STAT6 gain-of-function variant exacerbates multiple allergic symptoms.

BACKGROUND: Allergic diseases were long considered to be complex multifactorial disorders. However, recent findings indicate that severe allergic inflammation can be caused by monogenic immune defects. OBJECTIVES: We sought to clarify the molecular pathogenesis of a patient with early-onset multiple allergic diseases, a high serum IgE level, hypereosinophilia, treatment-resistant severe atopic dermatitis with increased dermal collagen fiber deposition, and eosinophilic gastrointestinal disorder with numerous polypoid nodules. METHODS: A missense variant in STAT6 was identified, and its function was examined using peripheral blood, transfected HEK293 cells, lymphoblastoid cell lines, and knock-in mice with the corresponding mutation. RESULTS: Whole-exome sequencing identified a de novo heterozygous missense variant in signal transducer and activator of transcription 6 (STAT6) (p.Asp419Asn). Luciferase reporter assay revealed that the transcriptional activity of this STAT6 mutant was upregulated even without IL-4 stimulation. Phosphorylation of STAT6 was not observed in either the patient's TH2 cells or lymphoblastoid cell lines without stimulation, whereas it was induced more strongly in both by IL-4 stimulation compared with healthy controls. STAT6 protein was present in the nuclear fraction of the lymphoblastoid cell lines of the patient even in the absence of IL-4 stimulation. The patient's gastric mucosa showed upregulation of STAT6-, fibrosis-, and germinal center formation-related molecules. Some of the knock-in mice with the corresponding mutation spontaneously developed dermatitis with skin thickening and eosinophil infiltration. Moreover, serum IgE levels and mRNA expression of type 2 cytokines were increased in the knock-in mice-with or without development of spontaneous dermatitis-compared with the wild-type mice. CONCLUSIONS: A novel STAT6 gain-of-function variant is a potential cause of primary atopic disorders.

Relationships between Slc1a5 and Osteoclastogenesis.

Slc1a5 (ASCT2) encodes a small neutral amino-acid exchanger and is the most well-studied glutamine transporter in cancer cells. To investigate the role of Slc1a5 in osteoclastogenesis, we developed Slc1a5-deficient mice by using a conventional gene-targeting approach. The Slc1a5-/- mice showed no obvious abnormalities in growth. Glutamine uptake was assessed in Slc1a5+/+ and Slc1a5-/- bone marrow cells stimulated with RANKL. The rate of glutamine uptake in Slc1a5-/- bone marrow cells was reduced to 70% of that of cells from Slc1a5+/+ bone marrow. To confirm the involvement of Slc1a5 in osteoclast formation, bone marrow cells derived from Slc1a5+/+ or Slc1a5-/- mice were stimulated with RANKL and macrophage colony-stimulating factor and stained with tartrate-resistant acid phosphatase. The bone resorption activity and actin ring formation of stimulated cells were measured. The formation of multinucleated osteoclasts in bone marrow cells isolated from Slc1a5-/- mice was severely impaired compared with those from Slc1a5+/+ mice. RANKL-induced expression of ERK, NFκB, p70S6K, and NFATc1 was suppressed in Slc1a5-/- osteoclasts. These results show that Slc1a5 plays an important role in osteoclast formation.

Podocyte-specific deletion of tubular sclerosis complex 2 promotes focal segmental glomerulosclerosis and progressive renal failure.

Obesity can initiate and accelerate the progression of kidney diseases. However, it remains unclear how obesity affects renal dysfunction. Here, we show that a newly generated podocyte-specific tubular sclerosis complex 2 (Tsc2) knockout mouse model (Tsc2Δpodocyte) develops proteinuria and dies due to end-stage renal dysfunction by 10 weeks of age. Tsc2Δpodocyte mice exhibit an increased glomerular size and focal segmental glomerulosclerosis, including podocyte foot process effacement, mesangial sclerosis and proteinaceous casts. Podocytes isolated from Tsc2Δpodocyte mice show nuclear factor, erythroid derived 2, like 2-mediated increased oxidative stress response on microarray analysis and their autophagic activity is lowered through the mammalian target of rapamycin (mTOR)-unc-51-like kinase 1 pathway. Rapamycin attenuated podocyte dysfunction and extends survival in Tsc2Δpodocyte mice. Additionally, mTOR complex 1 (mTORC1) activity is increased in podocytes of renal biopsy specimens obtained from obese patients with chronic kidney disease. Our work shows that mTORC1 hyperactivation in podocytes leads to severe renal dysfunction and that inhibition of mTORC1 activity in podocytes could be a key therapeutic target for obesity-related kidney diseases.

Identification of targets of tumor suppressor microRNA-34a using a reporter library system.

miRNAs play critical roles in various biological processes by targeting specific mRNAs. Current approaches to identifying miRNA targets are insufficient for elucidation of a miRNA regulatory network. Here, we created a cell-based screening system using a luciferase reporter library composed of 4,891 full-length cDNAs, each of which was integrated into the 3' UTR of a luciferase gene. Using this reporter library system, we conducted a screening for targets of miR-34a, a tumor-suppressor miRNA. We identified both previously characterized and previously uncharacterized targets. miR-34a overexpression in MDA-MB-231 breast cancer cells repressed the expression of these previously unrecognized targets. Among these targets, GFRA3 is crucial for MDA-MB-231 cell growth, and its expression correlated with the overall survival of patients with breast cancer. Furthermore, GFRA3 was found to be directly regulated by miR-34a via its coding region. These data show that this system is useful for elucidating miRNA functions and networks.

Testicular dysgenesis/regression without campomelic dysplasia in patients carrying missense mutations and upstream deletion of SOX9.

SOX9 haploinsufficiency underlies campomelic dysplasia (CD) with or without testicular dysgenesis. Current understanding of the phenotypic variability and mutation spectrum of SOX9 abnormalities remains fragmentary. Here, we report three patients with hitherto unreported SOX9 abnormalities. These patients were identified through molecular analysis of 33 patients with 46,XY disorders of sex development (DSD). Patients 1-3 manifested testicular dysgenesis or regression without CD. Patients 1 and 2 carried probable damaging mutations p.Arg394Gly and p.Arg437Cys, respectively, in the SOX9 C-terminal domain but not in other known 46,XY DSD causative genes. These substitutions were absent from ~120,000 alleles in the exome database. These mutations retained normal transactivating activity for the Col2a1 enhancer, but showed impaired activity for the Amh promoter. Patient 3 harbored a maternally inherited ~491 kb SOX9 upstream deletion that encompassed the known 32.5 kb XY sex reversal region. Breakpoints of the deletion resided within nonrepeat sequences and were accompanied by a short-nucleotide insertion. The results imply that testicular dysgenesis and regression without skeletal dysplasia may be rare manifestations of SOX9 abnormalities. Furthermore, our data broaden pathogenic SOX9 abnormalities to include C-terminal missense substitutions which lead to target-gene-specific protein dysfunction, and enhancer-containing upstream microdeletions mediated by nonhomologous end-joining.

Complex genomic rearrangement in the SOX9 5' region in a patient with Pierre Robin sequence and hypoplastic left scapula.

Pierre Robin sequence (PRS) can occur as a component of campomelic dysplasia (CD) and acampomelic CD (ACD) caused by dysfunction or dysregulation of SOX9, although it can also take place as an isolated form. Recently, genomic alterations in the far upstream and the far downstream region of SOX9 have been identified in patients with isolated PRS. Here, we report on a male patient with PRS and a heterozygous genomic rearrangement in the 5' region of SOX9. Clinical analysis revealed PRS-compatible craniofacial anomalies, mild hypoplasia of the left scapula, and normal male external genitalia. Molecular analysis identified a paracentric inversion on the long arm of chromosome 17 with breakpoints at 17q21.31 and 17q24.3, and a microdeletion spanning from -4.15 to -1.16 Mb relative to SOX9. These findings indicate that the chromosomal region more than 1.16 Mb apart from SOX9 contains at least one developmental enhancer(s) for SOX9 that plays a critical role in the development of the mandible and a relatively small role in the development of the scapula. Moreover, the concept of exclusion mapping argues that putative CD/ACD loci are located within the 1.16 Mb region closest to SOX9 coding exons, which remain intact in this Non-CD/ACD patient. This study provides a novel example for long-range cis-regulatory mutations of SOX9.

Effects of possible endocrine disruptors on MyD88-independent TLR4 signaling.

Endocrine disrupting chemicals (EDCs) may potentially worsen infectious diseases because EDCs disturb human immune function by interfering with endocrine balance. To evaluate the influence of EDCs on the innate immune function of macrophages, we investigated the effects of 37 possible EDCs on lipopolysaccharide-induced activation of the IFN-beta promoter. Alachlor, atrazine, benomyl, bisphenol A, carbaryl, diethyl phthalate, dipropyl phthalate, kelthane, kepone, malathion, methoxychlor, octachlorostyrene, pentachlorophenol, nonyl phenol, p-octylphenol, simazine and ziram all inhibited the activation. Kepone and ziram showed strong inhibitory effects. Aldicarb, amitrole, benzophenone, butyl benzyl phthalate, 2,4-dichlorophenoxy acetic acid, dibutyl phthalate, 2,4-dichlorophenol, dicyclohexyl phthalate, diethylhexyl adipate, diethylhexyl phthalate, dihexyl phthalate, di-n-pentyl phthalate, methomyl, metribuzin, nitrofen, 4-nitrotoluene, permethrin, trifluralin, 2,4,5-trichlorophenoxyacetic acid and vinclozolin had no significant effects at 100 muM. These results indicate that some agrochemicals and resin-related chemicals may potentially inhibit macrophage function, which suggests that endocrine disruptors may influence the development of infectious diseases.

Effects of possible endocrine disrupting chemicals on bacterial component-induced activation of NF-kappaB.

Endocrine disrupting chemicals (EDCs) have a possibility to exacerbate infectious diseases because EDCs disturb the human immune system by interfering with endocrine balance. To assess the influence of EDCs on the innate immune function of macrophages, we investigated the effects of thirty-seven possible endocrine disruptors on lipopolysaccharide (LPS)- or bacterial lipopeptide (Pam3CSK4)-induced activation of nuclear factor kappa B (NF-kappaB). Alachlor, benomyl, bisphenol A, carbaryl, kelthane, kepone, octachlorostyrene, pentachlorophenol, nonyl phenol, p-octylphenol and ziram inhibited both LPS- and Pam3CSK4-induced activation of NF-kappaB. Simazine inhibited only LPS-induced activation. A strong inhibitory effect was observed with ziram and benomyl. On the other hand, diethylhexyl adipate and 4-nitrotoluene tended to enhance the activation induced by Pam3CSK4 and LPS, respectively. Aldicarb, amitrole, atrazine, benzophenone, butyl benzyl phthalate, 2,4-dichlorophenoxy acetic acid, dibutyl phthalate, 2,4-dichlorophenol, dicyclohexyl phthalate, diethylhexyl phthalate, diethyl phthalate, dihexyl phthalate, di-n-pentyl phthalate, dipropyl phthalate, malathion, methomyl, methoxychlor, metribuzin, nitrofen, permethrin, trifluralin, 2,4,5-trichlorophenoxyacetic acid and vinclozolin had no significant effects at 100 microM. These results indicate that some agrochemicals have the potential to inhibit macrophage function and suggest that endocrine disruptors may influence the development of bacterial infections.