Congenital adrenal hyperplasia due to two rare CYP21A2 variant alleles, including a novel attenuated CYP21A1P/CYP21A2 chimera.

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.

Pseudogene TNXA Variants May Interfere with the Genetic Testing of CAH-X.

CAH-X is a hypermobility-type Ehlers-Danlos syndrome connective tissue dysplasia affecting approximately 15% of patients with 21-hydroxylase deficiency (21-OHD) congenital adrenal hyperplasia (CAH) due to contiguous deletion of CYP21A2 and TNXB genes. The two most common genetic causes of CAH-X are CYP21A1P-TNXA/TNXB chimeras with pseudogene TNXA substitution for TNXB exons 35-44 (CAH-X CH-1) and TNXB exons 40-44 (CAH-X CH-2). A total of 45 subjects (40 families) from a cohort of 278 subjects (135 families of 21-OHD and 11 families of other conditions) were found to have excessive TNXB exon 40 copy number as measured by digital PCR. Here, we report that 42 subjects (37 families) had at least one copy of a TNXA variant allele carrying a TNXB exon 40 sequence, whose overall allele frequency was 10.3% (48/467). Most of the TNXA variant alleles were in cis with either a normal (22/48) or an In2G (12/48) CYP21A2 allele. There is potential interference with CAH-X molecular genetic testing based on copy number assessment, such as with digital PCR and multiplex ligation-dependent probe amplification, since this TNXA variant allele might mask a real copy number loss in TNXB exon 40. This interference most likely happens amongst genotypes of CAH-X CH-2 with an in trans normal or In2G CYP21A2 allele.

A TNXB splice donor site variant as a cause of hypermobility type Ehlers-Danlos syndrome in patients with congenital adrenal hyperplasia.

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.

Measurement of serum tenascin-X in patients with congenital adrenal hyperplasia at risk for Ehlers-Danlos contiguous gene deletion syndrome CAH-X.

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 < 0.05). TNX levels were similar in all subjects carrying a TNXB mutation. However, CAH patients who did not harbor a TNXB mutation also had reduced TNX compared to controls (P < 0.001). Thus, measuring serum TNX is not an effective screen for CAH-X amongst patients with CAH. TNXB genotyping is recommended for CAH patients who have symptoms of a connective tissue disorder. Epigenetic factors that influence TNX expression require further study.

High-Throughput Screening for CYP21A1P-TNXA/TNXB Chimeric Genes Responsible for Ehlers-Danlos Syndrome in Patients with Congenital Adrenal Hyperplasia.

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.

Characterization of the CYP11A1 Nonsynonymous Variant p.E314K in Children Presenting With Adrenal Insufficiency.

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.

Revisiting the association of HLA alleles and haplotypes with CYP21A2 mutations in a large cohort of patients with congenital adrenal hyperplasia.

The CYP21A2 gene encoding 21­hydroxylase 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 21­hydroxylase 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 21­hydroxylase 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 < 0.0001); p.I172N and DQB1*03 (P = 0.035); and of the chimeric genes caused by 30-kb deletions: CH-1 and A*03 (P = 0.033); CH-5 and C*06-DRB1*07 (P < 0.0001); and CAH-X CH-1 and DQB1*03 (P = 0.004). Our findings show that a number of associations between HLA alleles and haplotypes and CYP21A2 mutations, including large 30-kb deletions, exist commonly across ethnicities. These HLA associations may have clinical implications for patients with CAH and may provide insight into the genetics of this highly complex region of the human genome.

Complement component 4 variations may influence psychopathology risk in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

CYP21A2 defects result in congenital adrenal hyperplasia (CAH), an autosomal recessive disorder characterized by impaired adrenal steroidogenesis. CYP21A2 lies within the major histocompatibility complex in an area of the genome highly susceptible to genetic variation. Alterations in the neighboring complement component 4 isotypes C4A and C4B have been associated with psychiatric and autoimmune disease. The purpose of this study was to evaluate C4A and C4B in patients with CAH in relation to CYP21A2 genotype and psychiatric and autoimmune comorbidity. We determined the copy numbers of C4A and C4B in 145 patients with CAH (median age: 15.5 years, IQR: 16.8) and 108 carrier relatives (median age: 41.5 years, IQR: 12.0) and evaluated serum C4 concentrations. Comorbidity was determined by medical record review. Only 30% of subjects had the expected two copies each of the two C4 genes. C4B copy number determined total C4 copy number and serum C4 concentration, negatively correlated with carriership of a 30-kb deletion (P < 10- 5), and positively correlated with carriership of p.V281L (P < 10- 5). High C4A copy number (≥ 3) was associated with increased risk of having an externalizing psychiatric condition (relative risk: 2.67, 95% CI: 1.03-6.89, P = 0.04). No association was found between C4 copy number and autoimmune disease. Mutation-specific C4 structural variations commonly occur in patients with CAH and may have important clinical consequences, including increased risk of psychiatric morbidity. Trial registration NCT00250159 (November 7, 2005).

Switching of the relative dominance between feedback mechanisms in lipopolysaccharide-induced NF-κB signaling.

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.

Cannabinoids induce pancreatic ß-cell death by directly inhibiting insulin receptor activation.

Cannabinoid 1 (CB1) receptors have been previously detected in pancreatic ß 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 ß 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 ß-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 ß-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.

Insulin and glucagon regulate pancreatic α-cell proliferation.

Type 2 diabetes mellitus (T2DM) results from insulin resistance and ß-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 ß 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.

Cannabinoids inhibit insulin receptor signaling in pancreatic ß-cells.

OBJECTIVE: Optimal glucose homeostasis requires exquisitely precise adaptation of the number of insulin-secreting ß-cells in the islets of Langerhans. Insulin itself positively regulates ß-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 ß-cell proliferation and if they influence insulin action. RESEARCH DESIGN AND METHODS: We measured EC production in isolated human and mouse islets and ß-cell line in response to glucose and KCl. We evaluated human and mouse islets, several ß-cell lines, and CB1R-null (CB1R(-/-)) mice for the presence of a fully functioning EC system. We investigated if ECs influence ß-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 ß-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 ß-cells and leads to increased ß-cell proliferation and mass. CB1R antagonism in db/db mice results in reduced blood glucose and increased ß-cell proliferation and mass, coupled with enhanced IR signaling in ß-cells. Furthermore, CB1R activation impedes insulin-stimulated IR autophosphorylation on ß-cells in a Gα(i)-dependent manner. CONCLUSIONS: These findings provide direct evidence for a functional interaction between CB1R and IR signaling involved in the regulation of ß-cell proliferation and will serve as a basis for developing new therapeutic interventions to enhance ß-cell function and proliferation in diabetes.

Requirement of an IkappaB-beta COOH terminal region protein for acidic-adaptation in CHO cells.

We previously reported that an IkappaB-beta 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-beta 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-kappaB activity since p105, IL1-beta, and MnSOD were target genes of NF-kappaB. Our data demonstrated that CTIB functioned to prevent the over-accumulation of p65 in the nucleus, ensuring the appropriate composition of the NF-kappaB complex in the nucleus to respond to stimuli under acidic conditions.

An IkappaB-beta COOH terminal region protein is essential for the proliferation of CHO cells under acidic stress.

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 IkappaB-beta 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.