The anterior pituitary gland is comprised of specialized cell-types that produce and secrete polypeptide hormones in response to hypothalamic input and feedback from target organs. These specialized ...cells arise during embryonic development, from stem cells that express SOX2 and the pituitary transcription factor PROP1, which is necessary to establish the stem cell pool and promote an epithelial to mesenchymal-like transition, releasing progenitors from the niche. Human and mouse embryonic stem cells can differentiate into all major hormone-producing cell types of the anterior lobe in a highly plastic and dynamic manner. More recently human induced pluripotent stem cells (iPSCs) emerged as a viable alternative due to their plasticity and high proliferative capacity. This mini-review gives an overview of the major advances that have been achieved to develop protocols to generate pituitary hormone-producing cell types from stem cells and how these mechanisms are regulated. We also discuss their application in pituitary diseases, such as pituitary hormone deficiencies.
Prokineticin receptor 2 (
) encodes for a G-protein-coupled receptor that can bind PROK1 and PROK2. Mice lacking
have been shown to present abnormal olfactory bulb formation as well as defects in ...GnRH neuron migration. Patients carrying mutations in
typically present hypogonadotropic hypogonadism, anosmia/hyposmia or Kallmann Syndrome. More recently variants in
have been linked to several other endocrine disorders. In particular, several patients with pituitary disorders have been reported, ranging from mild phenotypes, such as isolated growth hormone deficiency, to more severe ones, such as septo-optic dysplasia. Here we summarize the changing landscape of
-related disease, the variants reported to date, and discuss their origin, classification and functional assessment.
Congenital hypopituitarism (CH) and its associated syndromes, septo-optic dysplasia (SOD) and holoprosencephaly (HPE), are midline defects that cause significant morbidity for affected people. ...Variants in 67 genes are associated with CH, but a vast majority of CH cases lack a genetic diagnosis. Whole exome and whole genome sequencing of CH patients identifies sequence variants in genes known to cause CH, and in new candidate genes, but many of these are variants of uncertain significance (VUS).
The International Mouse Phenotyping Consortium (IMPC) is an effort to establish gene function by knocking-out all genes in the mouse genome and generating corresponding phenotype data. We used mouse embryonic imaging data generated by the Deciphering Mechanisms of Developmental Disorders (DMDD) project to screen 209 embryonic lethal and sub-viable knockout mouse lines for pituitary malformations.
Of the 209 knockout mouse lines, we identified 51 that have embryonic pituitary malformations. These genes not only represent new candidates for CH, but also reveal new molecular pathways not previously associated with pituitary organogenesis. We used this list of candidate genes to mine whole exome sequencing data of a cohort of patients with CH, and we identified variants in two unrelated cases for two genes, MORC2 and SETD5, with CH and other syndromic features.
The screening and analysis of IMPC phenotyping data provide proof-of-principle that recessive lethal mouse mutants generated by the knockout mouse project are an excellent source of candidate genes for congenital hypopituitarism in children.
Pituitary hormone deficiency occurs in ∼1:4,000 live births. Approximately 3% of the cases are due to mutations in the alpha isoform of POU1F1, a pituitary-specific transcriptional activator. We ...found four separate heterozygous missense variants in unrelated individuals with hypopituitarism that were predicted to affect a minor isoform, POU1F1 beta, which can act as a transcriptional repressor. These variants retain repressor activity, but they shift splicing to favor the expression of the beta isoform, resulting in dominant-negative loss of function. Using a high-throughput splicing reporter assay, we tested 1,070 single-nucleotide variants in POU1F1. We identified 96 splice-disruptive variants, including 14 synonymous variants. In separate cohorts, we found two additional synonymous variants nominated by this screen that co-segregate with hypopituitarism. This study underlines the importance of evaluating the impact of variants on splicing and provides a catalog for interpretation of variants of unknown significance in POU1F1.
Oral-facial-digital (OFD) syndromes are genetically heterogeneous developmental disorders, caused by pathogenic variants in genes involved in primary cilia formation and function. We identified a ...previously undescribed type of OFD with brain anomalies, ranging from alobar holoprosencephaly to pituitary anomalies, in 6 unrelated families.
Exome sequencing of affected probands was supplemented with alternative splicing analysis in patient and control lymphoblastoid and fibroblast cell lines, and primary cilia structure analysis in patient fibroblasts.
In 1 family with 2 affected males, we identified a germline variant in the last exon of ZRSR2, NM_005089.4:c.1211_1212del NP_005080.1:p.(Gly404GlufsTer23), whereas 7 affected males from 5 unrelated families were hemizygous for the ZRSR2 variant NM_005089.4:c.1207_1208del NP_005080.1:p.(Arg403GlyfsTer24), either occurring de novo or inherited in an X-linked recessive pattern. ZRSR2, located on chromosome Xp22.2, encodes a splicing factor of the minor spliceosome complex, which recognizes minor introns, representing 0.35% of human introns. Patient samples showed significant enrichment of minor intron retention. Among differentially spliced targets are ciliopathy-related genes, such as TMEM107 and CIBAR1. Primary fibroblasts containing the NM_005089.4:c.1207_1208del ZRSR2 variant had abnormally elongated cilia, confirming an association between defective U12-type intron splicing, OFD and abnormal primary cilia formation.
We introduce a novel type of OFD associated with elongated cilia and differential splicing of minor intron-containing genes due to germline variation in ZRSR2.
The pituitary gland is key for childhood growth, puberty, and metabolism. Pituitary dysfunction is associated with a spectrum of phenotypes, from mild to severe. Congenital Hypopituitarism (CH) is ...the most commonly reported pediatric endocrine dysfunction with an incidence of 1:4000, yet low rates of genetic diagnosis have been reported.
We aimed to unveil the genetic etiology of CH in a large cohort of patients from Argentina.
We performed whole exome sequencing of 137 unrelated cases of CH, the largest cohort examined with this method to date.
Of the 137 cases, 19.1% and 16% carried pathogenic or likely pathogenic variants in known and new genes, respectively, while 28.2% carried variants of uncertain significance. This high yield was achieved through the integration of broad gene panels (genes described in animal models and/or other disorders), an unbiased candidate gene screen with a new bioinformatics pipeline (including genes high loss of function intolerance), and analysis of copy number variants. Three novel findings emerged. First, the most prevalent affected gene encodes the cell adhesion factor ROBO1. Affected children had a spectrum of phenotypes, consistent with a role beyond pituitary stalk interruption syndrome. Second, we found that CHD7 mutations also produce a phenotypic spectrum, not always associated with full CHARGE syndrome. Third, we add new evidence of pathogenicity in the genes PIBF1 and TBC1D32, and report 13 novel candidate genes associated with CH (e.g. PTPN6, ARID5B).
Overall, these results provide an unprecedented insight into the diverse genetic etiology of hypopituitarism.
Abstract
Hypopituitarism with deficiency of one or more pituitary hormones (combined pituitary hormone deficiency or CPHD) can vary in severity and age at presentation. Additionally, the hormone ...abnormalities may evolve with time necessitating frequent evaluation. These hormonal deficits can also be present as part of a syndrome, with patients showing extrapituitary abnormalities such as eye or forebrain malformations. Over the past decade, there has been an explosion in the knowledge of the genetic cascade that orchestrates hypothalamic and pituitary development. Several transcription factors and signaling molecules are critical for cell differentiation and proliferation at a very early stage of gestation. However, more genes remain to be identified in order to provide patients with a definitive aetiology. We studied 95 children with congenital hypopituitarism from seven pediatric hospitals from Argentina. Children with non-endocrine, non-familial idiopathic short stature served as a control group (n=100). A custom exon capture panel based on single-molecule molecular inversion probes sequencing (MIPS) was performed in probands and parents. This panel tests 104 genes: including a selection of reported and candidate genes chosen from our knowledge of pituitary development in mice and PROP1 interacting proteins. We found at least 1 variant in 50 probands. The prevalence of known variants in the pituitary transcription factor genes PROP1 and PIT1 (frequently mutated in CPHD) was low in our cohort. A significant number of disease-causing variants were found in LHX3, LHX4, GLI2 and OTX2. An important advance of our study is the identification of pathogenic variants in recently discovered and novel genes. We found an heterozygous variant in the Roundabout (ROBO) receptor homolog 1 gene: ROBO1;p.Arg1504* in a patient with CPHD and septo-optic dysplasia (SOD); two variants in Steroid Receptor SRA1: p. Ile179Thr and p.Val110delinsArgLeu in a patient with CPHD and hypogonadism hypogonadotropic and a missense variant in Semaphorin 3A SEMA3A: p.Val588Leu in a case of CPHD and aortic coarctation. Our findings support evidence that the etiology of congenital hypopituitarism is highly heterogeneous and may be infrequently monogenic with full penetrance, underlying a more complex pathogenesis.
Presentation: Sunday, June 12, 2022 12:36 p.m. - 12:41 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.
Abstract
Disclosure: J. Martinez Mayer: None. M. Hackbartt: None. L. Iglesias Garcia: None. S. Vishnopolska: None. J. Zaiat: None. C. Perticarari: None. D.G. Braslavsky: None. A.C. Keselman: None. I. ...Bergada: None. R. Marino: None. P. Ramirez: None. N. Perez Garrido: None. M. Ciaccio: None. M.I. Di Palma: None. A. Belgorosky: None. V. Forclaz: None. M.G. Benzrihen: None. S. D´Amato: None. G.P. Rojas: None. M. Miras: None. P. Alejandra: None. L. Castro: None. G.F. Alonso: None. S. Mallea Gil: None. C. Ballarino: None. L.V. Latorre Villacorta: None. V. Figueroa: None. A. Morin: None. Z. Guntsche: None. H. Lee: None. E. Lee: None. Y. Song: None. S. Hyun: None. M. Marti: None. M.I. Perez-Millan: None.
Hypopituitarism is the decreased (hypo) secretion of one or more of the eight hormones normally produced by the pituitary gland. When there is decreased secretion of one specific pituitary hormone, the condition is known as isolated hormone deficiency; when more than one hormone is affected it is called Combined pituitary hormone deficiency (CPHD). Congenital hypopituitarism is a rare disorder, with a frequency of 1 case in 4000 live births. More than 30 genes were linked to this disorder, most of them transcription factors expressed during the development of the head, hypothalamus and pituitary. However, the etiology of around 85% of the cases remains unknown. The aim of this study was to unveil the genetic etiology in a large cohort of patients with congenital hypopituitarism from Argentina, employing whole exome sequencing (WES). We sequenced 148 patients presenting hypopituitarism with and without other abnormalities. We found pathogenic variants in known genes (LHX4, GLI2, GH1 and PROP1) or in or less frequently affected genes in CPHD (FOXA2, ROBO1) or in novel genes (RPL5, CHD7, COL1A1, KDM6A, SOX2). Interestingly, frameshift variants in CHD7 found in this study were identified in 2 independent cases with CPHD (none of them met the full criteria for CHARGE syndrome), suggesting a new spectrum of phenotypes for this gene. Variants causing alternative-splicing defects were identified in 3 independent cases: GH1: c.291+1G>C was found in one IGHD case and RPL5: c.189+1G>C in a patient with IGHD combined with erythroblastopenia, megalocornea, palate fistula, chronic diarrhea and microcephaly. Lastly, a splicing variant PROP1: c.343-1G>A was found in compound heterozygous together with PROP1; p.Arg16* in a CPHD patient. Another interesting finding was the identification of microdeletions in 4 cases of CPHD, encompassing between 17 and 64 deleted genes, among them genes related to CPHD such as IGF1R, SOX2, LHX4 and TGIF1. Our results highlight the importance of an exhaustive analysis of candidate variants, which contemplates splicing variants as well as copy number variation (CNVs). Finally, these findings further support the hypothesis that CPHD is highly heterogeneous and may be frequently oligogenic, underlying a very complex pathogenesis for genetic diagnosis.
Presentation: Thursday, June 15, 2023
Abstract
Disclosure: M.L. Brinkmeier: None. J.J. Martínez Mayer: None. B.S. Ellsworth: None. L.T. Raetzman: None. L. Cheung: None. M.I. Perez-Millan: None. S.A. Camper: None. S.W. Davis: None.
...Congenital hypopituitarism (CH) and its associated syndromes, septo-optic dysplasia and holoprosencephaly, are midline defects that cause significant morbidity for affected people. Variants in 44 genes are associated with CH, but most cases of CH are caused by unknown variants. Whole exome (WES) and whole genome sequencing (WGS) of CH patient DNA identifies variants in genes known to cause CH and variants of uncertain significance (VUS) in genes that are not currently associated with CH. The functional significance of VUS in CH candidate genes can be established using mouse models. Many of the human CH genes were first discovered in mice, and in most cases the mouse phenotype was an excellent predictor of the human phenotype. The International Mouse Phenotyping Consortium (IMPC) is an invaluable resource of phenotyping data generated by the international effort to determine function of all protein coding genes in the mouse genome through targeted disruption. Using the mouse phenotyping data generated by the Deciphering Mechanisms of Developmental Disorders (DMDD) project to characterize targeted alleles that cause embryonic lethality or subviability, we have identified 51 genes that cause embryonic pituitary malformations. The malformations range in severity and include absent adenohypophysis, absent neurohypophysis, hypoplasia, and hyperplasia. Using the GenePaint RNA in situ hybridization data for mouse embryos and single cell RNA sequencing (scRNAseq) data from e14.5 dissected pituitary glands, we confirmed expression for all candidate genes in the tissues that contribute to the developing pituitary gland. Using gene ontology (GO) terms associated with these 51 genes, we identified molecular pathways not previously associated with pituitary gland organogenesis, including cilia formation, amino acid metabolism, and epigenetic regulation. We present here the candidate gene list, scRNAseq expression data, GO term analysis, select images from GenePaint, and select images of the observed malformations. These genes associated with mouse pituitary malformations significantly expand the list of candidate genes for screening in WES and WGS in human cases of CH.
Presentation: Thursday, June 15, 2023
The first stable version of the Proteomics Standards Initiative mzIdentML open data standard (version 1.1) was published in 2012—capturing the outputs of peptide and protein identification software. ...In the intervening years, the standard has become well-supported in both commercial and open software, as well as a submission and download format for public repositories. Here we report a new release of mzIdentML (version 1.2) that is required to keep pace with emerging practice in proteome informatics. New features have been added to support: (1) scores associated with localization of modifications on peptides; (2) statistics performed at the level of peptides; (3) identification of cross-linked peptides; and (4) support for proteogenomics approaches. In addition, there is now improved support for the encoding of de novo sequencing of peptides, spectral library searches, and protein inference. As a key point, the underlying XML schema has only undergone very minor modifications to simplify as much as possible the transition from version 1.1 to version 1.2 for implementers, but there have been several notable updates to the format specification, implementation guidelines, controlled vocabularies and validation software. mzIdentML 1.2 can be described as backwards compatible, in that reading software designed for mzIdentML 1.1 should function in most cases without adaptation. We anticipate that these developments will provide a continued stable base for software teams working to implement the standard. All the related documentation is accessible at http://www.psidev.info/mzidentml.
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