Context Congenital hypopituitarism is a genetically heterogeneous condition. Whole exome sequencing (WES) is a promising approach for molecular diagnosis of patients with this condition. Objectives The aim of this study is to conduct WES in a patient with congenital hypopituitarism born to consanguineous parents, CDH2 screening in a cohort of patients with congenital hypopituitarism, and functional testing of a novel CDH2 variant. Design Genomic DNA from a proband and her consanguineous parents was analyzed by WES. Copy number variants were evaluated. The genetic variants were filtered for population frequency (ExAC, 1000 genomes, gnomAD, and ABraOM), in silico prediction of pathogenicity, and gene expression in the pituitary and/or hypothalamus. Genomic DNA from 145 patients was screened for CDH2 by Sanger sequencing. Results One female patient with deficiencies in growth hormone, thyroid-stimulating hormone, adrenocorticotropic hormone, luteinizing hormone, and follicle-stimulating hormone and ectopic posterior pituitary gland contained a rare homozygous c.865G>A (p.Val289Ile) variant in CDH2. To determine whether the p.Val289Ile variant in CDH2 affects cell adhesion properties, we stably transfected L1 fibroblast lines, labeled the cells with lipophilic dyes, and quantified aggregation. Large aggregates formed in cells expressing wildtype CDH2 , but aggregation was impaired in cells transfected with variant CDH2 or non-transfected. Conclusion A homozygous CDH2 allelic variant was found in one hypopituitarism patient, and the variant impaired cell aggregation function in vitro . No disease-causing variants were found in 145 other patients screened for CDH2 variants. Thus, CDH2 is a candidate gene for hypopituitarism that needs to be tested in different populations. Significance statement A female patient with hypopituitarism was born from consanguineous parents and had a homozygous, likely pathogenic, CDH2 variant that impairs cell aggregation in vitro . No other likely pathogenic variants in CDH2 were identified in 145 hypopituitarism patients.

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.

Objective The transcription factor OTX2is implicated in ocular, craniofacial, and pituitary development. Design We aimed to establish the contribution of OTX2 mutations in congenital hypopituitarism patients with/without eye abnormalities, study functional consequences, and establish OTX2 expression in the human brain, with a view to investigate the mechanism of action. Methods We screened patients from the UK (n = 103), international centres (n = 24), and Brazil (n = 282); 145 were within the septo-optic dysplasia spectrum, and 264 had no eye phenotype. Transactivation ability of OTX2 variants was analysed in murine hypothalamic GT1-7 neurons. In situ hybridization was performed on human embryonic brain sections. Genetically engineered mice were generated with a series of C-terminal OTX2 variants. Results Two chromosomal deletions and six haploinsufficient mutations were identified in individuals with eye abnormalities; an affected relative of one patient harboured the same mutation without an ocular phenotype. OTX2 truncations led to significant transactivation reduction. A missense variant was identified in another patient without eye abnormalities; however, studies revealed it was most likely not causative. In the mouse, truncations proximal to aa219 caused anophthalmia, while distal truncations and the missense variant were tolerated. During human embryogenesis, OTX2 was expressed in the posterior pituitary, retina, ear, thalamus, choroid plexus, and partially in the hypothalamus, but not in the anterior pituitary. Conclusions OTX2 mutations are rarely associated with hypopituitarism in isolation without eye abnormalities, and may be variably penetrant, even within the same pedigree. Our data suggest that the endocrine phenotypes in patients with OTX2 mutations are of hypothalamic origin.

The COPII component SEC24 mediates the recruitment of transmembrane cargos or cargo adaptors into newly forming COPII vesicles on the ER membrane. Mammalian genomes encode four Sec24 paralogs (Sec24a-d), with two subfamilies based on sequence homology (SEC24A/B and C/D), though little is known about their comparative functions and cargo-specificities. Complete deficiency for Sec24d results in very early embryonic lethality in mice (before the 8 cell stage), with later embryonic lethality (E7.5) observed in Sec24c null mice. To test the potential overlap in function between SEC24C/D, we employed dual recombinase mediated cassette exchange to generate a Sec24cc-d allele, in which the C-terminal 90% of SEC24C has been replaced by SEC24D coding sequence. In contrast to the embryonic lethality at E7.5 of SEC24C-deficiency, Sec24cc-d/c-d pups survive to term, though dying shortly after birth. Sec24cc-d/c-d pups are smaller in size, but exhibit no other obvious developmental abnormality by pathologic evaluation. These results suggest that tissue-specific and/or stage-specific expression of the Sec24c/d genes rather than differences in cargo export function explain the early embryonic requirements for SEC24C and SEC24D.

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.

Abstract The transcription factor OTX2 is implicated in pituitary, ocular and craniofacial development. Mutations have been described in patients with variable congenital hypopituitarism (CH) ranging from isolated growth hormone deficiency (IGHD) to combined pituitary hormone deficiency (CPHD) with/without an ectopic posterior pituitary (EPP).We aimed (i) to establish the contribution of OTX2 mutations in the etiology of CH in a sub-cohort of patients and to study their functional consequences and (ii) establish a detailed human OTX2 expression profile in a hypothalamo-pituitary (HP) context. We screened 127 patients from national (n=103) and international centers (n=24) on the septo-optic dysplasia (SOD) spectrum with variable eye abnormalities. Eye abnormalities included micro/anophthalmia, retinal dystrophy and/or coloboma in 29 of these patients, with the rest having optic nerve hypoplasia (ONH). An EPP was reported on MRI in 35 patients. The cohort previously tested negative for mutations in HESX1, SOX2, SOX3, PROKR2 and GH1. Transactivation assays involved a dual-luciferase reporter in murine hypothalamic GT1-7 neurons transiently transfected with OTX2 constructs. In situ hybridization was performed to analyze human brain OTX2 expression during embryogenesis. Seven heterozygous OTX2 changes were identified: two chromosomal deletions spanning OTX2 in patients with micro/anophthalmia, GHD and an EPP, with one patient having cerebellar hypoplasia. Three missense substitutions resulting in truncated proteins: (i) the previously reported p.S138* and (ii) p.C170*, in two patients with retinal dystrophy, EPP and IGHD respectively, and (iii) the novel p.E79* in a patient with micro/anophthalmia, EPP and CPHD. A novel insertion-deletion resulting in a truncated protein p.S167* in a patient with microphthalmia, GHD, ONH, EPP and an enlarged abnormal pituitary, and a novel deletion resulting in a frameshift p.Val139Aspfs*39 in a patient with microcephaly, microphthalmia, ONH and an EPP were also identified. The human OTX2 variants caused a significant reduction in transactivation compared to wild type. Our gene expression data identified human OTX2 transcripts in the posterior pituitary, retina, ear, thalamus, choroid plexus, and in the hypothalamus during embryogenesis, but not in RP. To conclude, we identified OTX2 variants in 7 unrelated CH patients with eye abnormalities including 3 with retinal dystrophy and one with a cerebellar abnormality. As OTX2 is involved at multiple levels during HP development, these patients should be monitored for evolving endocrinopathies. Human OTX2 is expressed in the posterior pituitary, the retina and the ear at CS19 and 20 (between 6-7 weeks gestation), and in areas of the hindbrain at CS23, but not in RP at any stage analyzed in this study. The endocrine phenotypes in patients with OTX2 mutations are most likely of hypothalamic origin.

Combined pituitary hormone deficiency (CPHD) is a genetically heterogeneous disorder caused by mutations in over 30 genes. The loss-of-function mutations in many of these genes, including orthodenticle homeobox 2 (OTX2), can present with a broad range of clinical symptoms, which provides a challenge for predicting phenotype from genotype. Another challenge in human genetics is functional evaluation of rare genetic variants that are predicted to be deleterious. Zebrafish are an excellent vertebrate model for evaluating gene function and disease pathogenesis, especially because large numbers of progeny can be obtained, overcoming the challenge of individual variation. To clarify the utility of zebrafish for the analysis of CPHD-related genes, we analyzed the effect of OTX2 loss of function in zebrafish. The otx2b gene is expressed in the developing hypothalamus, and otx2b(hu3625/hu3625) fish exhibit multiple defects in the development of head structures and are not viable past 10 days post fertilization (dpf). Otx2b hu3625/hu3625 fish have a small hypothalamus and low expression of pituitary growth hormone and prolactin (prl). The gills of otx2b(hu3625/hu3625) fish have weak sodium influx, consistent with the role of prolactin in osmoregulation. The otx2b(hu3625/hu3625 )eyes are microphthalmic with colobomas, which may underlie the inability of the mutant fish to find food. The small pituitary and eyes are associated with reduced cell proliferation and increased apoptosis evident at 3 and 5 dpf, respectively. These observations establish the zebrafish as a useful tool for the analysis of CPHD genes with variable and complex phenotypes.

Abstract Background: Growth hormone deficiency (GHD) occurs in ~ 1/8000 individuals, and 14% of the patients have mutations in five major candidate genes. However, over 30 genes have been implicated in hypopituitarism. WES (Whole Exome Sequencing) is a promising approach for molecular diagnosis of patients with GHD because it offers the opportunity to screen for all known genes in addition to novel disease gene discovery. Methods: WES was performed for 13 unrelated patients with congenital hypopituitarism born from consanguineous parents. The variants were filtered assuming autosomal recessive inheritance, rare variants in population databases, in silico analysis predicted as deleterious and pituitary and/or hypothalamus gene expression. To determine whether variants in CDH2 that were predicted to be deleterious were functionally significant, L1 fibroblast lines that have no endogenous CDH2 protein were stably transfected with either human wild type or variant CDH2, the transfected cells were labelled with lipophilic dyes, and cell adhesion properties were assessed. Results: Homozygous pathogenic or likely pathogenic allelic variants were found in 2 of the 13 patients. First, a female patient with GH, TSH, ACTH and LH/ FSH deficiencies presenting ectopic posterior pituitary lobe, non-visualized stalk, and hypoplastic anterior pituitary lobe had two homozygous rare variants predicted as deleterious: PLA2G4A p.Asn703Lys and CDH2 c.865G>A (p.Val289Ile). Only CDH2 is known to be expressed in the pituitary, and Pla2g4a null mice have a pleiotropic phenotype without obvious hypopituitarism. The CDH2 variant is rare and classified as deleterious. Sanger sequencing of CDH2 in four family members of the affected proband revealed that the unaffected parents and two unaffected siblings were heterozygous carriers. The effect of the CDH2 variant on cell aggregation was assessed in cell culture. Large cell aggregates formed in cells transfected with wild type CDH2, but cell aggregates were small or absent in cells that were either non-transfected or transfected with the CDH2 variant. Second, a patient with isolated GHD and no MRI abnormalities was identified with a rare, likely deleterious, homozygous GH1 c.171delT (p. Phe 57Leufs*43) variant. He had a sister who died at the age of 5 and had features of GHD. Conclusion: In a cohort of congenital hypopituitarism from consanguineous parents we had 15% molecular diagnosis using WES. We identified a variant in a known gene, GH1 c.171delT and a variant in a novel gene, CDH2 p.Val289I.

Severe is rare. It may be caused by either benign or malignant neoplasms of the adrenal or ovary. We report a rare case of a post-menopausal woman with hirsutism associated with virilization due to Leydig cell tumors (LCT) of both ovaries. Case report 61 yo female presented for evaluation of hirsutism. She had also been experiencing increased facial hair growth, deepening of voice, clitoromegaly, alo-pecia, and acne. Physical examination: normal vital signs. Patient had signs of virilization, including coarse hair along her upper lip, chin, lower abdomen and inner thigh with Ferriman-Gallwey score of 8, acne, and clitoromegaly. She had no signs of acanthosis nigricans or Cushing syndrome. Base line labs: testosterone testosterone estradiol 77 pg/mL estrone mIU/mL mIU/mL androstenedione Background:Cribriform - morular variant is a rare variant of papillary cancer with 1–2 % prevalence. There are no established guidelines to screen for thyroid cancer in patients with APC gene mutations and FAP. We present a case of a young female

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.

Congenital pituitary hormone deficiency is a disabling condition. It is part of a spectrum of disorders including craniofacial midline developmental defects ranging from holoprosencephaly through septo-optic dysplasia to combined and isolated pituitary hormone deficiency. The first genes discovered in the human disease were based on mouse models of dwarfism due to mutations in transcription factor genes. High-throughput DNA sequencing technologies enabled clinicians and researchers to find novel genetic causes of hypopituitarism for the more than three quarters of patients without a known genetic diagnosis to date. Transcription factor (TF) genes are at the forefront of the functional analysis of novel variants of unknown significance due to the relative ease in in vitro testing in a research lab. Genetic testing in hypopituitarism is of high importance to the individual and their family to predict phenotype composition, disease progression and to avoid life-threatening complications such as secondary adrenal insufficiency.This chapter aims to highlight our current understanding about (1) the contribution of TF genes to pituitary development (2) the diversity of inheritance and phenotype features in combined and select isolated pituitary hormone deficiency and (3) provide an initial assessment on how to approach variants of unknown significance in human hypopituitarism. Our better understanding on how transcription factor gene variants lead to hypopituitarism is a meaningful step to plan advanced therapies to specific genetic changes in the future.

Abstract OTX2 is a homeobox transcription factor important for eye, craniofacial and midline development. Whole gene deletions and heterozygous mutations in OTX2 cause variable anomalies with incomplete penetrance, and most cases present with craniofacial / ocular abnormalities, and hypopituitarism, including either isolated or combined pituitary hormone deficiency. Patients with variants of unknown significance in OTX2 pose a dilemma for medical diagnosis. OTX2 is highly conserved evolutionarily: variable craniofacial defects are observed in heterozygous Otx2 null mice and zebrafish with knockdown of otx2b together with other genes. Thus, mouse and zebrafish offer opportunities for functional analysis of human patient variants. A patient diagnosed with combined pituitary hormone deficiency, including deficiencies in GH, ACTH, FSH, and LH, with no obvious ocular or craniofacial abnormalities, had an intriguing, rare, likely deleterious OTX2H230L/+ variant in a predicted protein-protein interaction domain within the C-terminus. We tested the functional significance of the variant allele. No differences in the transactivation properties were observed in cell culture transfections. To assess function in a more physiological context, we generated an Otx2 allelic series in mice using CRISPR/Cas9. This included nested C-terminal deletions, frameshifts, the OTX2H230L/+ allele and other missense mutations. Surprisingly, all Otx2 variants after codon 220 were tolerated; no gross ocular, craniofacial, or pituitary growth insufficiency phenotypes were noted. In contrast, ocular phenotypes were 100% penetrant in Otx2L219Pfs*17/+ mice, including unilateral or bilateral anophthalmia or microphthalmia. Given the time and cost of generating mouse models of human disease, we explored the utility of zebrafish to assess patient variants in OTX2. Although zebrafish have two otx2 genes, previous studies showed that morpholino knockdown of otx2b caused craniofacial defects that mimic those of human patients if an unrelated gene in the pathway was also knocked down. We sought to characterize the phenotypic consequences of a complete loss of function variant in zebrafish because it could offer a cleaner, more robust system for assessment of human variants than the use of multiple, gene-specific morpholinos. Zebrafish homozygous for an essential splice site mutation, otx2bc.507-2A>C (otx2bhu3625) died at 11 days post fertilization. Preliminary data suggest mutants have reduced growth hormone transcripts at day 5, and assessment of zebrafish mutant vision and eye morphology is underway. The mouse studies suggest that the patient OTX2H230L/+ variant is likely tolerated, as are other variants and deletions C-terminal to position 220 of OTX2. Analysis of zebrafish genetic loss of function mutants suggest they offer an avenue for assessing the functional significance of human OTX2 variants.

The COPII component SEC24 mediates the recruitment of transmembrane cargoes or cargo adaptors into newly forming COPII vesicles on the ER membrane. Mammalian genomes encode four Sec24 paralogs (Sec24a-d), with two subfamilies based on sequence homology (SEC24A/B and C/D), though little is known about their comparative functions and cargo-specificities. Complete deficiency for Sec24d results in very early embryonic lethality in mice (before the 8 cell stage), with later embryonic lethality (E 7.5) observed in Sec24c null mice. To test the potential overlap in function between SEC24C/D, we employed dual recombinase mediated cassette exchange to generate a Sec24cc-d allele, in which the C-terminal 90% of SEC24C has been replaced by SEC24D coding sequence. In contrast to the embryonic lethality at E7.5 of SEC24C deficiency, Sec24cc-d/c-d pups survive to term, though dying shortly after birth. Sec24cc d/c-d pups are smaller in size, but exhibit no obvious developmental abnormality. These results suggest that tissue-specific and/or stage-specific expression of the Sec24c/d genes rather than differences in cargo function explain the early embryonic requirements for SEC24C and SEC24D.

Developmental disorders affecting the hypothalamic-pituitary system can result in pituitary hormone deficiency showing a diverse clinical presentation. A significant majority of these disorders are closely linked to defects in transcription factor genes which play a major role in pituitary development. Those affecting the early phase of organogenesis typically lead to complex conditions affecting the pituitary as well as structures in the central nervous system. Transcription factors involved in the late phase can result in combined but rarely isolated pituitary hormone deficiency without extra-pituitary manifestation. Identifying the defects in these pituitary transcription factor genes may provide a useful tool in predicting disease progression as well as screening family members. Several pituitary transcription factors can be detected in the adult gland as well which is strongly emphasized in the World Health Organization's most recent guideline for pituitary tumor classification. Our review summarizes the current essential knowledge relevant for clinical endocrinologists. Orv Hetil. 2018; 159(7): 278-284.

Absztrakt: A hypothalamohypophysealis rendszer fejlődesi rendellenessegei klinikai megjelenesukben sokszinű hypophysiselegtelenseggel jarhatnak. Ezen fejlődesi rendellenessegek jelentős reszet a hypophysis organogeneziset szabalyozo transzkripcios faktorok genetikai hibai okozhatjak. Az agyalapi mirigy fejlődesenek korai szakaszaban expresszalodo transzkripcios faktorokat kodolo genek mutacioi olyan osszetett fejlődesi rendellenessegekhez vezethetnek, amelyekben a hypopituitarismushoz egyeb kozponti idegrendszeri malformaciok is tarsulnak. Az organogenezis kesőbbi szakaszat szabalyozo transzkripcios faktorok genetikai elteresei jellemzően tobbszoros, ritkan izolalt agyalapi mirigy hormonhianyt okoznak extrahypophysealis manifesztacio nelkul. A hypophysistranszkripcios faktorok genetikai defektusainak azonositasa segitseget adhat a hormonhianyok előrejelzeseben es az erintett csaladtagok szűreseben. Egyes hypophysistranszkripcios faktorok expresszioja felnőttkorban is kimutathato, aminek fontos klinikai jelentősege van a hypophysisadenomak WHO altal ajanlott uj rendszerű, ezen faktorok expressziojat is figyelembe vevő osztalyozasaban. Orv Hetil. 2018; 159(7): 278–284. | Abstract: Developmental disorders affecting the hypothalamic-pituitary system can result in pituitary hormone deficiency showing a diverse clinical presentation. A significant majority of these disorders are closely linked to defects in transcription factor genes which play a major role in pituitary development. Those affecting the early phase of organogenesis typically lead to complex conditions affecting the pituitary as well as structures in the central nervous system. Transcription factors involved in the late phase can result in combined but rarely isolated pituitary hormone deficiency without extra-pituitary manifestation. Identifying the defects in these pituitary transcription factor genes may provide a useful tool in predicting disease progression as well as screening family members. Several pituitary transcription factors can be detected in the adult gland as well which is strongly emphasized in the World Health Organization’s most recent guideline for pituitary tumor classification. Our review summarizes the current essential knowledge relevant for clinical endocrinologists. Orv Hetil. 2018; 159(7): 278–284.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The genetic basis for combined pituitary hormone deficiency (CPHD) is complex, involving 30 genes in a variety of syndromic and nonsyndromic presentations. Molecular diagnosis of this disorder is valuable for predicting disease progression, avoiding unnecessary surgery, and family planning. We expect that the application of high throughput sequencing will uncover additional contributing genes and eventually become a valuable tool for molecular diagnosis. For example, in the last 3 years, six new genes have been implicated in CPHD using whole-exome sequencing. In this review, we present a historical perspective on gene discovery for CPHD and predict approaches that may facilitate future gene identification projects conducted by clinicians and basic scientists. Guidelines for systematic reporting of genetic variants and assigning causality are emerging. We apply these guidelines retrospectively to reports of the genetic basis of CPHD and summarize modes of inheritance and penetrance for each of the known genes. In recent years, there have been great improvements in databases of genetic information for diverse populations. Some issues remain that make molecular diagnosis challenging in some cases. These include the inherent genetic complexity of this disorder, technical challenges like uneven coverage, differing results from variant calling and interpretation pipelines, the number of tolerated genetic alterations, and imperfect methods for predicting pathogenicity. We discuss approaches for future research in the genetics of CPHD.

Thyrotrope hyperplasia and hypertrophy are common responses to primary hypothyroidism. To understand the genetic regulation of these processes, we studied gene expression changes in the pituitaries of Cga(-/-) mice, which are deficient in the common α-subunit of TSH, LH, and FSH. These mice have thyrotrope hypertrophy and hyperplasia and develop thyrotrope adenoma. We report that cell proliferation is increased, but the expression of most stem cell markers is unchanged. The α-subunit is required for secretion of the glycoprotein hormone β-subunits, and mutants exhibit elevated expression of many genes involved in the unfolded protein response, consistent with dilation and stress of the endoplasmic reticulum. Mutants have elevated expression of transcription factors that are important in thyrotrope function, such as Gata2 and Islet 1, and those that stimulate proliferation, including Nupr1, E2f1, and Etv5. We characterized the expression and function of a novel, overexpressed gene, transcription elongation factor A (SII)-like 5 (Tceal5). Stable expression of Tceal5 in a pituitary progenitor cell line is sufficient to increase cell proliferation. Thus, Tceal5 may act as a proto-oncogene. This study provides a rich resource for comparing pituitary transcriptomes and an analysis of gene expression networks.

LIM-homeodomain (HD) transcription factors form a multimeric complex and assign neuronal subtype identities, as demonstrated by the hexameric ISL1-LHX3 complex which gives rise to somatic motor (SM) neurons. However, the roles of combinatorial LIM code in motor neuron diversification and their subsequent differentiation is much less well understood. In the present study, we demonstrate that the ISL1 controls postmitotic cranial branchiomotor (BM) neurons including the positioning of the cell bodies and peripheral axon pathfinding. Unlike SM neurons, which transform into interneurons, BM neurons are normal in number and in marker expression in Isl1 mutant mice. Nevertheless, the movement of trigeminal and facial BM somata is stalled, and their peripheral axons are fewer or misrouted, with ectopic branches. Among genes whose expression level changes in previous ChIP-seq and microarray analyses in Isl1-deficient cell lines, we found that Slit2 transcript was almost absent from BM neurons of Isl1 mutants. Both ISL1-LHX3 and ISL1-LHX4 bound to the Slit2 enhancer and drove endogenous Slit2 expression in SM and BM neurons. Our findings suggest that combinations of ISL1 and LHX factors establish cell-type specificity and functional diversity in terms of motor neuron identities and/or axon development.