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Many patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency have CAH-X syndrome, a connective tissue dysplasia consistent with hypermobility-type Ehlers-Danlos syndrome due to a contiguous gene deletion involving the adjacent CYP21A2 and TNXB genes. CAH-X syndrome is caused by carrying CYP21A1P-TNXA/TNXB chimeric genes [CAH-X chimera 1 (CH-1) and chimera 2 (CH-2)] on one or more alleles. Genetic analysis is cumbersome due to pseudogene interference. We developed a PCR-based CAH-X high-throughput screening method to assess the copy numbers of TNXB exons 35 and 40; this method is amenable to either real-time quantitative PCR or droplet digital PCR (ddPCR). The assay was validated in a cohort of 278 subjects from 146 unrelated CAH families. Results were confirmed by a validated Sanger sequencing platform. A total of 44 CAH-X-positive calls were made, with 42 (26 CH-1 and 16 CH-2) confirmed. The assay had 100% sensitivity (42 true/42 positives), 99.2% specificity (234 true/236 negatives), and an overall 99.3% accuracy (276/278). Calls made by real-time quantitative PCR and ddPCR were consistent (100%), and ddPCR offered easier data interpretation. The CAH-X prevalence was 15.6% (21/135 probands), higher than the previously estimated 8.5%, and was particularly high (29.2% or 21/72) in those with a 30-Kb deletion. This assay is suitable for high-throughput CAH-X screening, especially in subjects testing positive for CAH in neonatal screening.

The CYP21A2 gene encoding 21-hydroxylase is on chromosome 6p21.3 within the human leukocyte antigen (HLA) class III major histocompatibility complex and an association between congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency and HLA class I and II alleles has been shown in genetically isolated populations. One-third of CAH causing alleles are 30-kb deletions due to homologous recombination events between active and pseudogenes resulting in chimeric genes. The aim of this study was to re-visit the association between the CYP21A2 variants and HLA polymorphisms in a large ethnically diverse cohort of patients with CAH who underwent comprehensive CYP21A2 genotyping, including specification of chimeric gene subtypes (CAH CH-1 through CH-9 of CYP21A1P/CYP21A2 chimeras; CAH-X CH-1 through CH-3 of TNXA/TNXB chimeras) in alleles with 30-kb deletions. The study population included 201 patients (86 males, 115 females, age 3–75 years) with CAH due to 21-hydroxylase deficiency (159 classic, 42 nonclassic) and 194 parents. Based on the availability of parental genotype, we determined the haplotypes of CYP21A2 mutations and HLA types in 95 probands (190 alleles). Five prevalent haplotype associations were found: p.V281L and B*14-C*08 (P

Background Congenital adrenal hyperplasia (CAH) due to 21‐hydroxylase deficiency is an autosomal recessive disease of steroidogenesis that affects 1 in 15,000. Approximately, 10% of the CAH population also suffer from CAH‐X, a connective tissue dysplasia consistent with hypermobility type Ehlers–Danlos syndrome (EDS). Most patients with CAH‐X carry a contiguous gene deletion involving CYP21A2 encoding 21‐hydroxylase and TNXB encoding tenascin‐X (TNX), but some are of unknown etiology. Methods We conducted clinical evaluation and medical history review of EDS‐related manifestations in subjects from two unrelated CAH families who carry a heterozygous TNXB c.12463+2T>C variant that alters the splice donor site of intron 42. A next generation sequencing (NGS) based EDS panel composed of 45 genes was performed for index patients from each family. TNX expression in patient skin biopsy tissues and dermal fibroblasts was assessed by qRT‐PCR and Sanger sequencing. Results All three evaluated CAH patients carrying the TNXB splice site variant had moderate EDS manifestations. An NGS panel excluded involvement of other known EDS‐related variants. RNA assay on skin biopsies and dermal fibroblasts did not detect splicing errors in TNX mRNA; however, the removal of intron 42 was less efficient in the allele harboring the splice site variant as evidenced by the existence of a premature TNX RNA form, leading to an allele specific decrease in TNX mRNA. Conclusions Carrying a TNXB c.12463+2T>C variant at the intron 42 splice donor site causes an allele specific decrease in TNX expression, which can be associated with moderate EDS in CAH patients., About 10% of congenital adrenal hyperplasia patients suffer from Ehlers‐Danlos Syndrome, a connective tissue dysplasia. In addition to previously reported contiguous CYP21A2‐TNXB deletions, here we report a TNXB splice site variant c.12463+2T>C as causative by decreasing the expression of tenascin‐X.

Context Cholesterol side-chain cleavage enzyme (P450scc), encoded by CYP11A1, catalyzes the first step of steroidogenesis. Complete P450scc deficiency leads to primary adrenal insufficiency (PAI) and 46,XY disordered sexual development. Partial impairment can cause variable adrenal and gonadal dysfunction. Objective Our aim was to evaluate the effects of the CYP11A1 variant p.E314K, identified in patients with PAI, specifically on P450scc enzyme stability and function. Patients and methods We studied four boys from two unrelated families presenting with PAI during childhood (3.6 to 9 years old). All patients were compound heterozygous for c.940G>A (p.E314K), a CYP11A1 nonsynonymous variant likely to be pathogenic by some but not all in silico prediction models, and c.835delA (p.I79Yfs*10), a known pathogenic variant. HEK293T cells were transfected with wild type (WT) and p.E314K mutant vectors, and a cycloheximide chase assay was performed to analyze protein stability. Pregnenolone production was assayed from cells expressing WT and p.E314K-F2 fusion proteins. Results Two boys experienced spontaneous puberty but then developed evidence of primary gonadal failure at 14 and 18 years old. Two boys had testicular adrenal rest tumor (TART), detected by ultrasound at ages 8.6 and 16 years. Compared with WT, mutant protein synthesis was reduced (P = 0.0006) with increased protein turnover, and mutant P450scc half-life was decreased by ~50%. p.E314K mutant P450scc retained 60% of WT enzymatic activity (P = 0.007). Conclusions The CYP11A1 p.E314K variant impairs P450scc stability and is a possible cause of PAI in childhood. Pathogenic CYP11A1 variants potentially affect both adrenal and gonadal function, and male patients may develop TART.

Background: The clinical presentation of congenital hypoaldosteronism due to aldosterone synthase (aka corticosterone methyloxidase; CMO) deficiency varies with age and infants present with signs/symptoms of isolated mineralocorticoid deficiency. Classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) is characterized by combined glucocorticoid, mineralocorticoid deficiency and androgen excess. Clinical Case: A 6.8 y/o Female was referred for genetic testing for classic 21OHD CAH. Past History: Birth weight/length: 7.9 lbs/20 inches at term, uncomplicated pregnancy. Normal female genitalia. She presented within the 1st week of life with poor feeding and poor weight gain. Newborn screen - normal. Inpatient admission at 1 month for failure to thrive. Initial workup significant for Na 127 mg/dL (135 - 145), K 6.5 mg/dL (3.4 - 5.3), mildly elevated random 17hydroxyprogesterone (17OHP) 241 ng/dL (32 - 139) and cortisol 11.6 mcg/dL. She was diagnosed with CAH and initiated on hydrocortisone and fludrocortisone therapy. Medications: Hydrocortisone 2.5 mg twice daily (6.3 mg/m2/day), fludrocortisone 50 mcg daily. Family History: Non-contributory, mid-parental height 154.9 cm. Physical exam: Height 115.6 cm (20th %ile), weight 19.8 kg (18th %ile), BP 109/65 mmHg (95th/ 84th %ile). Well-appearing with no cushingoid features. Genital/pubertal exam: Prepubertal, normal female external genitalia. Laboratory tests: ACTH stimulation test was performed which showed - 0min: 17OHP 19 ng/dL, Cortisol 8 mcg/dL; 60min: 17OHP 155 ng/dL (18), ruling out the diagnosis of classic 21OHD CAH. Hydrocortisone was discontinued. Further workup (holding fludrocortisone for 48hrs) showed mildly elevated plasma renin activity 3.5 ng/mL/hr (0.8 - 2), low aldosterone 1.8 ng/dL (3.5 - 124), normal 18-hydroxycorticosterone 11 ng/dL (6 - 85). 18-hydroxycorticosterone/aldosterone ratio 6.1 (ratio

OBJECTIVE Optimal glucose homeostasis requires exquisitely precise adaptation of the number of insulin-secreting β-cells in the islets of Langerhans. Insulin itself positively regulates β-cell proliferation in an autocrine manner through the insulin receptor (IR) signaling pathway. It is now coming to light that cannabinoid 1 receptor (CB1R) agonism/antagonism influences insulin action in insulin-sensitive tissues. However, the cells on which the CB1Rs are expressed and their function in islets have not been firmly established. We undertook the current study to investigate if intraislet endogenous cannabinoids (ECs) regulate β-cell proliferation and if they influence insulin action. RESEARCH DESIGN AND METHODS We measured EC production in isolated human and mouse islets and β-cell line in response to glucose and KCl. We evaluated human and mouse islets, several β-cell lines, and CB1R-null (CB1R−/−) mice for the presence of a fully functioning EC system. We investigated if ECs influence β-cell physiology through regulating insulin action and demonstrated the therapeutic potential of manipulation of the EC system in diabetic (db/db) mice. RESULTS ECs are generated within β-cells, which also express CB1Rs that are fully functioning when activated by ligands. Genetic and pharmacologic blockade of CB1R results in enhanced IR signaling through the insulin receptor substrate 2-AKT pathway in β-cells and leads to increased β-cell proliferation and mass. CB1R antagonism in db/db mice results in reduced blood glucose and increased β-cell proliferation and mass, coupled with enhanced IR signaling in β-cells. Furthermore, CB1R activation impedes insulin-stimulated IR autophosphorylation on β-cells in a Gαi-dependent manner. CONCLUSIONS These findings provide direct evidence for a functional interaction between CB1R and IR signaling involved in the regulation of β-cell proliferation and will serve as a basis for developing new therapeutic interventions to enhance β-cell function and proliferation in diabetes.

In urothelial bladder cancer accurate identification of grade and stage is critical for optimal treatment to achieve robust disease control and long-term survival. However, among the initially superficial tumors, which are non-invasive and highly treatable, 20 to 25 % recur, progress to invasive tumors, and metastasize during the patients’ lifetime. Thus, the challenge is to provide risk stratification during the initial diagnosis in order to identify those patients who are unlikely to progress while offering radical therapy to those who are at risk. To address this issue, the field has heavily focused on the discovery of few mutations in potential driver genes. However, recent findings indicate that deregulation of enhancers can play a major role in cancer progression. In this study, we employed DNase-Seq to detect enhancer activities genome-wide in 16 bladder cancer cell lines (BLCs) that included three lineages (T24, UMUC3, 253J lineages) with different tumorigenic and metastatic potentials, and thus represent models of cancer progression and metastasis development. We analyzed the gain and loss of enhancer activity in each BLC lineages as well as in metastatic cell lines relative to non-metastatic cell lines. Our analysis in the T24 lineage revealed a striking feature, a dramatic loss of intronic and distal DHSs (DNase I Hypersensitive sites), during the initial transition to tumorigenic type. During metastatic progression, new enhancer classes emerged and there was an enrichment for association with target organ-specific genes. The analysis of differential DHSs between metastatic and non-metastatic BLCs identified an enhancer signature enriched with lost DHSs in metastatic BLCs where nearby genes were associated with cellular movement/invasion functions. Lost DHSs were also present near key factors such as PPARG, RXRA, FOXA1, TP63, and GATA3, which play a role in urothelial development and differentiation, and the transcription activity of those genes were correlated with the change of enhancer activity. This study identified a set of DHSs that are associated with cancer progression in bladder cancer, and provides a potential clinical application for developing prognostic markers to predict the risk of developing aggressive disease. Citation Format: Sohyoung Kim, Lyuba Varticovski, Qizong Lao, Songjoon Baek, Michael L. Nickerson, Myong-Hee Sung, Lars Grontved, Thompson Bethtrice, Dan Theodorescu, Piyush K. Agarwal, Michael Dean, Gordon Hager. Genome-wide enhancer identify signature predictive of metastatic phenotypes in bladder cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3352. doi:10.1158/1538-7445.AM2017-3352

A fundamental goal in biology is to gain a quantitative understanding of how appropriate cell responses are achieved amid conflicting signals that work in parallel. Through live, single-cell imaging, we monitored both the dynamics of nuclear factor κB (NF-κB) signaling and inflammatory cytokine transcription in macrophages exposed to the bacterial product lipopolysaccharide (LPS). Our analysis revealed a previously uncharacterized positive feedback loop involving induction of the expression of Rela , which encodes the RelA (p65) NF-κB subunit. This positive feedback loop rewired the regulatory network when cells were exposed to LPS above a distinct concentration. Paradoxically, this rewiring of NF-κB signaling in macrophages (a myeloid cell type) required the transcription factor Ikaros, which promotes the development of lymphoid cells. Mathematical modeling and experimental validation showed that the RelA positive feedback overcame existing negative feedback loops and enabled cells to discriminate between different concentrations of LPS to mount an effective innate immune response only at higher concentrations. We suggest that this switching in the relative dominance of feedback loops (“feedback dominance switching”) may be a general mechanism in immune cells to integrate opposing feedback on a key transcriptional regulator and to set a response threshold for the host.

Cannabinoid 1 (CB1) receptors have been previously detected in pancreatic β cells, where they attenuate insulin action. We now report that CB1 receptors form a heteromeric complex with insulin receptors and the heterotrimeric guanosine triphosphate-binding protein α subunit Gα(i). Gα(i) inhibited the kinase activity of the insulin receptor in β cells by directly binding to the activation loop in the tyrosine kinase domain of the receptor. Consequently, phosphorylation of proapoptotic protein Bad was reduced and its apoptotic activity was stimulated, leading to β-cell death. Pharmacological blockade or genetic deficiency of CB1 receptors enhanced insulin receptor signaling after injury, leading to reduced blood glucose concentrations and activation of Bad, which increased β-cell survival. These findings provide direct evidence of physical and functional interactions between CB1 and insulin receptors and suggest a mechanism whereby peripherally acting CB1 receptor antagonists improve insulin action in insulin-sensitive tissues independent of the other metabolic effects of CB1 receptors.

Type 2 diabetes mellitus (T2DM) results from insulin resistance and β-cell dysfunction, in the setting of hyperglucagonemia. Glucagon is a 29 amino acid peptide hormone, which is secreted from pancreatic α cells: excessively high circulating levels of glucagon lead to excessive hepatic glucose output. We investigated if α-cell numbers increase in T2DM and what factor (s) regulate α-cell turnover. Lepr(db)/Lepr(db) (db/db) mice were used as a T2DM model and αTC1 cells were used to study potential α-cell trophic factors. Here, we demonstrate that in db/db mice α-cell number and plasma glucagon levels increased as diabetes progressed. Insulin treatment (EC50 = 2 nM) of α cells significantly increased α-cell proliferation in a concentration-dependent manner compared to non-insulin-treated α cells. Insulin up-regulated α-cell proliferation through the IR/IRS2/AKT/mTOR signaling pathway, and increased insulin-mediated proliferation was prevented by pretreatment with rapamycin, a specific mTOR inhibitor. GcgR antagonism resulted in reduced rates of cell proliferation in αTC1 cells. In addition, blockade of GcgRs in db/db mice improved glucose homeostasis, lessened α-cell proliferation, and increased intra-islet insulin content in β cells in db/db mice. These studies illustrate that pancreatic α-cell proliferation increases as diabetes develops, resulting in elevated plasma glucagon levels, and both insulin and glucagon are trophic factors to α-cells. Our current findings suggest that new therapeutic strategies for the treatment of T2DM may include targeting α cells and glucagon.

The inside of the human body is generally maintained at near neutrality, but the surroundings of some cells are often acidic. For example, the poorly organized vasculature in tumor tissues and vessel damage in inflammation loci cause hypoxia and a shortage of nutrients. Such insufficient perfusion leads to the accumulation of metabolic wastes that acidify microenvironments around cells that alter various cellular functions. Two kinds of findings with opposite results have been reported. The antitumoral immune response of killer cells against solid tumors was impaired in acidic environments. Endocytosis of IL2 receptors was inhibited by cytosolic acidification. In contrast, it was reported that extracellular acidification increased the cytosolic free calcium level and surface expression of CD18 in human neutrophils, resulting in activation of these cells. Since immune cells often have to work under acidic conditions, the immune response would be activated under acidic conditions or at least its defects may be minimized, even if the immune response is impaired by acidification. Investigations concerning immune responses under acidic conditions are important for the development of new anti-cancer and anti-inflammation strategies, but only a few investigations have been reported to date. We have recently found that CTIB (C-Terminus Protein of IκB-β) was required for the maintenance of internal pH and proliferation under acidic adaptation in CHO cells. Since a CTIB homologue was found in human immune cells, it would be expected to open a new field of research for immune cell functions. In this chapter, we have

Bladder cancer (BCa) is the 5th most prevalent cancer in the US, and the metastatic disease has very poor prognosis with 150,000 deaths per year worldwide. The use of molecular markers to predict disease progression has been largely unsuccessful, with only few therapeutically approachable mutations. Over 2,300 alterations in the coding sequences have been described in high-grade bladder cancer, encompassing frequent alterations in genes involving chromatin modifiers. Thus, chromatin organization may play a critical role in this tumor type. The development of tumor progression models from transitional carcinoma samples that are poorly tumorigenic and non-metastatic to highly metastatic to the lung or liver by serial in vivo passaging allows analysis of the mechanisms associated with tumor progression. We examined whole exome sequencing to identify mutations, gene expression, and DNase I hypersensitivity followed by deep sequencing to identify sites of chromatin modification during tumor progression. Bioinformatic analysis of was performed using algorithms developed at LRBGE. Comparative analysis combining multiple data sets was performed using Ingenuity Pathways. Mutations in several previously identified genes associated with bladder cancer: TP53, RAS and TERT, and mutations/deletions in several chromatin modifiers KDM6A and MLL2/3 were found in parental cells. Only few additional mutations, none identifiable as cancer driver mutations marked the progression to metastatic phenotype. Changes in gene expression were already evident in progression to tumorigenic state, with high score in cells in cell-to-cell interaction pathway marked by a decrease in cell adhesion, increase in genes associated with EMT and genes associated with cancer. Changes in global chromatin landscape occurred at many known genes correlated with pathways detected by expression analysis, but many were in novel sites distant to promoters, suggestive of novel distant enhancer and confirm massive reprogramming of regulatory networks. Many of these sites were unique for each stage of tumor progression and have potential clinical application. Specific chromatin modifications were found in NFkB and inflammatory pathways. Thus, in the tumorigenic stage, these cells acquired not only the capacity for tumor formation, but also some of the characteristics associated with metastasis. Combination of exome sequencing with the gene expression and unbiased analysis of global chromatin landscape provided valuable new information on bladder cancer biology and tumor progression. Citation Format: Lyuba Varticovski, Sohyoung Kim, Michael L. Nickerson, Bethtrice Thompson, Qizong Lao, Lars Grøntved, Songjoon Baek, Myong-Hee Sung, Dan Theodorescu, Michael Dean, Gordon L. Hager. Novel molecular markers of bladder cancer progression identified by global chromatin profiling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4783. doi:10.1158/1538-7445.AM2015-4783

We previously reported that an IκB-β COOH terminal region protein (designated CTIB) was essential for the proliferation of CHO cells under acidic stress (Lao et al., 2005. J Cell Physiol 203(1):186–192). In order to investigate the mechanisms underlying the requirement of CTIB for acidic adaptation, CTIB was silenced with an RNAi technique in CHO cells. CTIB silencing resulted in those cells completely failing to proliferate and maintain intracellular pH (pHi) homeostasis at an extracellular pH (pHe) of 6.3. An increased activation of p38 MAP kinase was induced by CTIB silencing at the low pH value. CTIB was only present in the cytoplasm and co-immunoprecipitation of the cytoplasmic fraction revealed that the loss of CTIB led to a loss of p65 in the immunoprecipitate complex. CTIB silencing reduced both the decrease in p65 and the increase in p50 in the nucleus when the cells were incubated at pHe 6.3. In cells with CTIB silenced, the transcriptions of p65, p105, and IL1-β were suppressed, and decreases in both the transcription and activity of MnSOD were observed at pHe 6.3. Suppression of these genes suggested a suppressed NF-κB activity since p105, IL1-β, and MnSOD were target genes of NF-κB. Our data demonstrated that CTIB functioned to prevent the over-accumulation of p65 in the nucleus, ensuring the appropriate composition of the NF-κB complex in the nucleus to respond to stimuli under acidic conditions. J. Cell. Physiol. 207: 238–243, 2006. © 2005 Wiley-Liss, Inc.

CHO-K1 cells were able to proliferate and maintain pHi homeostasis at pH 6.3. A novel acidic sensitive mutant, AS-5B, which proliferated at pH 7.4 but failed to either proliferate or maintain pHi homeostasis at pH 6.3, was derived from CHO-K1 using a replica method. The acidic-sensitivity of AS-5B was not due to deficiencies in sodium proton exchangers, HCO (co)transporters or H+-ATPases. A cDNA clone encoding a COOH terminal region of IκB-β conferred partial acidic-resistance on AS-5B, and the encoded protein was present in CHO-K1, but was nearly absent from AS-5B. Our data demonstrated that the expression of this small protein was essential for the proliferation of CHO cells under acidic stress. © 2004 Wiley-Liss, Inc.

e15500 Background: Metastatic bladder cancer carries poor prognosis and, in spite of extensive sequencing efforts, only few known driving mutations have been identified for this tumor type. However...

Voltage-gated Cav1.2 calcium channels play a crucial role in Ca2+ signaling. The poreforming α1C subunit is regulated by accessory Cavβ and α2δ subunits. Cavβ's are cytoplasmic proteins of various size encoded by four different genes (Cavβ1 - β4). Here we investigated the effect of three major Cavβ types, β1b, β2d and β3, on the structure of Cav1.2 by measuring inter and intramolecular distances between α1C and β in the plasma membrane of COS1 cells using three-color FRET microscopy. The results show that Cav1.2 channels are in close proximity in the plasma membrane. The presence of different Cavβ's does not result in significant differences in intramolecular distance between the termini of α1C, but significantly affects intermolecular distance between the termini of neighbor α1C subunits, which varies from 67 A (β1b) to 79 A (β3). Thus, our results show conclusively that plasma-membrane density of Cav1.2 channels depends on the type of Cavβ's present, suggesting a possible mechanism contributing to differences in Ca2+ signaling between various cell types.