Explore the vast range of titles available.

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dual recombinase mediated cassette exchange to generate a Sec24c c-d allele, in which the C-terminal 90% of SEC24C has been replaced by SEC24D coding sequence.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, Sec24c c-d/c-d pups survive to term, though dying shortly after birth. Sec24c c-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.

Background Nearly half of all individuals with Down Syndrome (DS) have some type of congenital heart defect (CHD), suggesting that DS sensitizes to CHD but does not cause it. We used a common mouse model of DS, the Ts65Dn mouse, to study the contribution of Tbx5, a known modifier of CHD, to heart defects on a trisomic backgroun. Mice that were heterozygous for a Tbx5 null allele were crossed with Ts65Dn mice. Thoraxes of progeny were fixed in 10% formalin, embedded in paraffin, and sectioned for analysis of CHD. Gene expression in embryonic hearts was examined by quantitative PCR and in situ hybridization. A TBX5 DNA binding site was verified by luciferase assays. Methods Mice that were heterozygous for a Tbx5 null allele were crossed with Ts65Dn mice. Thoraxes of progeny were fixed in 10 % formalin, embedded in paraffin, and sectioned for analysis of CHD. Gene expression in embryonic hearts was examined by quantitative PCR and in situ hybridization. A TBX5 DNA binding site was verified by luciferase assays. Results We crossed mice that were heterozygous for a Tbx5 null allele with Ts65Dn mice. Mice that were trisomic and carried the Tbx5 mutation (Ts65Dn; Tbx5 +/− ) had a significantly increased incidence of overriding aorta compared to their euploid littermates. Ts65Dn; Tbx5 +/− mice also showed reduced expression of Pitx2, a molecular marker for the left atrium. Transcript levels of the trisomic Adamts1 gene were decreased in Tbx5 +/− mice compared to their euploid littermates. Evidence of a valid binding site for TBX5 upstream of the trisomic Adamts1 locus was also shown. Conclusion Haploinsufficiency of Tbx5 and trisomy affects alignment of the aorta and this effect may stem from deviations from normal left-right patterning in the heart. We have unveiled a previously unknown interaction between the Tbx5 gene and trisomy, suggesting a connection between Tbx5 and trisomic genes important during heart development.

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.

Multiple endocrine neoplasia syndromes (MEN) are genetic disorders with glandular hyperplasia and consecutive malignant neoplasia. MEN type 2B is the least common form of these tumor syndromes. It presents with typical dysmorphic features, mucosal neuromas, ganglioneuromatosis, medullary thyroid carcinoma (MTC) and phaeochromocytoma. The prognosis depends on the presence of MTC. We have surprisingly found two unrelated patients with this syndrome at our department within two weeks. In the medical history of a 17-year-old boy, Crohn’s disease had been considered because of abdominal pain and distention. He had marfanoid appearance and previously undergone minor surgeries for a large tongue with neuromas and hypertrophic gums. Two weeks later, a 10-year-old girl presented with a hard palpable mass on her neck. She had thickened lips, neuromas on the tongue and a solitary thyroid nodule. Genetic analysis was carried out in both patients and a heterozygous M918T mutation of the RET proto-oncogene was found. Laboratory tests and imaging studies were consistent with MTC. Phaeochromocytoma was not present. Both patients underwent total thyroidectomy and lymph node dissection. Histological examination confirmed the diagnosis of MTC. In conclusion, the initial diagnosis of MEN 2B should be suspected on the presence of typical facial/oral signs and gastrointestinal symptoms. Hormonal tests and imaging techniques of the thyroid and the adrenals can confirm the clinical diagnosis of MEN 2B and genetic analysis can prove its germline origin.

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.

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.

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.

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.

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 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.