Cargando…

MON-717 Novel GLI2 Mutations Identified in Pediatric Patients with Combined Pituitary Hormone Deficiency: One Gene, Various Genotypes

Combined pituitary hormone deficiency (CPHD) is an important clinical problem caused by mutations in more than 30 different genes. Six genes in the Sonic Hedgehog (SHH) signalling pathway are reported to cause CPHD. SHH signaling is essential to induce pituitary cell identity in cells of Rathke’s po...

Descripción completa

Detalles Bibliográficos
Autores principales: Vishnopolska, Sebastian A, Braslavsky, Debora, Keselman, Ana Claudia, Bergada, Ignacio, Marino, Roxana M, Ramirez, Pablo C, Natalia, Perez Garrido, Ciaccio, Marta, Palma, María I Di, Belgorosky, Alicia, Miras, Mirta, Nicola, Juan P, Mortensen, Amanda Helen, Martí, Marcelo A, Camper, Sally Ann, Kitzman, Jacob, Perez Millán, Maria I
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7208733/
http://dx.doi.org/10.1210/jendso/bvaa046.1778
_version_ 1783530915258957824
author Vishnopolska, Sebastian A
Braslavsky, Debora
Keselman, Ana Claudia
Bergada, Ignacio
Marino, Roxana M
Ramirez, Pablo C
Natalia, Perez Garrido
Ciaccio, Marta
Palma, María I Di
Belgorosky, Alicia
Miras, Mirta
Nicola, Juan P
Mortensen, Amanda Helen
Martí, Marcelo A
Camper, Sally Ann
Kitzman, Jacob
Perez Millán, Maria I
author_facet Vishnopolska, Sebastian A
Braslavsky, Debora
Keselman, Ana Claudia
Bergada, Ignacio
Marino, Roxana M
Ramirez, Pablo C
Natalia, Perez Garrido
Ciaccio, Marta
Palma, María I Di
Belgorosky, Alicia
Miras, Mirta
Nicola, Juan P
Mortensen, Amanda Helen
Martí, Marcelo A
Camper, Sally Ann
Kitzman, Jacob
Perez Millán, Maria I
author_sort Vishnopolska, Sebastian A
collection PubMed
description Combined pituitary hormone deficiency (CPHD) is an important clinical problem caused by mutations in more than 30 different genes. Six genes in the Sonic Hedgehog (SHH) signalling pathway are reported to cause CPHD. SHH signaling is essential to induce pituitary cell identity in cells of Rathke’s pouch by stimulating expression of the transcription factors Lhx3 and Lhx4. In the absence of SHH signaling, a repressive isoform of the transcription factor GLI2 (Gli-Kruppel family member 2) suppresses gene expression. In the presence of SHH signaling, the activating form of GLI2 gains access to the nucleus and induces expression of downstream target genes. Heterozygous GLI2 loss of function mutations are found in patients with holoprosencephaly (HPE), HPE-like phenotypes associated with pituitary anomalies, and combined pituitary hormone deficiency with or without other extra-pituitary findings. We sought to identify the cause of CPHD in 171 unrelated patients diagnosed with or without extra-pituitary manifestations that were recruited from several Argentinean medical centers. We conducted panel sequencing, and identified GLI2 heterozygous variants that were rare and predicted to be deleterious in two unrelated patients, (p.L761P and p.1404Lfs) and a single, heterozygous, rare, likely deleterious GLI2 variant identified by exome sequencing (p.A203T). p.L761P and p.A203T variants were previously reported as candidates for HPE/CPHD, no functional studies were carried out to determine the effect of the variants on the gene function. We performed functional analysis of these variants using a mammalian cell line (NIH/3T3-CG) previously engineered to be a sensor for SHH signaling. It was stably transfected with a reporter gene that expresses GFP in response to GLI2 activation by a SHH agonist. We modified this cell line to assay GLI2 variants. We created a homozygous knock out of both endogenous Gli2 genes using CRISPR-Cas9 editing, and individual cell clones were selected for loss of GFP expression in response to SHH agonist treatment by FACS. We verified that transfecting the knockout cells with wild type Gli2 restored SHH responsive GFP expression. We assayed the ability of three patient GLI2 variants to rescue GFP expression and SHH agonist responsiveness and found that all three failed to fully rescue to wild type levels. This supports the hypothesis that the GLI2 variants in three CPHD patients are likely pathogenic. Thus, we identified three likely pathogenic GLI2 mutations in CPHD patients from Argentina. The variable phenotype of patients with GLI2 mutations worldwide could be caused by variation in other genes, environmental exposures, maternal effects, and/or epigenetic factors.
format Online
Article
Text
id pubmed-7208733
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher Oxford University Press
record_format MEDLINE/PubMed
spelling pubmed-72087332020-05-13 MON-717 Novel GLI2 Mutations Identified in Pediatric Patients with Combined Pituitary Hormone Deficiency: One Gene, Various Genotypes Vishnopolska, Sebastian A Braslavsky, Debora Keselman, Ana Claudia Bergada, Ignacio Marino, Roxana M Ramirez, Pablo C Natalia, Perez Garrido Ciaccio, Marta Palma, María I Di Belgorosky, Alicia Miras, Mirta Nicola, Juan P Mortensen, Amanda Helen Martí, Marcelo A Camper, Sally Ann Kitzman, Jacob Perez Millán, Maria I J Endocr Soc Genetics and Development (including Gene Regulation) Combined pituitary hormone deficiency (CPHD) is an important clinical problem caused by mutations in more than 30 different genes. Six genes in the Sonic Hedgehog (SHH) signalling pathway are reported to cause CPHD. SHH signaling is essential to induce pituitary cell identity in cells of Rathke’s pouch by stimulating expression of the transcription factors Lhx3 and Lhx4. In the absence of SHH signaling, a repressive isoform of the transcription factor GLI2 (Gli-Kruppel family member 2) suppresses gene expression. In the presence of SHH signaling, the activating form of GLI2 gains access to the nucleus and induces expression of downstream target genes. Heterozygous GLI2 loss of function mutations are found in patients with holoprosencephaly (HPE), HPE-like phenotypes associated with pituitary anomalies, and combined pituitary hormone deficiency with or without other extra-pituitary findings. We sought to identify the cause of CPHD in 171 unrelated patients diagnosed with or without extra-pituitary manifestations that were recruited from several Argentinean medical centers. We conducted panel sequencing, and identified GLI2 heterozygous variants that were rare and predicted to be deleterious in two unrelated patients, (p.L761P and p.1404Lfs) and a single, heterozygous, rare, likely deleterious GLI2 variant identified by exome sequencing (p.A203T). p.L761P and p.A203T variants were previously reported as candidates for HPE/CPHD, no functional studies were carried out to determine the effect of the variants on the gene function. We performed functional analysis of these variants using a mammalian cell line (NIH/3T3-CG) previously engineered to be a sensor for SHH signaling. It was stably transfected with a reporter gene that expresses GFP in response to GLI2 activation by a SHH agonist. We modified this cell line to assay GLI2 variants. We created a homozygous knock out of both endogenous Gli2 genes using CRISPR-Cas9 editing, and individual cell clones were selected for loss of GFP expression in response to SHH agonist treatment by FACS. We verified that transfecting the knockout cells with wild type Gli2 restored SHH responsive GFP expression. We assayed the ability of three patient GLI2 variants to rescue GFP expression and SHH agonist responsiveness and found that all three failed to fully rescue to wild type levels. This supports the hypothesis that the GLI2 variants in three CPHD patients are likely pathogenic. Thus, we identified three likely pathogenic GLI2 mutations in CPHD patients from Argentina. The variable phenotype of patients with GLI2 mutations worldwide could be caused by variation in other genes, environmental exposures, maternal effects, and/or epigenetic factors. Oxford University Press 2020-05-08 /pmc/articles/PMC7208733/ http://dx.doi.org/10.1210/jendso/bvaa046.1778 Text en © Endocrine Society 2020. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Genetics and Development (including Gene Regulation)
Vishnopolska, Sebastian A
Braslavsky, Debora
Keselman, Ana Claudia
Bergada, Ignacio
Marino, Roxana M
Ramirez, Pablo C
Natalia, Perez Garrido
Ciaccio, Marta
Palma, María I Di
Belgorosky, Alicia
Miras, Mirta
Nicola, Juan P
Mortensen, Amanda Helen
Martí, Marcelo A
Camper, Sally Ann
Kitzman, Jacob
Perez Millán, Maria I
MON-717 Novel GLI2 Mutations Identified in Pediatric Patients with Combined Pituitary Hormone Deficiency: One Gene, Various Genotypes
title MON-717 Novel GLI2 Mutations Identified in Pediatric Patients with Combined Pituitary Hormone Deficiency: One Gene, Various Genotypes
title_full MON-717 Novel GLI2 Mutations Identified in Pediatric Patients with Combined Pituitary Hormone Deficiency: One Gene, Various Genotypes
title_fullStr MON-717 Novel GLI2 Mutations Identified in Pediatric Patients with Combined Pituitary Hormone Deficiency: One Gene, Various Genotypes
title_full_unstemmed MON-717 Novel GLI2 Mutations Identified in Pediatric Patients with Combined Pituitary Hormone Deficiency: One Gene, Various Genotypes
title_short MON-717 Novel GLI2 Mutations Identified in Pediatric Patients with Combined Pituitary Hormone Deficiency: One Gene, Various Genotypes
title_sort mon-717 novel gli2 mutations identified in pediatric patients with combined pituitary hormone deficiency: one gene, various genotypes
topic Genetics and Development (including Gene Regulation)
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7208733/
http://dx.doi.org/10.1210/jendso/bvaa046.1778
work_keys_str_mv AT vishnopolskasebastiana mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes
AT braslavskydebora mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes
AT keselmananaclaudia mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes
AT bergadaignacio mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes
AT marinoroxanam mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes
AT ramirezpabloc mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes
AT nataliaperezgarrido mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes
AT ciacciomarta mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes
AT palmamariaidi mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes
AT belgoroskyalicia mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes
AT mirasmirta mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes
AT nicolajuanp mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes
AT mortensenamandahelen mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes
AT martimarceloa mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes
AT campersallyann mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes
AT kitzmanjacob mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes
AT perezmillanmariai mon717novelgli2mutationsidentifiedinpediatricpatientswithcombinedpituitaryhormonedeficiencyonegenevariousgenotypes