scholarly journals SIX3 Variant Causes Pituitary Stalk Interruption Syndrome and Combined Pituitary Hormone Deficiency

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A530-A530
Author(s):  
Hironori Bando ◽  
Michelle Brinkmeier ◽  
Peter Gergics ◽  
Qing Fang ◽  
Amanda Helen Mortensen ◽  
...  
Keyword(s):  
Pituitary Gland ◽  
Genetic Interaction ◽  
Pituitary Stalk ◽  
Hormone Deficiency ◽  
Pituitary Hormone ◽  
Beta Catenin ◽  
Loss Of Function ◽  
Rathke’S Pouch ◽  
Pituitary Development ◽  
Rathke's Pouch

Abstract The genetic basis for congenital hypopituitarism and related disorders is beginning to emerge, and over 30 causal genes have been identified. Mutations in some of these genes can also cause holoprosencephaly (HPE) or septo-optic dysplasia. SIX3 is a homeodomain protein expressed in the developing brain, pituitary gland, and eye. Heterozygous mutations in SIX3 cause variable HPE in humans and mice. We identified two children with neonatal GH and TSH deficiency and stalk interruption who were doubly heterozygous for rare, likely deleterious variants in SIX3 and POU1F1. Functional studies demonstrated that both variants are disruptive. We used Six3 and Pou1f1 loss of function mice to assess the genetic interaction between Six3 and Pou1f1. Six3 heterozygotes have variable pituitary gland dysmorphology, while Pou1f1 heterozygotes are normal. A significant portion of the Six3+/-; Pou1f1+/dw doubly heterozygous mice have a more pronounced pituitary phenotype than Six3+/-, supporting the possibility of digenic pituitary disease. To understand the role of SIX3 in pituitary and hypothalamic development, we used Prop1-cre and Nkx2.1-cre to delete Six3. Disruption of Six3 expression in Rathke’s pouch caused poor activation of Lhx3 expression and arrested anterior pituitary development. The Nkx2.1-cre, Six3flox/flox embryos had no evidence of infundibulum evagination and failed to induce FGF and BMP signaling, which normally drive expansion of Rathke’s pouch. By E11.5 cells in Rathke’s pouch underwent apoptosis. The Nkx2.1-cre, Six3flox/flox embryos failed to activate expression of Lhx2 and Tbx3 in the neural ectoderm. These embryos had elevated CCND1, MYCN, and Axin2 expression in the area of the presumptive infundibulum. This indicates that SIX3 is necessary to repress cell proliferation and Wnt/beta-catenin signals to promote formation of the pituitary stalk. Thus, Six3 has essential roles in both the neural and oral ectoderm for hypothalamic and pituitary development, respectively. Heterozygous loss of function variants in SIX3 could be a contributor to multiple pituitary hormone deficiencies in children, especially if there are associated craniofacial abnormalities or PSIS.

scholarly journals SAT-288 Pituitary Developmental Defects Caused by Haploinsufficiency for the Transcription Factor SIX3 Are Worsened by POU1F1 Deficiency

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Michelle Brinkmeier ◽  
Sally Ann Camper
Keyword(s):  
Transcription Factor ◽  
Pituitary Gland ◽  
Clinical Features ◽  
Hormone Deficiency ◽  
Pituitary Hormone ◽  
Loss Of Function ◽  
Pituitary Hormone Deficiency ◽  
Rathke’S Pouch ◽  
Rathke's Pouch ◽  
Vasopressin Neurons

Abstract Advances in genomic technologies are revolutionizing the practice of medicine by delivering molecular diagnoses that can be informative for prognosis and treatment of genetic disorders. Most of the known genetic causes of multiple pituitary hormone deficiency have been investigated as monogenic disorders. It can be challenging to predict clinical features from genetic data, as loss of function mutations in some genes can present with a spectrum of phenotypes ranging from craniofacial abnormalities, intellectual disability, and neurosensory and neuroendocrine defects to pituitary hormone deficiency with no other abnormalities. Although maternal exposures could be contributing factors, the contribution of rare, deleterious variation in other genes is a likely contributor. In humans, loss of function mutations in the transcription factor SIX3 cause variable, autosomal dominant holoprosencephaly with incomplete penetrance, and mouse models recapitulate some of the clinical features. Because Six3 and Pou1f1 gene expression patterns overlap in pituitary development, we hypothesized that doubly heterozygous mice (Six3+/-; Pou1f1+/dw) might have pituitary anomalies not present in singly heterozygous mice. We intercrossed Six3+/- and Pou1f1+/dw mice to produce doubly heterozygous animals. At e11.5, both Six3+/- and Six3+/-; Pou1f1+/dw exhibited abnormal morphology of the developing infundibulum and Rathke’s pouch, although ventral diencephalon expression of Tle4, Fgf10, and Nkx2.1 appeared normal. Both newborn Six3+/- and Six3+/-; Pou1f1+/dw littermates had abnormal pituitary gland morphology that resembled that of Aes-/-. AES is a co-repressor that interacts with SIX3. Specification of vasopressin neurons and anterior lobe hormone cell types appeared normal. Mice of all genotypes were born in expected Mendelian ratios (N=144, p=0.49), and there were no significant differences in body weight at 3 wks. A portion of the Six3+/- and doubly heterozygous mice developed hydrocephalus, exhibited failure to thrive, and died (6-9% of N=82, 85, respectively). At 6 wks, 25% (N=61) of the Six3+/-; Pou1f1+/dw animals exhibited striking pituitary dysmorphology in which the rostral aspect of the pituitary penetrated the palate. This was not observed in single heterozygotes. These results reveal that haploinsufficiency for Six3 affects Rathke’s pouch formation, resulting in pituitary gland dysmorphology in and around the stem cell niche. A significant portion of the Six3+/-; Pou1f1+/dw doubly heterozygous mice have a more pronounced pituitary phenotype than Six3+/-, supporting the possibility of digenic pituitary disease and highlighting phenotypic variability. Genetically engineered mice provide an excellent tool for assessing the possibility of gene-gene interactions that could enhance the severity of hypopituitarism and associated craniofacial development.

scholarly journals SAT-291 SIX3 Is Essential for Hypothalamic and Pituitary Development

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Hironori Bando ◽  
Michelle L Brinkmeier ◽  
Frederic Castinetti ◽  
Peter Gergics ◽  
Amanda H Mortensen ◽  
...  
Keyword(s):  
Bmp Signaling ◽  
Pituitary Hormone ◽  
Homeodomain Protein ◽  
Loss Of Function ◽  
Shh Signaling ◽  
Rathke’S Pouch ◽  
Pituitary Development ◽  
Rathke's Pouch ◽  
Oral Ectoderm ◽  
Congenital Hypopituitarism

Abstract The genetic basis for congenital hypopituitarism and related disorders is beginning to emerge, and over causal 30 genes have been identified, including six in the SHH signaling pathway. Mutations in some of these genes can also cause holoprosencephaly (HPE) or septo-optic dysplasia. SIX3 is a homeodomain protein expressed in the developing brain, pituitary gland, and eye. It activates SHH signaling and represses BMP signaling. Heterozygous mutations in SIX3 cause variable HPE in humans and mice. We identified a rare, heterozygous variant in SIX3 in two children with neonatal GH and TSH deficiency and stalk interruption, p.P74R. Using transient transfection in 3T3 cells, we demonstrated that the variant reduced the ability of SIX3 to transactivate the SHH enhancer and promoter of FOXG1, suggesting that the variant could be deleterious. To understand the role of SIX3 in hypothalamic and pituitary development we used Nkx2.1-cre and Prop1-cre to delete Six3 in mice. The Nkx2.1-cre, Six3flox/flox embryos had no evidence of infundibulum evagination or expression of Fgf10 or Tcf7l2 at e11.5. The oral ectoderm invaginated in mutants, but no definitive Rathke’s pouch formed. There was no evidence of Lhx3 expression and only trace amounts of Pitx1, indicating that pituitary induction failed due to the lack of Six3 in the developing hypothalamus. Similarly, disruption of Six3 expression in Rathke’s pouch using Prop1-cre ablated pituitary development. Together, these data reveal essential roles of Six3 in both the neural and oral ectoderm for hypothalamic and pituitary development, respectively. Heterozygous loss of function variants in SIX3 could be a contributor to multiple pituitary hormone deficiencies in children, especially if there are associated craniofacial abnormalities.

scholarly journals SAT-067 Maternal Transmission of Pituitary Stalk Interruption Syndrome(PSIS)

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Snigdha Reddy Likki ◽  
Holley F Allen ◽  
Chelsea Gordner
Keyword(s):  
Pituitary Gland ◽  
Adrenal Insufficiency ◽  
Anterior Pituitary ◽  
Hormone Replacement ◽  
Growth Failure ◽  
Pituitary Stalk ◽  
Hormone Deficiency ◽  
Pituitary Hormone ◽  
Z Score ◽  
Central Hypothyroidism

Abstract BACKGROUND: Pituitary stalk interruption syndrome (PSIS) is a rare entity characterized by thin or absent pituitary stalk, hypoplastic/aplastic anterior pituitary and ectopic posterior pituitary (EPP) on magnetic resonance imaging (MRI). PSIS can be associated with variable degrees of pituitary insufficiency 1. Most cases of combined pituitary hormone deficiency are sporadic, however in familial cases, there can be AD or AR inheritance with more than 30 genes identified in association with combined pituitary hormone deficiency (CPHD). We describe how diagnosis of 2 children with PSIS led to the discovery of the condition in their mother. Clinical Case: Child 1 presented at age 3yrs with growth failure in 2003 with ht z score -4.24 SD. Subsequent work up revealed low IGF-1 (< 25 ng /mL) and MRI showed EPP, small anterior pituitary gland and absent pituitary stalk. No GH stim test was performed. He was started on GH supplementation and later was diagnosed with central hypothyroidism, central adrenal insufficiency and hypogonadotropic hypogonadism and is doing well on multiple hormone replacement at age 19 yrs. Child 2, a half-brother to child 1 (same mother), presented at age 1yr with growth failure in 2017 with ht z score -2.06. GH stimulation test with glucagon was abnormal and resulted in a very low GH response (peak GH 0.52 ng/mL). MRI showed EPP with small anterior pituitary gland and interruption of the stalk. Later he was found to have central hypothyroidism and mild central adrenal insufficiency. He is receiving standard hormone replacement at 3 yrs of age. Mother of above 2 patients presented 6 mos postpartum in 2017 after her 7th and last pregnancy with fatigue and amenorrhea. Laboratory evaluation revealed central hypothyroidism (FT4 0.76 ng/dL) and she was prescribed levothyroxine followed by resumption of her menses. She was unable to breastfeed her children due to lack of supply. There were no concerns for DI, amenorrhea or infertility. She was referred to Endocrinology in 2019 for persistent fatigue with a question of GH deficiency. IGF-1 level was normal 114 ng/mL(z score -0.39) and GH stimulation test (clonidine + glucagon) was abnormal with peak GH 1.85 ng/ml. MRI showed EPP with hypoplastic pituitary stalk. Genetic testing was done for CPHD Sequencing Panel at Prevention Genetics which includes GL12, HESX1, LHX3, LHX4, OTX2, POU1F1, PROP1F1, PROP1, SOX2, SOX3 genes and results were negative. She has 4 other children (21, 12, 11, 10yrs) who are currently being investigated for hormone deficiencies. One child died at 3 months of age due to SIDS. Conclusion: We present 3 family members with PSIS. This family highlights the variable clinical phenotype of PSIS and importance of careful family history when evaluating children with congenital pituitary abnormalities and supports the need for more extensive gene panels for evaluation of CPHD. Reference:. Acta Endocrinologica, 2017. 13(1):96–105

scholarly journals SUN-287 A Case of Ectopic Neurohypophysis

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
kehinde Matilda folawewo ◽  
MaKenzie Hodge ◽  
Nada Osman ◽  
Anteneh Woldetensay Zenebe ◽  
Vijaya A Ganta ◽  
...  
Keyword(s):  
Pituitary Gland ◽  
Pituitary Stalk ◽  
Hormone Deficiency ◽  
Breast Development ◽  
Pituitary Hormone ◽  
Thyroid Function Tests ◽  
Mineral Density ◽  
Dxa Scan ◽  
Ectopic Neurohypophysis

Abstract Pituitary stalk interruption syndrome (PSIS) is a congenital disorder of the pituitary gland. Symptoms at presentation may vary widely as this disease presents along a spectrum which includes; ectopic posterior pituitary, interrupted pituitary stalk or aplasia and hypoplasia of the pituitary gland. It is a heterogeneous disorder in terms of radiologic and clinical presentation. It can present clinically as an isolated pituitary hormone deficiency (most common being growth hormone deficiency) or as multihormonal deficiencies. CASE PRESENTATION Patient is a 34-year-old woman with history of primary amenorrhea who was evaluated by a gynecologist and was prescribed oral contraceptive pills which lead to her having a menstrual bleed for the first time in her life. She denied any difficulty with smell. She had undergone normal psychomotor milestones and highest level of education was high school. She had normal puberty with normal pubic and axillary hair growth, normal breast development but no menarche. Of note, patient has a short stature, height is 4 feet and 11 inches, and her biological parents are of normal adult height On evaluation, patient had normal am cortisol, prolactin and thyroid function tests. IGF-1 was significantly low for her age, FSH and LH were inappropriately low for her low estradiol level suggesting hypogonadotropic hypogonadism. Patient subsequently had an MRI of the pituitary and DXA scan. MRI findings were suggestive of ectopic neurohypophysis. DXA scan showed significant reduction in bone mineral density for age. Patient is currently being treated with hormonal replacement which is the main modality of treatment for ectopic neurohyphysis. She will need long term follow up as disease progression to pan-hypopituitarism is common. CONCLUSION PSIS is a rare syndrome with different phenotypic presentation depending on when the diagnosis is made; therefore, adequate follow up is indicated as the disease can progress from a single hormonal deficiency to pan-hypopituitarism.

A mutation of the β-domain in POU1F1 causes pituitary deficiency due to dominant PIT-1β expression

2021 ◽  
Author(s):  
Shigeru Suzuki ◽  
Kumihiro Matsuo ◽  
Yoshiya Ito ◽  
Atsushi Kobayashi ◽  
Takahide Kokumai ◽  
...  
Keyword(s):  
Hormone Deficiency ◽  
Luciferase Reporter ◽  
Heterozygous Mutation ◽  
Pituitary Hormone ◽  
Pituitary Cells ◽  
Transactivation Domain ◽  
Exon 2 ◽  
Pituitary Development ◽  
Long Term Follow Up ◽  
Alternatively Spliced

Background: POU1F1 encodes both PIT-1α, which plays pivotal roles in pituitary development and GH, PRL and TSHB expression, and the alternatively spliced isoform PIT-1β, which contains an insertion of 26-amino acids (β-domain) in the transactivation domain of PIT-1α due to the use of an alternative splice acceptor at the end of the first intron. PIT-1β is expressed at much lower levels than PIT-1α and represses endogenous PIT-1α transcriptional activity. Although POU1F1 mutations lead to combined pituitary hormone deficiency (CPHD), no patients with β-domain mutations have been reported. Results: Here, we report that a three-generation family exhibited different degrees of CPHD, including growth hormone deficiency with intrafamilial variability of prolactin/TSH insufficiency and unexpected prolactinoma occurrence. The CPHD was due to a novel POU1F1 heterozygous variant (c.143-69T>G) in intron 1 of PIT-1α (RefSeq number NM_000306) or as c.152T>G (p.Ile51Ser) in exon 2 of PIT-1β (NM_001122757). Gene splicing experiments showed that this mutation yielded the PIT-1β transcript without other transcripts. Lymphocyte PIT-1β mRNA expression was significantly higher in the patients with the heterozygous mutation than a control. A luciferase reporter assay revealed that the PIT-1β-Ile51Ser mutant repressed PIT-1α and abolished transactivation capacity for the rat prolactin promoter in GH3 pituitary cells. Conclusions: We describe, for the first time, that PIT-1β mutation can cause CPHD through a novel genetic mechanism, such as PIT-1β overexpression, and that POU1F1 mutation might be associated with a prolactinoma. Analysis of new patients and long-term follow-up are needed to clarify the characteristics of PIT-1β mutations.

scholarly journals Pituitary Morphology and Function in 43 Children with Central Diabetes Insipidus

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Wendong Liu ◽  
Limin Wang ◽  
Minghua Liu ◽  
Guimei Li
Keyword(s):  
Diabetes Insipidus ◽  
Central Diabetes Insipidus ◽  
Pituitary Function ◽  
Pituitary Stalk ◽  
Hormone Deficiency ◽  
Pituitary Hormone ◽  
Initial Manifestation ◽  
Stalk Diameter ◽  
And Function

Objective. In pediatric central diabetes insipidus (CDI), etiology diagnosis and pituitary function monitoring are usually delayed. This study aimed to illustrate the importance of regular follow-up and pituitary function monitoring in pediatric CDI.Methods. The clinical, hormonal, and neuroradiological characteristics of children with CDI at diagnosis and during 1.5–2-year follow-up were collected and analyzed.Results. The study included 43 CDI patients. The mean interval between initial manifestation and diagnosis was 22.29 ± 3.67 months (range: 2–108 months). The most common complaint was polyuria/polydipsia. Causes included Langerhans cell histiocytosis, germinoma, and craniopharyngioma in 2, 5, and 4 patients; the remaining were idiopathic. No significant changes were found during the 1.5–2 years after CDI diagnosis. Twenty-three of the 43 cases (53.5%) had ≥1 anterior pituitary hormone deficiency. Isolated growth hormone deficiency was the most frequent abnormality (37.5%) and was not associated with pituitary stalk diameter. Multiple pituitary hormone deficiencies were found in 8 cases with pituitary stalk diameter > 4.5 mm.Conclusion. Diagnosis of CDI is usually delayed. CDI with a pituitary stalk diameter > 4.5 mm carries a higher risk of multiple pituitary hormone deficiencies. Long-term MRI and pituitary function follow-ups are necessary for children with idiopathic CDI.

scholarly journals SAT-241 Pituitary Stalk Interruption Syndrome Presenting as Neonatal Hypotonia

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Nordie Anne Bilbao
Keyword(s):  
Pituitary Gland ◽  
Neonatal Period ◽  
Hormone Replacement ◽  
Brain Mri ◽  
Rare Condition ◽  
Pituitary Stalk ◽  
Pituitary Hormone ◽  
Thyroid Function Tests ◽  
Acth Stimulation ◽  
Central Hypothyroidism

Abstract Pituitary stalk interruption syndrome (PSIS) is a rare condition that include congenital anatomic abnormalities of the pituitary gland and hypopituitarism. There is a wide variety of clinical presentation, with the age at presentation encompassing from neonatal period to adulthood and including one or more pituitary hormone deficiencies. In recent literature there is increasing recognition of PSIS presenting in the neonatal period, mostly involving hypoglycemia. Our patient is a full-term male infant who presented in the newborn period with hypotonia and hypothermia. He also had hypoglycemia, which was initially thought to be associated to hyperinsulinism in the context of gestational diabetes. Micropenis was noted on physical exam. As part of the study for hypotonia, serial thyroid function tests were obtained revealing central hypothyroidism. A low dose ACTH stimulation test was performed which revealed adrenal insufficiency. The patient was started on cortisol and thyroid hormone replacement. Brain MRI showed an ectopic neurohypophysis located along the floor of the hypothalamus, a small anterior pituitary gland, and a partially absent infundibulum, findings consistent with pituitary stalk interruption syndrome. The patient received testosterone injections for micropenis and is being followed for development of other pituitary hormone deficiencies. PSIS is a rare congenital condition that is increasingly recognized in neonates manifesting with signs of hypopituitarism.

Lhx4 and Prop1 are required for cell survival and expansion of the pituitary primordia

Development ◽  
2002 ◽  
Vol 129 (18) ◽  
pp. 4229-4239 ◽  
Author(s):  
Lori T. Raetzman ◽  
Robert Ward ◽  
Sally A. Camper
Keyword(s):  
Cell Survival ◽  
Anterior Pituitary ◽  
Molecular Genetic ◽  
Cell Types ◽  
Hormone Deficiency ◽  
Pituitary Hormone ◽  
Pituitary Cells ◽  
Temporal Shift ◽  
Pituitary Development ◽  
Show Evidence

Deficiencies in the homeobox transcription factors LHX4 and PROP1 cause pituitary hormone deficiency in both humans and mice. Lhx4 and Prop1 mutants exhibit severe anterior pituitary hypoplasia resulting from limited differentiation and expansion of most specialized cell types. Little is known about the mechanism through which these genes promote pituitary development. In this study we determined that the hypoplasia in Lhx4 mutants results from increased cell death and that the reduced differentiation is attributable to a temporal shift in Lhx3 activation. In contrast, Prop1 mutants exhibit normal cell proliferation and cell survival but show evidence of defective dorsal-ventral patterning. Molecular genetic analyses reveal that Lhx4 and Prop1 have overlapping functions in early pituitary development. Double mutants exhibit delayed corticotrope specification and complete failure of all other anterior pituitary cell types to differentiate. Thus, Lhx4 and Prop1 have critical, but mechanistically different roles in specification and expansion of specialized anterior pituitary cells.

scholarly journals Evaluation of pituitary imaging in patients with PROP-1 gene mutation

Medicina ◽  
2009 ◽  
Vol 45 (9) ◽  
pp. 693 ◽  
Author(s):  
Natalija Tkačenko ◽  
Danutė Lašienė ◽  
Silvija Jakštienė ◽  
Algidas Basevičius ◽  
Rasa Verkauskienė
Keyword(s):  
Transcription Factor ◽  
Pituitary Gland ◽  
Gene Mutation ◽  
Anterior Pituitary ◽  
Hormone Deficiency ◽  
Pituitary Hormone ◽  
Imaging Studies ◽  
Pituitary Hyperplasia ◽  
Pituitary Hypoplasia ◽  
Genetically Determined

The most common genetically determined cause of multiple pituitary hormone deficiency is PROP-1 gene mutation. PROP-1 is a transcription factor involved in the development of pituitary gland and affects hormonal synthesis of anterior pituitary. The aim of our study was to evaluate radiological aspects of the pituitary region in patients with PROP-1 gene mutation. Pituitary imaging studies were performed in 12 patients with a confirmed PROP-1 gene mutation. Pituitary hyperplasia was found in 5 (42%) and pituitary hypoplasia in 4 (33%) patients. Changes in pituitary size were not associated with the type of PROP-1 gene mutation.

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

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Sebastian A Vishnopolska ◽  
Debora Braslavsky ◽  
Ana Claudia Keselman ◽  
Ignacio Bergada ◽  
Roxana M Marino ◽  
...  
Keyword(s):  
Cell Line ◽  
Target Genes ◽  
Hormone Deficiency ◽  
Pituitary Hormone ◽  
Loss Of Function ◽  
Wild Type ◽  
Gfp Expression ◽  
Shh Signaling ◽  
Pituitary Hormone Deficiency ◽  
Combined Pituitary Hormone Deficiency

Abstract 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.

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