Primary ciliary dyskinesia in Japan: systematic review and meta-analysis.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare genetic disorder. Although the genetic tests and new diagnostic algorithms have recently been recommended, clinical signs and electron microscope (EM) findings have historically been the mainstays of diagnosis in Asia. To characterize PCD previously reported in Japan, we conducted a systematic review and meta-analysis. METHODS: A search using MEDLINE, EMBASE, and Japana Centra Revuo Medicina (in Japanese) databases was carried out to identify articles reporting PCD, Kartagener syndrome, or immotile cilia syndrome in Japanese patients and published between 1985 and 2015. RESULTS: After excluding duplication from 334 reports, we extracted 316 patients according to the criteria. Diagnosis was most frequently made in adulthood (148 patients [46.8%] ≥ 18 years old, 24 patients [7.6%] < 1 year old, 68 patients [21.5%] 1-17 years old and 76 patients [24.1%] lacking information). Of the 230 patients (72.8%) who received EM examination, there were patients with inner dynein arm (IDA) defects (n = 55; 23.9%), outer dynein arm (ODA) defects (14; 6.1%), both ODA and IDA defects (57; 24.8%), other structural abnormalities (25; 10.9%), no abnormalities (4; 1.7%), and no detailed conclusion or description (75; 32.6%). CONCLUSION: Delayed diagnosis of this congenital disease with high frequency of IDA defects and low frequency of ODA defects appear to be historical features of PCD reported in Japan, when EM was a main diagnostic tool. This review highlights problems experienced in this field, and provides basic information to establish a modernized PCD diagnosis and management system in the future.

Circulating granulysin levels in healthcare workers and latent tuberculosis infection estimated using interferon-gamma release assays.

BACKGROUND: Granulysin (GNLY) is produced by human lymphocyte subpopulations and exhibits antimicrobial activity against Mycobacterium tuberculosis. We examined the association between GNLY levels in blood and latent tuberculosis (TB) infection. METHODS: Latency of TB infection among Vietnamese healthcare workers was estimated using interferon-gamma release assays (IGRA), and serum GNLY concentrations were measured using enzyme-linked immunosorbent assays. The levels of GNLY expression in whole blood and the presence of GNLY alleles with the exon-4 polymorphism rs11127 were also determined using PCR-based methods. RESULTS: Among 109 study participants, 41 (37.6 %) were IGRA positive and had significantly lower serum GNLY concentrations compared with IGRA-negative participants (adjusted mean, 95 % confidence interval; 2.03, 1.72-2.44 vs. 2.48, 2.10-2.92 ng/ml, P = 0.0127; analysis of covariance). Serum GNLY concentrations and TB antigen-stimulated interferon-gamma values were weakly inversely correlated (r = -0.20, P = 0.0333). Serum GNLY concentrations varied with GNLY genotypes even after adjustment for gender and age (adjusted P = 0.0015) and were moderately correlated with GNLY expression in blood cells (r = 0.40, P < 0.0001). In subsequent analyses, low serum GNLY concentrations were significantly associated with IGRA status (adjusted odds ratio and 95 % confidence interval, 0.55 and 0.31-0.98, respectively), although GNLY genotype and mRNA levels were not. CONCLUSIONS: Decreased GNLY, presumably at the protein level, is linked to the immunological condition of latent TB infection.

Primary Ciliary Dyskinesia Due to Compound Heterozygous Variants in CFAP221 with Obstructive Azoospermia: Young's Syndrome May Be a Phenotype of Primary Ciliary Dyskinesia.

We report the case of a 42-year-old man with bronchiectasis who had a history of infertility treatment for obstructive azoospermia. Young's syndrome was suspected based on the triad of obstructive azoospermia, sinusitis, and bronchiectasis. He had normal electron microscopy findings, normal nasal nitric oxide levels (116 nL/min), and no situs inversus. However, we found compound heterozygous variants in CFAP221. This led to a diagnosis of primary ciliary dyskinesia (PCD). Distinguishing PCD from Young's syndrome in patients with the triad of obstructive azoospermia, sinusitis, and bronchiectasis is challenging. Young's syndrome may be a phenotype of PCD.

Impact of primary ciliary dyskinesia: Beyond sinobronchial syndrome in Japan.

Primary ciliary dyskinesia (PCD) is a rare genetic disorder characterized by impaired motile cilia function, particularly in the upper and lower airways. To date, more than 50 causative genes related to the movement, development, and maintenance of cilia have been identified. PCD mostly follows an autosomal recessive inheritance pattern, in which PCD symptoms manifest only in the presence of pathogenic variants in both alleles. Several genes causing PCD have been recently identified that neither lead to situs inversus nor cause definitive abnormalities in ciliary ultrastructure. Importantly, the distribution of disease-causing genes and pathogenic variants varies depending on ethnicity. In Japan, homozygosity for a ∼27.7-kb deletion of DRC1 is estimated to be the most common cause of PCD, presumably as a founder mutation. The clinical picture of PCD is similar to that of sinobronchial syndrome, thus making its differentiation from diffuse panbronchiolitis and other related disorders difficult. Given the diagnostic challenges, many cases remain undiagnosed or misdiagnosed, particularly in adults. While no fundamental cure is currently available, lifelong medical subsidies are provided in Japan, and proper respiratory management, along with continued prevention and treatment of infections, is believed to mitigate the decline in respiratory function. Timely action will be necessary when specific treatments for PCD become available in the future. This narrative review focuses on variations in the disease status of PCD in a non-Western country.

Primary Ciliary Dyskinesia Caused by Homozygous DNAAF1 Mutations Resulting from a Consanguineous Marriage: A Case Report from Japan.

We present the case of a 58-year-old female patient with primary ciliary dyskinesia (PCD). She was born to parents with a consanguineous marriage. Chest computed tomography conducted at age 41 years indicated no situs inversus, and findings of bronchiectasis were limited to the middle and lingular lobes. Despite long-term macrolide therapy, bronchiectasis exacerbations frequently occurred. PCD was suspected because of the low nasal nitric oxide level (20.7 nL/min). Electron microscopy revealed outer and inner dynein arm defects, and a genetic analysis identified a homozygous single-nucleotide deletion in the DNAAF1 gene. Based on these results, the patient was diagnosed with PCD due to a biallelic DNAAF1 mutation.

Practical guide for the diagnosis and management of primary ciliary dyskinesia.

OBJECTIVE: Primary ciliary dyskinesia (PCD) is a relatively rare genetic disorder that affects approximately 1 in 20,000 people. Approximately 50 genes are currently known to cause PCD. In light of differences in causative genes and the medical system in Japan compared with other countries, a practical guide was needed for the diagnosis and management of Japanese PCD patients. METHODS: An ad hoc academic committee was organized under the Japanese Rhinologic Society to produce a practical guide, with participation by committee members from several academic societies in Japan. The practical guide including diagnostic criteria for PCD was approved by the Japanese Rhinologic Society, Japanese Society of Otolaryngology-Head and Neck Surgery, Japanese Respiratory Society, and Japanese Society of Pediatric Pulmonology. RESULTS: The diagnostic criteria for PCD consist of six clinical features, six laboratory findings, differential diagnosis, and genetic testing. The diagnosis of PCD is categorized as definite, probable, or possible PCD based on a combination of the four items above. Diagnosis of definite PCD requires exclusion of cystic fibrosis and primary immunodeficiency, at least one of the six clinical features, and a positive result for at least one of the following: (1) Class 1 defect on electron microscopy of cilia, (2) pathogenic or likely pathogenic variants in a PCD-related gene, or (3) impairment of ciliary motility that can be repaired by correcting the causative gene variants in iPS cells established from the patient's peripheral blood cells. CONCLUSION: This practical guide provides clinicians with useful information for the diagnosis and management of PCD in Japan.

MxA transcripts with distinct first exons and modulation of gene expression levels by single-nucleotide polymorphisms in human bronchial epithelial cells.

Myxovirus resistance A (MxA) is a major interferon (IFN)-inducible antiviral protein. Promoter single-nucleotide polymorphisms (SNPs) of MxA near the IFN-stimulated response element (ISRE) have been frequently associated with various viral diseases, including emerging respiratory infections. We investigated the expression profile of MxA transcripts with distinct first exons in human bronchial epithelial cells. For primary culture, the bronchial epithelium was isolated from lung tissues with different genotypes, and total RNA was subjected to real-time reverse transcription polymerase chain reaction. The previously reported MxA transcript (T1) and a recently registered transcript with a distinct 5' first exon (T0) were identified. IFN-ß and polyinosinic-polycytidylic acid induced approximately 100-fold higher expression of the T1 transcript than that of the T0 transcript, which also had a potential ISRE motif near its transcription start site. Even without inducers, the T1 transcript accounted for approximately two thirds of the total expression of MxA, levels of which were significantly associated with its promoter and exon 1 SNPs (rs17000900, rs2071430, and rs464138). Our results suggest that MxA observed in respiratory viral infections is possibly dominated by the T1 transcript and partly influenced by relevant 5' SNPs.

Potential function of granulysin, other related effector molecules and lymphocyte subsets in patients with TB and HIV/TB coinfection.

BACKGROUND: Host effector mechanism against Mycobacterium tuberculosis (Mtb) infection is dependent on innate immune response by macrophages and neutrophils and the alterations in balanced adaptive immunity. Coordinated release of cytolytic effector molecules from NK cells and effector T cells and the subsequent granule-associated killing of infected cells have been documented; however, their role in clinical tuberculosis (TB) is still controversy. OBJECTIVE: To investigate whether circulating granulysin and other effector molecules are associated with the number of NK cells, iNKT cells, Vγ9(+)Vδ2(+) T cells, CD4(+) T cells and CD8(+) T cells, and such association influences the clinical outcome of the disease in patients with pulmonary TB and HIV/TB coinfection. METHODS: Circulating granulysin, perforin, granzyme-B and IFN-γ levels were determined by ELISA. The isoforms of granulysin were analyzed by Western blot analysis. The effector cells were analyzed by flow cytometry. RESULTS: Circulating granulysin and perforin levels in TB patients were lower than healthy controls, whereas the granulysin levels in HIV/TB coinfection were much higher than in any other groups, TB and HIV with or without receiving HAART, which corresponded to the number of CD8(+) T cells which kept high, but not with NK cells and other possible cellular sources of granulysin. In addition, the 17kDa, 15kDa and 9kDa isoforms of granulysin were recognized in plasma of HIV/TB coinfection. Increased granulysin and decreased IFN-γ levels in HIV/TB coinfection and TB after completion of anti-TB therapy were observed. CONCLUSION: The results suggested that the alteration of circulating granulysin has potential function in host immune response against TB and HIV/TB coinfection. This is the first demonstration so far of granulysin in HIV/TB coinfection.

FOXJ1 Variants Causing Primary Ciliary Dyskinesia with Hydrocephalus: A Case Report from Japan.

Primary ciliary dyskinesia (PCD) is a genetic disease characterized by motile cilia dysfunction, mostly inherited in an autosomal recessive or X-linked manner. We herein report a 29-year-old woman with PCD caused by a heterozygous frameshift mutation due to a single nucleotide deletion in exon 3 of FOXJ1. Heterozygous de novo mutations in FOXJ1 have been reported as an autosomal-dominant cause of PCD. The patient had situs inversus, congenital heart disease, infertility, and hydrocephalus. However, the nasal nitric oxide level was normal. Long-term macrolide therapy was remarkably effective. This is the first case report of PCD caused by a FOXJ1 variant in Japan.

Host-pathogen relationship in retreated tuberculosis with major rifampicin resistance-conferring mutations.

Introduction: It is assumed that host defense systems eliminating the pathogen and regulating tissue damage make a strong impact on the outcome of tuberculosis (TB) disease and that these processes are affected by rifampicin (RIF) resistance-conferring mutations of Mycobacterium tuberculosis (Mtb). However, the host responses to the pathogen harboring different mutations have not been studied comprehensively in clinical settings. We analyzed clinico-epidemiological factors and blood transcriptomic signatures associated with major rpoB mutations conferring RIF resistance in a cohort study. Methods: Demographic data were collected from 295 active pulmonary TB patients with treatment history in Hanoi, Vietnam. When recruited, drug resistance-conferring mutations and lineage-specific variations were identified using whole-genome sequencing of clinical Mtb isolates. Before starting retreatment, total RNA was extracted from the whole blood of HIV-negative patients infected with Mtb that carried either the rpoB H445Y or rpoB S450L mutation, and the total RNA was subjected to RNA sequencing after age-gender matching. The individual RNA expression levels in the blood sample set were also measured using real-time RT-PCR. Logistic and linear regression models were used to assess possible associations. Results: In our cohort, rpoB S450L and rpoB H445Y were major RIF resistance-conferring mutations [32/87 (36.8%) and 15/87 (17.2%), respectively]. H445Y was enriched in the ancient Beijing genotype and was associated with nonsynonymous mutations of Rv1830 that has been reported to regulate antibiotic resilience. H445Y was also more frequently observed in genetically clustered strains and in samples from patients who had received more than one TB treatment episode. According to the RNA sequencing, gene sets involved in the interferon-γ and-α pathways were downregulated in H445Y compared with S450L. The qRT-PCR analysis also confirmed the low expression levels of interferon-inducible genes, including BATF2 and SERPING1, in the H445Y group, particularly in patients with extensive lesions on chest X-ray. Discussion: Our study results showed that rpoB mutations as well as Mtb sublineage with additional genetic variants may have significant effects on host response. These findings strengthen the rationale for investigation of host-pathogen interactions to develop countermeasures against epidemics of drug-resistant TB.

Association of IFNGR2 gene polymorphisms with pulmonary tuberculosis among the Vietnamese.

Interferon-γ (IFN-γ) is a key molecule of T helper 1 (Th1)-immune response against tuberculosis (TB), and rare genetic defects of IFN-γ receptors cause disseminated mycobacterial infection. The aim of the present study was to investigate whether genetic polymorphisms found in the Th1-immune response genes play a role in TB. In our study, DNA samples were collected from two series of cases including 832 patients with new smear-positive TB and 506 unrelated individuals with no history of TB in the general population of Hanoi, Vietnam. Alleles of eight microsatellite markers located around Th1-immune response-related genes and single nucleotide polymorphisms near the promising microsatellites were genotyped. A set of polymorphisms within the interferon gamma receptor 2 gene (IFNGR2) showed a significant association with protection against TB (P = 0.00054). Resistant alleles tend to be less frequently found in younger age at diagnosis (P = 0.011). Luciferase assays revealed high transcriptional activity of the promoter segment in linkage disequilibrium with resistant alleles. We conclude that the polymorphisms of IFNGR2 may confer resistance to the TB development of newly infected individuals. Contribution of the genetic factors to TB appeared to be different depending on age at diagnosis.

Inter-rater agreement in the assessment of abnormal chest X-ray findings for tuberculosis between two Asian countries.

BACKGROUND: Inter-rater agreement in the interpretation of chest X-ray (CXR) films is crucial for clinical and epidemiological studies of tuberculosis. We compared the readings of CXR films used for a survey of tuberculosis between raters from two Asian countries. METHODS: Of the 11,624 people enrolled in a prevalence survey in Hanoi, Viet Nam, in 2003, we studied 258 individuals whose CXR films did not exclude the possibility of active tuberculosis. Follow-up films obtained from accessible individuals in 2006 were also analyzed. Two Japanese and two Vietnamese raters read the CXR films based on a coding system proposed by Den Boon et al. and another system newly developed in this study. Inter-rater agreement was evaluated by kappa statistics. Marginal homogeneity was evaluated by the generalized estimating equation (GEE). RESULTS: CXR findings suspected of tuberculosis differed between the four raters. The frequencies of infiltrates and fibrosis/scarring detected on the films significantly differed between the raters from the two countries (P < 0.0001 and P = 0.0082, respectively, by GEE). The definition of findings such as primary cavity, used in the coding systems also affected the degree of agreement. CONCLUSIONS: CXR findings were inconsistent between the raters with different backgrounds. High inter-rater agreement is a component necessary for an optimal CXR coding system, particularly in international studies. An analysis of reading results and a thorough discussion to achieve a consensus would be necessary to achieve further consistency and high quality of reading.

Transcription factor MAFB controls type I and II interferon response-mediated host immunity in Mycobacterium tuberculosis-infected macrophages.

MAFB, v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B, has been identified as a candidate gene for early tuberculosis (TB) onset in Thai and Japanese populations. Here, we investigated the genome-wide transcriptional profiles of MAFB-knockdown (KD) macrophages infected with Mycobacterium tuberculosis (Mtb) to highlight the potential role of MAFB in host immunity against TB. Gene expression analysis revealed impaired type I and type II interferon (IFN) responses and enhanced oxidative phosphorylation in MAFB-KD macrophages infected with Mtb. The expression of inflammatory chemokines, including IFN-γ-inducible genes, was confirmed to be significantly reduced by knockdown of MAFB during Mtb infection. A similar effect of MAFB knockdown on type I and type II IFN responses and oxidative phosphorylation was also observed when Mtb-infected macrophages were activated by IFN-γ. Taken together, our results demonstrate that MAFB is involved in the immune response and metabolism in Mtb-infected macrophages, providing new insight into MAFB as a candidate gene to guide further study to control TB.

Novel Screening System of Virulent Strains for the Establishment of a Mycobacterium avium Complex Lung Disease Mouse Model Using Whole-Genome Sequencing.

The establishment of animal models reflecting human Mycobacterium avium complex (MAC) lung disease (LD) pathology has the potential to expand our understanding of the disease pathophysiology. However, inducing sustained infection in immunocompetent mice is difficult since MAC generally shows less virulence and higher genetic variability than M. tuberculosis. To overcome this hurdle, we developed a screening system for identifying virulent MAC strains using whole-genome sequencing (WGS). We obtained nine clinical strains from Mycobacterium avium complex lung disease (MAC-LD) patients and divided them into two groups to make the mixed strain inocula for infection. Intranasal infection with the strain mixture of both groups in BALB/c mice resulted in progressive infection and extensive granuloma formation in the lungs, suggesting the existence of highly pathogenic strains in each group. We hypothesized that the change in the abundance of strain-specific single-nucleotide variants (SNVs) reflects the change in bacterial number of each strain in infected lungs. Based on this hypothesis, we quantified individual strain-specific SNVs in bacterial DNA from infected lungs. Specific SNVs for four strains were detected, suggesting the pathogenicity of these four strains. Consistent with these results, individual infection with these four strains induced a high lung bacterial burden, forming extensive peribronchial granuloma, while the other strains showed a decreased lung bacterial burden. The current method combining mixed infection and WGS accurately identified virulent strains that induced sustained infection in mice. This method will contribute to the establishment of mouse models that reflect human MAC-LD and lead to antimycobacterial drug testing. IMPORTANCE To promote research on Mycobacterium avium complex (MAC) pathogenicity, animal models reflecting human progressive MAC lung disease (MAC-LD) are needed. Because there is high genetic and virulence diversity among clinical MAC strains, choosing a suitable strain is an important process for developing a mouse model. In this study, we developed a screening system for virulent strains in mice by combining mixed infection and whole-genome sequencing analysis. This approach is designed on the hypothesis that in vivo virulence of MAC strains can be examined simultaneously by comparing changes in the abundance of strain-specific single-nucleotide variants in the mouse lungs after infection with mixed strains. The identified strains were shown to induce high bacterial burdens and cause extensive peribronchial granuloma resembling the pulmonary pathology of human MAC-LD. The current method will help researchers develop mouse models that reflect human MAC-LD and will lead to further investigation of MAC pathogenicity.

Multidrug-Resistant Tuberculosis Outbreak among Immigrants in Tokyo, Japan, 2019-2021.

In mid-September 2019, a teenage Chinese male student and part-time waiter in Tokyo was diagnosed with multidrug-resistant (MDR) sputum smear-positive pulmonary tuberculosis (TB). This study describes the outbreak investigation of his friends and colleagues at the restaurant. We investigated 6 friends and 15 colleagues; 5 friends and 13 colleagues underwent interferon-γ release assay (IGRA). Of these, 3 friends (60.0%) and 4 colleagues (30.8%) were IGRA-positive. Each of the friends and colleagues was found to have MDR-TB (20% and 7.7%, respectively). Challenges during the investigation were the unavailability of regimens for latent TB infection (LTBI) for contacts with MDR-TB, budgetary constraints concerning implementing computed tomography (CT) scans for the contacts, frequent address changes of foreign-born patients and contacts, investigation during the coronavirus disease pandemic, and variations of alphabetical expression of the names of the patients and contacts, particularly for those from China. It is recommended that the national government officially adopt prophylaxis regimens for LTBI with MDR-TB, address the budgetary constraints regarding CT scans, and deploy liaison officers for coordinating investigations involving many foreign-born patients and contacts scattered in multiple municipalities. The names of foreign-born persons could more accurately be identified using both the alphabet and Chinese characters.

Spatial multiomic profiling reveals the novel polarization of foamy macrophages within necrotic granulomatous lesions developed in lungs of C3HeB/FeJ mice infected with Mycobacterium tuberculosis.

Infection with Mycobacterium tuberculosis leads to the development of tuberculosis (TB) with the formation of granulomatous lesions. Foamy macrophages (FM) are a hallmark of TB granulomas, because they provide the primary platform of M. tuberculosis proliferation and the main source of caseous necrosis. In this study, we applied spatial multiomic profiling to identify the signatures of FM within the necrotic granulomas developed in a mouse model resembling human TB histopathology. C3HeB/FeJ mice were infected with M. tuberculosis to induce the formation of necrotic granulomas in the lungs. Using laser microdissection, necrotic granulomas were fractionated into three distinct regions, including the central caseous necrosis, the rim containing FM, and the peripheral layer of macrophages and lymphocytes, and subjected to proteomic and transcriptomic analyses. Comparison of proteomic and transcriptomic analyses of three distinct granulomatous regions revealed that four proteins/genes are commonly enriched in the rim region. Immunohistochemistry confirmed the localization of identified signatures to the rim of necrotic granulomas. We also investigated the localization of the representative markers for M1 macrophages in granulomas because the signatures of the rim included M2 macrophage markers. The localization of both macrophage markers suggests that FM in necrotic granulomas possessed the features of M1 or M2 macrophages. Gene set enrichment analysis of transcriptomic profiling revealed the upregulation of genes related to M2 macrophage activation and mTORC1 signaling in the rim. These results will provide new insights into the process of FM biogenesis, leading to further understanding of the pathophysiology of TB granulomas.

Molecular cloning of two novel mucin-like genes in the disease-susceptibility locus for diffuse panbronchiolitis.

Diffuse panbronchiolitis (DPB) is a rare complex genetic disease affecting East Asians and is strongly associated with the class I human leukocyte antigens (HLA)-B54 in Japanese and HLA-A11 in Koreans. We recently showed that an HLA-associated major susceptibility gene for DPB is probably located within the 200 kb in the class I region 300 kb telomeric of the HLA-B locus on the chromosome 6p21.3. We cloned two novel mucin-like genes designated panbronchiolitis related mucin-like 1 and 2 (PBMUCL1 and PBMUCL2) in the candidate region, which form a mucin-like gene cluster together with two adjacent genes, MUC21 and DPCR1. PBMUCL1 gene expression was remarkably upregulated by polyinosine-polycytidylic acid [poly(I:C)] stimulation in normal human bronchial epithelial cells redifferentiated at the air-liquid interface. We found genetic polymorphisms in PBMUCL1 gene which were associated with DPB: the A-allele of the PBMUCL1 intron 2 single nucleotide polymorphism (SNP) was positively associated and variable numbers of tandem repeats (VNTR) polymorphism in exon 3 (1,890-base pair deletion) was negatively associated. Despite a strong association with HLA-B in the Japanese, the mucin-like gene PBMUCL1 is also one of the candidate genes of DPB susceptibility.