title: Identification of a duplication within the GDF9 gene and novel candidate genes for primary ovarian insufficiency (POI) by a customized high-resolution array comparative genomic hybridization platform.
abstract: OBJECTIVE
  Can high-resolution array comparative genomic hybridization (CGH) analysis of DNA samples from women with primary ovarian insufficiency (POI) improve the diagnosis of the condition and identify novel candidate genes for POI?
  
  
  CONCLUSIONS
  A mutation affecting the regulatory region of growth differentiation factor 9 (GDF9) was identified for the first time together with several novel candidate genes for POI.
  
  
  BACKGROUND
  Most patients with POI do not receive a molecular diagnosis despite a significant genetic component in the pathogenesis.
  
  
  METHODS
  We performed a case-control study. Twenty-six patients were analyzed by array CGH for identification of copy number variants. Novel changes were investigated in 95 controls and in a separate population of 28 additional patients with POI. The experimental procedures were performed during a 1-year period.
  
  
  METHODS
  DNA samples from 26 patients with POI were analyzed by a customized 1M array-CGH platform with whole genome coverage and probe enrichment targeting 78 genes in sex development. By PCR amplification and sequencing, the breakpoint of an identified partial GDF9 gene duplication was characterized. A multiplex ligation-dependent probe amplification (MLPA) probe set for specific identification of deletions/duplications affecting GDF9 was developed. An MLPA probe set for the identification of additional cases or controls carrying novel candidate regions identified by array-CGH was developed. Sequencing of three candidate genes was performed.
  
  
  RESULTS
  Eleven unique copy number changes were identified in a total of 11 patients, including a tandem duplication of 475 bp, containing part of the GDF9 gene promoter region. The duplicated region contains three NOBOX-binding elements and an E-box, important for GDF9 gene regulation. This aberration is likely causative of POI. Fifty-four patients were investigated for copy number changes within GDF9, but no additional cases were found. Ten aberrations constituting novel candidate regions were detected, including a second DNAH6 deletion in a patient with POI. Other identified candidate genes were TSPYL6, SMARCC1, CSPG5 and ZFR2.
  
  
  CONCLUSIONS
  This is a descriptive study and no functional experiments were performed.
  
  
  CONCLUSIONS
  The study illustrates the importance of analyzing small copy number changes in addition to sequence alterations in the genetic investigation of patients with POI. Also, promoter regions should be included in the investigation.
  
  
  BACKGROUND
  The study was supported by grants from the Swedish Research council (project no 12198 to A.W. and project no 20324 to A.L.H.), Stockholm County Council (E.I., A.W. and K.R.W.), Foundation Frimurare Barnhuset (A.N., A.W. and M.B.), Karolinska Institutet (A.N., A.L.H., E.I., A.W. and M.B.), Novo Nordic Foundation (A.W.) and Svenska Läkaresällskapet (M.B.). The funding sources had no involvement in the design or analysis of the study. The authors have no competing interests to declare.
  
  
  BACKGROUND
  Not applicable.
authors.affiliation: Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm 171 76, Sweden Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital, Stockholm 171 76, Sweden Centre for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden ameli.norling@ki.com.
authors.forename: A
authors.initials: A
authors.lastname: Norling
authors.affiliation: Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital, Stockholm 171 76, Sweden.
authors.forename: A L
authors.initials: AL
authors.lastname: Hirschberg
authors.affiliation: Department of Clinical Science, Intervention and Technology, Section for Obstetrics and Gynaecology and Fertility Unit, Karolinska University Hospital, Stockholm, Sweden.
authors.forename: K A
authors.initials: KA
authors.lastname: Rodriguez-Wallberg
authors.affiliation: Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm 171 76, Sweden Centre for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: E
authors.initials: E
authors.lastname: Iwarsson
authors.affiliation: Centre for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden Department of Molecular Medicine and Surgery, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden Centre for Inherited Metabolic Diseases (CMMS), Karolinska University Hospital, Stockholm 171 76, Sweden.
authors.forename: A
authors.initials: A
authors.lastname: Wedell
authors.affiliation: Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm 171 76, Sweden Centre for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden Centre for Inherited Metabolic Diseases (CMMS), Karolinska University Hospital, Stockholm 171 76, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Barbaro
mesh_terms: D000293:Adolescent; D000328:Adult; D016022:Case-Control Studies; D002648:Child; D015331:Cohort Studies; D055028:Comparative Genomic Hybridization / Q000379:methods; D056915:DNA Copy Number Variations; D005260:Female; D020440:Gene Duplication; D055429:Growth Differentiation Factor 9 / Q000235:genetics*; D006801:Humans; D016649:Primary Ovarian Insufficiency / Q000235:genetics*
chemical_list: C525897:GDF9 protein, human; D055429:Growth Differentiation Factor 9
keywords: DNAH6; GDF9; TSPYL6; ZFR2; array-CGH