Gene-specific somatic epigenetic mosaicism of FDFT1 underlies a non-hereditary localized form of porokeratosis.

Porokeratosis is a clonal keratinization disorder characterized by solitary, linearly arranged, or generally distributed multiple skin lesions. Previous studies showed that genetic alterations in MVK, PMVK, MVD, or FDPS-genes in the mevalonate pathway-cause hereditary porokeratosis, with skin lesions harboring germline and lesion-specific somatic variants on opposite alleles. Here, we identified non-hereditary porokeratosis associated with epigenetic silencing of FDFT1, another gene in the mevalonate pathway. Skin lesions of the generalized form had germline and lesion-specific somatic variants on opposite alleles in FDFT1, representing FDFT1-associated hereditary porokeratosis identified in this study. Conversely, lesions of the solitary or linearly arranged localized form had somatic bi-allelic promoter hypermethylation or mono-allelic promoter hypermethylation with somatic genetic alterations on opposite alleles in FDFT1, indicating non-hereditary porokeratosis. FDFT1 localization was uniformly diminished within the lesions, and lesion-derived keratinocytes showed cholesterol dependence for cell growth and altered expression of genes related to cell-cycle and epidermal development, confirming that lesions form by clonal expansion of FDFT1-deficient keratinocytes. In some individuals with the localized form, gene-specific promoter hypermethylation of FDFT1 was detected in morphologically normal epidermis adjacent to methylation-related lesions but not distal to these lesions, suggesting that asymptomatic somatic epigenetic mosaicism of FDFT1 predisposes certain skin areas to the disease. Finally, consistent with its genetic etiology, topical statin treatment ameliorated lesions in FDFT1-deficient porokeratosis. In conclusion, we identified bi-allelic genetic and/or epigenetic alterations of FDFT1 as a cause of porokeratosis and shed light on the pathogenesis of skin mosaicism involving clonal expansion of epigenetically altered cells.

Exploring the genetic diversity of the Japanese population: Insights from a large-scale whole genome sequencing analysis.

The Japanese archipelago is a terminal location for human migration, and the contemporary Japanese people represent a unique population whose genomic diversity has been shaped by multiple migrations from Eurasia. We analyzed the genomic characteristics that define the genetic makeup of the modern Japanese population from a population genetics perspective from the genomic data of 9,287 samples obtained by high-coverage whole-genome sequencing (WGS) by the National Center Biobank Network. The dataset comprised populations from the Ryukyu Islands and other parts of the Japanese archipelago (Hondo). The Hondo population underwent two episodes of population decline during the Jomon period, corresponding to the Late Neolithic, and the Edo period, corresponding to the Early Modern era, while the Ryukyu population experienced a population decline during the shell midden period of the Late Neolithic in this region. Haplotype analysis suggested increased allele frequencies for genes related to alcohol and fatty acid metabolism, which were reported as loci that had experienced positive natural selection. Two genes related to alcohol metabolism were found to be 12,500 years out of phase with the time when they began to increase in the allele frequency; this finding indicates that the genomic diversity of Japanese people has been shaped by events closely related to agriculture and food production.

Chromosomal microdeletion leading to pituitary gigantism through hormone-gene overexpression.

Pituitary gigantism is a rare endocrinopathy characterized by tall stature due to growth hormone (GH) hypersecretion. This condition is generally linked to a genetic predisposition to tumors that produce GH or GH-releasing hormone (GHRH). Here, we report a Japanese woman who exhibited prominent body growth from infancy to reach an adult height of 197.4 cm (+7.4 standard deviation). Her blood GH levels were markedly elevated. She carried no pathogenic variants in known growth-controlling genes but had a hitherto unreported 752 kb heterozygous deletion at 20q11.23. The microdeletion was located 8.9 kb upstream of GHRH and encompassed exons 2-9 of a ubiquitously expressed gene TTI1 together with 12 other genes, pseudogenes and non-coding RNAs. Transcript analyses of the patient's leukocytes showed that the microdeletion produced chimeric mRNAs consisting of exon 1 of TTI1 and all coding exons of GHRH. In silico analysis detected promoter-associated genomic features around TTI1 exon 1. Genome-edited mice carrying the same microdeletion recapitulated accelerated body growth from a few weeks after birth. The mutant mice developed pituitary hyperplasia and exhibited ectopic Ghrh expression in all tissues examined. Thus, the extreme phenotype of pituitary gigantism in the patient likely reflects GHRH overexpression driven by an acquired promoter. The results of this study indicate that germline submicroscopic deletions have the potential to cause conspicuous developmental abnormalities due to gene overexpression. Furthermore, this study provides evidence that constitutive expression of a hormone-encoding gene can result in congenital disease.

Congenital anaemia associated with loss-of-function variants in DNA polymerase epsilon 1.

DNA polymerase epsilon (Pol ε), a component of the core replisome, is involved in DNA replication. Although genetic defects of Pol ε have been reported to cause immunodeficiency syndromes, its role in haematopoiesis remains unknown. Here, we identified compound heterozygous variants (p.[Asp1131fs];[Thr1891del]) in POLE, encoding Pol ε catalytic subunit A (POLE1), in siblings with a syndromic form of severe congenital transfusion-dependent anaemia. In contrast to Diamond-Blackfan anaemia, marked reticulocytopenia or marked erythroid hypoplasia was not found. Their bone marrow aspirates during infancy revealed erythroid dysplasia with strongly positive TP53 in immunostaining. Repetitive examinations demonstrated trilineage myelodysplasia within 2 years from birth. They had short stature and facial dysmorphism. HEK293 cell-based expression experiments and analyses of patient-derived induced pluripotent stem cells (iPSCs) disclosed a reduced mRNA level of Asp1131fs-POLE1 and defective nuclear translocation of Thr1891del-POLE1. Analysis of iPSCs showed compensatory mRNA upregulation of the other replisome components and increase of the TP53 protein, both suggesting dysfunction of the replisome. We created Pole-knockout medaka fish and found that heterozygous fishes were viable, but with decreased RBCs. Our observations expand the phenotypic spectrum of the Pol ε defect in humans, additionally providing unique evidence linking Pol ε to haematopoiesis.

Predictive factors and treatment outcomes associated with difficult-to-treat rheumatoid arthritis conditions: the ANSWER cohort study.

OBJECTIVES: To investigate the predictive factors for difficult-to-treat rheumatoid arthritis (D2T RA) and assess the efficacy of biological disease-modifying antirheumatic drugs (bDMARDs) and Janus kinase inhibitors (JAKi). METHODS: Retrospective analysis was conducted on data from the ANSWER cohort comprising 3,623 RA patients treated with bDMARDs or JAKi in Japan. Multivariate Cox proportional hazards modelling was used to analyse the hazard ratios (HRs) for treatment retention. RESULTS: Of these, 450 (12.4%) met the first two criteria of EULAR D2T RA definition (defined as D2T RA in this study). Factors contributing to D2T RA included age over 75 (compared to those under 65, HR = 0.46, 95% CI: 0.31 to 0.69), higher rheumatoid factor (RF) titres (HR = 1.005, 95% CI: 1.00 to 1.01), higher clinical disease activity index (HR = 1.02, 95% CI: 1.01 to 1.03), lower methotrexate dosage (HR = 0.97, 95% CI: 0.95 to 0.99), and comorbidities like hypertension (HR = 1.53, 95% CI: 1.2 to 1.95) and diabetes (HR = 1.37, 95% CI: 1.09 to 1.73). Anti-interleukin 6 receptor antibodies (aIL-6R, HR = 0.53, 95% CI: 0.37 to 0.75) and JAKi (HR = 0.64, 95% CI: 0.46 to 0.90) were associated with fewer discontinuations due to ineffectiveness compared to tumour necrosis factor inhibitors. Oral glucocorticoids usage (HR = 1.65, 95% CI: 1.11 to 2.47) was linked to increased discontinuation due to toxic adverse events. CONCLUSION: Younger onset, higher RF titres, and comorbidities predicted D2T RA development. For managing D2T RA, aIL-6R and JAKi exhibited superior drug retention.

Comparison of retention of biologics in Japanese patients with elderly-onset rheumatoid arthritis-the ANSWER cohort study.

OBJECTIVES: This multicentre, retrospective study aimed to compare retention and reasons for discontinuation between Janus kinase inhibitors (JAKi) and biologic disease-modifying antirheumatic drugs in patients with elderly-onset rheumatoid arthritis (EORA). METHODS: Patients with RA enrolled in a Japanese multicentre observational registry between 2015 and 2022 were included. EORA was defined as RA with onset at 60 or over. To adjust confounding factors by indication for initiation of tumor necrosis factor inhibitors (TNFi), interleukin-6 inhibitors (IL-6i), cytotoxic T-lymphocyte associated antigen 4 immunoglobulin (CTLA4-Ig) blockers, or JAKi, a propensity score based on baseline characteristics was used to compare drug retention. To assess the reasons for discontinuation, retention rates for ineffectiveness, adverse events, and remission were analyzed as secondary outcomes. RESULTS: A total of 572 patients with 835 treatment courses were identified (314 TNFi, 175 IL-6i, 228 CTLA4-Ig, and 118 JAKi). After adjusting for differences in baseline characteristics, drug retention was significantly higher for IL-6i (HR = 0.38, 95%CI = 0.27-0.55, p< 0.01) as compared with TNFi. Discontinuation due to lack of effectiveness was lower with the JAKi (HR = 0.38, 95%CI = 0.22-0.66, p< 0.01) and the IL-6i (HR = 0.29, 95%CI = 0.19-0.46, p< 0.01) as compared with the TNFi although the CTLA4-Ig had a similar HR to TNFi. The adjusted incidence of discontinuation due to adverse event was higher in the JAKi (HR = 2.86, 95%CI = 1.46-5.59, p< 0.01) than the TNFi. CONCLUSIONS: In EORA patients, IL-6i and JAKi had longer retention and less discontinuation due to ineffectiveness than TNFi. The potential risks of JAKi should be approached with an individualized perspective.

Systematic molecular analyses for 115 karyotypically normal men with isolated non-obstructive azoospermia.

STUDY QUESTION: Do copy-number variations (CNVs) in the azoospermia factor (AZF) regions and monogenic mutations play a major role in the development of isolated (non-syndromic) non-obstructive azoospermia (NOA) in Japanese men with a normal 46, XY karyotype? SUMMARY ANSWER: Deleterious CNVs in the AZF regions and damaging sequence variants in eight genes likely constitute at least 8% and approximately 8% of the genetic causes, respectively, while variants in other genes play only a minor role. WHAT IS KNOWN ALREADY: Sex chromosomal abnormalities, AZF-linked microdeletions, and monogenic mutations have been implicated in isolated NOA. More than 160 genes have been reported as causative/susceptibility/candidate genes for NOA. STUDY DESIGN, SIZE, DURATION: Systematic molecular analyses were conducted for 115 patients with isolated NOA and a normal 46, XY karyotype, who visited our hospital between 2017 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied 115 unrelated Japanese patients. AZF-linked CNVs were examined using sequence-tagged PCR and multiplex ligation-dependent probe amplification, and nucleotide variants were screened using whole exome sequencing (WES). An optimized sequence kernel association test (SKAT-O), a gene-based association study using WES data, was performed to identify novel disease-associated genes in the genome. The results were compared to those of previous studies and our in-house control data. MAIN RESULTS AND THE ROLE OF CHANCE: Thirteen types of AZF-linked CNVs, including the hitherto unreported gr/gr triplication and partial AZFb deletion, were identified in 63 (54.8%) cases. When the gr/gr deletion, a common polymorphism in Japan, was excluded from data analyses, the total frequency of CNVs was 23/75 (30.7%). This frequency is higher than that of the reference data in Japan and China (11.1% and 14.7%, respectively). Known NOA-causative AZF-linked CNVs were found in nine (7.8%) cases. Rare damaging variants in known causative genes (DMRT1, PLK4, SYCP2, TEX11, and USP26) and hemizygous/multiple-heterozygous damaging variants in known spermatogenesis-associated genes (TAF7L, DNAH2, and DNAH17) were identified in nine cases (7.8% in total). Some patients carried rare damaging variants in multiple genes. SKAT-O detected no genes whose rare damaging variants were significantly accumulated in the patient group. LIMITATIONS, REASONS FOR CAUTION: The number of participants was relatively small, and the clinical information of each patient was fragmentary. Moreover, the pathogenicity of identified variants was assessed only by in silico analyses. WIDER IMPLICATIONS OF THE FINDINGS: This study showed that various AZF-linked CNVs are present in more than half of Japanese NOA patients. These results broadened the structural variations of AZF-linked CNVs, which should be considered for the molecular diagnosis of spermatogenic failure. Furthermore, the results of this study highlight the etiological heterogeneity and possible oligogenicity of isolated NOA. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Grants from the Japan Society for the Promotion of Science (21K19283 and 21H0246), the Japan Agency for Medical Research and Development (22ek0109464h0003), the National Center for Child Health and Development, the Canon Foundation, the Japan Endocrine Society, and the Takeda Science Foundation. The results of this study were based on samples and patient data obtained from the International Center for Reproductive Medicine, Dokkyo Medical University Saitama Medical Center, Koshigaya, Japan. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Examination of nailfold videocapillaroscopy findings in ANCA-associated vasculitis.

OBJECTIVE: The objective of this study was to evaluate nailfold videocapillaroscopy (NVC) as a useful tool for assessing the disease activity of ANCA-associated vasculitis (AAV). METHODS: This study enrolled 51 patients with AAV and 21 healthy controls. We scored NVC findings semiquantitatively, and compared them between AAV patients and controls. We examined the association of NVC findings with disease activity indicators, histopathological findings of skin biopsies, and high-resolution CT (HRCT) scores in AAV. Additionally, we repeatedly rated the NVC findings 3 months after immunosuppressive therapy. RESULTS: Of the 51 enrolled patients, 36 (70.6%) showed a microangiopathy pattern and 4 (7.8%) showed a scleroderma pattern in AAV. The scores for microhaemorrhage, capillary loss, neoangiogenesis, and tortuosity were significantly higher in the AAV group than in the control group. NVC abnormalities correlated with the severity of skin, lung and kidney involvement. The scores of giant capillaries significantly correlated with the total BVAS and the chest BVAS; the scores of capillary loss correlated with the chest BVAS and the renal BVAS. The scores of microhaemorrhage significantly correlated with perivascular inflammatory cell infiltrations in the upper dermis of the purpura and tended to correlate with the total ground-glass opacity and consolidation scores on HRCT. In addition, capillary loss scores had a significant positive correlation with serum creatinine levels. Additionally, the microhaemorrhage scores were significantly reduced after 3 months of immunosuppressive therapy. CONCLUSION: In AAV patients, NVC abnormalities are significantly associated with disease severity. This result suggests that NVC is a useful tool for assessing the disease activity and treatment response in AAV.

Comparative effectiveness of biological disease-modifying antirheumatic drugs and Janus kinase inhibitor monotherapy in rheumatoid arthritis.

OBJECTIVES: To examine the effectiveness and drug tolerability of biological disease-modifying antirheumatic drugs (bDMARDs) and Janus kinase inhibitor (JAKi) monotherapy in patients with rheumatoid arthritis (RA) in a multicentre cohort study. METHODS: Patients with RA initiated with bDMARD/JAKi monotherapy without conventional synthetic DMARDs were included. Monotherapy regimens were categorised as interleukin-6 receptor inhibitors (IL-6Ri), cytotoxic T-lymphocyte-associated protein 4 immunoglobulin (CTLA4Ig), JAKi, or tumour necrosis factor inhibitors (TNFi). Multiple propensity score-based inverse probability weighting (IPW) was used to reduce selection bias. Linear mixed-effect models with IPW were used to examine changes in the disease activity score in 28 joints (DAS28)-erythrocyte sedimentation rate (ESR) at 24 weeks, and drug retention was compared among monotherapy using IPW Cox proportional hazards models. RESULTS: A total of 849 treatment courses from 635 patients were included (IL-6Ri, 218; CTLA4Ig, 183; JAKi, 92; TNFi, 356). The difference in change in DAS28-ESR at week 24 as the primary outcome was -0.93 (95% CI: -1.20 to -0.66) lower in the IL-6Ri group than TNFi, while that of CTLA4Ig and JAKi was similar with that of TNFi (-0.20 [-0.48 to 0.08], -0.25 [-0.67 to 0.16], respectively). IL-6Ri use was associated with significantly lower overall drug discontinuation than TNFi use (hazard ratio = 0.55 [0.39-0.78], P = 0.001). Similar retention rates were identified among CTLA4Ig and JAKi compared to TNFi. CONCLUSION: In the analysis with IPW to reduce selection bias, IL-6Ri monotherapy was superior to TNFi monotherapy in terms of effectiveness and drug retention. No significant differences were identified between CTLA4Ig, JAKi, and TNFi monotherapy.

Real-world comparative study of the efficacy of Janus kinase inhibitors in patients with rheumatoid arthritis: the ANSWER cohort study.

OBJECTIVE: This multicentre, retrospective study compared the efficacy and safety of tofacitinib, baricitinib, peficitinib and upadacitinib in real-world clinical settings after minimizing selection bias and adjusting the confounding patient characteristics. METHOD: The 622 patients were selected from the ANSWER cohort database and treated with tofacitinib (TOF), baricitinib (BAR), peficitinib (PEF) or upadacitinib (UPA). The patient's background was matched using propensity score-based inverse probability of treatment weighting (IPTW) among four treatment groups. The values of Clinical Disease Activity Index (CDAI), C-reactive protein (CRP), and modified Health Assessment Questionnaire (mHAQ) after drug initiation and the remission or low disease activity (LDA) rates of CDAI at 6 months after drug initiation were compared among the four groups. Further, the predictive factor for TOF and BAR efficacy was analysed. RESULTS: The retention and discontinuation rates until 6 months after drug initiations were not significantly different among the four JAK inhibitors treatment groups. Mean CDAI value, CDAI remission rate, and CDAI-LDA rate at 6 months after drug initiation were not significantly different among treatment groups. Baseline CDAI (TOFA: OR 1.09, P < 0.001; BARI: OR 1.07, P < 0.001), baseline CRP (TOFA: OR 1.32, P = 0.049), baseline glucocorticoid dose (BARI: OR 1.18, 95% CI 1.01-1.38, P = 0.035), a number of previous biological or targeted synthetic disease-modifying antirheumatic drugs (biological/targeted synthetic DMARDs) (BARI: OR 1.36, P = 0.004) were predictive factors for resistance to CDAI-LDA achievement to JAK inhibitor treatment. CONCLUSION: The efficacy and safety of TOF, BAR, PEF and UPA were not significantly different for the treatment of patients with rheumatoid arthritis.

Rare sequence variants associated with the risk of non-syndromic biliary atresia.

AIM: The etiology of non-syndromic biliary atresia (BA) remains largely unknown. In this study, we performed genome-wide screening of genes associated with the risk of non-syndromic BA. METHODS: We analyzed exome data of 15 Japanese patients with non-syndromic BA and 509 control individuals using an optimal sequence kernel association test (SKAT-O), a gene-based association study optimized for small-number subjects. Furthermore, we examined the frequencies of known BA-related single-nucleotide polymorphisms in the BA and control groups. RESULTS: SKAT-O showed that rare damaging variants of MFHAS1, a ubiquitously expressed gene encoding a Toll-like receptor-associated protein, were more common in the BA group than in the control group (Bonferroni corrected p-value = 0.0097). Specifically, p.Val106Gly and p.Arg556Cys significantly accumulated in the patient group. These variants resided within functionally important domains. SKAT-O excluded the presence of other genes significantly associated with the disease risk. Of 60 known BA-associated single-nucleotide polymorphisms, only eight were identified in the BA group. In particular, p.Ile3421Met of MYO15A and p.Ala421Thr of THOC2 were more common in the BA group than in the control group. However, the significance of these two variants is questionable, because MYO15A has been linked to deafness, but not to BA, and the p.Ala421Thr of THOC2 represents a relatively common single-nucleotide polymorphism in Asia. CONCLUSIONS: The results of this study indicate that rare damaging variants in MFHAS1 may constitute a risk factor for non-syndromic BA, whereas the contribution of other monogenic variants to the disease predisposition is limited.

Clinical features of fetal hydrothorax associated with mucopolysaccharidosis-VII.

Mucopolysaccharidosis (MPS)-VII, called Sly disease, is a lysosomal storage disorder that can cause fetal hydrops, including fetal hydrothorax (FHT). We describe two fetal cases that received thoracoamniotic shunting for FHT, which was later found to be associated with MPS-VII by exome sequencing. Bilateral FHT accompanied by skin edema and ascites was found before 20 weeks of gestation in both cases. One fetus died in utero at 35 weeks of gestation, and the other survived with preterm delivery at 30 weeks of gestation. Both cases inherited compound pathogenic variants of GUSB from parents. Comparison with previously reported primary FHT cases revealed distinct clinical features in MPS-VII-associated FHT: early gestational age at diagnosis (<26 weeks), bilateral effusion, skin edema with ascites, and poor survival. A genetic analysis would be considered for FHT cases, with consideration of shunting when they show early-onset bilateral effusions with skin edema and ascites.

Serum Complement C4 Levels Are a Useful Biomarker for Predicting End-Stage Renal Disease in Microscopic Polyangiitis.

This study aimed to evaluate the risk factors for end-stage renal disease (ESRD) in microscopic polyangiitis (MPA). In total, 74 patients with MPA were enrolled, and we compared the baseline clinical characteristics and disease activity between MPA patients who have progressed to ESRD and those without ESRD to select predictive factors for ESRD. Out of 74 patients, 12 patients (16.2%) had ESRD during follow-up. Serum C4 levels were significantly higher in MPA patients who have progressed to ESRD than in those without ESRD (p = 0.009). Multivariate analyses revealed that high serum creatinine levels (odds ratio (OR) 4.4, 95% confidence interval (CI) 1.25-15.5) and high serum C4 levels (OR 1.24, 95% CI 1.03-1.49) were risk factors for ESRD. Using receiver operating characteristic analysis, the cut-off value for initial serum C4 levels and serum creatinine levels were 29.6 mg/dL and 3.54 mg/dL, respectively. Patients with MPA with a greater number of risk factors (serum C4 levels > 29.6 mg/dL and serum creatinine levels > 3.54 mg/dL) had a higher ESRD progression rate. Serum C4 levels were significantly positively correlated with serum creatinine levels and kidney Birmingham vasculitis activity score (p = 0.02 and 0.04, respectively). These results suggest that serum C4 levels are useful tools for assessing renal disease activity and prognosis in MPA.

Prognostic Factors Affecting Death in Patients with Rheumatoid Arthritis Complicated by Pneumocystis jirovecii Pneumonia and One-Year Clinical Course: The ANSWER Cohort Study.

This multicenter retrospective study aimed to clarify the prognostic factors for mortality and changes in treatment modalities and disease activities after the onset of Pneumocystis jirovecii pneumonia (PCP) in patients with rheumatoid arthritis (RA). Data regarding the clinical background, treatment modalities, and disease activity indicators of RA at the onset of PCP (baseline), and 6 months and 12 months after treatment were extracted. Of the 37 patients with RA-PCP (median age, 69 years; 73% female), chemical prophylaxis was administered to 8.1%. Six patients died during PCP treatment. The serum C-reactive protein (CRP) levels and the prednisolone (PDN) dose at baseline in the PCP death group were significantly higher than those in the survivor group. Multivariate analysis using a Cox regression model showed that PDN dose at baseline was a predictor of death from PCP in patients with RA. During the 12 months from baseline, the RA disease activity significantly decreased. A high dose of corticosteroids for RA may result in a poor prognosis when PCP is complicated. In the future, preventive administration techniques must be established for patients with RA who need PCP prevention.

Quantitative assessment of copy number alterations by liquid biopsy for neuroblastoma.

Liquid biopsy, a method of detecting genomic alterations using blood specimens, has recently attracted attention as a noninvasive alternative to surgical tissue biopsy. We attempted quantitative analysis to detect amplification of MYCN (MYCNamp) and loss of heterozygosity at 11q (11qLOH), which are clinical requisites as prognostic factors of neuroblastoma (NB). In this study, cell-free DNA (cfDNA) was extracted from plasma samples from 24 NB patients at diagnosis. Copy numbers of MYCN and NAGK genes were quantitatively analyzed by droplet digital PCR (ddPCR). 11qLOH was also assessed by detecting allelic imbalances of heterozygous single nucleotide polymorphisms in the 11q region. The results obtained were compared to those of specimens from tumor tissues. The correlation coefficient of MYCN copy number of cfDNA and tumor DNA was 0.88 (p < 0.00001). 11qLOH was also accurately detected from cfDNA, except for one case with localized NB. Given the high accuracy of liquid biopsy, to investigate components of cfDNA, the proportion of tumor-derived DNA was estimated by examining the variant allele frequency of tumor-specific mutations in cfDNA. The proportion of tumor-derived DNA in cfDNA was 42.5% (range, 16.9%-55.9%), suggesting sufficient sensitivity of liquid biopsy for NB. In conclusion, MYCN copy number and 11qLOH could be quantitatively analyzed in plasma cfDNA by ddPCR assay. These results suggest that plasma cfDNA can be substituted for tumor DNA and can also be applied for comprehensive genomic profiling analysis.

Exome-based genome-wide screening of rare variants associated with the risk of polycystic ovary syndrome.

Purpose: Genetic factors associated with the risk of polycystic ovary syndrome (PCOS) remain largely unknown. Here, we conducted an optimal sequence kernel association test (SKAT-O), an exome-based rare variant association study, to clarify whether rare variants in specific genes contribute to the development of PCOS. Methods: SKAT-O was performed using exome data of 44 Japanese patients with PCOS and 301 control women. We analyzed frequencies of rare probably damaging variants in the genome. Results: Rare variants of GSTO2 were more commonly identified in the patient group than in the control group (6/44 vs. 1/301; Bonferroni-corrected p-value, 0.028), while the frequencies of variants in other genes were comparable between the two groups. The identified GSTO2 variants were predicted to affect the function, structure, stability, hydrophobicity, and/or the formation of intrinsically disordered regions of the protein. GSTO2 encodes a glutathione transferase that mediates the oxidative stress response and arsenic metabolism. Previously, common variants in GSTO2 and its paralog GSTO1 were associated with the risk of PCOS. Conclusions: The results indicate that there are no genes whose rare variants account for a large fraction of the etiology of PCOS, although rare damaging variants in GSTO2 may constitute a risk factor in some cases.

Prognostic factors affecting respiratory-related death in patients with rheumatoid arthritis complicated by interstitial lung disease: An ANSWER cohort study.

The aim of this multi-centre retrospective study was to clarify the prognostic factors for respiratory-related death in patients with interstitial lung disease (ILD) complicated rheumatoid arthritis (RA). Patient background data, treatment regimen, and disease activity indicators of RA and ILD at baseline, 6 months after the diagnosis of ILD, and at the last follow-up visit were extracted. A total of 312 patients with RA-ILD (17 patients who died from respiratory-related causes and 295 survivors) were included. Patients who died from respiratory-related causes had an older median age, a higher proportion of being male, and a higher anti-cyclic citrullinated peptide antibody positivity rate than survivors (p = .0001, .038, and .016, respectively); they also had significantly higher baseline serum levels of Krebs von den Lungen-6 (KL-6) than survivors (p = .013). Patients who died from respiratory-related causes showed significantly greater changes in serum KL-6 levels between the 6-month time point and the last visit [ΔKL-6 (6 months - last)] than survivors (p = .011). Multivariate analysis showed that the ΔKL-6 (6 months - last) corrected by disease duration was a predictor of respiratory-disease-related death in patients with RA-ILD (p < .0001). Long-term increase in serum KL-6 levels is associated with respiratory-disease related death in patients with RA-ILD.

Novel biallelic mutations in the DUOX2 gene underlying very early-onset inflammatory bowel disease: A case report.

Genetic variants affecting the function of dual oxidase 2 (DUOX2), the catalytic subunit of membrane-bound enzymes that produce hydrogen peroxide, are associated with very early-onset inflammatory bowel disease (VEO-IBD). We report the case of a 1-year-old boy diagnosed with VEO-IBD after presenting with bloody diarrhea. He had pancolitis and an extensive small intestinal ulcerative lesion at age 4 years. Infliximab treatment was successful but was discontinued due to delayed reaction. At age 7 years, treatment with ustekinumab was started, and remission has been maintained for more than 2 years. Whole-exome sequencing identified compound heterozygous missense DUOX2 variants of unknown significance (p.[R1212H];[F1490Y]). Protein expression in the whole-cell lysate and plasma membrane was lower in F1490Y-DUOX2 than in wild-type (WT)-DUOX2. Hydrogen peroxide generation upon ionomycin stimulation was lower in cells expressing R1212H-DUOX2 and F1490Y-DUOX2 than in those expressing WT-DUOX2. The novel, inherited, biallelic DUOX2 mutations may be molecular risk factors of VEO-IBD.

Identification of unique DNA methylation sites in Kabuki syndrome using whole genome bisulfite sequencing and targeted hybridization capture followed by enzymatic methylation sequencing.

BACKGROUND: Kabuki syndrome (KS) is a congenital malformation syndrome caused by mutations in the KMT2D and KDM6A genes that encode histone modification enzymes. Although KS is considered a single gene disorder, its symptoms vary widely. Recently, disease-specific DNA methylation patterns, or episignatures, have been recognized and used as a diagnostic tool for KS. Because of various crosstalk mechanisms between histone modifications and DNA methylation, DNA methylation analysis may have high potential for investigations into the pathogenesis of KS. RESULTS: In this study, we investigated altered CpG-methylation sites that were specific to KS to find important genes associated with the various phenotypes or pathogenesis of KS. Whole genome bisulfite sequencing (WGBS) was performed to select target CpG islands, and enzymatic conversion technology was applied after hybridization capture to confirm KS-specific episignatures of 130 selected differently methylated target regions (DMTRs) in DNA samples from the 65 participants, 31 patients with KS and 34 unaffected individuals, in this study. We identified 26 candidate genes in 22 DMTRs that may be associated with KS. Our results indicate that disease-specific methylation sites can be identified from a small number of WGBS samples, and hybridization capture followed by enzymatic methylation sequencing can simultaneously test the sites. CONCLUSIONS: Although DNA methylation can be tissue-specific, our results suggest that methylation profiling of DNA extracted from peripheral blood may be a powerful approach to study the pathogenesis of diseases.

Direct platelet adhesion potentiates group 2 innate lymphoid cell functions.

BACKGROUND: Platelets are thought to be involved in the pathophysiology of asthma, presumably through direct adhesion to inflammatory cells, including group 2 innate lymphoid cells (ILC2s). Here, we tried to elucidate the effects of platelet adhesion to ILC2s in vitro and in vivo, as well as the mechanisms involved. METHODS: Alternaria-induced ILC2-dependent airway inflammation models using wild-type and c-mpl-/- mice were evaluated. Both purified CD41+ and CD41- ILC2s were cultured with IL-2 and IL-33 to determine in vitro Type 2 (T2) cytokine production and cell proliferation. RNA-seq data of flow-cytometry-sorted CD41+ and CD41- ILC2s were used to isolate ILC2-specific genes. Flow cytometry was performed to determine the expression of CD41 and adhesion-related molecules on ILC2s in both mouse and human tissues. RESULTS: T2 inflammation and T2 cytokine production from ILC2s were significantly reduced in the c-mpl-/- mice compared to wild-type mice. Platelet-adherent ILC2s underwent significant proliferation and showed enhanced T2 cytokine production when exposed to IL-2 and IL-33. The functions of ILC2-specific genes were related to cell development and function. Upstream regulator analysis identified 15 molecules, that are thought to be involved in ILC2 activation. CD41 expression levels were higher in ILC2s from human PBMCs and mouse lung than in those from secondary lymphoid tissues, but they did not correlate with the P-selectin glycoprotein ligand-1 or CD24 expression level. CONCLUSION: Platelets spontaneously adhere to ILC2s, probably in the peripheral blood and airways, thereby potentiating ILC2s to enhance their responses to IL-33.