Genetic Rare Variants Affecting Multiple Pathways in Japanese Patients with Palindromic Rheumatism.

Palindromic rheumatism (PR) is a type of cryptogenic paroxysmal arthritis. Several genes may be involved in PR pathogenesis; however, conducting comprehensive case-control genetic studies for PR poses challenges owing to its rarity as a disease. Moreover, case-control studies may overlook rare variants that occur infrequently but play a significant role in pathogenesis. This study aimed to identify disease-related genes in Japanese patients with PR using whole-genome sequencing (WGS) and rare-variant analysis. Genomic DNA was obtained from two familial cases and one sporadic case, and it was subjected to WGS. WGS data of 104 healthy individuals obtained from a public database were used as controls. We performed data analysis for rare variants on detected variants using SKAT-O, KBAC, and SKAT, and subsequently defined significant genes. Significant genes combined with variants shared between the cases were defined as disease-related genes. We also performed pathway analysis for disease-related genes using Reactome. We identified 2,695,244 variants shared between cases; after excluding polymorphisms and noise, 74,640 variants were detected. We identified 540 disease-related genes, including 1,893 variants. Furthermore, we identified 32 significant pathways. Our results indicate that the detected genes and pathways in this study may be involved in PR pathogenesis.

Expression of ASC splice variant found in Japanese patients with palindromic rheumatism is regulated by rs8056505 single nucleotide polymorphism and interleukin-1 beta.

BACKGROUND: Palindromic rheumatism (PR) is an infrequent form of periodic arthritis. Based on the similarity of the pathogenesis of PR to autoinflammatory syndromes, we previously found that the dominant-active splice variant of the inflammasome adaptor protein, apoptosis-associated speck-like protein containing a CARD (ASC), which lacks exon 2 (Δexon2), is expressed in Japanese patients with PR. OBJECTIVE: Elucidation of the mechanism of Δexon2 ASC production and the effect of IL-1ß on splicing. METHODS: The genomic DNA of Japanese patients with PR was sequenced. The effect of the observed single nucleotide polymorphisms (SNPs) on ASC splicing was determined via exon trapping using THP-1 cells stimulated with interleukin-1 beta (IL-1ß) or ceramide. To investigate the genes that affect alternative splicing via IL-1ß, we analyzed the transcriptome of IL-1ß-treated THP-1 cells using RNA sequencing. RESULTS: We found the rs8056505 A->G SNP located in the 5'-untranslated region of the genomic ASC gene in patients and that Δexon2 expression was induced by this SNP, whereas it was suppressed by IL-1ß or ceramide. We detected 131,426 transcripts and identified 52 differentially expressed genes (DEGs) consisting of 41 downregulated genes and 11 upregulated genes in IL-1ß-stimulated THP-1 cells. The splicing-related gene MASCRNA was the most significantly induced gene by IL-1ß. CONCLUSIONS: We propose a cyclic expression model in which ASC alternates between wild-type and Δexon2 expression regulated by the rs8056505 G allele and splicing factors induced by IL-1ß. This cycle may be correlated with the formation of periodic PR pathologies.

Expression of a PYCARD/ASC variant lacking exon 2 in Japanese patients with palindromic rheumatism increases interleukin-1ß secretion.

BACKGROUND: Palindromic rheumatism (PR) is a rare periodic arthritis characterized by relapsing short episodes of arthritis. Although the pathogenesis of PR is still unclear, the clinical condition is similar to that of autoinflammatory diseases caused by dysregulation of inflammasome-related genes. OBJECTIVE: We analyzed the inflammasome adapter PYD and CARD domain-containing protein/apoptosis-associated speck-like protein containing a CARD (PYCARD/ASC) in Japanese patients with PR. METHODS: Serum interleukin (IL)-1ß concentrations in three Japanese patients with PR were measured. We also cloned PYCARD/ASC cDNA variants and expressed them in THP-1 cells to determine their effects on inflammasome activity following stimulation with phorbol 12-myristate 13-acetate and monosodium urate. Lysates of recombinant THP-1 cells were subjected to co-immunoprecipitation assays. RESULTS: Serum IL-1ß concentrations were significantly elevated in patients with PR, and a splice variant of PYCARD/ ASC mRNA lacking exon 2 (Δexon2) was dominantly expressed compared with that in controls. Moreover, IL-1ß secretion was significantly increased in THP-1 cells expressing Δexon2PYCARD/ASC compared with that in cells expressing the wild-type protein. The amount of NLRP3 bound to Δexon2PYCARD/ASC was increased after stimulation, whereas that bound to the wild-type protein was decreased. There were no differences in caspase-1 binding. CONCLUSIONS: Δexon2 PYCARD/ASC was associated with the pathogenesis of PR.

CD44v8-10 and CD44s Are Age-dependently Expressed in Primary Cultured Papillary Thyroid Carcinoma Cells and Are Associated with Cell Proliferation.

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, arising from follicular cells, and accounts for more than 80% of all thyroid malignant tumors. Although age is the strongest prognostic factor of PTC, and various cut-off ages (40-55 years) were suggested in previous studies, the molecular mechanisms causing age-related changes of PTC cell proliferation remain unclear. CD44 is a major cell surface receptor for hyaluronate and is known as a cancer stem cell marker. However, the association between CD44 and PTC is still unknown. Therefore, we determined the proliferation of primary cultured cells obtained from patients with PTC, and the CD44 mRNA expression profile to elucidate age-related association of CD44 with PTC. The results showed that cell proliferation was significantly decreased according to age. We also found that CD44v8-10 and CD44 splice variants were expressed dominantly in patients with PTC. Moreover, the CD44v8-10/CD44s mRNA expression ratio was significantly increased according to age, and there was a significant negative correlation between this expression ratio and cell proliferation. Our findings suggest that the CD44v8-10/CD44s expression ratio in PTC cells is useful for screening for aggressive PTC and may provide clinically valuable information.

A variant of death-receptor 3 associated with rheumatoid arthritis interferes with apoptosis-induction of T cell.

Rheumatoid arthritis (RA) is a chronic polyarthritis of unknown etiology. To unravel the molecular mechanisms in RA, we performed targeted DNA sequencing analysis of patients with RA. This analysis identified a variant of the death receptor 3 (DR3) gene, a member of the family of apoptosis-inducing Fas genes, which contains four single-nucleotide polymorphisms (SNPs) and a 14-nucleotide deletion within exon 5 and intron 5. We found that the deletion causes the binding of splicing regulatory proteins to DR3 pre-mRNA intron 5, resulting in a portion of intron 5 becoming part of the coding sequence, thereby generating a premature stop codon. We also found that this truncated DR3 protein product lacks the death domain and forms a heterotrimer complex with wildtype DR3 that dominant-negatively inhibits ligand-induced apoptosis in lymphocytes. Myelocytes from transgenic mice expressing the human DR3 variant produced soluble truncated DR3, forming a complex with TNF-like ligand 1A (TL1A), which inhibited apoptosis induction. In summary, our results reveal that a DR3 splice variant that interferes with ligand-induced T cell responses and apoptosis may contribute to RA pathogenesis.

Electrochemical Synthesis of Dihydrobenzofurans and Evaluation of Their Insect Antifeedant Activities.

Electrochemically synthesized dihydrobenzofurans were evaluated for their insect antifeedant activities against phytophagous insects. They were prepared through the coupling reactions of various alkenes with a phenoxy cation generated by oxidation near the cathode in the electrolytic reaction. The insect antifeedant activities of these synthetic dihydrobenzofurans were evaluated in the common cutworm (Spodoptera litura) and diamond back moth (Plutella xylostella) with the dual choice leaf disk bioassay method. The insect antifeedant activities of most of the acetophenone-type dihydrobenzofurans were strong, while those of derivatives with a t-butyl group were weaker. The biological activities in insect species differed with the structural features of the compounds.

Role of the MICA polymorphism in systemic lupus erythematosus.

OBJECTIVE: To study the genetic contribution of major histocompatibility complex class I polypeptide-related sequence A (MICA), important in natural killer (NK) cell function, in patients with systemic lupus erythematosus (SLE). METHODS: Japanese patients with SLE (n=716), those with rheumatoid arthritis (RA) (n=327), and healthy control subjects (n=351) were genotyped for the Val129 Met polymorphism (rs1051792) and transmembrane (TM) alanine-encoding GCT repeats, termed A4, A5, A5.1, A6, and A9, in the MICA gene. Recombinant human MICA-GST fusion proteins were tested on the NK cell line NK92MI for the expression of NK group 2, member D (NKG2-D), NK cell-mediated cytotoxicity, and interferon-γ (IFNγ) production. RESULTS: The MICA 129Met allele, TMA9 allele, and 129Met/Met genotype were positively associated with SLE (corrected P [Pcorr]=0.01 and odds ratio [OR] 1.3, Pcorr=0.003 and OR 1.6, and Pcorr=0.02 and OR 1.8, respectively), while the MICA 129Val allele was negatively associated with SLE (Pcorr=0.01, OR 0.8). The MICA 129Met;A9 haplotype was also associated with SLE (Pcorr=0.0006, OR 1.8), and there was an additive genetic effect between the MICA 129Met;A9 haplotype and HLA-DRB1*15:01. When NK92MI cells were incubated in vitro with recombinant human disease-associated 129Met;A9 (the combination of polymorphisms at 129Met and TMA9), expression of NKG2-D on NK92MI cells and cytotoxicity of the NK cells were inhibited, but production of IFNγ from NK92MI cells was enhanced. CONCLUSION: The MICA polymorphism is genetically associated with SLE, and MICA appears to contribute to the pathogenesis of SLE by modulating NK cell function.

Mathematical basis of affected sib-pair analysis: an extension to X-linked loci.

Most clinical genetic studies are done without knowing their mathematical basis. However, because the results of such studies rely on the correctness of the mathematical calculations involved, we cannot ignore the mathematics of genetic studies. In this study, the mathematical basis of the sib-pair method, which has been widely used in recent genome-wide studies, was extended to studies focusing on the X chromosome, and then applied to study a clinical microsatellite marker on the X chromosome. Our calculation involves classifying marker types on the X chromosome for an affected sib-pair and an unaffected sib, together with their sexual information and the possible parental marker types applicable to a genome-wide genetic study. The method proposed in this study was then applied to 41 Japanese rheumatoid families, and the locus DXS984 was found to be most likely to be linked with rheumatoid arthritis (RA). This locus, which we found nicely, was also highlighted in a previous study that used the Mapmaker/sibs program, so we can conclude that our calculation provides a solid foundation for understanding and confirming the results obtained using the sib-pair method.

Mammalian clock gene Cryptochrome regulates arthritis via proinflammatory cytokine TNF-alpha.

The mammalian clock genes, Period and Cryptochrome (Cry), regulate circadian rhythm. We show that circadian rhythmicity and rhythmic expression of Period in the nuclei of inflammatory synovial cells and spleen cells are disturbed in mouse models of experimental arthritis. Expressions of other clock genes, Bmal1 and Dbp, are also disturbed in spleen cells by arthritis induction. Deletion of Cry1 and Cry2 results in an increase in the number of activated CD3(+) CD69(+) T cells and a higher production of TNF-alpha from spleen cells. When arthritis is induced, Cry1(-/-)Cry2(-/-) mice develop maximal exacerbation of joint swelling, and upregulation of essential mediators of arthritis, including TNF-alpha, IL-1beta and IL-6, and matrix metalloproteinase-3. Wee-1 kinase is solely upregulated in Cry1(-/-)Cry2(-/-) mice, in line with upregulation of c-Fos and Wee-1 kinase in human rheumatoid arthritis. The treatment with anti-TNF-alpha Ab significantly reduced the severity and halted the progression of the arthritis of Cry1(-/-)Cry2(-/-) mice and vice versa, ectopic expression of Cry1 in the mouse embryonic fibroblast from Cry1(-/-)Cry2(-/-) mice significantly reduced the trans activation of TNF-alpha gene. Thus, the biological clock and arthritis influence each other, and this interplay can influence human health and disease.

Prospective study of methotrexate treatment for rheumatoid arthritis treated legitimately according to the government recommended 8 mg/week dose.

Clinical squeal of the treatment of rheumatoid arthritis patients with methotrexate (MTX) according to the Japanese government recommended dose of 8 mg/week was evaluated prospectively. A total of 176 patients with active RA attending Konan Kakogawa Hospital and Kobe University Hospital were enrolled. Patients' profile at the start of study was Class 2.0 +/- 1.1 and X-ray stage 2.6 +/- 1.0. The effects of MTX treatment were evaluated by the American College of Rheumatology (ACR) core set, disease activity score of 28 joints (DAS28), and European League Against Rheumatism (EULAR) response criteria. A modified Sharp method was used to evaluate the radiographs. The improvement in the clinical signs and symptoms of the ACR core set was maintained for a 24-month period (p < 0.05). The ACR20/50/70 and DAS28 were also improved at the 12- and 24-month assessments. However, 82 of 130 patients (63.5%) were found to be nonresponders at 24 months of MTX therapy, as evaluated by EULAR response criteria. The X-ray study showed that joint destruction progressed despite the treatment. Thus, long-term MTX treatment performed in accordance with the Japanese 8 mg/week regimen appears to be favorable in terms of the signs and symptoms of RA; however, it is clearly insufficient for and cannot halt the progression of rheumatic joint destruction.

Sjögren's syndrome patients presenting with hypergammaglobulinemia are relatively unresponsive to cevimeline treatment.

Sjögren's syndrome (SS) is a systemic autoimmune disease characterized by sicca symptoms, including dry eyes and dry mouth. Cevimeline is used for the treatment of dry mouth in patients with SS. Here we prospectively tested the clinical effectiveness of cevimeline at increasing saliva secretion in patients with SS, and the results were compared with the clinical parameters of the patients. Saliva secretion was increased >160% in 17 of 30 (56.7%) patients (P < 0.005). When the clinical parameters were compared between the patients who responded to cevimeline treatment and those who did not respond to the treatment, the frequency of patients presenting with hypergammaglobulinemia was significantly higher in the nonresponder group (P < 0.05). It thus appears that cevimeline is effective in SS patients with milder disease activity.

Angiopoietin 1 directly induces destruction of the rheumatoid joint by cooperative, but independent, signaling via ERK/MAPK and phosphatidylinositol 3-kinase/Akt.

OBJECTIVE: To determine whether angiopoietin 1 (Ang-1) potentiates overgrowth of the synovium and joint degradation in rheumatoid arthritis (RA), and to clarify the cell-signaling mechanisms of Ang-1 in the rheumatoid joint. METHODS: Expression of Ang-1, TIE-2 (a receptor for Ang-1), and matrix metalloproteinase 3 (MMP-3) was studied by immunohistochemistry. Activation of the ERK/MAPK and phosphatidylinositol (PI) 3-kinase/Akt pathways and of NF-kappaB was determined by Western blotting and an NF-kappaB p65 DNA binding activity assay, respectively. Induction of apoptosis was evaluated by nuclear staining, cell viability assay, and Western blotting of caspases. Synovial cell migration was evaluated by actin polymerization, Western blotting of Rho family proteins, and affinity purification with Rhotekin-Rho and p21-activated kinase 1. Matrix degradation was examined by induction of proMMP-3 secretion from synovial cells followed by in vitro cartilaginous matrix degradation assay. RESULTS: Ang-1 stimulated the ERK/MAPK and PI 3-kinase/Akt pathways in a cooperative but independent manner, which enhanced rheumatoid synovium overgrowth and joint destruction. In addition, Ang-1 activated NF-kappaB via Akt to promote cell growth, but also inhibited cell apoptosis via ERK and Akt. Ang-1 directly potentiated the extension of synovial cells in an ERK- and Akt-dependent manner by up-regulating Rho family proteins, which attenuated Rac signaling and led to membrane ruffling. Ang-1 induced proMMP-3 secretion from synovial cells, which resulted in direct degradation of the cartilaginous matrix. CONCLUSION: Ang-1 stimulates the ERK/MAPK and PI 3-kinase/Akt pathways cooperatively, but in a manner independent of each other, to directly potentiate synovium overgrowth and joint destruction in RA. In addition to inflammatory cytokines, Ang-1/TIE-2 signaling appears to be an independent factor that contributes to the destruction of the rheumatoid joint.

Synthesis and insect antifeedant activity of aurones against Spodoptera litura larvae.

A series of aurones were prepared from various phenols via phenoxy acetic acids and coumaranones and evaluated for insect antifeedant activity against the common cutworm (Spodoptera litura). The naturally occurring aurone was most active at an ED50 of 0.12 micromol/cm2. The synthetic precursor, coumaranones, showed that the introduction of methoxyl and methyl groups to the benzene ring increased insect antifeedant activity. Similarly, the tested aurones showed that the introduction of methoxyl group to the A and/or B rings increased the insect antifeedant activity, but 4,5,6- and 3',4',5'-trisubstituted compounds did not show this activity in this test. The hydroxylation of aurones in the B ring should be disadvantageous for insect antifeedant activity against S. litura. Although the melting points did not correlate well with the insect antifeedant activity, compounds that were nearly inactive had high melting points. A significant correlation was noted between biological activity (pED50) and a hydrogen-bonding parameter calculated from the Rf value obtained from SiOH thin-layer chromatography and a lipophilicity parameter (log k) calculated from the retention time in ODS high-performance liquid chromatography. The respective correlation coefficients (r) were -0.83 and -0.70. The introduction of alkoxy and alkyl groups along with adequate hydrogen bonding seems to contribute to the antifeedant activity of the compounds tested.

Insect antifeedants, pterocarpans and pterocarpol, in heartwood of Pterocarpus macrocarpus Kruz.

The insect antifeedant activities of pterocarpans and a sesquiterpene alcohol from the dichloromethane extract of Pterocarpus macrocarpus Kruz. (Leguminosae) were evaluated against the common cutworm, Spodoptera litura F. (Noctuidae), and the subterranean termite, Reticulitermes speratus (Kolbe)(Rhinotermitidae). Three pterocarpans, (-)-homopterocarpin (1), (-)-pterocarpin (2), and (-)-hydroxyhomopterocarpin (3) and the sesquiterpene alcohol, (+)-pterocarpol (5), were isolated from the dichloromethane extract of the heartwood of P. macrocarpus under guidance by a biological assay. Among these natural products, the most active insect antifeedant against both S. litura and R. speratus was 1. On the other hand, sesquiterpene alcohol 5 showed less insect antifeedant activity than the other pterocarpans against both insect species. While its methylated derivative, (-)-methoxyhomopterocarpin (4), showed high biological activity, 3 showed less insect antifeedant activity in this study. Interestingly, racemic 1 did not show insect antifeedant activity against S. litura. However, all of the test pterocarpans and isoflavones showed antifeedant activity against the test termites. Additionally, since these compounds were major constituents of P. macrocarpus, these antifeedant phenolics may act as chemical defense factors in this tree. In Thailand, lumber made from this tree is used to make furniture and in building construction due to its resistance to termite attack.

Hypermethylated promoter region of DR3, the death receptor 3 gene, in rheumatoid arthritis synovial cells.

OBJECTIVE: To examine the promoter activity and protein expression of the death receptor 3 gene DR3, a member of the apoptosis-inducing Fas gene family, with particular reference to the methylation status of its promoter region in rheumatoid arthritis (RA). METHODS: Genomic DNA was prepared from peripheral blood mononuclear cells obtained from healthy individuals and from patients with RA and synovial cells obtained from patients with RA and osteoarthritis. The methylation status of the DR3 promoter was analyzed by bisulfite genomic sequencing and methylation-specific polymerase chain reaction techniques. Gene promoter activity and protein expression were examined using the luciferase reporter and Western blotting techniques. RESULTS: The promoter region of the DR3 gene contained many CpG motifs, including one CpG island that was specifically hypermethylated in synovial cells from patients with RA. Promoter assays showed that the promoter CpG island was essential for the transactivation of the DR3 gene and that forced hypermethylation of the CpG island with the bacterial methylase Sss I in vitro resulted in inhibition of the DR3 gene expression. Furthermore, the expression of DR-3 protein was down-modulated in association with methylation of the promoter CpG island in RA synovial cells. CONCLUSION: The CpG island in the DR3 gene promoter was specifically methylated to down-modulate the expression of DR-3 protein in rheumatoid synovial cells, which may provide resistance to apoptosis in RA synovial cells.

Whole genome association study of rheumatoid arthritis using 27 039 microsatellites.

A major goal of current human genome-wide studies is to identify the genetic basis of complex disorders. However, the availability of an unbiased, reliable, cost efficient and comprehensive methodology to analyze the entire genome for complex disease association is still largely lacking or problematic. Therefore, we have developed a practical and efficient strategy for whole genome association studies of complex diseases by charting the human genome at 100 kb intervals using a collection of 27,039 microsatellites and the DNA pooling method in three successive genomic screens of independent case-control populations. The final step in our methodology consists of fine mapping of the candidate susceptible DNA regions by single nucleotide polymorphisms (SNPs) analysis. This approach was validated upon application to rheumatoid arthritis, a destructive joint disease affecting up to 1% of the population. A total of 47 candidate regions were identified. The top seven loci, withstanding the most stringent statistical tests, were dissected down to individual genes and/or SNPs on four chromosomes, including the previously known 6p21.3-encoded Major Histocompatibility Complex gene, HLA-DRB1. Hence, microsatellite-based genome-wide association analysis complemented by end stage SNP typing provides a new tool for genetic dissection of multifactorial pathologies including common diseases.

Biosynthesis of pyrethrin I in seedlings of Chrysanthemum cinerariaefolium.

The biosynthetic pathway to natural pyrethrins in Chrysanthemum cinerariaefolium seedlings was studied using [1-13C]d-glucose as a precursor, with pyrethrin I isolated using HPLC from a leaf extract. The 13C NMR spectrum of pyrethrin I from the precursor-administered seedlings indicated that the acid moiety was biosynthesized from d-glucose via 2-C-methyl-d-erythritol 4-phosphate, whereas the alcohol moiety was possibly biosynthesized from linolenic acid.

Inhibitory actions of several natural products on proliferation of rat vascular smooth muscle cells induced by Hsp60 from Chlamydia pneumoniae J138.

Atherosclerosis is a vascular disorder involving inflammation, a narrowed vascular lumen in the entire tunica intima, and reduced elasticity of the arterial wall. It has been found that Hsp60 from Chlamydia pneumoniae, an obligate bacterial pathogen associated with atheroma lesions, mimics human Hsp60, thereby causing attacks by immune cells on stressed endothelial cells expressing endogenous Hsp60 on their surface. Furthermore, Hsp60 from C. pneumoniae has been shown to promote the growth of vascular smooth muscle cells (VSMCs). To explore probes that can be used for studying signal transduction elicited by the chlamydial Hsp60, we have tested several natural products for their inhibitory actions on the Hsp60-induced proliferation of rat arterial smooth muscle cells. Sesamol, vanillyl alcohol, and trans-ferulic acid exhibited moderate inhibitory actions on the Hsp60-induced cell proliferation; zerumbone, humulene, and caryophylene effectively inhibited it at low concentrations with IC(50) values of 529, 122, and 110 nM, respectively. The results indicated that the 11-membered alicyclic ring is favorable for interactions with receptors involved in the Hsp60-induced VSMC proliferation.

Evaluation of calcium-alginate gel as an artificial diet medium for bioassays on common cutworms.

A calcium-alginate gel diet was developed for Spodoptera litura larvae, and its reliability as a carrier for incorporating antifeedants as well as insecticides was investigated. The alginate gel diet was prepared with a simple protocol, which does not involve any heating process. When tested using this diet, acephate, a Bacillus thuringiensis endotoxin formulation and rotenone reproducibly showed insecticidal activity against the larvae, while neem oil and scabequinone deterred the larval feeding effectively. However, not only the insecticidal activity of acephate but also the antifeedant activity of neem oil was reduced by replacing the alginate component by agar in the diet, suggesting the usefulness of the alginate gel diet as an assay tool for testing a broad range of samples against the larvae.