The Molecular Basis of Congenital Hypopituitarism and Related Disorders

CONTEXT: Congenital hypopituitarism (CH) is characterized by the presence of deficiencies in one or more of the six anterior pituitary (AP) hormones secreted from the five different specialized cell types of the AP. During human embryogenesis, hypothalamo-pituitary (HP) development is controlled by a complex spatio-temporal genetic cascade of transcription factors and signaling molecules within the hypothalamus and Rathke’s pouch, the primordium of the AP. / EVIDENCE ACQUISITION: This mini-review discusses the genes and pathways involved in HP development and how mutations of these give rise to CH. This may present in the neonatal period or later on in childhood, and may be associated with craniofacial midline structural abnormalities such as cleft lip/palate, visual impairment due to eye abnormalities such as optic nerve hypoplasia and microphthalmia or anophthalmia, or midline forebrain neuroradiological defects including agenesis of the septum pellucidum or corpus callosum or the more severe holoprosencephaly. / EVIDENCE SYNTHESIS: Mutations give rise to an array of highly variable disorders ranging in severity. There are many known causative genes in HP developmental pathways that are routinely screened in CH patients; however, over the last 5 years this list has rapidly increased due to the identification of variants in new genes and pathways of interest by next generation sequencing. / CONCLUSION: The majority of patients with these disorders do not have an identified molecular basis, often making management challenging. This mini-review aims to guide clinicians in making a genetic diagnosis based on patient phenotype, which in turn may impact on clinical management

Investigation of new candidate genes in a cohort of patients with familial congenital hypopituitarism and associated disorders

Congenital hypopituitarism is a complex variable genetic disorder that is known to be caused by multiple mutated genes, both in isolation and in variably penetrant cases of digenic inheritance. In only <10% of cases, a mutation in a known causative gene has been identified in the patient, leaving the vast majority of patients yet to have a genetic mutation detected that is responsible for the pathogenicity and that has functional significance to their condition. This study investigates novel genes and pathways involved in hypothalamo-pituitary development. Our large cohort of consanguineous and non-consanguineous pedigrees with hypothalamo-pituitary disease are routinely screened for variants in the known causative genes. In pedigrees where there are no variants in these particular genes, exome sequencing in collaboration with GOSgene is carried out to uncover novel genes and regions of interest that are abnormal in the individual. Upon the identification of any novel variant in known or novel genes, functional assays are conducted to further show the significance of the change. Firstly this study identifies the first novel homozygous mutation in the LHX4 gene, p.T126M, in two deceased brothers from a pedigree with combined pituitary hormone deficiency with subsequent fatal consequences. Functional luciferase assays showed that there was no significant difference between mutant p.T126M and WT constructs in transactivating the αGSU and prolactin promoters, and that mutant LHX4 could synergise with POU1F1 similar to WT LHX4. Secondly, six new candidate genes; CTPS2, RNPC3, PRMT6, FASN, APEX2 and EIF2S3, were identified in phenotypically unique pedigrees submitted to GOSgene for exome sequencing. The human embryonic expression profiles of these novel candidate genes were analysed in this study in a hypothalamo-pituitary context, as well as in related tissues that are affected in the individuals. Thirdly, the role of eIF2γ, encoded by EIF2S3, which was found to be mutated in an X-linked pedigree with congenital hypopituitarism, hypothyroidism and hyperinsulinism causing hypoglycaemia, was investigated in this study. A lentiviral shRNA knock out of the EIF2S3 gene in human pancreatic cells resulted in significantly higher apoptosis compared to WT cells. This study has used both a Sanger sequencing and an exome sequencing approach to identify novel variants in known and novel candidate genes respectively

Fractal dimension of large aggregates under different flocculation conditions

The two-dimensional fractal dimension (Df) of large aggregates of kaolin (> 540 μm) during the shear flocculation process for kaolin solution was investigated using non-intrusive in situ image-based acquisition system. Separate experiments were also carried out for three different sized sub-ranges of large aggregates (0.540–1.125 mm; 1.125–1.750 mm; 1.750–2.375 mm). Digital images were taken at a frequency of 10 Hz for 10 s for each different pairs of gradients of velocity (Gf) of 20 and 60 s− 1 and flocculation times of 2; 3; 4; 5; 10; 20; 30; 60; 120 and 180 min. For the same conditions, particle size distribution (PSD) was also determined. Under the investigated conditions, the lowest Gf produced the greatest Df (1.69) at a flocculation time of 30 min for the whole range of aggregates. Also, the evolution of the longest length of aggregate (l) and Df with time, showed that the dynamic steady-state was reached at different times for each shear rate and l ranges. However, Df varied for each size sub-range (ca. 1.1 to 1.8). Finally, the behavior of the aggregate structure may be understood by the predominance of different aggregation mechanisms such as cluster-cluster for Gf of 60 s− 1 and particle-cluster for Gf of 20 s− 1

Upregulation of the cell-cycle regulator RGC-32 in Epstein-Barr virus-immortalized cells

Epstein-Barr virus (EBV) is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III). The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15), is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL) cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator

The phenotypic spectrum associated with OTX2 mutations in humans

OBJECTIVE: The transcription factor OTX2 is 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 in the human brain, with a view to investigating the mechanism of action. METHODS: We screened patients from the UK (n=103), international centers (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

Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors

Activating mutations in BRAF disrupt the hypothalamo-pituitary axis leading to hypopituitarism in mice and humans

Germline mutations in BRAF and other components of the MAPK pathway are associated with the congenital syndromes collectively known as RASopathies. Here, we report the association of Septo-Optic Dysplasia (SOD) including hypopituitarism and Cardio-Facio-Cutaneous (CFC) syndrome in patients harbouring mutations in BRAF. Phosphoproteomic analyses demonstrate that these genetic variants are gain-of-function mutations leading to activation of the MAPK pathway. Activation of the MAPK pathway by conditional expression of the BrafV600E/+ allele, or the knock-in BrafQ241R/+ allele (corresponding to the most frequent human CFC-causing mutation, BRAF p.Q257R), leads to abnormal cell lineage determination and terminal differentiation of hormone-producing cells, causing hypopituitarism. Expression of the BrafV600E/+ allele in embryonic pituitary progenitors leads to an increased expression of cell cycle inhibitors, cell growth arrest and apoptosis, but not tumour formation. Our findings show a critical role of BRAF in hypothalamo-pituitary-axis development both in mouse and human and implicate mutations found in RASopathies as a cause of endocrine deficiencies in humans