title: Identifying the Deleterious Effect of Rare LHX4 Allelic Variants, a Challenging Issue.
abstract: LHX4 is a LIM homeodomain transcription factor involved in the early steps of pituitary ontogenesis. To date, 8 heterozygous LHX4 mutations have been reported as responsible of combined pituitary hormone deficiency (CPHD) in Humans. We identified 4 new LHX4 heterozygous allelic variants in patients with congenital hypopituitarism: W204X, delK242, N271S and Q346R. Our objective was to determine the role of LHX4 variants in patients' phenotypes. Heterologous HEK293T cells were transfected with plasmids encoding for wild-type or mutant LHX4. Protein expression was analysed by Western Blot, and DNA binding by electro-mobility shift assay experiments. Target promoters of LHX4 were cotransfected with wild type or mutant LHX4 to test the transactivating abilities of each variant. Our results show that the W204X mutation was associated with early GH and TSH deficiencies and later onset ACTH deficiency. It led to a truncated protein unable to bind to alpha-Gsu promoter binding consensus sequence. W204X was not able to activate target promoters in vitro. Cotransfection experiments did not favour a dominant negative effect. In contrast, all other mutants were able to bind the promoters and led to an activation similar as that observed with wild type LHX4, suggesting that they were likely polymorphisms. To conclude, our study underlines the need for functional in vitro studies to ascertain the role of rare allelic variants of LHX4 in disease phenotypes. It supports the causative role of the W204X mutation in CPHD and adds up childhood onset ACTH deficiency to the clinical spectrum of the various phenotypes related to LHX4 mutations.
authors.affiliation: Aix Marseille University, CNRS UMR7286, CRN2M, Faculté de médecine, Marseille, France and Reference Center for Rare Pituitary Diseases DEFHY, La Timone Hospital, Marseille, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Rochette
authors.affiliation: Aix Marseille University, CNRS UMR7286, CRN2M, Faculté de médecine, Marseille, France and Reference Center for Rare Pituitary Diseases DEFHY, La Timone Hospital, Marseille, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Jullien
authors.affiliation: Aix Marseille University, CNRS UMR7286, CRN2M, Faculté de médecine, Marseille, France and Reference Center for Rare Pituitary Diseases DEFHY, La Timone Hospital, Marseille, France; Laboratory of Molecular Endocrinology, La Conception Hospital, Assistance Publique Hôpitaux de Marseille, Marseille, France.
authors.forename: Alexandru
authors.initials: A
authors.lastname: Saveanu
authors.affiliation: CHU Nantes, Department of pediatric endocrinology, Nantes, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Caldagues
authors.affiliation: Centro de Investigaciones Endocrinologicas (CEDIE) « Dr. César Bergada » Division de Endocrinologia, Hospital de Ninos Ricardo Guttierrez, Buenos Aires, Argentina.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Bergada
authors.affiliation: Centro de Investigaciones Endocrinologicas (CEDIE) « Dr. César Bergada » Division de Endocrinologia, Hospital de Ninos Ricardo Guttierrez, Buenos Aires, Argentina.
authors.forename: Debora
authors.initials: D
authors.lastname: Braslavsky
authors.affiliation: Univ med center Ljubjana, Department Endocrinology, Zaloska 7, Ljubjana, Slovenia.
authors.forename: Marija
authors.initials: M
authors.lastname: Pfeifer
authors.affiliation: Aix Marseille University, CNRS UMR7286, CRN2M, Faculté de médecine, Marseille, France and Reference Center for Rare Pituitary Diseases DEFHY, La Timone Hospital, Marseille, France.
authors.forename: Rachel
authors.initials: R
authors.lastname: Reynaud
authors.affiliation: Aix Marseille University, CNRS UMR7286, CRN2M, Faculté de médecine, Marseille, France and Reference Center for Rare Pituitary Diseases DEFHY, La Timone Hospital, Marseille, France.
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Herman
authors.affiliation: Aix Marseille University, CNRS UMR7286, CRN2M, Faculté de médecine, Marseille, France and Reference Center for Rare Pituitary Diseases DEFHY, La Timone Hospital, Marseille, France; Laboratory of Molecular Endocrinology, La Conception Hospital, Assistance Publique Hôpitaux de Marseille, Marseille, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Barlier
authors.affiliation: Aix Marseille University, CNRS UMR7286, CRN2M, Faculté de médecine, Marseille, France and Reference Center for Rare Pituitary Diseases DEFHY, La Timone Hospital, Marseille, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Brue
authors.affiliation: Aix Marseille University, CNRS UMR7286, CRN2M, Faculté de médecine, Marseille, France and Reference Center for Rare Pituitary Diseases DEFHY, La Timone Hospital, Marseille, France; Laboratory of Molecular Endocrinology, La Conception Hospital, Assistance Publique Hôpitaux de Marseille, Marseille, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Enjalbert
authors.affiliation: Aix Marseille University, CNRS UMR7286, CRN2M, Faculté de médecine, Marseille, France and Reference Center for Rare Pituitary Diseases DEFHY, La Timone Hospital, Marseille, France.
authors.forename: Frederic
authors.initials: F
authors.lastname: Castinetti
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000172:deficiency; D000483:Alleles; D002648:Child; D002675:Child, Preschool; D005260:Female; D013006:Growth Hormone / Q000172:deficiency; D057809:HEK293 Cells; D006579:Heterozygote; D006801:Humans; D007018:Hypopituitarism / Q000150:complications / Q000235:genetics*; D007223:Infant; D060850:LIM-Homeodomain Proteins / Q000235:genetics*; D008297:Male; D009154:Mutation; D013972:Thyrotropin / Q000172:deficiency; D014157:Transcription Factors / Q000235:genetics*
chemical_list: C486362:LHX4 protein, human; D060850:LIM-Homeodomain Proteins; D014157:Transcription Factors; D000324:Adrenocorticotropic Hormone; D013972:Thyrotropin; D013006:Growth Hormone