Exome-wide benchmark of difficult-to-sequence regions using short-read next-generation DNA sequencing.

Next-generation DNA sequencing (NGS) in short-read mode has recently been used for genetic testing in various clinical settings. NGS data accuracy is crucial in clinical settings, and several reports regarding quality control of NGS data, primarily focusing on establishing NGS sequence read accuracy, have been published thus far. Variant calling is another critical source of NGS errors that remains unexplored at the single-nucleotide level despite its established significance. In this study, we used a machine-learning-based method to establish an exome-wide benchmark of difficult-to-sequence regions at the nucleotide-residue resolution using 10 genome sequence features based on real-world NGS data accumulated in The Genome Aggregation Database (gnomAD) of the human reference genome sequence (GRCh38/hg38). The newly acquired metric, designated the 'UNMET score,' along with additional lines of structural information from the human genome, allowed us to assess the sequencing challenges within the exonic region of interest using conventional short-read NGS. Thus, the UNMET score could provide a basis for addressing potential sequential errors in protein-coding exons of the human reference genome sequence GRCh38/hg38 in clinical sequencing.

Isolated Chronic Mucocutaneous Candidiasis due to a Novel Duplication Variant of IL17RC.

PURPOSE: Inborn errors of the IL-17A/F-responsive pathway lead to chronic mucocutaneous candidiasis (CMC) as a predominant clinical phenotype, without other significant clinical manifestations apart from mucocutaneous staphylococcal diseases. Among inborn errors affecting IL-17-dependent immunity, autosomal recessive (AR) IL-17RC deficiency is a rare disease with only three kindreds described to date. The lack of an in vitro functional evaluation system of IL17RC variants renders its diagnosis difficult. We sought to characterize a 7-year-old Japanese girl with CMC carrying a novel homozygous duplication variant of IL17RC and establish a simple in vitro system to evaluate the impact of this variant. METHODS: Flow cytometry, qPCR, RNA-sequencing, and immunoblotting were conducted, and an IL17RC-knockout cell line was established for functional evaluation. RESULTS: The patient presented with oral and mucocutaneous candidiasis without staphylococcal diseases since the age of 3 months. Genetic analysis showed that the novel duplication variant (Chr3: 9,971,476-9,971,606 dup (+131bp)) involving exon 13 of IL17RC results in a premature stop codon (p.D457Afs*16 or p.D457Afs*17). Our functional evaluation system revealed this duplication to be loss-of-function and enabled discrimination between loss-of-function and neutral IL17RC variants. The lack of response to IL-17A by the patient's SV40-immortalized fibroblasts was restored by introducing WT-IL17RC, suggesting that the genotype identified is responsible for her clinical phenotype. CONCLUSIONS: The clinical and cellular phenotype of the current case of AR IL-17RC deficiency supports a previous report on this rare disorder. Our newly established evaluation system will be useful for the diagnosis of AR IL-17RC deficiency, providing accurate validation of unknown IL17RC variants.

Isolated chronic mucocutaneous candidiasis due to a novel duplication variant of IL17RC.

Purpose: Inborn errors of the IL-17A/F-responsive pathway lead to chronic mucocutaneous candidiasis (CMC) as a predominant clinical phenotype, without other significant clinical manifestations apart from mucocutaneous staphylococcal diseases. Amongst inborn errors affecting IL-17-dependent immunity, autosomal recessive (AR) IL-17RC deficiency is a rare disease with only three kindreds described to date. The lack of an in vitro functional evaluation system of IL17RC variants renders its diagnosis difficult. We sought to characterize a seven-year-old Japanese girl with CMC carrying a novel homozygous duplication variant of IL17RC and establish a simple in vitro system to evaluate the impact of this variant. Methods: Flow cytometry, qPCR, RNA-sequencing, and immunoblotting were conducted, and an IL17RC-knockout cell line was established for functional evaluation. Results: The patient presented with oral and mucocutaneous candidiasis without staphylococcal diseases since the age of three months. Genetic analysis showed that the novel duplication variant (Chr3: 9,971,476-9,971,606 dup (+ 131bp)) involving exon 13 of IL17RC results in a premature stop codon (p.D457Afs*16 or p.D457Afs*17). Our functional evaluation system revealed this duplication to be loss-of-function and enabled discrimination between loss-of-function and neutral IL17RC variants. The lack of response to IL-17A by the patient's SV40-immortalized fibroblasts was restored by introducing WT-IL17RC, suggesting that the genotype identified is responsible for her clinical phenotype. Conclusions: The clinical and cellular phenotype of the current case of AR IL-17RC deficiency supports a previous report on this rare disorder. Our newly established evaluation system will be useful for diagnosis of AR IL-17RC deficiency, providing accurate validation of unknown IL17RC variants.

A case of VEXAS syndrome (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) with decreased oxidative stress levels after oral prednisone and tocilizumab treatment.

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome has recently been described as an autoinflammatory disease associated with severe adult-onset inflammatory manifestations. The various clinical manifestations include recurrent high-grade fever, neutrophilic dermatoses, cutaneous vasculitis, chondritis of the ear and nose, pulmonary infiltrates, cytopenia, uveitis, gastrointestinal pain or inflammation, aortitis, hepatosplenomegaly, and hematological disorders. VEXAS syndrome is caused by somatic mutations of the ubiquitin-like modifier activating enzyme 1 (UBA1) gene in myeloid-lineage cells. It is characterized by vacuolated myeloid and erythroid progenitor cells seen by bone marrow biopsy. We report the case of a 64-year-old Japanese man with VEXAS syndrome. At age 63, he was referred to us with a recurrent erythema on the hands associated with a general fever of 38-40°C that had persisted for 4 or 5 days and had recurred about once a month for a year. The skin rash appeared 2 or 3 days after the onset of each fever episode. Computed tomography (CT) of the chest revealed bilateral hilar lymphadenopathy (BHL), and the mediastinal lymph nodes were swollen. Sarcoidosis was suspected but was ruled out by several tests. Laboratory examinations showed elevated inflammatory markers. Bone marrow examination showed the vacuolization of myeloid precursor cells. A skin biopsy revealed dense dermal, predominantly perivascular, infiltrates. These consisted of mature neutrophils admixed with myeloperoxidase-positive CD163-positive myeloid cells, lymphoid cells and eosinophils. Sequencing analysis identified the somatic UBA1 variant c.122T > C, which results in p.Met41Thr. Treatment with oral prednisone (15 mg/day) and monthly intravenous tocilizumab injections (400 mg) completely resolved the symptoms. Neutrophils are a major source of reactive oxygen species, and the present case demonstrated numerous neutrophilic infiltrates. We hypothesize that the patient might have had elevated derivatives of reactive oxygen metabolites (d-ROMs). d-ROM quantification is a simple method for detecting hydroperoxide levels, and clinical trials have proven it useful for evaluating oxidative stress. In this study, we measured serum d-ROM before and after oral prednisone and tocilizumab treatment. The levels decreased significantly during treatment.

Clinical characteristics and treatment of 50 cases of Blau syndrome in Japan confirmed by genetic analysis of the NOD2 mutation.

OBJECTIVES: To collect clinical information and NOD2 mutation data on patients with Blau syndrome and to evaluate their prognosis. METHODS: Fifty patients with NOD2 mutations were analysed. The activity of each NOD2 mutant was evaluated in HEK293 cells by reporter assay. Clinical information was collected from medical records through the attending physicians. RESULTS: The study population comprised 26 males and 24 females aged 0-61 years. Thirty-two cases were sporadic, and 18 were familial from 9 unrelated families. Fifteen different mutations in NOD2 were identified, including 2 novel mutations (p.W490S and D512V); all showed spontaneous nuclear factor kappa B activation, and the most common mutation was p.R334W. Twenty-six patients had fever at relatively early timepoints in the disease course. Forty-three of 47 patients had a skin rash. The onset of disease in 9 patients was recognised after BCG vaccination. Forty-five of 49 patients had joint lesions. Thirty-eight of 50 patients had ocular symptoms, 7 of which resulted in blindness. After the diagnosis of Blau syndrome, 26 patients were treated with biologics; all were antitumour necrosis factor agents. Only 3 patients were treated with biologics alone; the others received a biologic in combination with methotrexate and/or prednisolone. None of the patients who became blind received biologic treatment. CONCLUSIONS: In patients with Blau syndrome, severe joint contractures and blindness may occur if diagnosis and appropriate treatment are delayed. Early treatment with a biologic agent may improve the prognosis.

SOCS3 Expressed in M2 Macrophages Attenuates Contact Hypersensitivity by Suppressing MMP-12 Production.

Numerous studies have clarified the immunological mechanisms of contact hypersensitivity (CHS). In addition, we have recently shown that M2 macrophages play key roles in the development of CHS by producing matrix metalloproteinase-12 (MMP-12). However, regulatory mechanisms of the elicitation phase in CHS remain largely unknown. To determine the roles of suppressor of cytokine signaling (SOCS) family members in M2 macrophages in the regulation of CHS, we investigated the expression of SOCS family members in M2 macrophages at the inflammatory sites of CHS. Transcriptome analysis revealed that among SOCS family members, SOCS3 was highly expressed in M2 macrophages at the site of CHS, and SOCS3 induction was reduced by IFN-? neutralization. 2,4-Dinitrofluorobenzene-induced CHS was significantly enhanced and prolonged in mice lacking SOCS3 expression in monocytes/macrophages (SOCS3(?/?) mice) compared with that in control mice. Importantly, expression of MMP-12 in M2 macrophages was significantly increased in SOCS3(?/?) mice at the site of CHS, and deletion of the MMP-12 gene reduced the exacerbated CHS in SOCS3(?/?) mice. Finally, IFN-? inhibited IL-4-induced MMP-12 expression in a SOCS3-dependent manner. Taken together, these results suggest that SOCS3 expressed in M2 macrophages is involved in the attenuation and/or resolution of CHS, presumably by suppressing MMP-12 production.

Role of Bcl-3 in the development of follicular helper T cells and in the pathogenesis of rheumatoid arthritis.

OBJECTIVE: We have previously shown that expression of the Bcl-3 gene, a member of the IκB family, is down-regulated in CD4+ T cells from patients with rheumatoid arthritis (RA) following tocilizumab therapy. The objective of this study was to examine the role of Bcl-3 in the pathogenesis of RA. METHODS: DNA microarray analysis was used to compare the signal intensity of Bcl-3 in CD4+ T cells from untreated RA patients and healthy controls. We examined the roles of interleukin-6 (IL-6)/STAT-3 signaling in the induction of Bcl-3. In addition, we analyzed the gene expression profiles of Bcl-3-transduced CD4+ T cells by RNA sequencing. The effects of enforced expression as well as gene silencing of Bcl-3 on the development of follicular helper T (Tfh) cells were evaluated. Finally, we examined correlations between the signal intensities of Bcl-3 and Tfh cell-related genes in CD4+ T cells from untreated RA patients. RESULTS: Bcl-3 levels were significantly higher in RA patients than in healthy controls. IL-6 induced Bcl-3 expression in CD4+ T cells in a STAT-3-dependent manner. Transcriptome analysis revealed that the expression of Bcl-6, a master regulator of Tfh cell differentiation, was significantly up-regulated by the enforced Bcl-3 expression. The enforced Bcl-3 expression increased, but Bcl-3 silencing decreased, the numbers of IL-21-producing Tfh-like cells. Bcl-3 levels in CD4+ T cells from RA patients correlated positively with the levels of Tfh cell-related genes CXCR5, inducible costimulator, and achaete-scute homolog 2. CONCLUSION: Bcl-3 is involved in the development of Tfh cells and the pathogenesis of RA, presumably by inducing IL-21 production.

Roles of mast cells in the pathogenesis of inflammatory myopathy.

INTRODUCTION: In addition to the pivotal roles of mast cells in allergic diseases, recent data suggest that mast cells play crucial roles in a variety of autoimmune responses. However, their roles in the pathogenesis of autoimmune skeletal muscle diseases have not been clarified despite their distribution in skeletal muscle. Therefore, the objective of this study is to determine the roles of mast cells in the development of autoimmune skeletal muscle diseases. METHODS: The number of mast cells in the affected muscle was examined in patients with dermatomyositis (DM) or polymyositis (PM). The susceptibility of mast cell-deficient WBB6F1-Kit(W)/Kit(Wv) mice (W/W(v) mice) to a murine model of polymyositis, C protein-induced myositis (CIM), was compared with that of wild-type (WT) mice. The effect of mast cell reconstitution with bone marrow-derived mast cells (BMMCs) on the susceptibility of W/W(v) mice to CIM was also evaluated. RESULTS: The number of mast cells in the affected muscle increased in patients with PM as compared with patients with DM. W/W(v) mice exhibited significantly reduced disease incidence and histological scores of CIM as compared with WT mice. The number of CD8⁺ T cells and macrophages in the skeletal muscles of CIM decreased in W/W(v) mice compared with WT mice. Engraftment of BMMCs restored the incidence and histological scores of CIM in W/W(v) mice. Vascular permeability in the skeletal muscle was elevated in WT mice but not in W/W(v) mice upon CIM induction. CONCLUSION: Mast cells are involved in the pathogenesis of inflammatory myopathy.

Prediction of relapse after discontinuation of biologic agents by ultrasonographic assessment in patients with rheumatoid arthritis in clinical remission: high predictive values of total gray-scale and power Doppler scores that represent residual synovial inflammation before discontinuation.

OBJECTIVE: This prospective study aimed to determine whether the comprehensive ultrasonographic assessment of synovial inflammation predicts relapse after discontinuation of treatment with a biologic agent in patients with rheumatoid arthritis (RA) in clinical remission. METHODS: RA patients in clinical remission (Disease Activity Score in 28 joints [DAS28] <2.6) receiving treatment with a biologic agent who agreed to discontinue the treatment were recruited. Patients underwent a comprehensive ultrasound scan on 134 synovial sites in 40 joints and were prospectively followed up for 6 months. Physicians who evaluated the patients during the study period were blinded to the baseline ultrasound findings. RESULTS: Forty-two patients receiving either a tumor necrosis factor antagonist or tocilizumab were enrolled. Using the optimal cutoff values determined by receiver operating characteristic curve analysis, relapse rates were significantly higher in patients whose total ultrasound scores at discontinuation were high than in those whose total ultrasound scores were low (P < 0.001 for both total gray-scale and power Doppler scores), whereas the difference between high and low DAS28 was not statistically significant (P = 0.158 by log rank test). Positive and negative predictive values were 80.0% and 73.3% for the total gray-scale score and 88.9% and 74.2% for the total power Doppler score, respectively. CONCLUSION: In RA patients in clinical remission receiving treatment with a biologic agent, residual synovial inflammation determined by comprehensive ultrasound assessment predicted relapse within a short term after discontinuation of the treatment. Our data provide a rationale and groundwork to conduct a large-scale study for establishment of ultrasound-based strategies to optimize the period of treatment with a biologic agent.

Efficacy of abatacept for arthritis in patients with an overlap syndrome between rheumatoid arthritis and systemic lupus erythematosus.

INTRODUCTION: This study aimed to investigate the efficacy of abatacept for arthritis in patients with rhupus, an overlap syndrome between rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). METHODS: Patients who fulfilled both the 2010 ACR/EULAR criteria for RA classification and the 1997 ACR revised criteria for classification of SLE and received abatacept treatment for arthritis were retrospectively studied. RESULTS: Six rhupus patients who fulfilled the inclusion criteria above were identified. All patients had active arthritis despite receiving antirheumatic drugs including methotrexate when abatacept was initiated. Clinical Disease Activity Index (CDAI) significantly decreased between baseline and 12 weeks (P = 0.028) and remained low through 24 weeks. All patients achieved either a good or moderate response according to the EULAR response criteria at 24 weeks. Health Assessment Questionnaire-Disability Index (HAQ-DI) also significantly decreased between baseline and 24 weeks (P = 0.043). In addition, the levels of immunoglobulin G and anti-DNA antibody significantly decreased between baseline and 24 weeks (P = 0.028 and P = 0.043, resp.). CONCLUSIONS: Treatment with abatacept is likely to be efficacious in patients with rhupus whose arthritis is refractory to methotrexate. In addition, abatacept may have a moderate effect on abnormal antibody production in rhupus patients.

Role of calcium ionophore A23187-induced activation of IkappaB kinase 2 in mast cells.

BACKGROUND: Mast cells are known to play a pivotal role in allergic diseases by releasing granules containing histamine and other preformed chemical mediators. Cross-linking of high-affinity receptors for IgE (FcεRI) on mast cells results in rapid increases in intracellular free calcium concentration [Ca(2+)]i and consequent activation of many transcription factors, including NFAT, NF-κB, JNK and CREB. Ca(2+) signaling is essential for many cellular activities such as proliferation, gene expression and degranulation in mast cells. In addition to Ca(2+) signaling, previous reports have shown that IkappaB kinase 2 (IKK2 or IKKß), a central component of the IKK complex mediating NF-κB activation, also plays a crucial role in FcεRI-mediated degranulation and cytokine production. Moreover, it has been demonstrated that activation of PKCß, a calcium-dependent PKC isoform, leads to IKK2 activation in many cell types. However, the roles of Ca(2+) signaling and PKCß in the activation of IKK2 in mast cells remain largely unknown. METHODS: We investigated the effect of PKC inhibitor Gö6976 on calcium ionophore A23187-induced activation of IKK2 in mast cells. We also examined the role of IKK2 in A23187-induced NF-κB-dependent gene induction, degranulation, proinflammatory cytokine production and extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation by using IKK2-deficient (IKK2(-/-)) fetal liver-derived mast cells (FLMCs). RESULTS: A23187 activated IKK2 and NF-κB even in the presence of Gö6976 in mast cells. A23187-induced degranulation, cytokine production and activation of ERK1/2 were diminished in IKK2(-/-) FLMCs compared to those in wild-type FLMCs. CONCLUSIONS: Ca(2+)-IKK2 signaling is involved in the degranulation and cytokine production in activated mast cells by a mechanism independent of PKCß.

Therapeutic potential of B and T lymphocyte attenuator expressed on CD8+ T cells for contact hypersensitivity.

In the past decade, mechanisms underlying allergic contact dermatitis have been intensively investigated by using contact hypersensitivity (CHS) models in mice. However, the regulatory mechanisms, which could be applicable for the treatment of allergic contact dermatitis, are still largely unknown. To determine the roles of B and T lymphocyte attenuator (BTLA), a CD28 family coinhibitory receptor, in hapten-induced CHS, BTLA-deficient (BTLA(-/-)) mice and littermate wild-type (WT) mice were subjected to DNFB-induced CHS, severe combined immunodeficient (SCID) mice were injected with CD4(+) T cells, and CD8(+) T cells from either WT mice or BTLA(-/-) mice were subjected to CHS. BTLA(-/-) mice showed enhanced DNFB-induced CHS and proliferation and IFN-γ production of CD8(+) T cells as compared with WT mice. SCID mice injected with WT CD4(+) T cells and BTLA(-/-) CD8(+) T cells exhibited more severe CHS as compared with those injected with WT CD4(+) T cells and WT CD8(+) T cells. On the other hand, SCID mice injected with BTLA(-/-) CD4(+) T cells and WT CD8(+) T cells exhibited similar CHS to those injected with WT CD4(+) T cells and WT CD8(+) T cells. Finally, to evaluate the therapeutic potential of an agonistic agent for BTLA on CHS, the effects of an agonistic anti-BTLA antibody (6A6) on CHS were examined. In vivo injection of 6A6 suppressed DNFB-induced CHS and IFN-γ production of CD8(+) T cells. Taken together, these results suggest that stimulation of BTLA with agonistic agents has therapeutic potential in CHS.