Functional Analyses of a Novel Splice Variant in the CHD7 Gene, Found by Next Generation Sequencing, Confirm Its Pathogenicity in a Spanish Patient and Diagnose Him with CHARGE Syndrome
Mutations in CHD7 have been shown to be a major cause of CHARGE syndrome, which presents many symptoms and features common to other syndromes making its diagnosis difficult. Next generation sequencing (NGS) of a panel of intellectual disability related genes was performed in an adult patient without...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2018-01-01
|
| Series: | Frontiers in Genetics |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/article/10.3389/fgene.2018.00007/full |
| id |
doaj-874715fc58564df0a90263bad61671ec |
|---|---|
| record_format |
Article |
| spelling |
doaj-874715fc58564df0a90263bad61671ec2020-11-25T01:05:23ZengFrontiers Media S.A.Frontiers in Genetics1664-80212018-01-01910.3389/fgene.2018.00007329516Functional Analyses of a Novel Splice Variant in the CHD7 Gene, Found by Next Generation Sequencing, Confirm Its Pathogenicity in a Spanish Patient and Diagnose Him with CHARGE SyndromeOlatz Villate0Olatz Villate1Nekane Ibarluzea2Eugenia Fraile-Bethencourt3Alberto Valenzuela4Eladio A. Velasco5Detelina Grozeva6F. L. Raymond7María P. Botella8María-Isabel Tejada9María-Isabel Tejada10María-Isabel Tejada11Biocruces Health Research Institute, Barakaldo, SpainMolecular Genetics Laboratory, Genetics Service, Cruces University Hospital, Barakaldo, SpainBiocruces Health Research Institute, Barakaldo, SpainSplicing and Cancer Laboratory, Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas, Universidad de Valladolid, Valladolid, SpainSplicing and Cancer Laboratory, Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas, Universidad de Valladolid, Valladolid, SpainSplicing and Cancer Laboratory, Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas, Universidad de Valladolid, Valladolid, SpainDepartment of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United KingdomDepartment of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United KingdomDepartment of Pediatrics, Araba University Hospital, Vitoria, SpainBiocruces Health Research Institute, Barakaldo, SpainMolecular Genetics Laboratory, Genetics Service, Cruces University Hospital, Barakaldo, SpainClinical Group, Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid, SpainMutations in CHD7 have been shown to be a major cause of CHARGE syndrome, which presents many symptoms and features common to other syndromes making its diagnosis difficult. Next generation sequencing (NGS) of a panel of intellectual disability related genes was performed in an adult patient without molecular diagnosis. A splice donor variant in CHD7 (c.5665 + 1G > T) was identified. To study its potential pathogenicity, exons and flanking intronic sequences were amplified from patient DNA and cloned into the pSAD® splicing vector. HeLa cells were transfected with this construct and a wild-type minigene and functional analysis were performed. The construct with the c.5665 + 1G > T variant produced an aberrant transcript with an insert of 63 nucleotides of intron 28 creating a premature termination codon (TAG) 25 nucleotides downstream. This would lead to the insertion of 8 new amino acids and therefore a truncated 1896 amino acid protein. As a result of this, the patient was diagnosed with CHARGE syndrome. Functional analyses underline their usefulness for studying the pathogenicity of variants found by NGS and therefore its application to accurately diagnose patients.http://journal.frontiersin.org/article/10.3389/fgene.2018.00007/fullCHD7next generation sequencingalternative splicingminigeneCHARGE syndrome |
| collection |
DOAJ |
| language |
English |
| format |
Article |
| sources |
DOAJ |
| author |
Olatz Villate Olatz Villate Nekane Ibarluzea Eugenia Fraile-Bethencourt Alberto Valenzuela Eladio A. Velasco Detelina Grozeva F. L. Raymond María P. Botella María-Isabel Tejada María-Isabel Tejada María-Isabel Tejada |
| spellingShingle |
Olatz Villate Olatz Villate Nekane Ibarluzea Eugenia Fraile-Bethencourt Alberto Valenzuela Eladio A. Velasco Detelina Grozeva F. L. Raymond María P. Botella María-Isabel Tejada María-Isabel Tejada María-Isabel Tejada Functional Analyses of a Novel Splice Variant in the CHD7 Gene, Found by Next Generation Sequencing, Confirm Its Pathogenicity in a Spanish Patient and Diagnose Him with CHARGE Syndrome Frontiers in Genetics CHD7 next generation sequencing alternative splicing minigene CHARGE syndrome |
| author_facet |
Olatz Villate Olatz Villate Nekane Ibarluzea Eugenia Fraile-Bethencourt Alberto Valenzuela Eladio A. Velasco Detelina Grozeva F. L. Raymond María P. Botella María-Isabel Tejada María-Isabel Tejada María-Isabel Tejada |
| author_sort |
Olatz Villate |
| title |
Functional Analyses of a Novel Splice Variant in the CHD7 Gene, Found by Next Generation Sequencing, Confirm Its Pathogenicity in a Spanish Patient and Diagnose Him with CHARGE Syndrome |
| title_short |
Functional Analyses of a Novel Splice Variant in the CHD7 Gene, Found by Next Generation Sequencing, Confirm Its Pathogenicity in a Spanish Patient and Diagnose Him with CHARGE Syndrome |
| title_full |
Functional Analyses of a Novel Splice Variant in the CHD7 Gene, Found by Next Generation Sequencing, Confirm Its Pathogenicity in a Spanish Patient and Diagnose Him with CHARGE Syndrome |
| title_fullStr |
Functional Analyses of a Novel Splice Variant in the CHD7 Gene, Found by Next Generation Sequencing, Confirm Its Pathogenicity in a Spanish Patient and Diagnose Him with CHARGE Syndrome |
| title_full_unstemmed |
Functional Analyses of a Novel Splice Variant in the CHD7 Gene, Found by Next Generation Sequencing, Confirm Its Pathogenicity in a Spanish Patient and Diagnose Him with CHARGE Syndrome |
| title_sort |
functional analyses of a novel splice variant in the chd7 gene, found by next generation sequencing, confirm its pathogenicity in a spanish patient and diagnose him with charge syndrome |
| publisher |
Frontiers Media S.A. |
| series |
Frontiers in Genetics |
| issn |
1664-8021 |
| publishDate |
2018-01-01 |
| description |
Mutations in CHD7 have been shown to be a major cause of CHARGE syndrome, which presents many symptoms and features common to other syndromes making its diagnosis difficult. Next generation sequencing (NGS) of a panel of intellectual disability related genes was performed in an adult patient without molecular diagnosis. A splice donor variant in CHD7 (c.5665 + 1G > T) was identified. To study its potential pathogenicity, exons and flanking intronic sequences were amplified from patient DNA and cloned into the pSAD® splicing vector. HeLa cells were transfected with this construct and a wild-type minigene and functional analysis were performed. The construct with the c.5665 + 1G > T variant produced an aberrant transcript with an insert of 63 nucleotides of intron 28 creating a premature termination codon (TAG) 25 nucleotides downstream. This would lead to the insertion of 8 new amino acids and therefore a truncated 1896 amino acid protein. As a result of this, the patient was diagnosed with CHARGE syndrome. Functional analyses underline their usefulness for studying the pathogenicity of variants found by NGS and therefore its application to accurately diagnose patients. |
| topic |
CHD7 next generation sequencing alternative splicing minigene CHARGE syndrome |
| url |
http://journal.frontiersin.org/article/10.3389/fgene.2018.00007/full |
| work_keys_str_mv |
AT olatzvillate functionalanalysesofanovelsplicevariantinthechd7genefoundbynextgenerationsequencingconfirmitspathogenicityinaspanishpatientanddiagnosehimwithchargesyndrome AT olatzvillate functionalanalysesofanovelsplicevariantinthechd7genefoundbynextgenerationsequencingconfirmitspathogenicityinaspanishpatientanddiagnosehimwithchargesyndrome AT nekaneibarluzea functionalanalysesofanovelsplicevariantinthechd7genefoundbynextgenerationsequencingconfirmitspathogenicityinaspanishpatientanddiagnosehimwithchargesyndrome AT eugeniafrailebethencourt functionalanalysesofanovelsplicevariantinthechd7genefoundbynextgenerationsequencingconfirmitspathogenicityinaspanishpatientanddiagnosehimwithchargesyndrome AT albertovalenzuela functionalanalysesofanovelsplicevariantinthechd7genefoundbynextgenerationsequencingconfirmitspathogenicityinaspanishpatientanddiagnosehimwithchargesyndrome AT eladioavelasco functionalanalysesofanovelsplicevariantinthechd7genefoundbynextgenerationsequencingconfirmitspathogenicityinaspanishpatientanddiagnosehimwithchargesyndrome AT detelinagrozeva functionalanalysesofanovelsplicevariantinthechd7genefoundbynextgenerationsequencingconfirmitspathogenicityinaspanishpatientanddiagnosehimwithchargesyndrome AT flraymond functionalanalysesofanovelsplicevariantinthechd7genefoundbynextgenerationsequencingconfirmitspathogenicityinaspanishpatientanddiagnosehimwithchargesyndrome AT mariapbotella functionalanalysesofanovelsplicevariantinthechd7genefoundbynextgenerationsequencingconfirmitspathogenicityinaspanishpatientanddiagnosehimwithchargesyndrome AT mariaisabeltejada functionalanalysesofanovelsplicevariantinthechd7genefoundbynextgenerationsequencingconfirmitspathogenicityinaspanishpatientanddiagnosehimwithchargesyndrome AT mariaisabeltejada functionalanalysesofanovelsplicevariantinthechd7genefoundbynextgenerationsequencingconfirmitspathogenicityinaspanishpatientanddiagnosehimwithchargesyndrome AT mariaisabeltejada functionalanalysesofanovelsplicevariantinthechd7genefoundbynextgenerationsequencingconfirmitspathogenicityinaspanishpatientanddiagnosehimwithchargesyndrome |
| _version_ |
1725194788696752128 |