Publications by authors named "Yasushi Okazaki"

225 Publications

Valine metabolites analysis in ECHS1 deficiency.

Mol Genet Metab Rep 2021 Dec 9;29:100809. Epub 2021 Oct 9.

Department of Pediatrics, Jichi Medical University, Tochigi, Japan.

Short-chain enoyl-CoA hydratase (ECHS1) is involved in amino acid and fatty acid catabolism in mitochondria and its deficiency causes Leigh syndrome or exercise-induced dystonia. More than 60 patients with this condition have been reported till date. The accumulation of intermediate metabolites of valine is assumed to be responsible for the cytotoxicity. Since protein restriction, including valine reportedly improves neurological symptoms, it is essential to consider the possible incidence of and diagnose ECHS1 syndrome in the earlier stages. This study reported the liquid chromatography with tandem mass spectrometry (LC-MS/MS) urine and plasma metabolite analysis in six cases, including four new cases with ECHS1 deficiency. The values of urine cysteine/cysteamine conjugates from valine metabolites, S-(2-carboxypropyl) cysteine/cysteamine from methacrylyl-CoA, and S-(2-carboxyethyl) cysteine/cysteamine from acryloyl-CoA were separated between six patients and six normal controls. The LC-MS/MS analysis revealed that these metabolites can be used for the early diagnosis and evaluation of diet therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymgmr.2021.100809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8507190PMC
December 2021

Neonatal-onset mitochondrial disease: clinical features, molecular diagnosis and prognosis.

Arch Dis Child Fetal Neonatal Ed 2021 Oct 7. Epub 2021 Oct 7.

Department of Metabolism, Chiba Children's Hospital, Chiba, Japan

Objective: Neonatal-onset mitochondrial disease has not been fully characterised owing to its heterogeneity. We analysed neonatal-onset mitochondrial disease in Japan to clarify its clinical features, molecular diagnosis and prognosis.

Design: Retrospective observational study from January 2004 to March 2020.

Setting: Population based.

Patients: Patients (281) with neonatal-onset mitochondrial disease diagnosed by biochemical and genetic approaches.

Interventions: None.

Main Outcome Measures: Disease types, initial symptoms, biochemical findings, molecular diagnosis and prognosis.

Results: Of the 281 patients, multisystem mitochondrial disease was found in 194, Leigh syndrome in 26, cardiomyopathy in 38 and hepatopathy in 23 patients. Of the 321 initial symptoms, 236 occurred within 2 days of birth. Using biochemical approaches, 182 patients were diagnosed by mitochondrial respiratory chain enzyme activity rate and 89 by oxygen consumption rate. The remaining 10 patients were diagnosed using a genetic approach. Genetic analysis revealed 69 patients had nuclear DNA variants in 36 genes, 11 of 15 patients had mitochondrial DNA variants in five genes and four patients had single large deletion. The Cox proportional hazards regression analysis showed the effects of Leigh syndrome (HR=0.15, 95% CI 0.04 to 0.63, p=0.010) and molecular diagnosis (HR=1.87, 95% CI 1.18 to 2.96, p=0.008) on survival.

Conclusions: Neonatal-onset mitochondrial disease has a heterogenous aetiology. The number of diagnoses can be increased, and clarity regarding prognosis can be achieved by comprehensive biochemical and molecular analyses using appropriate tissue samples.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/archdischild-2021-321633DOI Listing
October 2021

Trigenic // mutations in myelodysplasia with Usher syndrome.

Heliyon 2021 Aug 14;7(8):e07804. Epub 2021 Aug 14.

Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan.

Trio-next generation sequencing is useful to identify undiagnosed inherited diseases. We have attended a patient with trigenic // pathogenic variants, which caused two distinct diseases, myelodysplastic syndrome and Usher syndrome. Whole genome sequencing of peripheral blood from the patient and his parents were applied to identify disease-causing genes. Sanger sequencing was performed to validate the identified // variants. Our results identified disease-associated variants in (disease inheritance autosomal recessive) and in (disease inheritance also autosomal recessive) and a variant in (disease inheritance autosomal dominant). Although the variants identified in and have been reported, their co-existence in association with disease-causing variation in a third gene has not. They broaden the spectrum of in Usher syndrome. Findings on next generation sequencing guided rapid and accurate diagnosis, resulting in patient-tailored therapeutic intervention.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.heliyon.2021.e07804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8379464PMC
August 2021

Ngn3-Positive Cells Arise from Pancreatic Duct Cells.

Int J Mol Sci 2021 Aug 9;22(16). Epub 2021 Aug 9.

Department of Nutritional Science and Food Safety, Faculty of Applied Bioscience, Tokyo University of Agriculture, Tokyo 156-8502, Japan.

The production of pancreatic β cells is the most challenging step for curing diabetes using next-generation treatments. Adult pancreatic endocrine cells are thought to be maintained by the self-duplication of differentiated cells, and pancreatic endocrine neogenesis can only be observed when the tissue is severely damaged. Experimentally, this can be performed using a method named partial duct ligation (PDL). As the success rate of PDL surgery is low because of difficulties in identifying the pancreatic duct, we previously proposed a method for fluorescently labeling the duct in live animals. Using this method, we performed PDL on neurogenin3 (Ngn3)-GFP transgenic mice to determine the origin of endocrine precursor cells and evaluate their potential to differentiate into multiple cell types. Ngn3-activated cells, which were marked with GFP, appeared after PDL operation. Because some GFP-positive cells were aligned proximally to the duct, we hypothesized that Ngn3-positive cells arise from the pancreatic duct. Therefore, we next developed an in vitro pancreatic duct culture system using Ngn3-GFP mice and examined whether Ngn3-positive cells emerge from this duct. We observed GFP expressions in ductal organoid cultures. GFP expressions were correlated with Ngn3 expressions and endocrine cell lineage markers. Interestingly, tuft cell markers were also correlated with GFP expressions. Our results demonstrate that in adult mice, Ngn3-positive endocrine precursor cells arise from the pancreatic ducts both in vivo and in vitro experiments indicating that the pancreatic duct could be a potential donor for therapeutic use.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22168548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8395223PMC
August 2021

Genome sequencing and RNA-seq analyses of mitochondrial complex I deficiency revealed Alu insertion-mediated deletion in NDUFV2.

Hum Mutat 2021 Nov 2;42(11):1422-1428. Epub 2021 Sep 2.

Diagnostics and Therapeutics of Intractable Diseases, Intractable Disease Research Center, Graduate School of Medicine, Juntendo University, Tokyo, Japan.

Isolated complex I deficiency is the most common cause of pediatric mitochondrial disease. Exome sequencing (ES) has revealed many complex I causative genes. However, there are limitations associated with identifying causative genes by ES analysis. In this study, we performed multiomics analysis to reveal the causal variants. We here report two cases with mitochondrial complex I deficiency. In both cases, ES identified a novel c.580G>A (p.Glu194Lys) variant in NDUFV2. One case additionally harbored c.427C>T (p.Arg143*), but no other variants were observed in the other case. RNA sequencing showed aberrant exon splicing of NDUFV2 in the unsolved case. Genome sequencing revealed a novel heterozygous deletion in NDUFV2, which included one exon and resulted in exon skipping. Detailed examination of the breakpoint revealed that an Alu insertion-mediated rearrangement caused the deletion. Our report reveals that combined use of transcriptome sequencing and GS was effective for diagnosing cases that were unresolved by ES.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/humu.24274DOI Listing
November 2021

Immunohistochemical staining patterns of p53 predict the mutational status of TP53 in oral epithelial dysplasia.

Mod Pathol 2021 Aug 17. Epub 2021 Aug 17.

Department of Pathology, Saitama Medical Center, Saitama Medical University, Saitama, Japan.

Next-generation sequencing of oral squamous cell carcinoma (OSCC) has revealed TP53 as the most frequently mutated gene in OSCC mutually exclusive with human papillomavirus infection. Oral epithelial dysplasia (OED) is defined as a precancerous lesion of OSCC by the current World Health Organization (WHO) classification; therefore, it is assumed that TP53 mutations occur in early precancerous conditions such as OED. Here, we conducted an integrated analysis of TP53, including whole coding sequencing of TP53, FISH analysis of the 17p13.1 locus, and immunohistochemical analysis for p53 (p53-IHC), in 40 OED cases. We detected 20 mutations in 16 (40%) OED cases, and four cases, each harbored two mutations. FISH analysis revealed six of 24 cases (25%) had a deletion on 17p13.1, and four cases had concurrent TP53 mutations and 17p13.1 deletion (2-hit). Also, the increased frequency of TP53 mutations in higher degrees of OED implies acquisition of the mutation is a major event toward OSCC. p53-IHC revealed that overall cases could be categorized into four patterns that correlate well with the mutational status of TP53. Especially, two patterns, broad p53 expression type (pattern HI) and p53 null type (pattern LS), strongly correlated with a missense mutation and nonsense mutation, respectively. Furthermore, seven of the 40 cases progressed to SCC, and six of these seven cases presented pattern HI or LS. Therefore, patterns HI and LS have a high risk for malignant transformation if excisional treatment is not performed irrespective of the dysplasia grade. Although the current WHO classification mainly focuses on morphological criteria for the diagnosis of OED, interobserver discrepancy appears in some instances of the OED diagnosis. Our immunohistochemical analysis supports a more accurate pathological diagnosis for OED in cases of low dysplastic changes or of differential diagnosis with non-dysplastic lesions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41379-021-00893-9DOI Listing
August 2021

Interleukin-4 Promotes Tuft Cell Differentiation and Acetylcholine Production in Intestinal Organoids of Non-Human Primate.

Int J Mol Sci 2021 Jul 24;22(15). Epub 2021 Jul 24.

Department of Nutritional Science and Food Safety, Faculty of Applied Bioscience, Tokyo University of Agriculture, Tokyo 156-8502, Japan.

In the intestine, the innate immune system excludes harmful substances and invading microorganisms. Tuft cells are taste-like chemosensory cells found in the intestinal epithelium involved in the activation of group 2 innate lymphoid cells (ILC2). Although tuft cells in other tissues secrete the neurotransmitter acetylcholine (ACh), their function in the gut remains poorly understood. In this study, we investigated changes in the expression of genes and cell differentiation of the intestinal epithelium by stimulation with interleukin-4 (IL-4) or IL-13 in macaque intestinal organoids. Transcriptome analysis showed that tuft cell marker genes were highly expressed in the IL-4- and IL-13-treated groups compared with the control, and the gene expression of choline acetyltransferase (ChAT), a synthesis enzyme of ACh, was upregulated in IL-4- and IL-13-treated groups. ACh accumulation was observed in IL-4-induced organoids using high-performance liquid chromatography-mass spectrometry (HPLC/MS), and ACh strongly released granules from Paneth cells. This study is the first to demonstrate ACh upregulation by IL-4 induction in primates, suggesting that IL-4 plays a role in Paneth cell granule secretion via paracrine stimulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22157921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8348364PMC
July 2021

Long-term prognosis and genetic background of cardiomyopathy in 223 pediatric mitochondrial disease patients.

Int J Cardiol 2021 Oct 21;341:48-55. Epub 2021 Jul 21.

Diagnostics and Therapeutics of Intractable Diseases, Intractable Disease Research Center, Graduate School of Medicine, Juntendo University, Tokyo, Japan; Laboratory for Comprehensive Genomic Analysis, RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan. Electronic address:

Background: Cardiomyopathy is a risk factor for poor prognosis in pediatric patients with mitochondrial disease. However, other risk factors including genetic factors related to poor prognosis in mitochondrial disease has yet to be fully elucidated.

Methods And Results: Between January 2004 and September 2019, we enrolled 223 consecutive pediatric mitochondrial disease patients aged <18 years with a confirmed genetic diagnosis, including 114 with nuclear gene mutations, 89 patients with mitochondrial DNA (mtDNA) point mutations, 11 with mtDNA single large-scale deletions and 9 with chromosomal aberrations. Cardiomyopathy at baseline was observed in 46 patients (21%). Hazard ratios (HR) and 95% confidence intervals (CI) were calculated for all-cause mortality. Over a median follow-up of 36 months (12-77), there were 85 deaths (38%). The overall survival rate was significantly lower in patients with cardiomyopathy than in those without (p < 0.001, log-rank test). By multivariable analysis, left ventricular (LV) hypertrophy (HR = 4.6; 95% CI: 2.8-7.3), neonatal onset (HR = 2.9; 95% CI: 1.8-4.5) and chromosomal aberrations (HR = 2.9; 95% CI: 1.3-6.5) were independent predictors of all-cause mortality. Patients with LV hypertrophy with neonatal onset and/or chromosomal aberrations had higher mortality (100% in 21 patients) than those with LV hypertrophy alone (71% in 14 patients).

Conclusion: In pediatric patients with mitochondrial disease, cardiomyopathy was common (21%) and was associated with increased mortality. LV hypertrophy, neonatal onset and chromosomal aberrations were independent predictors of all-cause mortality. Prognosis is particularly unfavorable if LV hypertrophy is combined with neonatal onset and/or chromosomal aberrations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijcard.2021.06.042DOI Listing
October 2021

Identification of Lynch syndrome-associated DNA mismatch repair-deficient bladder cancer in a Japanese hospital-based population.

Int J Clin Oncol 2021 Aug 2;26(8):1524-1532. Epub 2021 Jul 2.

Department of Digestive Tract and General Surgery, Saitama Medical Center, Saitama Medical University, Saitama, Japan.

Background: The prevalence of Lynch syndrome (LS)-associated DNA mismatch repair (MMR)-deficient bladder cancer (BC) has scarcely been investigated.

Methods: Immunohistochemistry for four MMR proteins (MLH1, MSH2, MSH6, and PMS2) was performed in formalin-fixed paraffin-embedded (FFPE) sections prepared from the resected specimens of 618 consecutive newly diagnosed BC cases. Genetic/epigenetic analyses were performed in patients displaying the loss of any MMR proteins in the tumor.

Results: Of the 618 patients, 9 (1.5%) showed the loss of MMR protein expression via immunohistochemistry; specifically, 3, 3, 2, and 1 patients displayed the loss of MLH1/PMS2, PMS2, MSH6, and MSH2/MSH6, respectively. All nine patients were male with a median age of 68 years (63-79 years). One had been previously diagnosed as having LS with an MSH2 variant. Genetic testing demonstrated the presence of a pathogenic PMS2 variant (n = 1), a variant of uncertain significance in MSH2 (n = 1), and no pathogenic germline variants of the MMR genes (n = 1). One patient with MSH6-deficient BC did not complete the genetic testing because of severe degradation of DNA extracted from the FFPE specimen, but the patient was strongly suspected to have LS because of their history of colon cancer and MSH6-deficient upper urinary tract cancer. There remained a possibility that the remaining four patients who refused genetic testing had LS.

Conclusions: The prevalence of LS-associated MMR-deficient BC was estimated to be 0.6-1.1% among unselected BC cases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10147-021-01922-yDOI Listing
August 2021

APC germline variant analysis in the adenomatous polyposis phenotype in Japanese patients.

Int J Clin Oncol 2021 Sep 9;26(9):1661-1670. Epub 2021 Jun 9.

Department of Digestive Tract and General Surgery, Saitama Medical Center, Saitama Medical University, Saitama, Japan.

Background: Familial adenomatous polyposis (FAP), an autosomal dominant disorder characterized by multiple colonic polyps, is caused by a germline pathogenic variant of the APC gene. However, this variant is not detected in up to 30% of patients with the adenomatous polyposis phenotype.

Methods: We performed next-generation sequencing (NGS) to identify the causative genes in FAP patients with 10 or more polyps. For patients in whom the APC germline variant was not able to be identified, we screened for APC mosaicism using high-coverage NGS of APC with DNA from leucocytes and/or frozen tissue.

Results: The pathogenic APC germline variant was found in 93.3%, 71.6%, and 17.1% of patients with profuse-type polyposis, sparse-type polyposis, and oligo-polyposis, respectively. The APC germline variant detection rate in patients with FAP-related diseases was 69.7% for fundic gland polyposis, 79.7% for duodenal adenoma, 94.7% for desmoid tumor, and 71.4% for thyroid cancer, with increasing numbers of extracolonic lesions associated with an increasing APC germline variant detection rate. A mosaic test detected nine patients with APC mosaicism. A comparison of APC-associated polyposis with APC mosaicism showed that patients with APC mosaicism had a low frequency of duodenal adenoma and a family history of colonic polyposis.

Conclusions: We determined the detection rate of the APC germline variant by phenotype and identified APC mosaicism. Genetic testing of FAP patients is important because it can help with surgical decision-making, monitoring, and genetic counseling. Furthermore, genetic testing by NGS proved to be an effective method of detecting APC germline variants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10147-021-01946-4DOI Listing
September 2021

Clinical heterogeneity in patients with m.4412G > A MT-TM mutation and different heteroplasmy levels.

Mitochondrion 2021 07 3;59:214-215. Epub 2021 Jun 3.

Diagnostics and Therapeutics of Intractable Diseases, Intractable Disease Research Center, Graduate School of Medicine, Juntendo University, Tokyo, Japan; Laboratory for Comprehensive Genomic Analysis, RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan. Electronic address:

The identification of the m.4412G > A MT-TM (mt-tRNA) mutation was first reported in 2019. The affected individual presented with childhood-onset seizures and myopathy and bilateral basal ganglia changes, with heteroplasmy levels in muscle as high as 90%. Here, we describe another adult-onset patient with the same mutation and additional phenotypes, including hearing impairment, cerebellar ataxia, progressive dementia, and myopathy. The 10% heteroplasmy level observed in skin fibroblasts from this patient are lower than those in the previously reported patient. Our report suggests possible clinical heterogeneity in patients with mitochondrial tRNA mutations based on heteroplasmy levels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mito.2021.06.001DOI Listing
July 2021

A case of ATR-X syndrome with mitochondrial respiratory chain dysfunction.

Eur J Med Genet 2021 Aug 27;64(8):104251. Epub 2021 May 27.

Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, Aichi, Japan.

Alpha-thalassemia X-linked intellectual disability (ATR-X) syndrome is caused by a mutation in ATRX, which is essential for proper chromatin remodeling. ATRX dysfunction leads to dysregulation of many genes due to abnormal chromatin remodeling, and causes a multisystem disorder in patients with ATR-X. Because mitochondrial disorders also show multisystem involvement, whether mitochondrial function is affected in patients with ATR-X is of interest. Here, we report a case of a 4-year-old male with a mutation (NM_000489.4: c.736C > T p.Arg246Cys) in ATRX, who showed mitochondrial dysfunction with complex I deficiency. The results from our study suggest that target genes of the ATRX protein may include those responsible for mitochondrial function, and mitochondrial dysfunction may contribute to some ATR-X phenotypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejmg.2021.104251DOI Listing
August 2021

A high mutation load of m.14597A>G in MT-ND6 causes Leigh syndrome.

Sci Rep 2021 05 27;11(1):11123. Epub 2021 May 27.

Diagnostics and Therapeutics of Intractable Diseases, Intractable Disease Research Center, Juntendo University, Graduate School of Medicine, Tokyo, Japan.

Leigh syndrome (LS) is an early-onset progressive neurodegenerative disorder associated with mitochondrial deficiency. m.14597A>G (p.Ile26Thr) in the MT-ND6 gene was reported to cause Leber's hereditary optic neuropathy (LHON) or dementia/dysarthria. In previous reports, less than 90% heteroplasmy was shown to result in adult-onset disease. Here, by whole mitochondrial sequencing, we identified m.14597A>G mutation of a patient with LS. PCR-RFLP analysis on fibroblasts from the patient revealed a high mutation load (> 90% heteroplasmy). We performed functional assays using cybrid cell models generated by fusing mtDNA-less rho0 HeLa cells with enucleated cells from patient fibroblasts carrying the m.14597A>G variant. Cybrid cell lines bearing the m.14597A>G variant exhibited severe effects on mitochondrial complex I activity. Additionally, impairment of cell proliferation, decreased ATP production and reduced oxygen consumption rate were observed in the cybrid cell lines bearing the m.14597A>G variant when the cells were metabolically stressed in medium containing galactose, indicating mitochondrial respiratory chain defects. These results suggest that a high mutation load of m.14597A>G leads to LS via a mitochondrial complex I defect, rather than LHON or dementia/dysarthria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-90196-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8160132PMC
May 2021

Germline deletion of chromosome 2p16-21 associated with Lynch syndrome.

Hum Genome Var 2021 May 19;8(1):19. Epub 2021 May 19.

Department of Digestive Tract and General Surgery, Saitama Medical Center, Saitama Medical University, Kawagoe, Japan.

We identified a Japanese patient with Lynch syndrome with a novel large germline deletion of chromosome 2p16-21, including the EPCAM, MSH2, and KCNK12 genes. The proband was a 46-year-old man with ascending colon cancer. The clinical significance of germline KCNK12 gene deletion, which encodes one of the subfamilies of two-pore-domain potassium channels, is still unknown.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41439-021-00152-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8134480PMC
May 2021

CD82 is a marker to isolate β cell precursors from human iPS cells and plays a role for the maturation of β cells.

Sci Rep 2021 05 5;11(1):9530. Epub 2021 May 5.

Institute for Quantitative Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

Generation of pancreatic β cells from pluripotent stem cells is a key technology to develop cell therapy for insulin-dependent diabetes and considerable efforts have been made to produce β cells. However, due to multiple and lengthy differentiation steps, production of β cells is often unstable. It is also desirable to eliminate undifferentiated cells to avoid potential risks of tumorigenesis. To isolate β cell precursors from late stage pancreatic endocrine progenitor (EP) cells derived from iPS cells, we have identified CD82, a member of the tetraspanin family. CD82 cells at the EP stage differentiated into endocrine cells more efficiently than CD82 EP stage cells. We also show that CD82 cells in human islets secreted insulin more efficiently than CD82 cells. Furthermore, knockdown of CD82 expression by siRNA or inhibition of CD82 by monoclonal antibodies in NGN3 cells suppressed the function of β cells with glucose-stimulated insulin secretion, suggesting that CD82 plays a role in maturation of EP cells to β cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-88978-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100138PMC
May 2021

Unique and abnormal subependymal pseudocysts in a newborn with mitochondrial disease.

Sci Prog 2021 Apr-Jun;104(2):368504211011873

Department of Pediatrics and Child Health, Nihon University School of Medicine, Tokyo, Japan.

Neonatal mitochondrial disease is occasionally observed in patients with intraventricular cysts in the brain. Atypical morphology is rarely seen in these cysts. Here, we report a case of neonatal lethal mitochondrial disease with gene mutation. We have, for the first time, described a subependymal pseudocyst (SEPC) with a fluctuating membrane. Our findings suggest that SEPCs with fluctuating membranes can be a potential diagnostic indicator of neonatal mitochondrial disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/00368504211011873DOI Listing
April 2021

Comprehensive analysis of DNA mismatch repair-deficient gastric cancer in a Japanese hospital-based population.

Jpn J Clin Oncol 2021 May;51(6):886-894

Department of Digestive Tract and General Surgery, Saitama Medical Center, Saitama Medical University, Saitama, Japan.

Background: The attention on mismatch repair-deficient (dMMR) gastric cancer has increased in this era of anti-PD-1 blockade therapy; however, the prevalence and molecular genetics of patients with dMMR gastric cancer have not been completely investigated.

Methods: Immunohistochemistry of MMR proteins (MLH1, MSH2, MSH6 and PMS2) was performed on formalin-fixed paraffin-embedded sections prepared from resected primary gastric cancers of 513 consecutive patients. Genetic and/or epigenetic alterations of the MMR genes were also investigated.

Results: Loss of expression of one or more MMR proteins was observed in 58 patients (11.3%); 54 patients showed loss of MLH1/PMS2, 3 patients showed loss of MLH1/PMS2/MSH6 and 1 patient showed loss of PMS2 alone. Among these 58 patients, 55 showed hypermethylation of the promoter region of MLH1. Genetic testing revealed that the remaining three patients had Lynch syndrome (n = 1) or Lynch-like syndrome (n = 2). A total of 15 patients (25.9% of all patients with dMMR gastric cancer and 2.9% of all patients with gastric cancer), including 11 patients with stage I-III dMMR gastric cancer who had recurrence and 4 patients with stage IV dMMR gastric cancer, are potential candidates for the use of anti-PD-1 blockades.

Conclusions: This is the first study to investigate the frequency and molecular genetic mechanisms of dMMR gastric cancer comprehensively, focusing on the benefit of using PD-1 blockades. Our observations will be beneficial in the clinical practice of metastatic gastric cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jjco/hyab026DOI Listing
May 2021

Fatal perinatal mitochondrial cardiac failure caused by recurrent duplications in the locus.

Med (N Y) 2021 Jan 9;2(1):49-73. Epub 2020 Jul 9.

Genetic Metabolic Disorders Service, Sydney Children's Hospital Network, The Children's Hospital at Westmead, Sydney, NSW 2145, Australia.

Background: In about half of all patients with a suspected monogenic disease, genomic investigations fail to identify the diagnosis. A contributing factor is the difficulty with repetitive regions of the genome, such as those generated by segmental duplications. The locus is one such region, in which recessive deletions and dominant duplications have recently been reported to cause lethal perinatal mitochondrial diseases characterized by pontocerebellar hypoplasia or cardiomyopathy, respectively.

Methods: Whole exome, whole genome and long-read DNA sequencing techniques combined with studies of RNA and quantitative proteomics were used to investigate 17 subjects from 16 unrelated families with suspected mitochondrial disease.

Findings: We report six different duplications in the gene locus causing a distinctive presentation including lethal perinatal cardiomyopathy, persistent hyperlactacidemia, and frequently corneal clouding or cataracts and encephalopathy. The recurrent 68 Kb duplications are identifiable from genome and exome sequencing but usually missed by microarrays. The duplications result in the formation of identical chimeric ATAD3A/ATAD3C proteins, altered ATAD3 complexes and a striking reduction in mitochondrial oxidative phosphorylation complex I and its activity in heart tissue.

Conclusions: duplications appear to act in a dominant-negative manner and the inheritance infers a low recurrence risk for families, unlike most pediatric mitochondrial diseases. More than 350 genes underlie mitochondrial diseases. In our experience the locus is now one of the five most common causes of nuclear-encoded pediatric mitochondrial disease but the repetitive nature of the locus means diagnoses may be frequently missed by current genomic strategies.

Funding: Australian NHMRC, US Department of Defense, Japanese AMED and JSPS agencies, Australian Genomics Health Alliance and Australian Mito Foundation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.medj.2020.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7875323PMC
January 2021

Prenatal diagnosis of severe mitochondrial diseases caused by nuclear gene defects: a study in Japan.

Sci Rep 2021 Feb 11;11(1):3531. Epub 2021 Feb 11.

Center for Medical Genetics, Chiba Children's Hospital, Chiba, Japan.

Prenatal diagnoses of mitochondrial diseases caused by defects in nuclear DNA (nDNA) or mitochondrial DNA have been reported in several countries except for Japan. The present study aimed to clarify the status of prenatal genetic diagnosis of mitochondrial diseases caused by nDNA defects in Japan. A comprehensive genomic analysis was performed to diagnose more than 400 patients, of which, 13 families (16 cases) had requested prenatal diagnoses. Eight cases diagnosed with wild type homozygous or heterozygous variants same as either of the heterozygous parents continued the pregnancy and delivered healthy babies. Another eight cases were diagnosed with homozygous, compound heterozygous, or hemizygous variants same as the proband. Of these, seven families chose to terminate the pregnancy, while one decided to continue the pregnancy. Neonatal- or infantile-onset mitochondrial diseases show severe phenotypes and lead to lethality. Therefore, such diseases could be candidates for prenatal diagnosis with careful genetic counseling, and prenatal testing could be a viable option for families.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-81015-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878886PMC
February 2021

[Cancer Risk in Lynch Syndrome-Associated Endometrial Cancer Patients and Their Relatives].

Gan To Kagaku Ryoho 2020 Dec;47(13):2257-2259

Dept. of Digestive Tract and General Surgery, Saitama Medical Center, Saitama Medical University.

Endometrial cancer(EC)is often the sentinel cancer in women with Lynch syndrome(LS), but the actual incidence of EC as the sentinel cancer in patients with LS is not well-known in Japan. We investigated the history of malignancies and incidence of sentinel cancers in patients with LS-associated EC and their relatives. We examined 8 patients with LS-associated EC between 2005 and 2019. Five of them(63%)had suffered from a cancer other than EC, while 5(63%)had developed a cancer after EC. Seven patients(88%)had EC as the sentinel cancer, while 1(13%)developed colorectal cancer before EC. Among first-degree relatives(15 men and 23 women), 15(40%)had a history of cancer, of whom 7 were women (30%). Five women(22%)had EC, all sentinel. Among second-degree relatives(40 men, 44 women, 14 unknown), 16 (16%)had cancer. Four women(9%)had a history of cancer, of whom 2(5%)had EC, all sentinel. Although we only investigated a few LS cases, the importance of EC as the sentinel cancer was highlighted in Japanese women with LS.
View Article and Find Full Text PDF

Download full-text PDF

Source
December 2020

[A Family with Lynch Syndrome Diagnosed after a Proband of Elderly Multiple Colorectal Cancers].

Gan To Kagaku Ryoho 2020 Dec;47(13):1909-1912

Dept. of Digestive Tract and General Surgery, Saitama Medical Center, Saitama Medical University.

The proband was a 77-year-old man who had been admitted to a local hospital for fecal occult blood. He was diagnosed with descending colon carcinoma, T4a, N1, M0, Stage Ⅲb, and rectal adenoma. He had undergone surgeries for rectal cancer at 52 years of age and cecum colon cancer at 57 years of age. Regarding his family history, 5 first-degree and 3 second- degree relatives had a history of gastrointestinal and gynecological cancers, thus meeting 2 of the 5 criteria of the revised Bethesda guidelines. The microsatellite-instability(MSI)test performed using preoperative biopsy tissues demonstrated high-frequency MSI(MSI-H). Hartmann's procedure was performed for MSI-H colon cancer under a strong suspicion of Lynch syndrome. Pathological findings were consistent with descending colon carcinoma, tub2, pT3, pN0, M0, pStage Ⅱa. He was then referred to our hospital. We performed the immunohistochemistry(IHC)analysis of the mismatch repair protein using surgical specimens. The IHC analysis revealed defective expression of the MSH2/MSH6 protein. We found a pathogenic variant in the mismatch repair gene, MSH2(c.1510+2T>G), through genetic testing and finally diagnosed the patient with Lynch syndrome. After disclosure of the results to the proband, 7 relatives underwent genetic testing for the MSH2 variant. Four relatives had the same variant and were also diagnosed with Lynch syndrome. They subsequently underwent surveillance for Lynch syndrome-associated cancers. In 2 variant carriers with a history of early colorectal cancer, an early colon cancer was identified and successfully resected endoscopically. Surveillance for Lynch syndrome-associated cancer is ongoing for the proband and variant carriers.
View Article and Find Full Text PDF

Download full-text PDF

Source
December 2020

[A Family of Attenuated Familial Adenomatous Polyposis].

Gan To Kagaku Ryoho 2020 Dec;47(13):1905-1908

Dept. of Digestive Tract and General Surgery, Saitama Medical Center, Saitama Medical University.

The proband was a 49-year-old woman who had undergone total colectomy, ileorectostomy, and bilateral ovariectomy for the treatment of cecal(T3N0)and sigmoid colon(T4a, N2b, M1c2[Ova], Stage Ⅳc)cancers. Pathological findings revealed 6 adenomas and 2 adenocarcinoma-in-adenomas in the right colon, other than advanced colon cancers. She had a family history of colorectal cancer meeting the Amsterdam Criteria I, but none of her relatives had definite polyposis. Considering the possibility of Lynch syndrome, the microsatellite-instability test and immunohistochemistry(IHC)examination of the mismatch repair protein were performed, leading to the results of microsatellite stable and proficient mismatch repair protein expression. Therefore, we performed the multigene panel test containing 26 genes using the next-generation sequencing technology. In the APC(5q22.2)gene, a pathogenic variant(exon 12 c.994C>T/p.Arg332*)was identified, leading to a diagnosis of attenuated familial adenomatous polyposis(AFAP). After disclosure of the results to the proband, the single-site variant analysis was performed on her 3 daughters. In her second and third daughters, the same variant was confirmed, and laparoscopic total colectomy was performed 23 and 35 months after the disclosure of the genetic analysis results, respectively. Currently, we are conducting periodical surveillance for the residual rectum.
View Article and Find Full Text PDF

Download full-text PDF

Source
December 2020

Prevalence and clinicopathological/molecular characteristics of mismatch repair protein-deficient tumours among surgically treated patients with prostate cancer in a Japanese hospital-based population.

Jpn J Clin Oncol 2021 Apr;51(4):639-645

Department of Digestive Tract and General Surgery, Saitama Medical Center, Saitama Medical University, Saitama, Japan.

Background: The prevalence and molecular characteristics of deficient mismatch repair prostate cancer in the Japanese population have scarcely been investigated.

Methods: Immunohistochemistry for mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) was performed in formalin-fixed paraffin-embedded sections prepared from resected primary prostate cancers in patients who underwent prostatectomy at our institution between January 2001 and May 2016. Genetic and/or epigenetic alterations of mismatch repair genes were investigated in patients with any loss of mismatch repair protein expression in the tumour.

Results: Of the 337 patients, four (1.2%) showed loss of mismatch repair protein expression on immunohistochemistry. All four patients showed loss of both MSH2 and MSH6 protein expression. Genetic testing was performed in two of the four patients, demonstrating no pathogenic germline alterations were present. In each of these two patients, at least one somatic alteration inactivating MSH2 without MSH2 hypermethylation was identified, leading to the diagnosis of supposed 'Lynch-like syndrome'. Patients with deficient mismatch repair prostate cancer were at a significantly higher stage (pT2pN0 vs. pT3-4pN0/pTanypN1, P = 0.02) and had a greater Gleason score (<8 vs. ≥8, P < 0.01) than those with proficient mismatch repair prostate cancer.

Conclusions: The prevalence of deficient mismatch repair prostate cancer in the Japanese hospital-based prostatectomized population was extremely low. To improve screening efficacy for deficient mismatch repair prostate cancer, screening candidates can be limited to patients with locally advanced, node-positive and/or Gleason score of 8 or greater prostate cancer. Universal tumour screening for Lynch syndrome seems ineffective in patients with prostate cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jjco/hyaa207DOI Listing
April 2021

Leigh Syndrome Due to Mutations Initially Presenting as LBSL.

Genes (Basel) 2020 11 9;11(11). Epub 2020 Nov 9.

Diagnostics and Therapeutics of Intractable Diseases, Intractable Disease Research Center, Graduate School of Medicine, Juntendo University, Bunkyo-ku, Tokyo 113-8421, Japan.

Leigh syndrome (LS) is most frequently characterized by the presence of focal, bilateral, and symmetric brain lesions Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare condition, characterized by progressive pyramidal, cerebellar, and dorsal column dysfunction. We describe a case with infantile-onset neurodegeneration, psychomotor retardation, irritability, hypotonia, and nystagmus. Brain MRI demonstrated signal abnormalities in the deep cerebral white matter, corticospinal and dorsal column tracts, and pyramids, which resemble the MRI pattern of a severe form of LBSL, and involvement of basal ganglia and thalamus that resemble the radiological features of LS. We identified biallelic loss-of-function mutations, one novel (c.756delC, p.Thr253Glnfs*44) and another reported (c.1156C > T, p.Arg386Cys), in (NADH:Ubiquinone Oxidoreductase Core Subunit V1) by exome sequencing. Biochemical and functional analyses revealed lactic acidosis, complex I (CI) assembly and enzyme deficiency, and a loss of NDUFV1 protein. Complementation assays restored the NDUFV1 protein, CI assembly, and CI enzyme levels. The clinical and radiological features of this case are compatible with the phenotype of LS and LBSL associated with mutations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/genes11111325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697158PMC
November 2020

Prevalence and Molecular Characterization of Defective DNA Mismatch Repair in Small-bowel Carcinoma in a Japanese Hospital-based Population.

J Anus Rectum Colon 2020 29;4(4):165-173. Epub 2020 Oct 29.

Diagnosis and Therapeutics of Intractable Disease, Juntendo University Graduate School of Medicine, Tokyo, Japan.

Objectives: To investigate the prevalence and molecular characteristics of defective DNA mismatch repair (dMMR) in small-bowel carcinoma (SBC) in a Japanese-hospital population.

Methods: Immunohistochemistry was performed to evaluate the expression of MMR proteins (MLH1, MSH2, MSH6, and PMS2) in formalin-fixed paraffin-embedded sections prepared from surgically resected primary SBCs from 30 patients during March 2002 to March 2017. Genetic testing for Lynch syndrome was performed in patients who demonstrated MMR protein loss.

Results: Two of 30 patients (6.7%) demonstrated concomitant loss of MSH2/MSH6 protein expression. Further genetic testing identified a pathogenic variant in one of these patients.

Conclusions: The prevalence of dMMR SBCs in a Japanese hospital-based population seems lower than that reported in previous studies. To determine whether dMMR SBCs might be strongly linked to Lynch syndrome, there is a need for further investigation with a larger sample size.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.23922/jarc.2020-026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595680PMC
October 2020

A case report of adult-onset COQ8B nephropathy presenting focal segmental glomerulosclerosis with granular swollen podocytes.

BMC Nephrol 2020 08 28;21(1):376. Epub 2020 Aug 28.

Department of Nephrology, Hiroshima University Hospital, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551, Japan.

Background: Primary coenzyme Q10 (CoQ10) deficiency of genetic origin is one of a few treatable focal segmental glomerulosclerosis (FSGS). Renal morphologic evidence for COQ8B mutation and CoQ10 deficiencies of other gene mutations is assessed using electron microscopy with marked increase of abnormal-shaped mitochondria in podocytes. However, light microscopic morphologic features of deficiencies other than FSGS have not been reported.

Case Presentation: A 30-year-old woman was admitted to our hospital because proteinuria was found during four consecutive medical checkups. She had no medical history or family history of proteinuria and severe renal dysfunction. The swollen podocytes were stained to the same extent as mitochondria-rich proximal tubular cells under both Masson's trichrome and hematoxylin-eosin staining, whereas no mitochondrial abnormalities were detected under the first electron microscopic views. As proteinuria and estimated glomerular filtration rate (eGFR) deteriorated after pregnancy, we reevaluated the additional electron microscopic views and detected mitochondrial abnormalities. Genetic testing revealed COQ8B mutation (c.532C > T, p.R178W); therefore, we diagnosed COQ8B nephropathy. CoQ10 supplementation improved proteinuria and stopped eGFR reduction.

Conclusions: This is the first report of granular swollen podocytes due to mitochondrial diseases detected under light microscopy. We propose that this finding can be the clue for the diagnosis of both COQ8B nephropathy and the other CoQ10 deficiencies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12882-020-02040-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456044PMC
August 2020

Prevalence and molecular characteristics of DNA mismatch repair deficient endometrial cancer in a Japanese hospital-based population.

Jpn J Clin Oncol 2021 Jan;51(1):60-69

Department of Digestive Tract and General Surgery, Saitama Medical Center, Saitama Medical University, Saitama, Japan.

Background: The prevalence and molecular characteristics of defective DNA mismatch repair endometrial cancers in the Japanese population have been underexplored. Data supporting clinical management of patients with Lynch-like syndrome and germline variant of uncertain significance of mismatch repair genes are still lacking.

Methods: Immunohistochemistry of mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) was performed on formalin-fixed paraffin-embedded sections prepared from resected primary endometrial cancers in 395 women with a median age of 59 years. Genetic and/or epigenetic alterations of the mismatch repair genes were also investigated.

Results: Loss of expression of one or more mismatch repair proteins was observed in 68 patients (17.2%). A total of 17 out of 68 patients (25%, 4.3% of all cases) were identified as candidates for genetic testing for Lynch syndrome after excluding 51 patients with MLH1 hypermethylated cancer. Fourteen of these 17 patients subjected to genetic testing were found to have Lynch syndrome (n = 5), germline variant of uncertain significance (n = 2) or Lynch-like syndrome (n = 7). Compared with patients with Lynch syndrome, those with germline variant of uncertain significance and Lynch-like syndrome tended to demonstrate an older age at the time of endometrial cancer diagnosis (P = 0.07), less fulfillment of the revised Bethesda guidelines (P = 0.09) and lower prevalence of Lynch syndrome-associated tumors in their first-degree relatives (P = 0.01).

Conclusions: This study provides useful information for management in patients with DNA mismatch repair endometrial cancer. Specifically, cancer surveillance as recommended in patients with Lynch syndrome might not be necessary in patients with germline variant of uncertain significance and Lynch-like syndrome and their relatives.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/jjco/hyaa142DOI Listing
January 2021

Advanced pathological study for definite diagnosis of mitochondrial cardiomyopathy.

J Clin Pathol 2020 Aug 17. Epub 2020 Aug 17.

Department of Research, Charles R Drew University of Medicine and Science, Los Angeles, California, USA.

Aims: Mitochondrial cardiomyopathy (MCM) is difficult to make a definite diagnosis because of various cardiovascular phenotypes and no diagnostic criteria in the pathology examination. We aim to add myocardial pathology to the diagnostic criteria for mitochondrial respiratory chain disorders.

Methods: Quantitative analysis of mitochondria using electron microscopy and immunohistopathological analysis with respiratory chain enzyme antibodies were performed in 11 patients with hypertrophic or restrictive cardiomyopathy who underwent endomyocardial biopsy for possible MCM . Respiratory chain enzymatic assay in biopsied myocardium and genetic studies were also performed in all the subjects to define MCM.

Results: Four patients were diagnosed with MCM according to the recent criteria of mitochondrial respiratory chain disorders. Using electron microscopy with quantitative analysis, the volume density of mitochondria within cardiac muscle cells was significantly increased in the MCM group compared with the non-MCM group (p=0.007). Immunohistopathological results were compatible with the result of the respiratory chain enzymatic assay.

Conclusions: Pathological diagnosis of MCM could be confirmed by a quantitative study of electron microscopy and immunohistopathological analysis using the mitochondrial respiratory chain enzyme subunit antibody.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/jclinpath-2020-206801DOI Listing
August 2020

A novel homozygous variant in MICOS13/QIL1 causes hepato-encephalopathy with mitochondrial DNA depletion syndrome.

Mol Genet Genomic Med 2020 10 4;8(10):e1427. Epub 2020 Aug 4.

Diagnostics and Therapeutics of Intractable Diseases, Intractable Disease Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan.

Background: Mitochondrial DNA depletion syndrome (MTDPS) is part of a group of mitochondrial diseases characterized by a reduction in mitochondrial DNA copy number. Most MTDPS is caused by mutations in genes that disrupt deoxyribonucleotide metabolism.

Methods: We performed the whole-exome sequencing of a hepato-encephalopathy patient with MTDPS and functional analyses to determine the clinical significance of the identified variant.

Results: Here, whole-exome sequencing of a patient presenting with hepato-encephalopathy and MTDPS identified a novel homozygous frameshift variant, c.13_29del (p.Trp6Profs*71) in MICOS13. MICOS13 (also known as QIL1, MIC13, or C19orf70) is a component of the MICOS complex, which plays crucial roles in the maintenance of cristae junctions at the mitochondrial inner membrane. We found loss of MICOS13 protein and fewer cristae structures in the mitochondria of fibroblasts derived from the patient. Stable expression of a wild-type MICOS13 cDNA in the patients fibroblasts using a lentivirus system rescued mitochondrial respiratory chain complex deficiencies.

Conclusion: Our findings suggest that the novel c.13_29del (p.Trp6Profs*71) MICOS13 variant causes hepato-encephalopathy with MTDPS. We propose that MICOS13 is classified as the cause of MTDPS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/mgg3.1427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549589PMC
October 2020

A Model for Predicting DNA Mismatch Repair-deficient Colorectal Cancer.

Anticancer Res 2020 Aug;40(8):4379-4385

Department of Digestive Tract and General Surgery, Saitama Medical Center, Saitama Medical University, Saitama, Japan.

Background/aim: Identifying patients with DNA mismatch repair-deficient (dMMR) colorectal cancer (CRC) is vital to improve treatment and identify patients with Lynch syndrome (LS). We developed a prediction model for dMMR CRC using clinicopathologic features.

Patients And Methods: We reviewed the medical records of 1,147 patients who underwent resection of stage I-IV CRC in whom universal screening for LS using immunohistochemistry for MMR proteins had performed. Univariate and multivariate logistic regression analyses were used to build a prediction model of dMMR CRC.

Results: The prevalence of dMMR CRC was 5.2%. Age (≥75 years), tumor location (right-sided colon), main histologic features (poor differentiation), maximum tumor size (≥65 mm), and stage (I/II) were independent significant variables related to dMMR. We created a formula for predicting the likelihood of dMMR, and the probability ranged from 0.2% to 83%.

Conclusion: dMMR CRC can be identified efficiently using clinicopathologic features obtained in daily clinical practice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.21873/anticanres.14441DOI Listing
August 2020
-->