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Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by ciliary structural abnormalities and dysfunction, leading to chronic rhinosinusitis, otitis media with effusion, bronchiectasis, and infertility. Approximately half of Japanese PCD cases are attributed to variants in the dynein regulatory complex subunit 1 (DRC1) gene, predominantly featuring homogeneous deletions of exons 1-4 spanning 27,748 base pairs on chromosome 2. Here, we report 10 new PCD cases (9 families) in addition to 29 previously reported cases (24 families) caused by DRC1 variants. Among these 39 cases, biallelic DRC1 exon 1-4 deletions were detected in 38 (97.4%). These DRC1 deletions exhibited an identical breakpoint in all PCD cases in the Japanese and Korean populations, strongly suggesting a founder effect. In this study, we performed haplotype analysis, using a whole-exome sequencing dataset of 18 Japanese PCD patients harboring large biallelic DRC1 deletions. We estimated that the founder allele likely emerged 115.1 generations ago (95% confidence interval: 33.7-205.1), suggesting an origin of approximately 3050 years ago, coinciding with the transition from the Jomon period to the early Yayoi period in Japan. Considering the formation of the modern Japanese population, the founder with the DRC1 exon 1-4 deletion likely lived on the Korean peninsula, with the allele later transmitted to Japan through migration. This study provides insights into the origin of the DRC1 copy number variant, the most frequent PCD variant in the Japanese and Korean populations, highlighting the importance of understanding population-specific genetic variations in the context of human migration and disease prevalence.
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Primary ciliary dyskinesia (PCD) is a hereditary disease caused by genes related to motile cilia. We report two male pediatric cases of PCD caused by hemizygous pathogenic variants in the OFD1 centriole and centriolar satellite protein (OFD1) gene. The variants were NM_003611.3: c.[2789_2793delTAAAA] (p.[Ile930LysfsTer8]) in Case 1 and c.[2632_2635delGAAG] (p.[Glu878LysfsTer9]) in Case 2. Both cases had characteristic recurrent respiratory infections. Neither case had symptoms of oral-facial-digital syndrome type I. We identified a variant (c.2632_2635delGAAG) that has not been previously reported in any case of OFD1-PCD.
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[This corrects the article DOI: 10.3389/fcell.2023.1290876.].
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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.
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Pruebas Genéticas , Síndrome de Kartagener , Humanos , Síndrome de Kartagener/diagnóstico , Síndrome de Kartagener/terapia , Síndrome de Kartagener/genética , Diagnóstico Diferencial , Cilios/ultraestructura , Cilios/patología , Japón , Dineínas Axonemales/genética , ProteínasRESUMEN
The emergence of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) variants necessitated a rapid evaluation system for their pathogenesis. Lung epithelial cells are their entry points; however, in addition to their limited source, the culture of human alveolar epithelial cells is especially complicated. Induced pluripotent stem cells (iPSCs) are an alternative source of human primary stem cells. Here, we report a model for distinguishing SARS-CoV-2 variants at high resolution, using separately induced iPSC-derived alveolar and airway cells in micro-patterned culture plates. The position-specific signals induced the apical-out alveolar type 2 and multiciliated airway cells at the periphery and center of the colonies, respectively. The infection studies in each lineage enabled profiling of the pathogenesis of SARS-CoV-2 variants: infection efficiency, tropism to alveolar and airway lineages, and their responses. These results indicate that this culture system is suitable for predicting the pathogenesis of emergent SARS-CoV-2 variants.
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COVID-19 , Células Madre Pluripotentes Inducidas , Humanos , SARS-CoV-2/fisiología , PulmónRESUMEN
Alveolar type 2 (AT2) epithelial cells are tissue stem cells capable of differentiating into alveolar type 1 (AT1) cells for injury repair and maintenance of lung homeostasis. However, the factors involved in human AT2-to-AT1 cell differentiation are not fully understood. Here, we established SFTPCGFP and AGERmCherry-HiBiT dual-reporter induced pluripotent stem cells (iPSCs), which detected AT2-to-AT1 cell differentiation with high sensitivity and identified factors inducing AT1 cell differentiation from AT2 and their progenitor cells. We also established an "on-gel" alveolar epithelial spheroid culture suitable for medium-throughput screening. Among the 274 chemical compounds, several single compounds, including LATS-IN-1, converted AT1 cells from AT2 and their progenitor cells. Moreover, YAP/TAZ signaling activation and AKT signaling suppression synergistically recapitulated the induction of transcriptomic, morphological, and functionally mature AT1 cells. Our findings provide novel insights into human lung development and lung regenerative medicine.
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Células Epiteliales Alveolares , Células Madre Pluripotentes Inducidas , Humanos , Células Cultivadas , Pulmón , Diferenciación Celular , Células EpitelialesRESUMEN
The lung is regarded as having limited regenerative capacity, and there are few treatment options for refractory lung diseases, such as interstitial pneumonia. Lung transplantation is the final option available in some scenarios. Research in this area has been slow owing to the complex structure of the lung for efficient gas exchange between the alveolar spaces and capillaries as well as the difficulty in obtaining specimens from patients with progressive lung disease. However, basic research over the past decade in the field of mouse and human embryology using genetic lineage tracing techniques and stem cell biology using primary and pluripotent stem cell-derived alveolar organoids has begun to clarify the tissue response in various intractable lung diseases and the mechanisms underlying remodeling. Advancement in this area may expand potential therapeutic targets for alveolar regeneration, providing alternatives to lung transplantation, and contribute to the development of effective therapeutic methods that activate or repopulate stem cells in the lung. In this review, we cover research focused on alveolar epithelial cells and discuss methods expected to regenerate lungs that are damaged by diseases.
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Organoides , Medicina Regenerativa , Organoides/citología , Humanos , Medicina Regenerativa/métodos , Animales , Pulmón/citología , Regeneración/fisiología , Alveolos Pulmonares/citología , Enfermedades Pulmonares/terapia , Enfermedades Pulmonares/patologíaRESUMEN
For mucociliary clearance of pathogens, tracheal multiciliated epithelial cells (MCCs) organize coordinated beating of cilia, which originate from basal bodies (BBs) with basal feet (BFs) on one side. To clarify the self-organizing mechanism of coordinated intracellular BB-arrays composed of a well-ordered BB-alignment and unidirectional BB-orientation, determined by the direction of BB to BF, we generated double transgenic mice with GFP-centrin2-labeled BBs and mRuby3-Cep128-labeled BFs for long-term, high-resolution, dual-color live-cell imaging in primary-cultured tracheal MCCs. At early timepoints of MCC differentiation, BB-orientation and BB-local alignment antecedently coordinated in an apical microtubule-dependent manner. Later during MCC differentiation, fluctuations in BB-orientation were restricted, and locally aligned BB-arrays were further coordinated to align across the entire cell (BB-global alignment), mainly in an apical intermediate-sized filament-lattice-dependent manner. Thus, the high coordination of the BB-array was established for efficient mucociliary clearance as the primary defense against pathogen infection, identifying apical cytoskeletons as potential therapeutic targets.
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Cuerpos Basales , Citoesqueleto , Ratones , Animales , Microtúbulos , Cilios , Células EpitelialesRESUMEN
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, continues to spread around the world with serious cases and deaths. It has also been suggested that different genetic variants in the human genome affect both the susceptibility to infection and severity of disease in COVID-19 patients. Angiotensin-converting enzyme 2 (ACE2) has been identified as a cell surface receptor for SARS-CoV and SARS-CoV-2 entry into cells. The construction of an experimental model system using human iPS cells would enable further studies of the association between viral characteristics and genetic variants. Airway and alveolar epithelial cells are cell types of the lung that express high levels of ACE2 and are suitable for in vitro infection experiments. Here, we show that human iPS cell-derived airway and alveolar epithelial cells are highly susceptible to viral infection of SARS-CoV-2. Using gene knockout with CRISPR-Cas9 in human iPS cells we demonstrate that ACE2 plays an essential role in the airway and alveolar epithelial cell entry of SARS-CoV-2 in vitro. Replication of SARS-CoV-2 was strongly suppressed in ACE2 knockout (KO) lung cells. Our model system based on human iPS cell-derived lung cells may be applied to understand the molecular biology regulating viral respiratory infection leading to potential therapeutic developments for COVID-19 and the prevention of future pandemics.
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Interstitial lung disease (ILD) represents a large group of diseases characterized by chronic inflammation and fibrosis of the lungs, for which therapeutic options are limited. Among several causative genes of familial ILD with autosomal dominant inheritance, the mutations in the BRICHOS domain of SFTPC cause protein accumulation and endoplasmic reticulum stress by misfolding its proprotein. Through a screening system using these two phenotypes in HEK293 cells and evaluation using alveolar epithelial type 2 (AT2) cells differentiated from patient-derived induced pluripotent stem cells (iPSCs), we identified Cryptotanshinone (CPT) as a potential therapeutic agent for ILD. CPT decreased cell death induced by mutant SFTPC overexpression in A549 and HEK293 cells and ameliorated the bleomycin-induced contraction of the matrix in fibroblast-dependent alveolar organoids derived from iPSCs with SFTPC mutation. CPT and this screening strategy can apply to abnormal protein-folding-associated ILD and other protein-misfolding diseases.
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Although recent decades have seen the identification, classification and discovery of the genetic basis of many children's interstitial and rare lung disease (chILD) disorders, detailed understanding of pathogenesis and specific therapies are still lacking for most of them. Fortunately, a revolution of technological advancements has created new opportunities to address these critical knowledge gaps. High-throughput sequencing has facilitated analysis of transcription of thousands of genes in thousands of single cells, creating tremendous breakthroughs in understanding normal and diseased cellular biology. Spatial techniques allow analysis of transcriptomes and proteomes at the subcellular level in the context of tissue architecture, in many cases even in formalin-fixed, paraffin-embedded specimens. Gene editing techniques allow creation of "humanized" animal models in a shorter time frame, for improved knowledge and preclinical therapeutic testing. Regenerative medicine approaches and bioengineering advancements facilitate the creation of patient-derived induced pluripotent stem cells and their differentiation into tissue-specific cell types which can be studied in multicellular "organoids" or "organ-on-a-chip" approaches. These technologies, singly and in combination, are already being applied to gain new biological insights into chILD disorders. The time is ripe to systematically apply these technologies to chILD, together with sophisticated data science approaches, to improve both biological understanding and disease-specific therapy.
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Primary ciliary dyskinesia (PCD) is a hereditary disease caused by pathogenic variants in genes associated with motile cilia. Some variants responsible for PCD are reported to be ethnic-specific or geographical-specific. To identify the responsible PCD variants of Japanese PCD patients, we performed next-generation sequencing of a panel of 32 PCD genes or whole-exome sequencing in 26 newly identified Japanese PCD families. We then combined their genetic data with those from 40 Japanese PCD families reported previously, for an overall analysis of 66 unrelated Japanese PCD families. We conducted Genome Aggregation Database and TogoVar database analyses to reveal the PCD genetic spectrum of the Japanese population and compare with other ethnic groups worldwide. We identified 22 unreported variants among the 31 patients in the 26 newly identified PCD families, including 17 deleterious variants estimated to cause lack of transcription or nonsense-mediated mRNA decay and 5 missense mutations. In all 76 PCD patients from the 66 Japanese families, we identified 53 variants on 141 alleles in total. Copy number variation in DRC1 is the most frequent variant in Japanese PCD patients, followed by DNAH5 c.9018C>T. We found 30 variants specific to the Japanese population, of which 22 are novel. Furthermore, 11 responsible variants in the Japanese PCD patients are common in East Asian populations, while some variants are more frequent in other ethnic groups. In conclusion, PCD is genetically heterogeneous between different ethnicities, and Japanese PCD patients have a characteristic genetic spectrum.
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Trastornos de la Motilidad Ciliar , Variaciones en el Número de Copia de ADN , Pueblos del Este de Asia , Humanos , Trastornos de la Motilidad Ciliar/genética , Trastornos de la Motilidad Ciliar/patología , Variaciones en el Número de Copia de ADN/genética , Genómica , MutaciónRESUMEN
Mesenchymal cells are necessary for organ development. In the lung, distal tip fibroblasts contribute to alveolar and airway epithelial cell differentiation and homeostasis. Here, we report a method for generating human induced pluripotent stem cell (iPSC)-derived mesenchymal cells (iMESs) that can induce human iPSC-derived alveolar and airway epithelial lineages in organoids via epithelial-mesenchymal interaction, without the use of allogenic fetal lung fibroblasts. Through a transcriptome comparison of dermal and lung fibroblasts with their corresponding reprogrammed iPSC-derived iMESs, we found that iMESs had features of lung mesenchyme with the potential to induce alveolar type 2 (AT2) cells. Particularly, RSPO2 and RSPO3 expressed in iMESs directly contributed to AT2 cell induction during organoid formation. We demonstrated that the total iPSC-derived alveolar organoids were useful for characterizing responses to the influenza A (H1N1) virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, demonstrating their utility for disease modeling.
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COVID-19 , Células Madre Pluripotentes Inducidas , Subtipo H1N1 del Virus de la Influenza A , Humanos , SARS-CoV-2 , COVID-19/metabolismo , OrganoidesRESUMEN
Primary ciliary dyskinesia (PCD) is a rare hereditary disease. We herein report two sisters in their 20s with suspected PCD. They were both born at full term and did not have situs inversus. Chest computed tomography showed similar signs of bronchiectasis in both siblings. Genetic examinations of the family confirmed that the sisters both harbored a homozygous variant in the growth-arrest-specific 2-like 2 (GAS2L2) gene. This is the third report of a family with PCD caused by a GAS2L2 variant.
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Bronquiectasia , Trastornos de la Motilidad Ciliar , Situs Inversus , Bronquiectasia/diagnóstico por imagen , Bronquiectasia/genética , Trastornos de la Motilidad Ciliar/diagnóstico por imagen , Trastornos de la Motilidad Ciliar/genética , Femenino , Humanos , Proteínas de Microfilamentos , Proteínas Asociadas a Microtúbulos/genética , Hermanos , Tomografía Computarizada por Rayos XRESUMEN
Mixed tumour of the skin or chondroid syringoma (CS) is a rare and mostly benign neoplasm of the sweat glands. Although CS is frequently located on varied parts of the head and neck region, the lower lip is a rarely reported site. The present report describes a case of CS of the lower lip in a 58-year-old male as an expository case to further emphasise the need for proper diagnosis, appropriate treatment and prognostic evaluation. The patient presented with a round, non-tender, slightly hard and mobile mass beneath the mucocutaneous junction of his left lateral side of the lower lip. Radiology revealed a mass measuring 11x11x7 mm3 in size at a depth of ~2 mm. Furthermore, magnetic resonance T1- and T2-weighted images showed slightly low and high signal intensities, respectively. A provisional diagnosis of benign tumour of the lower lip was made, and surgical excision biopsy taken under local anaesthesia, while considering the patient's cosmetic appearance. Histopathology demonstrated features akin to apocrine gland, chondroid and myxoid stroma consistent with the diagnosis of benign CS. No evidence of recurrence or satellites were recorded after a follow-up of nearly 2 years. Although rare, a high index of suspicion for CS among other cutaneous adnexal tumours of the lower lip is necessary. In addition, interprofessional collaboration in the management of such oral tumours could enhance patient satisfaction amid prevailing intraoral and aesthetic concerns.
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The absence of in vitro platforms for human pulmonary toxicology studies is becoming an increasingly serious concern. The respiratory system has a dynamic mechanical structure that extends from the airways to the alveolar region. In addition, the epithelial, endothelial, stromal, and immune cells are highly organized in each region and interact with each other to function synergistically. These cells of varied lineage, particularly epithelial cells, have been difficult to use for long-term culture in vitro, thus limiting the development of useful experimental tools. This limitation has set a large distance between the bench and the bedside for analyzing the pathogenic mechanisms, the efficacy of candidate therapeutic agents, and the toxicity of compounds. Several researchers have proposed solutions to these problems by reporting on methods for generating human lung epithelial cells derived from pluripotent stem cells (PSCs). Moreover, the use of organoid culture, organ-on-a-chip, and material-based techniques have enabled the maintenance of functional PSC-derived lung epithelial cells as well as primary cells. The aforementioned technological advances have facilitated the in vitro recapitulation of genetic lung diseases and the detection of ameliorating or worsening effects of genetic and chemical interventions, thus indicating the future possibility of more sophisticated preclinical compound assessments in vitro. In this review, we will update the recent advances in lung cell culture methods, principally focusing on human PSC-derived lung epithelial organoid culture systems with the hope of their future application in toxicology studies.
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Pulmón/citología , Células Madre Pluripotentes/citología , Toxicología/métodos , Animales , Técnicas de Cultivo de Célula , Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/tendencias , Humanos , Dispositivos Laboratorio en un Chip , Organoides/citología , Toxicología/tendenciasRESUMEN
Mucoepidermoid carcinomas (MECs) are rare head and neck malignant tumours that were originally considered to be benign. It has been estimated that ~20% of MECs in the major salivary glands, such as the parotid gland, and 50% in the several minor salivary glands found in the oral cavity, are malignant. The diagnosis of MECs is mainly based on ancillary and immunohistochemistry testing. However, owing to the difficulty in harvesting adequate material for histological examination, the histopathological diagnosis of intraoral MECs may be particularly challenging. We herein report a rare case of an 82-year-old patient who presented to the Department of Oral and Maxillofacial Surgery of Ryukyu University Hospital with complaints of a progressive swelling and pain in the ventral surface of the apex of the tongue. The patient had previously undergone needle biopsy and the histopathological analysis of the tumour suggested a diagnosis of irritation fibroma. To ensure a more accurate histopathological assessment, an incisional biopsy was performed, in addition to the haematological and radiological assessments. Examination of the obtained surgical specimen confirmed low-grade MEC of the anterior lingual gland. The tumour was surgically excised, the patient healed uneventfully and no recurrence was detected on the regular 3-year follow-up. Although MECs are relatively more common in the minor salivary glands of the oral cavity, they are a rare occurrence in the anterior lingual gland. Therefore, adequate histological material should be surgically harvested to perform a complete evaluation of the morphology and cytology of the tumour and ensure the accuracy of diagnosis.
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An 11-month-old boy with productive cough was referred to our hospital. He had nasal obstruction immediately after birth, and wheezing, wet cough, and rhinorrhea were observed daily after the neonatal period. Clinical and imaging findings revealed secretory otitis media, chronic sinusitis, and bronchiectasis. Primary ciliary dyskinesia was suspected. Transmission electron microscopy of nasal cilia showed defects of the outer and inner dynein arms. Genetic examinations of the family revealed copy number variation in PIH1 domain-containing 3 (PIH1D3) in the proband and mother. This is the first report of a Japanese patient with primary ciliary dyskinesia caused by copy number variation in PIH1D3.
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Trastornos de la Motilidad Ciliar , Síndrome de Kartagener , Cilios , Trastornos de la Motilidad Ciliar/genética , Tos , Variaciones en el Número de Copia de ADN/genética , Humanos , Lactante , Síndrome de Kartagener/genética , Masculino , NarizRESUMEN
OBJECTIVE: Primary ciliary dyskinesia (PCD) is a rare hereditary disease. Most reports of PCD in Japan are case reports, and clinical analysis has not been performed. Differences in the causative genes might affect the clinical features in different ethnic groups. The purpose of this study was to clarify the clinical features of Japanese patients with PCD. METHODS: We performed a retrospective chart review of PCD patients seen at Mie University Hospital and patients whose blood samples were sent to us for genetic analysis from 2011 to 2020. Data on the following items were collected and analyzed: age at first visit to the hospital, age at diagnosis of PCD, process of referral to our facility, chief complaint, situs status, PrImary CiliARy DyskinesiA Rule (PICADAR) score, nasal nitric oxide concentration, otoscopic findings, rhinoscopic findings, and paranasal computed tomography scan findings. RESULTS: Sixty-seven patients (24 male, 43 female) were diagnosed with PCD during the study period. Age at diagnosis ranged from 2 months to 69 years (median, 17 years). Respiratory symptoms (77%) were the most common complaint, followed by nasal (15%) and aural (8%) symptoms. Situs inversus was present in 17 (25%) cases. Only 2 cases had congenital cardiac anomalies. The mean PICADAR score was 7.3 (range, 3-14) points. Approximately 50% of tympanic membranes showed retraction, suggesting otitis media with effusion. The mean Lund-Mackay score was 12.8 (range, 7-17) points, suggesting that the radiographic findings were not always severe. There was no significant difference in the total Lund-Mackay score between patients with and without situs inversus (12.7 vs. 12.6, respectively). CONCLUSION: Situs inversus was present in 25% of Japanese PCD patients, which is much lower than observed in other countries. This is a result of differences in the major disease-causing genes. The general rule that "situs inversus is observed in approximately 50% of PCD patients" cannot be applied, at least, in Japanese PCD patients.