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Abstract Background Congenital adrenal hyperplasia (CAH) due to 21‐hydroxylase (21OH) deficiency is an autosomal recessive inborn error of cortisol biosynthesis, with varying degrees of aldosterone production. There is a continuum of phenotypes which generally correlate with genotype and the expected residual 21OH activity of the less severely impaired allele. CYP21A1P/CYP21A2 chimeric genes caused by recombination between CYP21A2 and its highly homologous CYP21A1P pseudogene are common in CAH and typically associated with salt‐wasting CAH, the most severe form. Nine chimeras have been described (CH‐1 to CH‐9). Aims The aim of this study was to genetically evaluate two variant alleles carried by a 22‐year‐old female with the non‐salt‐wasting simple virilizing form of CAH and biallelic 30‐kb deletions. Methods The haplotypes of the CYP21A2 heterozygous variants, as well as the chimeric junction sites, were determined by Sanger sequencing TA clones of an allele‐specific PCR product. Results Genetic testing revealed two rare CYP21A1P/CYP21A2 chimeras: allele 1 matches the previously described CAH CH‐1 chimera but without the P30L variant, and allele 2, termed here as novel CAH CH‐10, has a junction site between c.293‐37 and c.29314, which is expected to retain partial 21OH activity. Conclusion These two variant alleles further document the complex nature of RCCX modules and highlight that not all CYP21A1P/CYP21A2 chimera severely impair 21OH activity.

Abstract Objective Approximately 10% of patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency carry a mutation that disrupts CYP21A2 and the flanking TNXB gene resulting in CAH-X, a contiguous gene deletion syndrome. TNXB encodes tenascin-X (TNX), an extracellular matrix glycoprotein that plays an important role in collagen organization. TNXB impairment is associated with Ehlers–Danlos syndrome. Symptoms include joint hypermobility, hernias and cardiac defects. We measured serum TNX using an antibody targeting the amino-terminal of the TNX protein in 161 subjects, including extensively genotyped and phenotyped CAH patients, their relatives, and healthy controls. Results We evaluated the potential of serum TNX as a screening tool for CAH-X. CAH-X patients, especially haploinsufficient patients carrying the TNXA-TNXB chimeric gene CAH-X-CH-1 showed reduced TNX levels compared to controls (P

Abstract 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.

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.

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 (CBIR) agonism/antagonism influences insulin action in insulin-sensitive tissues. However, the cells on which the CBIRs 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 KC1. 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 CBIRs that are fully functioning when activated by ligands. Genetic and pharmacologic blockade of CBIR 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. CBIR 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, CBIR 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. [ABSTRACT FROM AUTHOR]

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, HCO3- (co)transporters or H+-ATPases. A cDNA clone encoding a COOH terminal region of I?B- 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. [ABSTRACT FROM AUTHOR]