Published papers

The relapse of tongue cancer (TC) after chemotherapy is caused by minimal residual disease (MRD), which is a few remaining cancer cells after chemotherapy. To understand the mechanism of MRD in TC, we created a library of TC organoids (TCOs) from 28 untreated TC patients at diverse ages and cancer stages. These TCOs reproduced the primary TC tissues both in vitro and in a xenograft model, and several TCO lines survived after cisplatin treatment (chemo-resistant TCOs). Of note, the chemo-resistant TCOs showed “heritable” embryonic diapause-like features before treatment and activation of the autophagy and cholesterol biosynthetic pathways. Importantly, inhibiting these pathways with specific inhibitors converted the chemo-resistant TCOs into chemo-sensitive TCOs. Conversely, autophagy activation with mTOR inhibitors conferred chemo-resistance on the chemo-sensitive TCOs. This unique model provides insights into the mechanism of MRD formation in TCs, leading to effective therapeutic approaches to reduce the recurrence of TC.

Nonsense-mediated mRNA decay represents a biologic clearing system against aberrant mRNAs harboring nonsense and frameshift mutations and depends on three factors, UPF1, UPF2, and UPF3 (UPF3A, UPF3B). While germline pathogenic variants of UPF3B and UPF2 are known to be associated with neurodevelopmental disorders, germline variants in UPF1 have not been reported, until date, as being associated with any human disorders. Herein, we report two unrelated patients with de novo UPF1 variants. Patient 1 was a 5-year-old girl with intellectual disabilities, frontal bossing, hypertelorism, high frontal hairline, and thin upper lip. Patient 2 was a 2-year-old female child with intellectual disabilities and similar features. Trio exome analysis revealed a de novo heterozygous variant in UPF1 in both the patients (Patient 1: NM_002911.4): c.949_951del, p.(Asp317del); Patient 2: c.1984G>A, p.(Asp662Asn)). We conducted experiments using Drosophila models to evaluate the functional relevance of these UPF1 variants. Enforced expression of the wild-type Upf1 allele under the control of the pan-neuronal nSyb-GAL4 driver caused mortality, mostly at the pupal stage, but still yielded adult flies. By contrast, expression of the Asp294del (Asp317del in humans) variant caused embryonic or early larval lethality and that of the Asp643Asn (Asp662Asn in humans) caused third instar larval lethality; neither produced pupa nor adult fly. Thus, the developmental defects caused by the variants, especially Asp294del, were more severe than those caused by the wild-type allele. These observations suggest that both variants are deleterious mutations. In conclusion, germline variants in UPF1 are associated with intellectual disabilities in humans.

Germline variants in ARID1A have been associated with the so-called BAFopathies, including Coffin-Siris syndrome, which is characterized by hypertrichosis, short fifth finger, thin upper lip, and thick lower lip, is associated with a unique episignature. Hydrocephalus has not been considered part of BAFopathy until recently, when ARID1A variants were implicated in prenatal-onset hydrocephalus. It remains unknown whether ARID1A-associated hydrocephalus is linked to a specific class of variants and whether it exhibits an episignature comparable to that of BAFopathies. We conducted genomic and epigenomic analyses on a fetus diagnosed with severe hydrocephalus on prenatal ultrasound at 19 weeks. After detailed genetic counseling, the pregnancy was terminated at 21 weeks. The delivered fetus exhibited short fifth fingers, thin upper lip, and thick lower lip. Postmortem exome sequencing using umbilical cord blood identified a de novo heterozygous frameshift variant in the last exon of ARID1A (NM_006015.6:c.5259_5262dupGTCT, p.(Ser1755Valfs∗2)). The frameshift variant in the last exon was expected to escape nonsense-mediated mRNA decay (NMD), and we did confirm this through RNA-seq. Concurrent episignature analysis by nanopore sequencing and a support vector machine-based classifier showed that the fetus maps to the BAFopathy group rather than a separate position on the UMAP. Genotype-phenotype correlation analysis of unpublished data from previous reports regarding hydrocephalus and potential NMD escape, with input from the original authors, indicated that the association remains ambiguous. Hence, ARID1A-associated hydrocephalus occurs within the broader clinical and epigenomic spectrum of BAFopathies, but a distinct genetic mechanism caused by NMD escape is unlikely to play a role.

In standard short-read whole-exome sequencing (WES), capture probes are typically designed to target the protein-coding regions (CDS), and regions outside the exons—except for adjacent intronic sequences—are rarely sequenced. Although the majority of known pathogenic variants reside within the CDS as nonsynonymous variants, some disease-causing variants are located in regions that are difficult to detect by WES alone, such as deep intronic variants and structural variants, often requiring whole-genome sequencing (WGS) for detection. Moreover, WES has limitations in reliably identifying pathogenic variants within mitochondrial DNA or repetitive regions. Here, we propose a strategy to improve the diagnostic yield in a cost-effective manner by expanding the target design of WES beyond the CDS. As an illustrative example, we experimentally validated an extended WES approach covering intronic and untranslated regions (UTRs) of 188 genes listed in the Japanese public health insurance-covered multiple gene testing, intronic and UTRs of 81 genes listed in ACMG Secondary Findings (SF) v3.2, and 70 repeat regions associated with diseases. Additionally, the entire mitochondrial genome was targeted. We demonstrate the coverage of these extended regions based on experimental data and present case examples in which previously diagnosed pathogenic variants located outside the CDS were successfully detected using this approach. This strategy enables a substantial increase in the chance of achieving a definitive diagnosis for patients using WES alone, without requiring WGS, at a cost comparable to conventional WES. Our method has the potential to significantly shorten the diagnostic odyssey and represents a valuable approach in clinical genomics.

Background Pulmonary non-tuberculous mycobacterial (NTM) disease is a respiratory infection with an increasing incidence worldwide, including Japan. Host factors may also be involved in the establishment of pulmonary NTM disease. Cystic fibrosis transmembrane conductance regulator (CFTR) variants are associated with pulmonary NTM disease and bronchiectasis. However, data on CFTR variants in the Japanese population remain limited. Objectives We aimed to determine the frequency of CFTR variants and the impact on the clinical features of pulmonary NTM disease and bronchiectasis in the Japanese population. Methods We analysed 458 patients with either pulmonary NTM disease, non-cystic fibrosis bronchiectasis or both at Keio University Hospital from February 2016 to March 2019. CFTR variants were identified using exome sequencing, Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). These variants were determined to be deleterious using CFTR2 and in silico tools. Clinical characteristics of patients with and without CFTR variants were compared in a 1:8 age-matched and sex-matched ratio. Additionally, exome sequencing was performed for the family of a patient with a family history of pulmonary NTM disease. Results Deleterious CFTR variants were identified in 16 patients (3.5%). One variant was identified by MLPA, and 15 by Sanger sequencing. All patients harboured a CFTR variant in one allele. Compared with matched controls, these patients had lower sputum culture conversion rates and higher rates of macrolide resistance. In one family cluster, members with pulmonary NTM disease were found to carry the same CFTR variant. Conclusions We defined the frequency and clinical characteristics of CFTR variants among the Japanese population with either pulmonary NTM disease, noncystic fibrosis bronchiectasis or both and found that patients with CFTR variants may be refractory to pulmonary Mycobacterium avium complex disease. Further comprehensive research is needed to assess the impact of CFTR variants on pulmonary NTM disease and bronchiectasis in non-European populations.

Repeated oncogenic mutations and polyclonal proliferation are evident in cancers. However, little is known about the polyclonal principles governing the systemic cancerous lineage during immunotherapy. Here, we examine a unique autopsy case of metastatic urothelial carcinoma that exhibits different treatment responses to anti-PD-1 therapy at each tumor site. By performing in-depth analyses of different multiregional bulk tumor masses, we reveal that subsets of subclones acquire potential driver mutations under treatment selection pressure. Spatial transcriptomics analysis reveals that subclones resistant to immunotherapy form distinct immunosuppressive environments consistent with their habitats. Furthermore, different cancer hallmarks are identified in each of the subclones that expand under immunotherapy at single-cell level; for example, one subclone is more proliferative, and another is more stem-cell-like. In summary, this study provides an overall picture of the polyclonal competition and changes in the immune microenvironment that are related to resistance to immunotherapy in patients with malignancies.

CUL3 forms Cullin-Ring ubiquitin ligases (CRL) with Ring-box protein and BTB-adaptor proteins. A variety of BTB-adaptor proteins have been reported to interact with the N-terminus of CUL3, which makes it possible to recognize various substrates for degradation. Regarding the association of CUL3 with neurodevelopmental disorders, a recent study reported three patients with global developmental delay, who carried de novo variants in CUL3. Here, we describe a novel de novo CUL3 variant (c.158G > A, p.Ser53Asn) identified in a patient with global developmental delay, who presented some novel dysmorphic features, including macrocephaly, characteristic facial features, and cutis marmorata. Immunoprecipitation and immunoblot analyses identified significantly weaker binding ability to some BTB proteins in CUL3-S53N compared to wild-type. Interestingly, label-free quantification proteomics analysis of samples immunoprecipitated by CUL3-S53N showed a significantly decreased interaction with some BTB proteins, while almost equal interaction or significantly increased interaction was observed with other BTB proteins. The binding between CUL3 and BTB proteins is essential for CRL substrate recognition, and alteration of their interaction is thought to result in the quantitative alteration in substrate proteins. It is possible that the difference of dysmorphic features between the present case and previously reported cases is caused by the distinctive effect of each CUL3 variant on substrate proteins. The clinical information of the present case will expand the picture of CUL3-related global developmental disorders, and subsequent cell biological analysis of the novel mutation will provide insight into the underlying molecular mechanism of how CUL3 pathogenic variants cause neurological disorders.

Esophageal squamous cell carcinoma (ESCC) is a deadly cancer with a poor prognosis and a high recurrence rate after chemotherapy, posing a significant clinical challenge. To elucidate the molecular basis of chemotherapy (chemo)-resistance and to develop methods to effectively eliminate chemo-resistant tumor clones, we established an ESCC organoid (ESCCO) library from 24 ESCC patients of various stages, ages, and treatments. These ESCCOs faithfully recapitulate the oncogenic mutations observed in the original ESCC tissues and manifest tumorigenic properties when xenografted. The ESCCOs respond differently to cisplatin and 5-fluorouracil, chemotherapeutic agents commonly used to treat ESCC patients, with 7 ESCCOs exhibiting potent chemo-resistance. Notably, the chemo-resistant ESCCOs show higher genes involved in antioxidant stress response pathways and more accessible chromatin at their loci than the sensitive ESCCOs. These genes can serve as valuable biomarkers to stratify chemo-resistant ESCCs in histopathological specimens. Through drug screening using the ESCCO library, we reveal that fedratinib effectively induces cell death in chemo-resistant ESCCOs. Collectively, our human ESCCO model offers novel insights into the mechanism of chemo-resistance in ESCCs, which is critical for developing effective therapeutic approaches to eradicate the recurrence of ESCCs.

Pogo transposable element with zinc finger domain(POGZ)遺伝子は近年、本遺伝子の変異と自閉症スペクトラム障害および知的障害の関連性が指摘されている。これまでに40例の報告があり、言語発達遅滞、視力障害、小頭症、肥満傾向といった表現型が指摘されている。脳波異常は20%に報告されているが、てんかんとして治療を要した症例の報告はない。今回、これまでに報告のなかった難治性のてんかんと退行を有する症例にde novoのPOGZ遺伝子短縮型変異(c.2102del;p.Pro701Leufs*18)をヘテロ接合性に同定した。POGZ変異が原因となる表現型は自閉症スペクトラム障害に止まらず、多彩な神経症状を示し、進行性のてんかん症候群の原因となる可能性が示唆された。(著者抄録)

COL4A1変異は,IV型コラーゲンの異常により血管などの基底膜を障害し,多臓器に多彩な症状を呈する.てんかんを合併した場合は難治なことが多い.症例は5歳4ヵ月の男児.先天性の眼病変を認め,Peters異常と診断された.発達遅滞を伴い,頭部画像所見に異常を認めた.10ヵ月時にてんかん性スパズムを発症し,West症候群と診断された.ACTH療法を含む初期治療で発作は一時消失したが再発した.1歳9ヵ月時にvigabatrin(VGB)を眼病変の悪化を考慮し慎重に導入し,VGBは適量(100mg/kg/day)で発作は抑制された.眼科的検査を複数回実施し,網膜電図に異常は認めなかった.3歳6ヵ月にVGBを中止後も発作は抑制されている.COL4A1にde novoの既知の病的変異NM_001845.6:c.3620G>A,p.(G1207E)を認めた.COL4A1関連疾患の難治West症候群に対しては先天性眼球異常を伴う例でもVGBを試みる意義があると考えられた.(著者抄録)

West症候群(WS)では近年多くの原因遺伝子が知られるようになった。今回、WSの原因遺伝子としては一般的ではないPTEN(Phosphatase and Tensin Homolog)にバリアントをもつWSの女児例を経験した。周産期歴には特に異常なく、生後7ヵ月時にシリーズ形成する癲癇性スパズムを発症した。初診時に筋緊張低下と運動発達遅延および頭囲拡大を認め、発作間欠期脳波検査でヒプサリズミアがみられWSと診断した。頭部MRIでは脳回の構造異常や過誤腫はみられず、ACTH療法とその後の抗発作薬内服治療によって発作は消失し、脳波異常も改善したが、中等度～重度の精神運動発達遅滞がみられた。遺伝子解析でPTENに既知の病的バリアント(NM 000314:exon6:c.517C>T:p.(R173C))が同定された。一般に生殖細胞系列のPTEN病的バリアントを原因とする疾患の表現型としてCowden症候群やBannayan-Riley-Ruvalcaba症候群、Proteus症候群などが知られており、これらはいずれも過誤腫を伴う疾患で、PTEN Hamartoma Tumor Syndromeと総称されている。既報ではPTENバリアントによって脳形成障害をきたした場合でも癲癇を発症する割合は低く、本例のように脳回の構造異常を伴わずにWSをきたした症例の報告はこれまでになかった。