Functional analysis of novel A20 variants in patients with atypical inflammatory diseases

Background A20 haploinsufficiency (HA20) is an early-onset autoinflammatory disease caused by mutations in the TNFAIP3 gene, which encodes the protein A20. Numerous truncating mutations in the TNFAIP3 gene have been reported in HA20 patients, whereas fewer missense variants have had their pathogenicity confirmed. Here, we evaluated the pathogenic significance of three previously unreported missense variants of the TNFAIP3 gene in suspected cases of HA20. Methods We obtained the clinical features and immunological data of three patients with missense variants (Glu192Lys, Ile310Thr, and Gln709Arg) of unknown significance of TNFAIP3. We then performed in vitro functional assays including analysis of nuclear factor (NF)-κB reporter gene activity, detection of A20 expression and phosphorylation of A20 by IκB kinase β (IKKβ), and K63-deubiquitination assay using TNFAIP3-deficient HEK293 cells. Three known pathogenic missense mutations reported previously were also investigated. Results The inhibitory effect on NF-κB reporter gene activity was significantly disrupted by A20 Glu192Lys and the three known mutations. The variants Ile310Thr and Gln709Arg did not show a difference from the wild type in any of the assays performed in this study. Conclusions Among the three variants in the TNFAIP3 gene, Glu192Lys was interpreted as being likely pathogenic, but Ile310Thr and Gln709Arg as being not pathogenic (uncertain significance and likely benign, respectively), based on the American College of Medical Genetics and Genomics standards and guidelines. Our study highlights the necessity of performing in vitro functional assays, notably, NF-κB reporter gene assay, to evaluate the pathogenicity of TNFAIP3 missense variants for the accurate diagnosis of HA20.

Abstract 322: Postnatal Islet-1 Cardioblasts Are of Neural Crest and Not Second Heart-Field Lineage

Introduction: The transcription factor Islet-1 (Isl1) is expressed in cardiac mesodermal and neural crest (CNC) lineages during cardiogenesis. A pool of Isl1 + cells persist in the perinatal heart (Isl1 + CPCs), some of which are touted as residual second heart-field (SHF)- derived cardioblasts. However, direct lineage-tracing evidence, supporting a SHF over a CNC origin of Isl1 + CPCs, are lacking. Hypothesis: Isl1 + CPCs are of CNC and not SHF lineage. Methods: The Isl1-nLacZ, Wnt1-Cre;tdTomato and Wnt1::FlpE;RC::Fela mice, and iPSCs derived from Wnt1-Cre;tdTomato mice (iPSC Wnt1 ) were employed to lineage-trace Isl1 + CPCs. Results: Temporal analysis of Isl1-nLacZ embryos illustrated a transient, stage-specific reporter gene activity in mesendodermal and neuroectodermal cells. Particularly, at embryonic day (E)9.5, reporter gene activity (x-gal), was strong in the outflow tract (OFT), and exhibited a weak, spotty pattern in the heart. At E12.5, x-gal was strong in the neural tube (NT); reduced in the OFT; and undetectable in the heart. Isl1 immunohistochemistry (IHC) illustrated similar activity of x-gal with endogenous Isl1 expression. Importantly, x-gal remained permanently undetected in the heart. Isl1 IHC in E12.5 Wnt1-Cre;tdTomato and Wnt1::FlpE;RC::Fela embryos indicated that Isl1 + cells in the OFT and NT colocalized with Wnt1 reporter transgenes. At E18.5 and postnatal day 1 (PN1), Isl1 + cells were exclusively of Wnt1 lineage, indicating that Isl1 + CPCs are CNC- and not SHF-derived. Since CNCs minimally contribute cardiomyocytes in mammals, a presumptive full cardiomyogenic capacity of Isl1 + CPCs potentially contrasts with a CNC origin. To address this controversy, we differentiated iPSC Wnt1 toward the CNC lineage, using a previously established embryoid body (EB) differentiation protocol involving transient BMP antagonism. At ~EB-day 9, tdTomato + /Isl1 + CNCs emerged, which progressively differentiated into spontaneously beating, Nkx2.5 + cardiomyocytes. Conclusions: Our findings clarify that Isl1 + CPCs are of CNC and not SHF origin, and suggest a novel role of the mammalian CNC, as a contributor of long-lived myocardial progenitors that could be targeted for heart-related therapeutic purposes.

Angiotensin II-Dependent Transcriptional Activation of Human Steroidogenic Acute Regulatory Protein Gene by a 25-kDa cAMP-Responsive Element Modulator Protein Isoform and Yin Yang 1

Transcriptional activation of the steroidogenic acute regulatory protein (STAR) gene is a critical component in the angiotensin II (Ang II)-dependent increase in aldosterone biosynthesis in the adrenal gland. The purpose of this study was to define the molecular mechanisms that mediate the Ang II-dependent increase in STARD1 gene (STAR) expression in H295R human adrenocortical cells. Mutational analysis of the STAR proximal promoter revealed that a nonconsensus cAMP-responsive element located at −78 bp relative to the transcription start site (−78CRE) is required for the Ang II-stimulated STAR reporter gene activity. DNA immunoaffinity chromatography identified a 25-kDa cAMP-responsive element modulator isoform and Yin Yang 1 (YY1) as −78CRE DNA-binding proteins, and Ang II treatment of H295R cells increased expression of that 25-kDa CREM isoform. Small interfering RNA silencing of CREM and YY1 attenuated the Ang II-dependent increases in STAR reporter gene activity and STAR mRNA levels. Conversely, overexpression of CREM and YY1 in COS-1 cells resulted in transactivation of STAR reporter gene activity. Chromatin immunoprecipitation analysis demonstrated recruitment of CREM and YY1 to the STAR promoter along with increased association of the coactivator cAMP response element-binding protein-binding protein (CBP) and increased phosphorylated RNA polymerase II after Ang II treatment. Together our data reveal that the Ang II-stimulated increase in STAR expression in H295R cells requires 25 kDa CREM and YY1. The recruitment of these transcription factors to the STAR proximal promoter results in association of CBP and activation of RNA polymerase II leading to increased STAR transcription.

The Phytoestrogen Genistein Is a Tissue-Specific Androgen Receptor Modulator

To enable studies of androgen signaling in different tissues in vivo, we generated an androgen receptor (AR) reporter mouse line by inserting a luciferase gene construct into the murine genome. The construct is driven by four copies of androgen-responsive elements from the mouse sex-limited protein gene (slp-HRE2) and a minimal thymidine kinase promoter. Luciferase activity was readily measurable in a number of murine tissues, including prostate, lung, testis, brain, and skeletal muscle, and testosterone administration elicited a significant increase in reporter gene activity in these tissues. Consumption of isoflavonoid genistein is linked to reduced risk of prostate cancer, but direct effects of genistein on the AR pathway are not well understood. To examine androgen-modulating activity of genistein in vivo, male mice received daily doses of genistein (10 mg/kg) for 5 d. In intact males, genistein was antiandrogenic in testis, prostate, and brain, and it attenuated reporter gene activity by 50–80%. In castrated males, genistein exhibited significant androgen agonistic activity in prostate and brain by increasing reporter gene activity over 2-fold in both tissues. No antiandrogenic action was seen in lung or skeletal muscle of intact males. Gene expression profiling of the murine prostate under the same experimental conditions revealed that genistein modulates androgen-dependent transcription program in prostate in a fashion similar to that observed in reporter mice by luciferase expression. In conclusion, genistein is a partial androgen agonist/antagonist in some but not in all mouse tissues and should be considered as a tissue-specific AR modulator.