoise Kuhne</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Conne, Beatrice" sort="Conne, Beatrice" uniqKey="Conne B" first="Béatrice" last="Conne">Béatrice Conne</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Santoni, Federico" sort="Santoni, Federico" uniqKey="Santoni F" first="Federico" last="Santoni">Federico Santoni</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Lambert, Sandy" sort="Lambert, Sandy" uniqKey="Lambert S" first="Sandy" last="Lambert">Sandy Lambert</name><affiliation><nlm:aff id="aff2"><addr-line>FHU-TRANSLAD, Département de Génétique, Hôpital Le Bocage, CHU, Dijon, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Huet, Frederic" sort="Huet, Frederic" uniqKey="Huet F" first="Frederic" last="Huet">Frederic Huet</name><affiliation><nlm:aff id="aff2"><addr-line>FHU-TRANSLAD, Département de Génétique, Hôpital Le Bocage, CHU, Dijon, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"><addr-line>EA 4271 GAD Génétique des Anomalies du Développement, Université de Bourgogne, Dijon, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Mugneret, Francine" sort="Mugneret, Francine" uniqKey="Mugneret F" first="Francine" last="Mugneret">Francine Mugneret</name><affiliation><nlm:aff id="aff2"><addr-line>FHU-TRANSLAD, Département de Génétique, Hôpital Le Bocage, CHU, Dijon, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Jaruzelska, Jadwiga" sort="Jaruzelska, Jadwiga" uniqKey="Jaruzelska J" first="Jadwiga" last="Jaruzelska">Jadwiga Jaruzelska</name><affiliation><nlm:aff id="aff10"><addr-line>Polish Academy of Sciences, Institute of Human Genetics, Poznań, Poland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Faivre, Laurence" sort="Faivre, Laurence" uniqKey="Faivre L" first="Laurence" last="Faivre">Laurence Faivre</name><affiliation><nlm:aff id="aff2"><addr-line>FHU-TRANSLAD, Département de Génétique, Hôpital Le Bocage, CHU, Dijon, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"><addr-line>EA 4271 GAD Génétique des Anomalies du Développement, Université de Bourgogne, Dijon, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Wilhelm, Dagmar" sort="Wilhelm, Dagmar" uniqKey="Wilhelm D" first="Dagmar" last="Wilhelm">Dagmar Wilhelm</name><affiliation><nlm:aff id="aff7"><addr-line>Department of Anatomy and Developmental Biology, Monash University, Clayton, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Jegou, Bernard" sort="Jegou, Bernard" uniqKey="Jegou B" first="Bernard" last="Jégou">Bernard Jégou</name><affiliation><nlm:aff id="aff9"><addr-line>Institut National de la Santé et de la Recherche Médicale (Inserm) U1085-IRSET, Université de Rennes 1, Structure Fédérative Recherche Biosit, Campus de Beaulieu, Rennes, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff11"><addr-line>EHESP School of Public Health, Rennes, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Trainor, Paul A" sort="Trainor, Paul A" uniqKey="Trainor P" first="Paul A." last="Trainor">Paul A. Trainor</name><affiliation><nlm:aff id="aff8"><addr-line>Stowers Institute for Medical Research, Kansas City, Missouri, United States of America</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff12"><addr-line>Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Resh, Marilyn D" sort="Resh, Marilyn D" uniqKey="Resh M" first="Marilyn D." last="Resh">Marilyn D. Resh</name><affiliation><nlm:aff id="aff4"><addr-line>Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff5"><addr-line>Gerstner Sloan-Kettering Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff6"><addr-line>Graduate Program in Pharmacology, Weill Graduate School of Medical Sciences of Cornell University, New York, New York, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Antonarakis, Stylianos E" sort="Antonarakis, Stylianos E" uniqKey="Antonarakis S" first="Stylianos E." last="Antonarakis">Stylianos E. Antonarakis</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff13"><addr-line>iGE3, Institute of Genetics and Genomics of Geneva, University of Geneva, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Nef, Serge" sort="Nef, Serge" uniqKey="Nef S" first="Serge" last="Nef">Serge Nef</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff13"><addr-line>iGE3, Institute of Genetics and Genomics of Geneva, University of Geneva, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24784881</idno><idno type="pmc">4006744</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4006744</idno><idno type="RBID">PMC:4006744</idno><idno type="doi">10.1371/journal.pgen.1004340</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000587</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000587</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Loss of Function Mutation in the Palmitoyl-Transferase HHAT Leads to Syndromic 46,XY Disorder of Sex Development by Impeding Hedgehog Protein Palmitoylation and Signaling</title><author><name sortKey="Callier, Patrick" sort="Callier, Patrick" uniqKey="Callier P" first="Patrick" last="Callier">Patrick Callier</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff2"><addr-line>FHU-TRANSLAD, Département de Génétique, Hôpital Le Bocage, CHU, Dijon, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"><addr-line>EA 4271 GAD Génétique des Anomalies du Développement, Université de Bourgogne, Dijon, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Calvel, Pierre" sort="Calvel, Pierre" uniqKey="Calvel P" first="Pierre" last="Calvel">Pierre Calvel</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Matevossian, Armine" sort="Matevossian, Armine" uniqKey="Matevossian A" first="Armine" last="Matevossian">Armine Matevossian</name><affiliation><nlm:aff id="aff4"><addr-line>Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff5"><addr-line>Gerstner Sloan-Kettering Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff6"><addr-line>Graduate Program in Pharmacology, Weill Graduate School of Medical Sciences of Cornell University, New York, New York, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Makrythanasis, Periklis" sort="Makrythanasis, Periklis" uniqKey="Makrythanasis P" first="Periklis" last="Makrythanasis">Periklis Makrythanasis</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Bernard, Pascal" sort="Bernard, Pascal" uniqKey="Bernard P" first="Pascal" last="Bernard">Pascal Bernard</name><affiliation><nlm:aff id="aff7"><addr-line>Department of Anatomy and Developmental Biology, Monash University, Clayton, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Kurosaka, Hiroshi" sort="Kurosaka, Hiroshi" uniqKey="Kurosaka H" first="Hiroshi" last="Kurosaka">Hiroshi Kurosaka</name><affiliation><nlm:aff id="aff8"><addr-line>Stowers Institute for Medical Research, Kansas City, Missouri, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Vannier, Anne" sort="Vannier, Anne" uniqKey="Vannier A" first="Anne" last="Vannier">Anne Vannier</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Thauvin Robinet, Christel" sort="Thauvin Robinet, Christel" uniqKey="Thauvin Robinet C" first="Christel" last="Thauvin-Robinet">Christel Thauvin-Robinet</name><affiliation><nlm:aff id="aff2"><addr-line>FHU-TRANSLAD, Département de Génétique, Hôpital Le Bocage, CHU, Dijon, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"><addr-line>EA 4271 GAD Génétique des Anomalies du Développement, Université de Bourgogne, Dijon, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Borel, Christelle" sort="Borel, Christelle" uniqKey="Borel C" first="Christelle" last="Borel">Christelle Borel</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Mazaud Guittot, Severine" sort="Mazaud Guittot, Severine" uniqKey="Mazaud Guittot S" first="Séverine" last="Mazaud-Guittot">Séverine Mazaud-Guittot</name><affiliation><nlm:aff id="aff9"><addr-line>Institut National de la Santé et de la Recherche Médicale (Inserm) U1085-IRSET, Université de Rennes 1, Structure Fédérative Recherche Biosit, Campus de Beaulieu, Rennes, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Rolland, Antoine" sort="Rolland, Antoine" uniqKey="Rolland A" first="Antoine" last="Rolland">Antoine Rolland</name><affiliation><nlm:aff id="aff9"><addr-line>Institut National de la Santé et de la Recherche Médicale (Inserm) U1085-IRSET, Université de Rennes 1, Structure Fédérative Recherche Biosit, Campus de Beaulieu, Rennes, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Desdoits Lethimonier, Christele" sort="Desdoits Lethimonier, Christele" uniqKey="Desdoits Lethimonier C" first="Christèle" last="Desdoits-Lethimonier">Christèle Desdoits-Lethimonier</name><affiliation><nlm:aff id="aff9"><addr-line>Institut National de la Santé et de la Recherche Médicale (Inserm) U1085-IRSET, Université de Rennes 1, Structure Fédérative Recherche Biosit, Campus de Beaulieu, Rennes, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Guipponi, Michel" sort="Guipponi, Michel" uniqKey="Guipponi M" first="Michel" last="Guipponi">Michel Guipponi</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Zimmermann, Celine" sort="Zimmermann, Celine" uniqKey="Zimmermann C" first="Céline" last="Zimmermann">Céline Zimmermann</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Stevant, Isabelle" sort="Stevant, Isabelle" uniqKey="Stevant I" first="Isabelle" last="Stévant">Isabelle Stévant</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Kuhne, Francoise" sort="Kuhne, Francoise" uniqKey="Kuhne F" first="Françoise" last="Kuhne">Françoise Kuhne</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Conne, Beatrice" sort="Conne, Beatrice" uniqKey="Conne B" first="Béatrice" last="Conne">Béatrice Conne</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Santoni, Federico" sort="Santoni, Federico" uniqKey="Santoni F" first="Federico" last="Santoni">Federico Santoni</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Lambert, Sandy" sort="Lambert, Sandy" uniqKey="Lambert S" first="Sandy" last="Lambert">Sandy Lambert</name><affiliation><nlm:aff id="aff2"><addr-line>FHU-TRANSLAD, Département de Génétique, Hôpital Le Bocage, CHU, Dijon, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Huet, Frederic" sort="Huet, Frederic" uniqKey="Huet F" first="Frederic" last="Huet">Frederic Huet</name><affiliation><nlm:aff id="aff2"><addr-line>FHU-TRANSLAD, Département de Génétique, Hôpital Le Bocage, CHU, Dijon, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"><addr-line>EA 4271 GAD Génétique des Anomalies du Développement, Université de Bourgogne, Dijon, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Mugneret, Francine" sort="Mugneret, Francine" uniqKey="Mugneret F" first="Francine" last="Mugneret">Francine Mugneret</name><affiliation><nlm:aff id="aff2"><addr-line>FHU-TRANSLAD, Département de Génétique, Hôpital Le Bocage, CHU, Dijon, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Jaruzelska, Jadwiga" sort="Jaruzelska, Jadwiga" uniqKey="Jaruzelska J" first="Jadwiga" last="Jaruzelska">Jadwiga Jaruzelska</name><affiliation><nlm:aff id="aff10"><addr-line>Polish Academy of Sciences, Institute of Human Genetics, Poznań, Poland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Faivre, Laurence" sort="Faivre, Laurence" uniqKey="Faivre L" first="Laurence" last="Faivre">Laurence Faivre</name><affiliation><nlm:aff id="aff2"><addr-line>FHU-TRANSLAD, Département de Génétique, Hôpital Le Bocage, CHU, Dijon, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"><addr-line>EA 4271 GAD Génétique des Anomalies du Développement, Université de Bourgogne, Dijon, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Wilhelm, Dagmar" sort="Wilhelm, Dagmar" uniqKey="Wilhelm D" first="Dagmar" last="Wilhelm">Dagmar Wilhelm</name><affiliation><nlm:aff id="aff7"><addr-line>Department of Anatomy and Developmental Biology, Monash University, Clayton, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Jegou, Bernard" sort="Jegou, Bernard" uniqKey="Jegou B" first="Bernard" last="Jégou">Bernard Jégou</name><affiliation><nlm:aff id="aff9"><addr-line>Institut National de la Santé et de la Recherche Médicale (Inserm) U1085-IRSET, Université de Rennes 1, Structure Fédérative Recherche Biosit, Campus de Beaulieu, Rennes, France</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff11"><addr-line>EHESP School of Public Health, Rennes, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Trainor, Paul A" sort="Trainor, Paul A" uniqKey="Trainor P" first="Paul A." last="Trainor">Paul A. Trainor</name><affiliation><nlm:aff id="aff8"><addr-line>Stowers Institute for Medical Research, Kansas City, Missouri, United States of America</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff12"><addr-line>Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Resh, Marilyn D" sort="Resh, Marilyn D" uniqKey="Resh M" first="Marilyn D." last="Resh">Marilyn D. Resh</name><affiliation><nlm:aff id="aff4"><addr-line>Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff5"><addr-line>Gerstner Sloan-Kettering Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff6"><addr-line>Graduate Program in Pharmacology, Weill Graduate School of Medical Sciences of Cornell University, New York, New York, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Antonarakis, Stylianos E" sort="Antonarakis, Stylianos E" uniqKey="Antonarakis S" first="Stylianos E." last="Antonarakis">Stylianos E. Antonarakis</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff13"><addr-line>iGE3, Institute of Genetics and Genomics of Geneva, University of Geneva, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author><author><name sortKey="Nef, Serge" sort="Nef, Serge" uniqKey="Nef S" first="Serge" last="Nef">Serge Nef</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff13"><addr-line>iGE3, Institute of Genetics and Genomics of Geneva, University of Geneva, Geneva, Switzerland</addr-line></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS Genetics</title><idno type="ISSN">1553-7390</idno><idno type="eISSN">1553-7404</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (<italic>HHAT</italic>) gene. This mutation occurred in the conserved membrane bound <italic>O</italic>-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and <italic>Hhat</italic> loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Hughes, Ia" uniqKey="Hughes I">IA Hughes</name></author><author><name sortKey="Houk, C" uniqKey="Houk C">C Houk</name></author><author><name sortKey="Ahmed, Sf" uniqKey="Ahmed S">SF Ahmed</name></author><author><name sortKey="Lee, Pa" uniqKey="Lee P">PA Lee</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Das, Dk" uniqKey="Das D">DK Das</name></author><author><name sortKey="Sanghavi, D" uniqKey="Sanghavi D">D Sanghavi</name></author><author><name sortKey="Gawde, H" uniqKey="Gawde H">H Gawde</name></author><author><name sortKey="Idicula Thomas, S" uniqKey="Idicula Thomas S">S Idicula-Thomas</name></author><author><name sortKey="Vasudevan, L" uniqKey="Vasudevan L">L 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<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS Genet</journal-id><journal-id journal-id-type="iso-abbrev">PLoS Genet</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosgen</journal-id><journal-title-group><journal-title>PLoS Genetics</journal-title></journal-title-group><issn pub-type="ppub">1553-7390</issn><issn pub-type="epub">1553-7404</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24784881</article-id><article-id pub-id-type="pmc">4006744</article-id><article-id pub-id-type="publisher-id">PGENETICS-D-13-03093</article-id><article-id pub-id-type="doi">10.1371/journal.pgen.1004340</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Biology and Life Sciences</subject><subj-group><subject>Developmental Biology</subject><subj-group><subject>Morphogenesis</subject><subj-group><subject>Sex Determination</subject></subj-group></subj-group><subj-group><subject>Cell Differentiation</subject><subject>Cell Fate Determination</subject></subj-group></subj-group><subj-group><subject>Genetics</subject></subj-group></subj-group></article-categories><title-group><article-title>Loss of Function Mutation in the Palmitoyl-Transferase HHAT Leads to Syndromic 46,XY Disorder of Sex Development by Impeding Hedgehog Protein Palmitoylation and Signaling</article-title><alt-title alt-title-type="running-head">46,XY DSD due to Impaired HHAT Activity</alt-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes"><name><surname>Callier</surname><given-names>Patrick</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author" equal-contrib="yes"><name><surname>Calvel</surname><given-names>Pierre</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author" equal-contrib="yes"><name><surname>Matevossian</surname><given-names>Armine</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref><xref ref-type="aff" rid="aff5"><sup>5</sup></xref><xref ref-type="aff" rid="aff6"><sup>6</sup></xref></contrib><contrib contrib-type="author"><name><surname>Makrythanasis</surname><given-names>Periklis</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Bernard</surname><given-names>Pascal</given-names></name><xref ref-type="aff" rid="aff7"><sup>7</sup></xref></contrib><contrib contrib-type="author"><name><surname>Kurosaka</surname><given-names>Hiroshi</given-names></name><xref ref-type="aff" rid="aff8"><sup>8</sup></xref></contrib><contrib contrib-type="author"><name><surname>Vannier</surname><given-names>Anne</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Thauvin-Robinet</surname><given-names>Christel</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Borel</surname><given-names>Christelle</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Mazaud-Guittot</surname><given-names>Séverine</given-names></name><xref ref-type="aff" rid="aff9"><sup>9</sup></xref></contrib><contrib contrib-type="author"><name><surname>Rolland</surname><given-names>Antoine</given-names></name><xref ref-type="aff" rid="aff9"><sup>9</sup></xref></contrib><contrib contrib-type="author"><name><surname>Desdoits-Lethimonier</surname><given-names>Christèle</given-names></name><xref ref-type="aff" rid="aff9"><sup>9</sup></xref></contrib><contrib contrib-type="author"><name><surname>Guipponi</surname><given-names>Michel</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Zimmermann</surname><given-names>Céline</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Stévant</surname><given-names>Isabelle</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Kuhne</surname><given-names>Françoise</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Conne</surname><given-names>Béatrice</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Santoni</surname><given-names>Federico</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Lambert</surname><given-names>Sandy</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Huet</surname><given-names>Frederic</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Mugneret</surname><given-names>Francine</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Jaruzelska</surname><given-names>Jadwiga</given-names></name><xref ref-type="aff" rid="aff10"><sup>10</sup></xref></contrib><contrib contrib-type="author"><name><surname>Faivre</surname><given-names>Laurence</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Wilhelm</surname><given-names>Dagmar</given-names></name><xref ref-type="aff" rid="aff7"><sup>7</sup></xref></contrib><contrib contrib-type="author"><name><surname>Jégou</surname><given-names>Bernard</given-names></name><xref ref-type="aff" rid="aff9"><sup>9</sup></xref><xref ref-type="aff" rid="aff11"><sup>11</sup></xref></contrib><contrib contrib-type="author"><name><surname>Trainor</surname><given-names>Paul A.</given-names></name><xref ref-type="aff" rid="aff8"><sup>8</sup></xref><xref ref-type="aff" rid="aff12"><sup>12</sup></xref></contrib><contrib contrib-type="author"><name><surname>Resh</surname><given-names>Marilyn D.</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref><xref ref-type="aff" rid="aff5"><sup>5</sup></xref><xref ref-type="aff" rid="aff6"><sup>6</sup></xref></contrib><contrib contrib-type="author"><name><surname>Antonarakis</surname><given-names>Stylianos E.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff13"><sup>13</sup></xref></contrib><contrib contrib-type="author"><name><surname>Nef</surname><given-names>Serge</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff13"><sup>13</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib></contrib-group><aff id="aff1"><label>1</label><addr-line>Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland</addr-line></aff><aff id="aff2"><label>2</label><addr-line>FHU-TRANSLAD, Département de Génétique, Hôpital Le Bocage, CHU, Dijon, France</addr-line></aff><aff id="aff3"><label>3</label><addr-line>EA 4271 GAD Génétique des Anomalies du Développement, Université de Bourgogne, Dijon, France</addr-line></aff><aff id="aff4"><label>4</label><addr-line>Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America</addr-line></aff><aff id="aff5"><label>5</label><addr-line>Gerstner Sloan-Kettering Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America</addr-line></aff><aff id="aff6"><label>6</label><addr-line>Graduate Program in Pharmacology, Weill Graduate School of Medical Sciences of Cornell University, New York, New York, United States of America</addr-line></aff><aff id="aff7"><label>7</label><addr-line>Department of Anatomy and Developmental Biology, Monash University, Clayton, Australia</addr-line></aff><aff id="aff8"><label>8</label><addr-line>Stowers Institute for Medical Research, Kansas City, Missouri, United States of America</addr-line></aff><aff id="aff9"><label>9</label><addr-line>Institut National de la Santé et de la Recherche Médicale (Inserm) U1085-IRSET, Université de Rennes 1, Structure Fédérative Recherche Biosit, Campus de Beaulieu, Rennes, France</addr-line></aff><aff id="aff10"><label>10</label><addr-line>Polish Academy of Sciences, Institute of Human Genetics, Poznań, Poland</addr-line></aff><aff id="aff11"><label>11</label><addr-line>EHESP School of Public Health, Rennes, France</addr-line></aff><aff id="aff12"><label>12</label><addr-line>Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas, United States of America</addr-line></aff><aff id="aff13"><label>13</label><addr-line>iGE3, Institute of Genetics and Genomics of Geneva, University of Geneva, Geneva, Switzerland</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Mundlos</surname><given-names>Stefan</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1"><addr-line>Max Planck Institute for Molecular Genetics, Germany</addr-line></aff><author-notes><corresp id="cor1">* E-mail: <email>Serge.Nef@unige.ch</email></corresp><fn fn-type="conflict"><p>The authors have declared that no competing interests exist.</p></fn><fn fn-type="con"><p>Conceived and designed the experiments: PCalv SN SEA. Performed the experiments: PCalv AM PM PB CZ FK BC IS. Analyzed the data: PCalv PCall AV DW CB MG FS IS. Contributed reagents/materials/analysis tools: PCall AM MDR HK CTR SMG AR CDL SL FH FM JJ LF BJ PAT. Wrote the paper: PCalv PM SN MDR.</p></fn></author-notes><pub-date pub-type="collection"><month>5</month><year>2014</year></pub-date><pub-date pub-type="epub"><day>1</day><month>5</month><year>2014</year></pub-date><volume>10</volume><issue>5</issue><elocation-id>e1004340</elocation-id><history><date date-type="received"><day>8</day><month>11</month><year>2013</year></date><date date-type="accepted"><day>7</day><month>3</month><year>2014</year></date></history><permissions><copyright-statement>© 2014 Callier et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Callier et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.</license-p></license></permissions><abstract><p>The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (<italic>HHAT</italic>) gene. This mutation occurred in the conserved membrane bound <italic>O</italic>-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and <italic>Hhat</italic> loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development.</p></abstract><abstract abstract-type="summary"><title>Author Summary</title><p>Disorders of gonadal development represent a clinically and genetically heterogeneous class of DSD caused by defects in gonadal development and/or a failure of testis/ovarian differentiation. Unfortunately, in many cases the genetic aetiology of DSD is unknown, indicating that our knowledge of the factors mediating sex determination is limited. Using exome sequencing on a case of autosomal recessive syndromic 46,XY DSD with testicular dysgenesis and chondrodysplasia, we found a homozygous missense mutation (G287V) within the coding sequence of the O-acetyl-transferase <italic>HHAT</italic> gene. The <italic>HHAT</italic> gene encodes an enzyme required for the attachment of palmitoyl residues that are critical for multimerization and long range signaling potency of hedgehog secreted proteins. We found that <italic>HHAT</italic> is widely expressed in human organs during fetal development, including testes and ovaries around the time of sex determination. <italic>In vitro</italic> assays show that G287V mutation impairs HHAT palmitoyl-transferase activity and mice lacking functional <italic>Hhat</italic> exhibit testicular dysgenesis as well as other skeletal, neuronal and growth defects that recapitulate most aspects of the syndromic 46,XY DSD patient. These data provide the first clinical evidence of the essential role played by lipid modification of Hedgehog proteins in human testicular organogenesis and embryonic development.</p></abstract><funding-group><funding-statement>This work was supported by The Gerbert Rüf Stiftung, the Fondation Gertrude von Meissner, the Novartis Foundation for medical-biological research and by the Département de L'instruction Publique of the State of Geneva to SN; the US National Institutes of Health to MDR; the Bodossaki Foundation to PM; the regional council of Burgundy (grant STIC11) to PCall, CTR and LF; the Swiss National Science Foundation, the Gerbert Rüf Stiftung and ERC (EU) grants to SEA; and the Stowers Institute for Medical Research and the National Institute for Dental and Craniofacial Research (DE-01682) to PAT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</funding-statement></funding-group><counts><page-count count="12"></page-count></counts></article-meta></front><body><sec id="s1"><title>Introduction</title><p>Disorders of sex development (DSD) are rare “congenital conditions in which development of the chromosomal, gonadal or anatomical sex is atypical” <xref rid="pgen.1004340-Hughes1" ref-type="bibr">[1]</xref>, and which display a wide spectrum of phenotypes. One clinically and genetically heterogeneous class of DSD is partial or complete 46,XY gonadal dysgenesis <xref rid="pgen.1004340-Das1" ref-type="bibr">[2]</xref>, caused by a defect in gonadal development and/or a failure of testis differentiation. Individuals with 46,XY complete gonadal dysgenesis (46,XY CGD) are characterized by a 46,XY karyotype, normal female external genitalia, undeveloped (“streak”) gonads, no sperm production, and the presence of Müllerian structures. Despite considerable progress in understanding the genetic factors involved in gonadal differentiation, the causative mutation for individuals with 46,XY CGD remains unknown in 80% of the cases <xref rid="pgen.1004340-Hughes1" ref-type="bibr">[1]</xref>, <xref rid="pgen.1004340-Eggers1" ref-type="bibr">[3]</xref>, <xref rid="pgen.1004340-Hughes2" ref-type="bibr">[4]</xref>. The majority of resolved cases involve mutations or deletions in genes coding for SRY, desert hedgehog (DHH) , MAP3K1 <xref rid="pgen.1004340-Pearlman1" ref-type="bibr">[5]</xref> and NR5A1 (SF1) while the prevalence of duplications involving genes coding for NR0B1 (DAX1) and WNT4 represent ∼1% of the resolved cases <xref rid="pgen.1004340-Domenice1" ref-type="bibr">[6]</xref>. One characteristic of DSD with gonadal dysgenesis is their frequent association with other congenital malformations such as growth or mental retardation, conditions that can be referred to as syndromic DSD <xref rid="pgen.1004340-Hiorta1" ref-type="bibr">[7]</xref>. The large variation in cases of syndromic 46,XY DSD involving gonadal dysgenesis suggests that among the network of genes essential for proper development of testes and ovaries, some genes may have pleiotropic actions. The study of syndromic DSD thus provides an opportunity to discover new genes involved in human sex determination and improve the diagnosis and clinical management of DSD patients.</p><p>The hedgehog (Hh) family of signaling molecules is composed of three members, namely sonic hedgehog (SHH), desert hedgehog (DHH) and indian hedgehog (IHH). Hh molecules function as morphogens that signal at both short and long range through the patched 1 receptor (PTCH1) in a concentration dependent manner. All Hh ligands are initially synthesized as precursor proteins that undergo auto-proteolytic cleavage and dual lipid post-translational modifications. A cholesterol molecule is attached to the C-terminus of the Hh signaling moiety <xref rid="pgen.1004340-Porter1" ref-type="bibr">[8]</xref>, <xref rid="pgen.1004340-Porter2" ref-type="bibr">[9]</xref>, while the N-terminus is palmitoylated by the membrane-bound O-acyl-transferase hedgehog acyl-transferase (HHAT) <xref rid="pgen.1004340-Pepinsky1" ref-type="bibr">[10]</xref>, <xref rid="pgen.1004340-Buglino1" ref-type="bibr">[11]</xref>. These lipid modifications are required for efficient signaling at both long and short range <xref rid="pgen.1004340-Chen1" ref-type="bibr">[12]</xref>, <xref rid="pgen.1004340-Buglino2" ref-type="bibr">[13]</xref>. In particular, palmitate attachment to SHH is required for the formation of multimeric SHH complexes and increases its signaling potency <xref rid="pgen.1004340-Pepinsky1" ref-type="bibr">[10]</xref>, <xref rid="pgen.1004340-Chen1" ref-type="bibr">[12]</xref>, <xref rid="pgen.1004340-Goetz1" ref-type="bibr">[14]</xref>, <xref rid="pgen.1004340-Taylor1" ref-type="bibr">[15]</xref>.</p><p>Hedgehog ligands play a major role in embryonic patterning, growth and differentiation for a variety of organs and tissues. For instance, SHH acts on neural, cranial and skeletal patterning as well as kidney, lung, tooth and eye development. In the case of bone growth and pancreas development it acts in a synergistic manner with IHH (for review, read <xref rid="pgen.1004340-Varjosalo1" ref-type="bibr">[16]</xref>). Loss of <italic>SHH</italic> expression or function in humans results in pathologies as diverse as holoprosencephaly <xref rid="pgen.1004340-Belloni1" ref-type="bibr">[17]</xref>, preaxial polydactyly <xref rid="pgen.1004340-Lettice1" ref-type="bibr">[18]</xref> or non-syndromic colobomatous microphthalmia <xref rid="pgen.1004340-Schimmenti1" ref-type="bibr">[19]</xref>. Mutations in <italic>IHH</italic>, on the other hand, more specifically lead to bone growth defects (brachydactyly or acrocapitofemoral dysplasia <xref rid="pgen.1004340-Byrnes1" ref-type="bibr">[20]</xref>). Interestingly, several mutations of <italic>DHH</italic> have been described in patients with a non-syndromic form of 46,XY DSD with partial or complete gonadal dysgenesis <xref rid="pgen.1004340-Das1" ref-type="bibr">[2]</xref>, <xref rid="pgen.1004340-Canto1" ref-type="bibr">[21]</xref>, <xref rid="pgen.1004340-Canto2" ref-type="bibr">[22]</xref>, <xref rid="pgen.1004340-Umehara1" ref-type="bibr">[23]</xref>. Similarly, ablation of <italic>Dhh</italic> in mice leads to severe testis dysgenesis including apolar Sertoli cells, anastomotic testis cords, a reduced number of fetal Leydig cells and insufficient production of androgens, resulting in male infertility, hypogonadism, underdeveloped male accessory glands and feminized external genitalia <xref rid="pgen.1004340-Clark1" ref-type="bibr">[24]</xref>, <xref rid="pgen.1004340-PierucciAlves1" ref-type="bibr">[25]</xref>, <xref rid="pgen.1004340-Yao1" ref-type="bibr">[26]</xref>. In the fetal mouse testis, <italic>Dhh</italic> is expressed specifically in the Sertoli cells from E11.5 onwards <xref rid="pgen.1004340-Bitgood1" ref-type="bibr">[27]</xref> and thus is required for testis cord formation, Leydig cell differentiation and possibly germ cell survival <xref rid="pgen.1004340-Clark1" ref-type="bibr">[24]</xref>, <xref rid="pgen.1004340-PierucciAlves1" ref-type="bibr">[25]</xref>, <xref rid="pgen.1004340-Yao1" ref-type="bibr">[26]</xref>, <xref rid="pgen.1004340-Makela1" ref-type="bibr">[28]</xref>.</p><p>Taking advantage of next generation sequencing technologies, we identified by whole exome sequencing a missense mutation in the palmitoyl transferase gene <italic>HHAT</italic> in a unique case of autosomal recessive chondrodysplasia-46,XY DSD with gonadal dysgenesis (Nivelon-Nivelon-Mabille syndrome). These findings provide the first clinical evidence of the essential role that post-translational lipid modifications have on the functional performance of Hh proteins and hence on human testicular organogenesis and embryonic development.</p></sec><sec id="s2"><title>Results</title><sec id="s2a"><title>A familial case of syndromic 46,XY complete gonadal dysgenesis with chondrodysplasia</title><p>We studied a family with two children affected by a rare autosomal recessive syndrome associated with DSD and chondrodysplasia (OMIM 600092) <xref rid="pgen.1004340-Nivelon1" ref-type="bibr">[29]</xref>, <xref rid="pgen.1004340-ThauvinRobinet1" ref-type="bibr">[30]</xref> (<xref ref-type="fig" rid="pgen-1004340-g001">Fig. 1A</xref>). The first sibling displayed severe dwarfism with generalized chondrodysplasia, a narrow, bell-shaped thorax, micromelia, brachydactyly, severe microcephaly with cerebellar vermis hypoplasia, facial anomalies, hypoplastic irides, and coloboma of both optic discs. Follow up diagnostic tests at 16 years old confirmed previous clinical observations and identified mild mental retardation, muscular hypertrophy, myopia and other facial anomalies such as upslanting palpebral fissures, puffy eyelids, large mouth, and mild prognathism <xref rid="pgen.1004340-ThauvinRobinet1" ref-type="bibr">[30]</xref>. The karyotype was 46,XY but the patient exhibited clinical features of a 46,XY DSD with complete gonadal dysgenesis (CGD), including normal external female genitalia, lack of pubertal development, primary amenorrhea, and hypergonadotrophic hypogonadism. Histology confirmed the testicular dysgenesis and identified persistent Müllerian and Wolffian duct structures, in particular epididymal remnants. The left gonad contained large inclusions of immature testicular tissue mainly composed of dysplastic immature seminiferous tubules (<xref ref-type="fig" rid="pgen-1004340-g001">Fig. 1C</xref> - arrowhead). These structures were delimited with a dense interstitial compartment containing a greatly reduced proportion of cells expressing the cholesterol side-chain cleavage enzyme CYP11A1 compared to normal adult testis (<xref ref-type="supplementary-material" rid="pgen.1004340.s001">Fig. S1</xref>). The right gonad was hypoplastic and largely composed of fibroblastic and endothelial tissues. It contained some structures that resembled immature seminiferous tubules surrounded by myoid cells (<xref ref-type="fig" rid="pgen-1004340-g001">Fig. 1D</xref> - arrowheads). No CYP11A1-expressing cells were detected around these structures (data not shown). It is possible that a small group of functional steroidogenic cells may have developed and persisted in the dysgenic gonads of the patient, in a number sufficient to explain the presence of vestigial Wolffian duct derivatives, however insufficient to achieve complete and normal development of the male genitalia.</p><fig id="pgen-1004340-g001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pgen.1004340.g001</object-id><label>Figure 1</label><caption><title>HHAT mutation in 46,XY DSD with chondrodysplasia.</title><p>A) The familial case presented here consists of two non-consanguineous individuals with two siblings affected with chondrodysplasia. The histological analysis of the dysgenetic gonads revealed the presence of few immature seminiferous tubules in both left (B) and right (C) gonads (arrowheads). D) Sanger sequencing of the proband and parental genomic DNA confirmed the parental origin of the G>T substitution observed at position 860 of HHAT long isoform. E) The mutated Glycine 287 is conserved among vertebrates from zebrafish to humans, and is localized N-terminal to the 6th transmembrane domain of HHAT long isoform.</p></caption><graphic xlink:href="pgen.1004340.g001"></graphic></fig><p>The second sibling had a 46,XX karyotype with histologically normal ovaries, but otherwise exhibited similar phenotypic abnormalities including severe dwarfism and generalized chondrodysplasia <xref rid="pgen.1004340-Nivelon1" ref-type="bibr">[29]</xref>. Pregnancy was interrupted after ultrasound examination at 17 weeks of gestation. To date no other similar case has been reported.</p></sec><sec id="s2b"><title>Homozygous G287V missense mutation in the <italic>HHAT</italic> gene as a cause for Nivelon-Nivelon-Mabille syndrome</title><p>A 244K aCGH analysis performed on genomic DNA extracted from blood cells of the patient did not reveal any chromosomal rearrangements that could account for the pathology. All the copy number variations (CNV) identified during the analysis were annotated in the database of genomic variants (DGV) as benign (data not shown). We then performed targeted exon capture and next-generation sequencing on DNA from the 46,XY DSD proband and her parents (<xref ref-type="table" rid="pgen-1004340-t001">Table 1</xref>). Variants were evaluated using VariantMaster <xref rid="pgen.1004340-Santoni1" ref-type="bibr">[31]</xref>, for <italic>de novo</italic>, X-linked and recessive models (<xref ref-type="table" rid="pgen-1004340-t002">Table 2</xref>).</p><table-wrap id="pgen-1004340-t001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pgen.1004340.t001</object-id><label>Table 1</label><caption><title>Number of reads, coverage and variants identified per individual.</title></caption><alternatives><graphic id="pgen-1004340-t001-1" xlink:href="pgen.1004340.t001"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">father</td><td align="left" rowspan="1" colspan="1">mother</td><td align="left" rowspan="1" colspan="1">patient</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Total reads</td><td align="left" rowspan="1" colspan="1">174'432'036</td><td align="left" rowspan="1" colspan="1">204'738'642</td><td align="left" rowspan="1" colspan="1">200'725'004</td></tr><tr><td align="left" rowspan="1" colspan="1">Reads after removing duplicates</td><td align="left" rowspan="1" colspan="1">99'626'106</td><td align="left" rowspan="1" colspan="1">109'452'796</td><td align="left" rowspan="1" colspan="1">109'029'488</td></tr><tr><td align="left" rowspan="1" colspan="1">On target<xref ref-type="table-fn" rid="nt102">*</xref></td><td align="left" rowspan="1" colspan="1">60'463'056</td><td align="left" rowspan="1" colspan="1">65'192'523</td><td align="left" rowspan="1" colspan="1">64'962'546</td></tr><tr><td align="left" rowspan="1" colspan="1">Mean coverage<xref ref-type="table-fn" rid="nt102">*</xref></td><td align="left" rowspan="1" colspan="1">116.5</td><td align="left" rowspan="1" colspan="1">124.3</td><td align="left" rowspan="1" colspan="1">124.7</td></tr><tr><td align="left" rowspan="1" colspan="1">Coverage ×8<xref ref-type="table-fn" rid="nt102">*</xref></td><td align="left" rowspan="1" colspan="1">93.8%</td><td align="left" rowspan="1" colspan="1">93.9%</td><td align="left" rowspan="1" colspan="1">94.6%</td></tr><tr><td align="left" rowspan="1" colspan="1">Coverage ×20<xref ref-type="table-fn" rid="nt102">*</xref></td><td align="left" rowspan="1" colspan="1">87.1%</td><td align="left" rowspan="1" colspan="1">87.5%</td><td align="left" rowspan="1" colspan="1">88.6%</td></tr><tr><td align="left" rowspan="1" colspan="1">Synonymous SNV</td><td align="left" rowspan="1" colspan="1">10'446</td><td align="left" rowspan="1" colspan="1">10'635</td><td align="left" rowspan="1" colspan="1">10'648</td></tr><tr><td align="left" rowspan="1" colspan="1">Missense SNV</td><td align="left" rowspan="1" colspan="1">9'596</td><td align="left" rowspan="1" colspan="1">9'607</td><td align="left" rowspan="1" colspan="1">9'724</td></tr><tr><td align="left" rowspan="1" colspan="1">Total exonic</td><td align="left" rowspan="1" colspan="1">21'604</td><td align="left" rowspan="1" colspan="1">21'922</td><td align="left" rowspan="1" colspan="1">22'032</td></tr><tr><td align="left" rowspan="1" colspan="1">Splicing (±10 bp)</td><td align="left" rowspan="1" colspan="1">3'955</td><td align="left" rowspan="1" colspan="1">4'046</td><td align="left" rowspan="1" colspan="1">4'107</td></tr><tr><td align="left" rowspan="1" colspan="1">Total exonic + splicing</td><td align="left" rowspan="1" colspan="1">25'559</td><td align="left" rowspan="1" colspan="1">25'968</td><td align="left" rowspan="1" colspan="1">26'139</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt101"><label></label><p>SNV: Single Nucleotide Variant.</p></fn><fn id="nt102"><label></label><p>*The target is the total protein coding sequence of the human genes according to RefSeq.</p></fn></table-wrap-foot></table-wrap><table-wrap id="pgen-1004340-t002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pgen.1004340.t002</object-id><label>Table 2</label><caption><title>List of variants identified for the different models tested.</title></caption><alternatives><graphic id="pgen-1004340-t002-2" xlink:href="pgen.1004340.t002"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col width="115*" align="left" span="1"></col><col width="98*" align="center" span="1"></col><col width="97*" align="center" span="1"></col><col width="97*" align="center" span="1"></col><col width="97*" align="center" span="1"></col><col width="98*" align="center" span="1"></col><col width="97*" align="center" span="1"></col><col width="97*" align="center" span="1"></col><col width="98*" align="center" span="1"></col><col width="96*" align="center" span="1"></col><col width="98*" align="center" span="1"></col><col width="97*" align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Chr</td><td align="left" rowspan="1" colspan="1">Position</td><td align="left" rowspan="1" colspan="1">Gene</td><td align="left" rowspan="1" colspan="1">NCBI</td><td align="left" rowspan="1" colspan="1">cDNA</td><td align="left" rowspan="1" colspan="1">Protein change</td><td align="left" rowspan="1" colspan="1">dbSNP</td><td align="left" rowspan="1" colspan="1">MAF</td><td align="left" rowspan="1" colspan="1">SIFT/NNSplice<xref ref-type="table-fn" rid="nt104">*</xref></td><td align="left" rowspan="1" colspan="1">PolyPhen2/ESEfinder<xref ref-type="table-fn" rid="nt104">*</xref></td><td align="left" rowspan="1" colspan="1">Mutation Taster<xref ref-type="table-fn" rid="nt104">*</xref></td><td align="left" rowspan="1" colspan="1">GERP++</td></tr></thead><tbody><tr><td colspan="2" align="left" rowspan="1"><bold>De novo model</bold></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">chr1</td><td align="left" rowspan="1" colspan="1">1.1E+08</td><td align="left" rowspan="1" colspan="1">CELSR2</td><td align="left" rowspan="1" colspan="1">NM_001408.2</td><td align="left" rowspan="1" colspan="1">c.50T>C</td><td align="left" rowspan="1" colspan="1">p.(Leu17Pro)</td><td align="left" rowspan="1" colspan="1">rs200277265</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">0</td><td align="left" rowspan="1" colspan="1">−0.926</td></tr><tr><td colspan="2" align="left" rowspan="1"><bold>X-linked model</bold></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">chrX</td><td align="left" rowspan="1" colspan="1">10104754</td><td align="left" rowspan="1" colspan="1">WWC3</td><td align="left" rowspan="1" colspan="1">NM_015691.3</td><td align="left" rowspan="1" colspan="1">c.2845C>T</td><td align="left" rowspan="1" colspan="1">p.(Arg949Cys)</td><td align="left" rowspan="1" colspan="1">rs56399961</td><td align="left" rowspan="1" colspan="1">0.002226</td><td align="left" rowspan="1" colspan="1">0.013</td><td align="left" rowspan="1" colspan="1">0.999</td><td align="left" rowspan="1" colspan="1">0.9999</td><td align="left" rowspan="1" colspan="1">5.4</td></tr><tr><td align="left" rowspan="1" colspan="1">chrX</td><td align="left" rowspan="1" colspan="1">1.29E+08</td><td align="left" rowspan="1" colspan="1">XPNPEP2</td><td align="left" rowspan="1" colspan="1">NM_003399.5</td><td align="left" rowspan="1" colspan="1">c.644C>T</td><td align="left" rowspan="1" colspan="1">p.(Thr215Ile)</td><td align="left" rowspan="1" colspan="1">rs138365897</td><td align="left" rowspan="1" colspan="1">0.001908</td><td align="left" rowspan="1" colspan="1">0.004</td><td align="left" rowspan="1" colspan="1">0.838</td><td align="left" rowspan="1" colspan="1">0.9958</td><td align="left" rowspan="1" colspan="1">3.66</td></tr><tr><td align="left" rowspan="1" colspan="1">chrX</td><td align="left" rowspan="1" colspan="1">1.54E+08</td><td align="left" rowspan="1" colspan="1">F8</td><td align="left" rowspan="1" colspan="1">NM_000132.3</td><td align="left" rowspan="1" colspan="1">c.1272-5T>C</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">0.9>0.8</td><td align="left" rowspan="1" colspan="1">9.3>8.9</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">−4.07</td></tr><tr><td colspan="2" align="left" rowspan="1"><bold>Recessive model</bold></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">chr1</td><td align="left" rowspan="1" colspan="1">2.11E+08</td><td align="left" rowspan="1" colspan="1">HHAT</td><td align="left" rowspan="1" colspan="1">NM_001170564.1</td><td align="left" rowspan="1" colspan="1">c.449G>T</td><td align="left" rowspan="1" colspan="1">p.(Gly150Val)</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">0.001</td><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">0.9988</td><td align="left" rowspan="1" colspan="1">4.98</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt103"><label></label><p>MAF: Minimum Allele Frequency.</p></fn><fn id="nt104"><label></label><p>*Scores for the different pathogenicity prediction scores. For the splice variant NNSplice and ESEfinder were used instead of SIFT, PolyPhen2 and Mutation Taster.</p></fn></table-wrap-foot></table-wrap><p>One <italic>de novo</italic> variant was identified in <italic>CELSR2</italic> (NM_001408.2:c.50T>C:p.(Leu17Pro), OMIM: 604265) which is predicted as non-pathogenic by PolyPhen2 and Mutation Taster, and pathogenic by SIFT. RNAi-based knockouts of <italic>Celsr2</italic> in mice resulted in prominent simplification of the dendritic arbors of cortical pyramidal neurons and Purkinje neurons <xref rid="pgen.1004340-Shima1" ref-type="bibr">[32]</xref>, which in combination with the low pathogenicity score led us to not consider this variant further. Among the X-linked variants the NM_000132.3:c.1272-5T>C in <italic>F8</italic> (OMIM: 300841) was not considered, as both programs (i.e. NNSplice and ESEfinder) predicted a slight decrease of the acceptor splice site, which we did not consider as potentially pathogenic. No data were found in the literature that allowed us to evaluate the potential role of WWC3 in the patients' phenotype. <italic>XPNPEP2</italic> (OMIM: 300145) has been implicated in ACE inhibitor-induced angioedema <xref rid="pgen.1004340-CiliaLaCorte1" ref-type="bibr">[33]</xref>. The rather high allelic frequency for both <italic>WWC3</italic> and <italic>XPNPEP2</italic> variants (∼1/2000) coupled with the absence of a clear functional link between these genes and the patient phenotype led us to consider them as non-pathogenic or at least non-related to the patient's phenotype.</p><p>The NM_001170564.1:c.449G>T:p.(Gly150Val) in the <italic>HHAT</italic> short isoform, equivalent to NM_001122834:c.860G>T:p.(Gly287Val) in the <italic>HHAT</italic> long isoform (OMIM: 605743), was the only variant remaining after filtering variants for the recessive model. The variant is homozygous in the patient and heterozygous in the parents. Inheritance of the newly identified mutation was confirmed by Sanger sequencing (<xref ref-type="fig" rid="pgen-1004340-g001">Fig. 1D</xref>). All pathogenicity prediction algorithms, i.e. SIFT, PolyPhen and Mutation Taster, consider this mutation as deleterious, damaging or pathogenic (<xref ref-type="table" rid="pgen-1004340-t002">Table 2</xref>). G287V is located just next to the 6th predicted transmembrane domain and in the membrane bound <italic>O</italic>-acyltransferase (MBOAT) domain, two highly conserved regions of HHAT (<xref ref-type="fig" rid="pgen-1004340-g001">Fig. 1E</xref>). The G287 residue is also well conserved (GERP++: 4.98) across species from zebrafish to mice and humans (<xref ref-type="fig" rid="pgen-1004340-g001">Fig. 1E</xref>), suggesting an important role for this amino-acid in either HHAT protein folding, stability or the acyltransferase activity required for palmitoylation of Hh proteins. In addition, the pleiotropic phenotype including dwarfism, chondrodysplasia, brachydactyly and gonadal dysgenesis is consistent with previously described defects in SHH, DHH and IHH signaling <xref rid="pgen.1004340-Nieuwenhuis1" ref-type="bibr">[34]</xref>.</p></sec><sec id="s2c"><title><italic>HHAT</italic> is widely expressed in mouse and fetal human organs including ovaries and Sertoli cells in developing testes</title><p>In order to obtain insight into the potential role of HHAT during fetal development, we characterized its expression in several human tissues, including fetal testes and ovaries, just after the time of sex determination (gestation week (GW) 9). Expression analysis revealed that <italic>HHAT</italic> is expressed at various levels in most of the fetal human organs investigated, including testes and ovaries (<xref ref-type="fig" rid="pgen-1004340-g002">Fig. 2A</xref>). Interestingly, the different hedgehog genes showed more specific expression profiles. As expected, <italic>DHH</italic> is specifically expressed in the embryonic testis, whereas strong <italic>SHH</italic> expression is detected in the fetal lung and eye and is absent from the fetal heart at this developmental stage (<xref ref-type="fig" rid="pgen-1004340-g002">Fig. 2A</xref>). We observed a strong expression of <italic>IHH</italic> in the small intestine and digits, consistent with its well-established role in the development of these structures <xref rid="pgen.1004340-Gao1" ref-type="bibr">[35]</xref>, <xref rid="pgen.1004340-vandenBrink1" ref-type="bibr">[36]</xref>. In the adult testis, qRT-PCR from purified human adult Sertoli cells, expressing <italic>SOX9</italic>, and Leydig cells, expressing <italic>INSL3</italic>, indicated that <italic>HHAT</italic> is expressed at high levels in Sertoli cells, the unique gonadal source of Hh ligands <xref rid="pgen.1004340-Bitgood1" ref-type="bibr">[27]</xref>, <xref rid="pgen.1004340-Franco1" ref-type="bibr">[37]</xref> (<xref ref-type="fig" rid="pgen-1004340-g002">Fig. 2B</xref>). In mice, transcriptional analysis of XX and XY SF1-positive gonadal cells during sex determination confirmed that <italic>Dhh</italic> is strongly and specifically expressed in XY cells starting at E11.5 (<xref ref-type="fig" rid="pgen-1004340-g002">Fig. 2C</xref>), a profile consistent with its reported expression in Sertoli cells <xref rid="pgen.1004340-Bitgood1" ref-type="bibr">[27]</xref>. In contrast, <italic>Hhat</italic> transcripts are already present in the somatic progenitors of both XX and XY gonads at E10.5 prior to sex determination. Following testicular differentiation, there is a 2.5-fold increase in <italic>Hhat</italic> transcripts levels in XY SF1<sup>+</sup> cells by E13.5 (<xref ref-type="fig" rid="pgen-1004340-g002">Fig. 2C</xref>). Overall, the broad expression of <italic>HHAT</italic> during development is consistent with the wide-ranging functions of Hh in mediating organogenesis and embryonic patterning. In gonads, our expression analysis suggests that <italic>HHAT</italic> is expressed in the supporting cell lineage at the time of sex determination and later in Sertoli cells.</p><fig id="pgen-1004340-g002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pgen.1004340.g002</object-id><label>Figure 2</label><caption><title>HHAT is widely expressed during human fetal development and post-natal life.</title><p>A) qRT-PCR analysis of <italic>HHAT</italic> and Hedgehog protein coding genes (<italic>DHH</italic>, <italic>IHH</italic> and <italic>SHH</italic>) expression in human fetal tissues at Gestation Week 9. B) qRT-PCR analysis reveals that HHAT is preferentially expressed in human Sertoli cells (SOX9-expressing cells) compared to Leydig cells (INSL3-expressing cells). C) Evaluation of the expression of <italic>Hhat</italic> (left panel) and Hedgehog genes (right panel) by qRT-PCR in mouse SF1<sup>+</sup> somatic cells from developing XY (blue bars) and XX (red bars) gonads. Legend: L, lung; H, Heart; I, Intestine; T, Tongue; K, Kidney; D, anterior limb digits.</p></caption><graphic xlink:href="pgen.1004340.g002"></graphic></fig></sec><sec id="s2d"><title>G287V mutation impairs HHAT palmitoyl-transferase activity <italic>in vitro</italic></title><p>To further investigate the effect of the G287V mutation on HHAT function, plasmids encoding wild type and G287V HA-tagged HHAT were generated and expressed in COS-1 cells. Both wild type and G287V HHAT were expressed at equivalent levels in the presence or absence of co-transfected SHH, which in turn was expressed at the same level in the presence of either wild type or mutant HHAT (<xref ref-type="fig" rid="pgen-1004340-g003">Fig. 3A</xref>). Immunofluorescence staining revealed that the subcellular localization of wild type and G287V HHAT was similar; both proteins localized in the endoplasmic reticulum (ER) and the Golgi (<xref ref-type="fig" rid="pgen-1004340-g003">Fig. 3B</xref>). To test whether the G287V mutation altered protein stability, cells expressing wild type or mutant HHAT were treated with cycloheximide and chloramphenicol, and the amount of HHAT protein remaining at various time points was measured. The stability of the G287V mutant was comparable to that of wild type HHAT (<xref ref-type="fig" rid="pgen-1004340-g003">Fig. 3C</xref>).</p><fig id="pgen-1004340-g003" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pgen.1004340.g003</object-id><label>Figure 3</label><caption><title>G287V mutation results in loss of HHAT activity.</title><p>A) COS-1 cells were transfected with the indicated constructs and cell lysates were analyzed directly by Western blotting. B) COS-1 cells transfected with wild type or G287V HHAT-HA were fixed and processed for indirect immunofluoresence and stained with the antibodies indicated. C) COS-1 cells were transfected with the indicated HHAT construct and incubated in DMEM supplemented with 10% FBS, 100 µg/ml cyclohexamide, and 40 µg/ml chloramphenicol. At each indicated time point, cells were lysed and subjected to SDS-PAGE and Western blotting with anti-HA antibodies. The amount of HA signal at each time point was determined using GS-800 Calibrated Densitometer (BioRad). Experiments were carried out in duplicate and repeated three times. D) <italic>In vitro</italic> palmitoylation assay using SHH and membranes from cells expressing wild type or G287V HHAT. Top panel, incorporation of <sup>125</sup>I-iodopalmitate into SHH detected by phosphorimaging. Middle panel, Anti-SHH Western blot. Lower panel, Anti-HA Western blot. Experiments were carried out in duplicate and repeated three times. E) <italic>In vitro</italic> palmitoylation assay using DHH and membranes from cells expressing wild type or G287V HHAT. Top panel, incorporation of <sup>125</sup>I-iodopalmitate into DHH detected by phosphorimaging. Middle panel, Coomassie-staining of DHH. Lower panel, Anti-HA Western blot. Experiments were carried out in duplicate and repeated three times.</p></caption><graphic xlink:href="pgen.1004340.g003"></graphic></fig><p>We next used an <italic>in vitro</italic> assay for HHAT activity, which quantifies the incorporation of <sup>125</sup>I-iodopalmitate into Hh proteins, to compare the activity of G287V HHAT to that of wild type. Membranes from cells expressing either wild type or G287V HHAT were incubated with <sup>125</sup>I-iodo palmitoyl CoA and purified, recombinant SHH protein. In the presence of wild type HHAT, robust incorporation of <sup>125</sup>I-iodopalmitate into SHH was detected (<xref ref-type="fig" rid="pgen-1004340-g003">Fig. 3D</xref>). By contrast, no incorporation of <sup>125</sup>I-iodopalmitate into SHH was detected with G287V HHAT. DHH is the only hedgehog protein expressed in gonads and is likely the more relevant Hh family substrate in this system. To date, no study has tested the ability of HHAT to palmitoylate DHH. We purified a recombinant DHH protein encompassing the N-terminal signaling fragment, along with a C23A DHH mutant, which lacks the N-terminal Cys residue required for palmitoylation. Using the <italic>in vitro</italic> HHAT activity assay, we provide the first evidence that HHAT directly palmitoylates DHH and that the G287V mutation results in loss of activity (<xref ref-type="fig" rid="pgen-1004340-g003">Fig. 3E</xref>). Taken together, these results clearly indicate that G287V HHAT lacks the ability to palmitoylate hedgehog proteins.</p></sec><sec id="s2e"><title>Palmitoylation of hedgehog ligands is required for testis organogenesis in mice</title><p>In mice, <italic>Hhat</italic> loss of function recapitulates many of the skeletal and growth defects observed in the patients described above, as well as in human individuals bearing mutations in genes encoding Hh proteins <xref rid="pgen.1004340-Nieuwenhuis1" ref-type="bibr">[34]</xref>. Mice lacking functional HHAT developed to term but died soon after birth. Mutant mice displayed severe dwarfism, short-limbs, diminished chondrogenesis and osteogenesis, facial defects, and holoprosencephaly together with acrania and agnathia <xref rid="pgen.1004340-Chen1" ref-type="bibr">[12]</xref>, <xref rid="pgen.1004340-Dennis1" ref-type="bibr">[38]</xref>. Since defects in testicular differentiation and development have never been investigated in these mutant mice, we analyzed by histological means and immunofluorescence the pattern of expression of key somatic and germ cell markers in the developing gonads of wild type (WT) and <italic>Hhat<sup>Creface/Creface</sup></italic> mutant (Creface) mice <xref rid="pgen.1004340-Dennis1" ref-type="bibr">[38]</xref>. The developing XX mutant gonads (E11.5–E15.5) were histologically indistinguishable from XX control gonads (<xref ref-type="supplementary-material" rid="pgen.1004340.s002">Fig. S2A–H</xref>). As expected, none of the testis-specific markers including SOX9 and CYP11A1 were expressed (data not shown), but cells positive for the key ovarian-promoting factors FOXL2 were normally present in XX mutant gonads at E13.5 (<xref ref-type="supplementary-material" rid="pgen.1004340.s002">Fig. S2J</xref>). As expected, female germ cells were normal in numbers and entered meiosis at around E13.5, as shown by the expression of MVH and γH2AX, which were similar in both control and mutant XX gonads (<xref ref-type="supplementary-material" rid="pgen.1004340.s002">Fig. S2M</xref>,N & O,P). Thus, we conclude that ovarian differentiation occured normally in XX mutant gonads.</p><p>In XY embryos, no obvious differences were observed between mutant and control XY gonads at E11.5 (<xref ref-type="supplementary-material" rid="pgen.1004340.s003">Fig. S3A, B</xref>). SRY-positive pre-Sertoli cells were present (<xref ref-type="supplementary-material" rid="pgen.1004340.s003">Fig. S3C, D</xref>), indicating that the testicular program was normally initiated, despite the absence of functional HHAT. However, our analysis revealed that testis development was severely affected at subsequent stages, leading ultimately to testicular dysgenesis. We observed a drastic reduction in testis size, which was apparent from E12.5 to E15.5 (<xref ref-type="fig" rid="pgen-1004340-g004">Fig. 4A–F</xref>). In addition, H&E staining (<xref ref-type="fig" rid="pgen-1004340-g004">Fig. 4A–F</xref>) as well as immunofluorescence for marker proteins of germ cells (ECADH and MVH) and Sertoli cells (SOX9 and WT1) (<xref ref-type="fig" rid="pgen-1004340-g004">Fig. 4G–V</xref>), demonstrated not only a decrease in testis cord numbers between wild-type and mutant testes, but also an alteration of the size and shape of testis cords at all stages investigate, which appeared irregular and anastomotic (<xref ref-type="fig" rid="pgen-1004340-g004">Fig. 4</xref>). Interestingly, while testis cord formation was affected, the differentiation of Sertoli cells appeared to be normal. Both, the expression of SOX9 and WT1, two Sertoli cells markers, were present in XY mutant gonads at all stages investigated (<xref ref-type="fig" rid="pgen-1004340-g004">Fig. 4G–L & S–V</xref>). These data collectively suggest that HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation.</p><fig id="pgen-1004340-g004" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pgen.1004340.g004</object-id><label>Figure 4</label><caption><title>Testicular dysgenesis in mouse embryo lacking <italic>Hhat</italic>.</title><p>A–F) H&E staining of wild-type (WT) and <italic>Hhat<sup>Creface/Creface</sup></italic> mutant (Creface) XY gonads at E12.5 (A, B), E13.5 (C, D) and E15.5 (E, F). Note the reduction in testis size from E12.5 onward and the presence of dysgenetic testis cords (asterisk) with considerable variations in term of shape and size. Finally, the interstitial space (arrowhead) in the E13.5 and E15.5 mutant testes was abnormally dense and cellular in comparison of control mice testis. G–X) Expression of key testicular markers was assessed by double/single immunofluorescence at E12.5, E13.5 and E15.5 on both wild-type (WT) and <italic>Hhat<sup>Creface/Creface</sup></italic> mutant (Creface) XY gonads using either the Sertoli cell marker SOX9 (green) and the germ cell marker ECADH (red, G–L), the meiotic marker γH2AX (red) along with the germ cell marker MVH (green; M–R) or the Sertoli cell marker WT1 (green) with the Leydig cell marker CYP11A1 (red, S–X). Note the near complete absence of Leydig cells in mutant testes.</p></caption><graphic xlink:href="pgen.1004340.g004"></graphic></fig><p>The interstitial compartment of the developing mutant testes at E13.5 and E15.5 exhibited a high cellular density typical of irregular and dense connective tissues, in sharp contrast with the loose structure generally observed in control testes at these developmental ages (compare arrows in <xref ref-type="fig" rid="pgen-1004340-g004">Fig. 4 D&F with C&E</xref>). In fact, we found that the differentiation of fetal Leydig cells and steroidogenesis were not initiated in mutant gonads, indicated by the absence of the Leydig cell-specific marker CYP11A1 at E12.5 and E13.5 (<xref ref-type="fig" rid="pgen-1004340-g004">Fig. 4 T&V</xref>). Nevertheless, few rare CYP11A1-positive cells were observed at E15.5 (arrows in <xref ref-type="fig" rid="pgen-1004340-g004">Fig. 4X</xref>), an expression pattern reminiscent to what was found in the patient gonads (<xref ref-type="supplementary-material" rid="pgen.1004340.s001">Fig. S1D</xref>). Overall, the analysis of the <italic>Hhat<sup>Creface/Creface</sup></italic> mutant XY gonads confirmed that the loss of functional HHAT affects the development of fetal Leydig cells as well as the proper formation of testis cords, leading ultimately to testicular dysgenesis.</p></sec></sec><sec id="s3"><title>Discussion</title><p>We used whole-exome sequencing (WES) for the identification of the underlying genetic cause of a unique case of autosomal recessive syndromic 46,XY DSD with testicular dysgenesis and chondrodysplasia. Analysis of WES results using an autosomal recessive model revealed a homozygous G287V missense mutation in the hedgehog acyl-transferase (<italic>HHAT</italic>) gene. This gene encodes an endoplasmic reticulum protein, HHAT, which catalyzes the transfer of palmitate onto hedgehog ligands. The Gly287 residue is a highly conserved amino acid which, when mutated, leads to a non-functional HHAT protein that lacks the ability to palmitoylate hedgehog proteins. Consistent with the patient phenotype, HHAT is expressed in a wide variety of human fetal organs as well as in Sertoli cells, the unique source of DHH in the testis. In addition, <italic>Hhat</italic> loss-of-function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in the patient. Overall, these data emphasize the essential role played by post-translational lipid modification of Hh ligands in mediating patterning of many aspects of the body, including the testis, limbs, axial skeleton and the central nervous system.</p><sec id="s3a"><title>Role of Hedgehog palmitoylation in embryonic development</title><p>The patient familial history indicates that the patient and sibling were conceived by a healthy, non-consanguineous caucasian couple <xref rid="pgen.1004340-Nivelon1" ref-type="bibr">[29]</xref>. Despite an extensive search through large cohorts of patients suffering from skeletal disorders and/or DSD, we have not been able to identify a second familial case with a pathogenic mutation in the <italic>HHAT</italic> locus. This suggests that the frequency of pathogenic variants of <italic>HHAT</italic> is extremely low within the general population, which would explain the absence of other listed cases of Nivelon-Nivelon-Mabille syndrome. It is possible that other mutations in the <italic>HHAT</italic> locus may lead to severe defects in HHAT cellular localization, expression and/or activity that might cause lethality at the embryonic or fetal stages. Alternatively, additional variants may be found in the non-coding regulatory regions of the gene, as is the case for SHH and other genes <xref rid="pgen.1004340-Makrythanasis1" ref-type="bibr">[39]</xref>, <xref rid="pgen.1004340-Jeong1" ref-type="bibr">[40]</xref>.</p><p>The wide range of developmental and testicular anomalies observed in both the patient and mice lacking the capacity to palmitoylate Hh proteins is consistent with a perturbation of SHH, DHH and IHH signaling. When tested <italic>in vivo</italic> or with <italic>in vitro</italic> cell-based assays, palmitoylation of SHH has been shown to be required for the formation of soluble multimeric complexes which enhances its signaling potency <xref rid="pgen.1004340-Pepinsky1" ref-type="bibr">[10]</xref>, <xref rid="pgen.1004340-Chen1" ref-type="bibr">[12]</xref>, <xref rid="pgen.1004340-Goetz1" ref-type="bibr">[14]</xref>, <xref rid="pgen.1004340-Taylor1" ref-type="bibr">[15]</xref>. In agreement, mice with <italic>Hhat</italic> loss-of-function fail to properly establish a long-range Hh signaling gradient <xref rid="pgen.1004340-Dennis1" ref-type="bibr">[38]</xref> which results in testicular, neuronal, skeletal and limb patterning defects <xref rid="pgen.1004340-Chen1" ref-type="bibr">[12]</xref>, <xref rid="pgen.1004340-Dennis1" ref-type="bibr">[38]</xref> similar to those observed in <italic>Shh</italic><xref rid="pgen.1004340-Jeong2" ref-type="bibr">[41]</xref>, <xref rid="pgen.1004340-Chiang1" ref-type="bibr">[42]</xref>, <italic>Dhh</italic><xref rid="pgen.1004340-Clark1" ref-type="bibr">[24]</xref>, <xref rid="pgen.1004340-PierucciAlves1" ref-type="bibr">[25]</xref>, <xref rid="pgen.1004340-Yao1" ref-type="bibr">[26]</xref> and <italic>Ihh</italic><xref rid="pgen.1004340-Gao1" ref-type="bibr">[35]</xref>, <xref rid="pgen.1004340-StJacques1" ref-type="bibr">[43]</xref> knockout mice. Interestingly, mouse embryos expressing a non-palmitoylated form of SHH (<italic>Shh<sup>C25S</sup></italic>) exhibit similar defects to that of <italic>Hhat</italic> mutant embryos due to defective SHH signaling <xref rid="pgen.1004340-Chen1" ref-type="bibr">[12]</xref>. These similar phenotypes indicate that Hh proteins are indeed the major targets of HHAT.</p><p>Surprisingly, the clinical characteristics and defects observed in the patient bearing the G287V mutation are not as severe as the phenotypes found in <italic>Shh<sup>C25S</sup></italic> mutant or <italic>Hhat</italic> knockout mice <xref rid="pgen.1004340-Chen1" ref-type="bibr">[12]</xref>, <xref rid="pgen.1004340-Dennis1" ref-type="bibr">[38]</xref>. While homozygous <italic>Hhat</italic> mutant mice die around birth, the patient exhibits normal viability despite severe skeletal, craniofacial and neuronal defects. This could be explained by species differences and/or a small residual transferase activity in the G287V-HHAT mutant protein that is not detected in our <italic>in vitro</italic> assays.</p><p>The role of Hh proteins in regulating embryonic pattern formation and adult tissue maintenance in mammals and the deleterious effects that accompany their ablation has been extensively studied (for review see <xref rid="pgen.1004340-Varjosalo1" ref-type="bibr">[16]</xref>). In the present study, we face a novel and peculiar situation where Hh primary sequences are not affected, but rather the establishment of morphogen gradients and subsequent long range signaling actions of all three Hh ligands. This is reflected by the variable severity of the multiple Hh gene-related symptoms described in this patient. For instance, the impaired testicular development suffered by the patient reaches a severity that is close, if not identical, to what was observed in previous cases of DHH mutation <xref rid="pgen.1004340-Das1" ref-type="bibr">[2]</xref>, <xref rid="pgen.1004340-Canto1" ref-type="bibr">[21]</xref>, with the generation of streak gonads that do not contain functional reproductive structures. Similarly, the patient suffered a severe form of type A1 brachydactyly, which affects each of the five fingers – a situation typically observed in patients with IHH mutations <xref rid="pgen.1004340-Gao2" ref-type="bibr">[44]</xref>. In these particular cases, the severity of the symptoms might reflect the necessity to establish a proper long-range Hh gradient for gonadal organogenesis and finger development. On the other hand, the relatively mild abnormalities observed in optical disc and the absence of holoprosencephaly in the patient are in stark contrast with the severe craniofacial defect and abnormal forebrain development found in patients with SHH loss of function <xref rid="pgen.1004340-Belloni1" ref-type="bibr">[17]</xref>, <xref rid="pgen.1004340-Traiffort1" ref-type="bibr">[45]</xref>. These differences in severity might account for the fact that forebrain development into two separated hemispheres does not rely on the establishment of a long range SHH gradient as much as other patterning SHH-related processes.</p></sec><sec id="s3b"><title>Lipid modifications of Hh ligands are conserved during evolution</title><p>The core components required for Hh production, movement and signal transduction events are conserved between <italic>Drosophila</italic> and humans <xref rid="pgen.1004340-Ingham1" ref-type="bibr">[46]</xref>, <xref rid="pgen.1004340-Wilson1" ref-type="bibr">[47]</xref>. This is particularly true for the mechanisms of post-translational dual lipid modification of Hh ligands <xref rid="pgen.1004340-Porter2" ref-type="bibr">[9]</xref>, <xref rid="pgen.1004340-Pepinsky1" ref-type="bibr">[10]</xref>. This is required to generate soluble, multimeric forms of Hh, create the Hh-signaling gradient and to enhance Hh-signaling potency <xref rid="pgen.1004340-Buglino2" ref-type="bibr">[13]</xref>. Palmitoylation is one of the two critical lipid modifications <xref rid="pgen.1004340-Lee1" ref-type="bibr">[48]</xref> and is mediated by the hedgehog HHAT. This protein is highly conserved, particularly in transmembrane regions and MBOAT domains. It is noteworthy that the mutated G287 residue, one of the highly conserved residues in vertebrate evolution, is positioned adjacent to the predicted 6<sup>th</sup> transmembrane region and 2<sup>nd</sup> MBOAT domain (<xref ref-type="fig" rid="pgen-1004340-g001">Fig. 1E</xref>). Importantly, the G287V mutation does not affect the subcellular localisation and stability of mutant HHAT, both of which are comparable to that of wild type HHAT. It does, however, impair its ability to palmitoylate hedgehog ligands. Further work will be required to define the precise structural role of G287 in mediating HHAT protein folding and its palmitoyl-transferase activity.</p></sec><sec id="s3c"><title>HHAT is essential for testicular development and fetal Leydig cell differentiation</title><p>Our expression studies using human and mouse embryonic tissues as well as purified cells indicate that <italic>HHAT</italic> is expressed in developing gonads around the time of sex determination, probably in the supporting cell lineage, and later in Sertoli cells, which are the sole source of hedgehog ligand in the gonad <xref rid="pgen.1004340-Bitgood1" ref-type="bibr">[27]</xref>, <xref rid="pgen.1004340-Franco1" ref-type="bibr">[37]</xref>. The absence of functional HHAT leads to testicular dysgenesis both in humans and in the knockout mouse model. Careful analysis of testis development during the sex determination period revealed that early stages of gonad development occur normally up to E11.5 in <italic>Hhat<sup>Creface/Creface</sup></italic> mice, and that the initiation of testicular program is not affected. However, by E12.5 testicular development was significantly impaired in XY embryos with a massive reduction in testis size, a reduction in the number of testis cords that also appeared irregular in shape and diameter, and a almost complete absence of fetal Leydig cells. Thus HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of Leydig cells. In this context, it is interesting to note that <italic>Hhat<sup>Creface/Creface</sup></italic> testicular phenotype was similar to the one observed in <italic>Dhh</italic> knockout mice. Knockout mice for the <italic>Dhh</italic> gene display testis dysgenesis including apolar Sertoli cells, anastomotic testis cords, a decreased number of fetal Leydig cells and an insufficient production of androgens resulting in male infertility, hypogonadism and feminized external genitalia <xref rid="pgen.1004340-Clark1" ref-type="bibr">[24]</xref>, <xref rid="pgen.1004340-PierucciAlves1" ref-type="bibr">[25]</xref>, <xref rid="pgen.1004340-Yao1" ref-type="bibr">[26]</xref>. Consistent with its role in the mouse, several mutations of <italic>DHH</italic> have also been described in patients with a non-syndromic form of 46,XY DSD with partial or complete gonadal dysgenesis <xref rid="pgen.1004340-Das1" ref-type="bibr">[2]</xref>, <xref rid="pgen.1004340-Canto1" ref-type="bibr">[21]</xref>, <xref rid="pgen.1004340-Canto2" ref-type="bibr">[22]</xref>, <xref rid="pgen.1004340-Umehara1" ref-type="bibr">[23]</xref>. Although it is now clear that HHAT is essential for testis formation in both humans and mice, further work will be required to define the precise role of HHAT and Hh palmitoylation in mediating testis development and how it influences testis cord formation and the specification of fetal Leydig and peritubular myoid cells.</p></sec></sec><sec sec-type="materials|methods" id="s4"><title>Materials and Methods</title><sec id="s4a"><title>Patient management, sample collection and DNA extraction</title><p>All clinical investigations have been performed according to Declaration of Helsinki principles. The study was approved by the local French ethics committee (#DC2011-1332). The proposita was described in <xref rid="pgen.1004340-Nivelon1" ref-type="bibr">[29]</xref>, <xref rid="pgen.1004340-ThauvinRobinet1" ref-type="bibr">[30]</xref> and referred at 16 years of age for follow-up. The patient was seen at the Medical Genetics Center of Dijon in September 2004. She presented mild mental retardation, a dysmorphic face, an anteriorly bent thorax with a rigid spine, brachydactyly, muscular hypertrophy and myopia. Endocrine studies performed because of lack of pubertal development showed hypergonadotrophic hypogonadism. Gonadectomy was performed in order to prevent malignant transformation. The proband's parents had a normal phenotype. Genomic DNA from the proband and her parents were isolated from blood samples using the Qiagen DNA mini kit (Qiagen, Valencia, California). An array-CGH 244K (Agilent) has excluded pathogenic microdeletions and microduplications.</p></sec><sec id="s4b"><title>Exome capture and sequencing</title><p>Exome capture was performed using the SureSelect Human All Exon v3 kit (Agilent Inc). Sequencing was carried out on an Illumina HiSeq 2000 instrument. Fastq files were obtained using the Illumina CASAVA v1.8.1 software and processed using our “in house” bioinformatic pipeline running on the Vital-IT Center for high-performance computing of the Swiss Institute of Bioinformatics (SIB; <ext-link ext-link-type="uri" xlink:href="http://www.vital-it.ch">http://www.vital-it.ch</ext-link>). This pipeline utilizes published algorithms in a sequential manner (BWA <xref rid="pgen.1004340-Li1" ref-type="bibr">[49]</xref> for map reads, SAMtools <xref rid="pgen.1004340-Li2" ref-type="bibr">[50]</xref> for detection of variants, Pindel <xref rid="pgen.1004340-Wang1" ref-type="bibr">[51]</xref> for the detection of indels, ANNOVAR <xref rid="pgen.1004340-Ye1" ref-type="bibr">[52]</xref> for the annotation). The entire coding sequence corresponding to the RefSeq <xref rid="pgen.1004340-Pruitt1" ref-type="bibr">[53]</xref> coding genes was used as the reference for the calculation of coverage and reads on target. All experiments were performed using the manufacturer's recommended protocols without modifications.</p><p>Results were analyzed using the VariantMaster software <xref rid="pgen.1004340-Santoni1" ref-type="bibr">[31]</xref> in order to identify <italic>de novo</italic> variants as well as variants respecting different Mendelian inheritance models (dominant with reduced penetrance, recessive, X-linked). Variant validation was performed by targeted Sanger sequencing.</p></sec><sec id="s4c"><title>Human tissue collection and Sertoli and Leydig cell purification</title><p>Human fetal gonads (9 gestational weeks) were obtained from pregnant women referred to the Rennes University Hospital (France) for legal abortion in the first trimester as previously described <xref rid="pgen.1004340-MazaudGuittot1" ref-type="bibr">[54]</xref>. All women received information and gave verbal consent in accordance with national guidelines (Agence de la Biomédecine, authorization #PFS09-011) and protocols were approved by the local ethics committee of Rennes (#11-48). The termination of pregnancy was induced by treatment with Mifegyne (mifepristone) followed 48 h later by Cytotec treatment, and finalized by aspiration. None of the terminations were motivated by fetal abnormality. After recovery from the aspiration product under a binocular microscope (Olympus SZX7, Lille, France), gonads and other somatic organs were snap frozen and stored at −80°C until RNA extraction.</p><p>Adult human testes were collected either from prostate cancer patients undergoing orchiectomy who had not received any hormone therapy or from brain dead men with the authorization from the Agence de la Biomédecine (authorization # PFS09-015, “Etude de la spermatogenèse humaine normale et pathologique”). Leydig cells were isolated using a combination of mechanical dissociation, enzymatic digestion, filtration through nylon meshes and density Percoll gradient centrifugation, as previously described <xref rid="pgen.1004340-Willey1" ref-type="bibr">[55]</xref>. Human primary Sertoli cells were purchased from Lonza (Basel, Switzerland) following isolation procedures described elsewhere <xref rid="pgen.1004340-Egan1" ref-type="bibr">[56]</xref>, <xref rid="pgen.1004340-Lipshultz1" ref-type="bibr">[57]</xref>.</p></sec><sec id="s4d"><title>Isolation of mouse Sf1:GFP positive cells and RNA extraction</title><p>Urogenital ridges from individual embryos bearing a Sf1-BAC-eGFP transgene-positive urogenital ridges <xref rid="pgen.1004340-Nef1" ref-type="bibr">[58]</xref>, <xref rid="pgen.1004340-Stallings1" ref-type="bibr">[59]</xref> were dissected at relevant stages (E10.5, E11.5, E12.5 and E13.5), digested with trypsin/EDTA, filtered through a 40-mm cell strainer to generate single-cell suspensions and then sorted using a FACS Vantage SE with a purity of above 97% as previously described <xref rid="pgen.1004340-Nef1" ref-type="bibr">[58]</xref>.</p></sec><sec id="s4e"><title>Quantitative Reverse Transcriptase PCR (qRT-PCR)</title><p>Total RNA from human samples was isolated using Macherey Nagel RNA isolation kit, and 250 ng of total RNA were DNase-treated and converted to 1<sup>st</sup> strand cDNA using SuperScript II Reverse Transcriptase following the manufacturer's instructions (Invitrogen Life Technologies). RNA from Sf1<sup>+</sup> cells was extracted using RNeasy microkit from Qiagen according to the manufacturer's protocol and amplified RNAs were obtained and converted into double-stranded cDNA as previously described <xref rid="pgen.1004340-Nef1" ref-type="bibr">[58]</xref>. qPCR was carried out in optical 100-well plates and labeled by using the SYBR green master mix (Applied Biosystems). The fluorescence was quantified with a Prism 7900 HT sequence detection system (Applied Biosystems). The expression of each gene was assayed in triplicate as previously described <xref rid="pgen.1004340-Cederroth1" ref-type="bibr">[60]</xref>, with mouse <italic>Gapdh</italic> and human <italic>RPL19</italic> genes used as reference genes. Primers used for qRT-PCR are listed in <xref ref-type="supplementary-material" rid="pgen.1004340.s004">Table S1</xref> and were designed using the software PRIMER EXPRESS (Applied Biosystems). The statistical significance of fold-changes was determined by a paired Student's <italic>t</italic>-test.</p></sec><sec id="s4f"><title>Reagents and antibodies</title><p>Coenzyme A, CoA synthetase, and anti-HA antibodies were purchased from Sigma (St. Louis, MO). Anti-SHH and anti-CYP11A1 antibodies were purchased from Santa Cruz Biotechnology. Anti-γH2AX, anti-MVH and anti-ECADH antibodies were purchased from Cell Signaling Technology, R&D Systems and Becton Dickinson (USA), respectively. Anti-WT1 was purchased from Dakocytomation (CA, USA). Anti-SRY, anti-SOX9, anti-FOXL2 antibody were previously described <xref rid="pgen.1004340-Pitetti1" ref-type="bibr">[61]</xref>. Alexa-conjugated secondary antibodies were purchased from Invitrogen. [<sup>125</sup>I] NaI was obtained from Perkin Elmer.</p></sec><sec id="s4g"><title>Mammalian expression plasmids</title><p>The full length <italic>SHH</italic> and the wild type <italic>HHAT</italic> cDNA plasmids, in a pcDNA3.1 backbone with a C-terminal HA-tag, have been previously described <xref rid="pgen.1004340-Buglino1" ref-type="bibr">[11]</xref>. The G287V <italic>HHAT</italic> construct was generated from wild-type <italic>HHAT</italic> by site directed mutagenesis using the QuikChange mutagenesis kit (Stratagene). A plasmid encoding full length <italic>DHH</italic> was purchased from Origene. To generate the <italic>DHH</italic> (23–198) construct for purification, an <italic>Nde</italic>I restriction site was introduced preceding basepair 69 of DHH. A <italic>Bam</italic>HI site followed by a stop codon was introduced preceding basepair 597. The digested fragment was ligated into <italic>Nde</italic>I and <italic>Bam</italic>HI digested pET19b (Novagen). The CATATG sequence at the beginning of <italic>DHH</italic> was deleted using QuikChange to generate the N-terminally His6-tagged human <italic>DHH</italic> (23–198), with an enterokinase cleavage site immediately preceding residue 23 of <italic>DHH</italic>. The C23A <italic>DHH</italic> construct was generated by site directed mutagenesis using QuikChange. All constructs and mutations were confirmed by DNA sequencing.</p></sec><sec id="s4h"><title>Cell culture and transfection</title><p>COS-1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin. 293FT cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin, 50 µg/ml streptomycin, 500 µg/ml Geneticin, 1 mM GlutaMAX (Invitrogen), 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids. Transfections were carried out using Lipofectamine 2000 (Invitrogen).</p></sec><sec id="s4i"><title>Expression of purified recombinant DHH</title><p>His-tagged SHH, DHH (23–198) and C23A DHH (23–198) were expressed in <italic>Escherichia coli</italic> BL21(DE3)pLysS cells, purified on Ni-NTA-agarose resin, and dialyzed (20 mM Tris-HCL pH8.0, 350 mM NaCl, and 1 mM β-mercaptoethanol) in the presence of 6 ng/mL enterokinase. The protein concentration was measured using the DC protein assay (Bio-Rad). The N termini of both wild-type and mutant proteins were confirmed by Edman degradation.</p></sec><sec id="s4j"><title><italic>In vitro</italic> palmitoylation assay</title><p>293FT cells were transfected with plasmids encoding wild type or G287V HHAT. Cells were lysed in hypotonic lysis buffer (0.2 mM MgCl<sub>2</sub> and 10 mM HEPES, pH 7.3) 48 hours post transfection. After Dounce homogenization, sucrose was added to a final concentration of 0.25 M and the membrane fraction was pelleted by ultracentrifugation at 100,000 g for 45 min in a Beckman Ti-70.1 fixed angle rotor. The pellet was resuspended in the hypotonic lysis buffer containing 0.25 M sucrose and flash frozen. 10 µg of membranes were incubated with 10 µl of recombinant SHH or DHH (0.2 mg/ml in 20 mM MES (pH 6.5), 1 mM EDTA, and 1 mM DTT), followed by the addition of 30 µl of reaction buffer (167 mM MES (pH 6.5), 1.7 mM DTT, 0.083% Triton X-100, and 167 µM <sup>125</sup>I-iodopalmitoylCoA (synthesized using CoA synthetase as previously described <xref rid="pgen.1004340-Buglino3" ref-type="bibr">[62]</xref>). After one hour, the reaction was stopped by the addition of 50 µl of 2× sample buffer with 40 mM DTT. Samples were electrophoresed on SDS-PAGE gels, dried, and exposed to phosphorimaging for 12–18 h.</p></sec><sec id="s4k"><title>Protein stability assay</title><p>COS-1 cells were transfected with wild type or G287V <italic>HHAT</italic>. 48 hours post-transfection, cells were incubated in media supplemented with 100 µg/ml cyclohexamide and 40 µg/ml chloramphenicol for 0, 4, 8, 10, or 24 hours. Cells were lysed in 500 µl radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris (pH 7.4), 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA), electrophoresed on SDS-PAGE gels, transferred to PVDF membranes and probed with anti-HA antibody to determine protein levels.</p></sec><sec id="s4l"><title><italic>Hhat</italic> knockout mouse line</title><p>The <italic>Hhat+/Creface</italic> mouse line was established and maintained by Paul A. Trainor at the Stowers Institute for Medical Research. These mice were housed in the Laboratory Animal Services Facility at the Stowers Institute for Medical Research according to IACUC animal welfare guidelines. <italic>Hhat<sup>Creface/Creface</sup></italic> embryos and control littermates were generated and genotyped by classic PCR as described previously <xref rid="pgen.1004340-Dennis1" ref-type="bibr">[38]</xref>. Embryos were collected from timed matings and staged by designating noon of the day on which the mating plug was detected as E0.5. Embryos were fixed overnight in 4% paraformaldehyde (PFA) and embedded in paraffin. Sections (5-µm) were stained with hematoxylin and eosin (H&E) or processed for immunofluorescence (IF).</p></sec><sec id="s4m"><title>Histology and immunofluorescence</title><p>Cells were transfected with wild type or G827V <italic>HHAT</italic> and grown on coverslips for 24 hours. Cells on coverslips were fixed in 4% paraformaldehyde, permeabilized with 0.2% TritonX-100, and stained with indicated antibodies and Hoechst dye. Coverslips were mounted on slides using ProLong Gold mounting solution (Invitrogen). Images were collected on a Zeiss LSM 510 microscope using a 63× water immersion objective.</p><p>The patient gonads and healthy human adult testes were fixed in formol whereas mouse samples used for immunofluorescence were fixed in paraformaldehyde. Five µm-sections were cut from paraffin embedded samples and stained with hematoxylin and eosin (H&E), or processed for IF and stained with indicated antibodies and DAPI. All images were obtained with a Zeiss Axioscop or a Nikon C1 Upright microscope.</p></sec></sec><sec sec-type="supplementary-material" id="s5"><title>Supporting Information</title><supplementary-material content-type="local-data" id="pgen.1004340.s001"><label>Figure S1</label><caption><p>CYP11A1-expressing cells are reduced in the dysgenetic XY gonads of the patient with <italic>HHAT</italic> mutation. Haematoxylin and eosin staining (A,B) as well as immunostaining for the Leydig markers CYP11A1 (C,D) from testis of a fertile adult men (A,C) and the left gonads of the patient bearing G287V mutation in HHAT (B, D).</p><p>(PDF)</p></caption><media xlink:href="pgen.1004340.s001.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pgen.1004340.s002"><label>Figure S2</label><caption><p>Ovarian differentiation is normal in XX mice embryos lacking <italic>Hhat</italic>. A–H) H&E stained sections of wild-type (WT) and <italic>Hhat<sup>Creface/Creface</sup></italic> mutant (Creface) XX gonads at E11.5 (A, B), E12.5 (C, D), E13.5 (E, F) and E15.5 (G, H). I–P) Expression of key ovarian markers was assessed by double immunofluorescence at E12.5, E13.5 and E15.5 on both wild-type (WT) and <italic>Hhat<sup>Creface/Creface</sup></italic> mutant (Creface) XX gonads using either FOXL2 (green) and the germ cell marker ECADH (red, I–J) or the meiotic marker γH2AX (red) along with the germ cell marker MVH (green; K–P). Dotted lines mark the developing gonads at E11.5 (A,B).</p><p>(PDF)</p></caption><media xlink:href="pgen.1004340.s002.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pgen.1004340.s003"><label>Figure S3</label><caption><p>SRY is expressed in the genital ridges of XY embryos lacking <italic>Hhat</italic> gene. Haematoxylin and eosin staining (A,B) as well as immunostaining for SRY (C,D) from WT (A–C) and <italic>Hhat<sup>Creface/Creface</sup></italic> mutant (Creface, C–D) XY gonads at E11.5. Dotted lines mark the gonads (A,B). Note the similar pattern of SRY expression for both genotypes.</p><p>(PDF)</p></caption><media xlink:href="pgen.1004340.s003.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="pgen.1004340.s004"><label>Table S1</label><caption><p>Primers sequences for real-time PCR.</p><p>(XLSX)</p></caption><media xlink:href="pgen.1004340.s004.xlsx"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack><p>We thank Cécile Mutel for IF experiments, Nicolas Veillard for technical assistance, Isabelle Coiffec for help in human fetal gonad collection and the medical staffs of Rennes 1 hospitals for human organ collection. 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