title: A novel follicle-stimulating hormone receptor mutation causing primary ovarian failure: a fertility application of whole exome sequencing.
abstract: OBJECTIVE
  Can whole exome sequencing (WES) and in vitro validation studies be used to find the causative genetic etiology in a patient with primary ovarian failure and infertility?
  
  
  CONCLUSIONS
  A novel follicle-stimulating hormone receptor (FSHR) mutation was found by WES and shown, via in vitro flow cytometry studies, to affect membrane trafficking.
  
  
  BACKGROUND
  WES may diagnose up to 25-35% of patients with suspected disorders of sex development (DSD). FSHR mutations are an extremely rare cause of 46, XX gonadal dysgenesis with primary amenorrhea due to hypergonadotropic ovarian failure.
  
  
  METHODS
  A WES study was followed by flow cytometry studies of mutant protein function.
  
  
  METHODS
  The study subjects were two Turkish sisters with hypergonadotropic primary amenorrhea, their parents and two unaffected sisters. The affected siblings and both parents were sequenced (trio-WES). Transient transfection of HEK 293T cells was performed with a vector containing wild-type FSHR as well as the novel FSHR variant that was discovered by WES. Cellular localization of FSHR protein as well as FSH-stimulated cyclic AMP (cAMP) production was evaluated using flow cytometry.
  
  
  RESULTS
  Both affected sisters were homozygous for a previously unreported missense mutation (c.1222G>T, p.Asp408Tyr) in the second transmembrane domain of FSHR. Modeling predicted disrupted secondary structure. Flow cytometry demonstrated an average of 48% reduction in cell-surface signal detection (P < 0.01). The mean fluorescent signal for cAMP (second messenger of FSHR), stimulated by FSH, was reduced by 50% in the mutant-transfected cells (P < 0.01).
  
  
  CONCLUSIONS
  This is an in vitro validation. All novel purported genetic variants can be clinically reported only as 'variants of uncertain significance' until more patients with a similar phenotype are discovered with the same variant.
  
  
  CONCLUSIONS
  We report the first WES-discovered FSHR mutation, validated by quantitative flow cytometry. WES is a valuable tool for diagnosis of rare genetic diseases, and flow cytometry allows for quantitative characterization of purported variants. WES-assisted diagnosis allows for treatments aimed at the underlying molecular etiology of disease. Future studies should focus on pharmacological and assisted reproductive treatments aimed at the disrupted FSHR, so that patients with FSH resistance can be treated by personalized medicine.
  
  
  BACKGROUND
  E.V. is partially funded by the DSD Translational Research Network (NICHD 1R01HD068138). M.S.B. is funded by the Neuroendocrinology, Sex Differences and Reproduction training grant (NICHD 5T32HD007228). The authors have no competing interests to disclose.
authors.affiliation: Department of Human Genetics, David Geffen School of Medicine at the University of California Los Angeles, 695 Charles E Young Drive South, Los Angeles, CA 90095, USA.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Bramble
authors.affiliation: Department of Human Genetics, David Geffen School of Medicine at the University of California Los Angeles, 695 Charles E Young Drive South, Los Angeles, CA 90095, USA Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of California Los Angeles, 10833 Le Conte Avenue, Room 24-130 CHS, Los Angeles, CA 90095, USA.
authors.forename: Ellen H
authors.initials: EH
authors.lastname: Goldstein
authors.affiliation: Department of Human Genetics, David Geffen School of Medicine at the University of California Los Angeles, 695 Charles E Young Drive South, Los Angeles, CA 90095, USA.
authors.forename: Allen
authors.initials: A
authors.lastname: Lipson
authors.affiliation: Department of Human Genetics, David Geffen School of Medicine at the University of California Los Angeles, 695 Charles E Young Drive South, Los Angeles, CA 90095, USA.
authors.forename: Tuck
authors.initials: T
authors.lastname: Ngun
authors.affiliation: Department of Human Genetics, David Geffen School of Medicine at the University of California Los Angeles, 695 Charles E Young Drive South, Los Angeles, CA 90095, USA.
authors.forename: Ascia
authors.initials: A
authors.lastname: Eskin
authors.affiliation: Department of Human Genetics, David Geffen School of Medicine at the University of California Los Angeles, 695 Charles E Young Drive South, Los Angeles, CA 90095, USA.
authors.forename: Jason E
authors.initials: JE
authors.lastname: Gosschalk
authors.affiliation: Department of Human Genetics, David Geffen School of Medicine at the University of California Los Angeles, 695 Charles E Young Drive South, Los Angeles, CA 90095, USA.
authors.forename: Lara
authors.initials: L
authors.lastname: Roach
authors.affiliation: Department of Human Genetics, David Geffen School of Medicine at the University of California Los Angeles, 695 Charles E Young Drive South, Los Angeles, CA 90095, USA.
authors.forename: Neerja
authors.initials: N
authors.lastname: Vashist
authors.affiliation: Department of Human Genetics, David Geffen School of Medicine at the University of California Los Angeles, 695 Charles E Young Drive South, Los Angeles, CA 90095, USA.
authors.forename: Hayk
authors.initials: H
authors.lastname: Barseghyan
authors.affiliation: Department of Human Genetics, David Geffen School of Medicine at the University of California Los Angeles, 695 Charles E Young Drive South, Los Angeles, CA 90095, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Lee
authors.affiliation: Department of Human Genetics, David Geffen School of Medicine at the University of California Los Angeles, 695 Charles E Young Drive South, Los Angeles, CA 90095, USA.
authors.forename: Valerie A
authors.initials: VA
authors.lastname: Arboleda
authors.affiliation: Department of Development, Reproduction, and Cancer, Institut Cochin, U1016 Inserm, University Sorbonne Paris, CNRS UMR8104, Paris, France.
authors.forename: Daniel
authors.initials: D
authors.lastname: Vaiman
authors.affiliation: Department of Medical Genetics, Women and Children Hospital, Halkkent Mh. Fatih Sultan Mehmet Boulevard No. 23, Mersin 33240, Turkey.
authors.forename: Zafer
authors.initials: Z
authors.lastname: Yuksel
authors.affiliation: Department of Development, Reproduction, and Cancer, Institut Cochin, U1016 Inserm, University Sorbonne Paris, CNRS UMR8104, Paris, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Fellous
authors.affiliation: Department of Human Genetics, David Geffen School of Medicine at the University of California Los Angeles, 695 Charles E Young Drive South, Los Angeles, CA 90095, USA evilain@ucla.edu.
authors.forename: Eric
authors.initials: E
authors.lastname: Vilain
mesh_terms: D000328:Adult; D003241:Consanguinity; D059472:Exome; D005260:Female; D055106:Genome-Wide Association Study; D057809:HEK293 Cells; D006720:Homozygote; D006801:Humans; D008958:Models, Molecular; D020125:Mutation, Missense; D016649:Primary Ovarian Insufficiency / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D021381:Protein Transport; D011962:Receptors, FSH / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D035781:Siblings; D014421:Turkey; D055815:Young Adult
chemical_list: D011962:Receptors, FSH; D011994:Recombinant Proteins
keywords: follicle-stimulating hormone receptor; premature ovarian failure; primary amenorrhea; resistant ovary syndrome; whole exome sequencing