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Coincident with the blossoming of the sakura was the 14th annual CDB Symposium hosted by the RIKEN Center for Developmental Biology in Kobe, Japan. This year's meeting, 'Size in Development: Growth, Shape and Allometry' focused on the molecular and cellular mechanisms underlying differences in size and shape and how they have evolved. On display was the power of using diverse approaches ranging from the study of organoids to whole organisms., (© 2016. Published by The Company of Biologists Ltd.)

Background: Hypertrophic cardiomyopathy (HCM), which is inherited as an autosomal dominant trait, is the most prevalent hereditary cardiac disease. Although there are several reports on the systematic screening of mutations in the disease-causing genes in European and American populations, only limited information is available for Asian populations, including Japanese., Methods and Results: Genetic screening of disease-associated mutations in 8 genes for sarcomeric proteins, MYH7, MYBPC3, MYL2, MYL3, TNNT2, TNNI3, TPM1, and ACTC, was performed by direct sequencing in 112 unrelated Japanese proband patients with familial HCM; 37 different mutations, including 13 novel ones in 5 genes, MYH7, MYBPC3, TNNT2, TNNI3, and TPM1, were identified in 49 (43.8%) patients. Among them, 3 carried compound heterozygous mutations in MYBPC3 or TNNT2. The frequency of patients carrying the MYBPC3, MYH7, and TNNT2 mutations were 19.6%, 10.7%, and 8.9%, respectively, and the most frequently affected genes in the northeastern and southwestern parts of Japan were MYBPC3 and MYH7, respectively. Several mutations were found in multiple unrelated proband patients, for which the geographic distribution suggested founder effects of the mutations., Conclusions: This study demonstrated the frequency and distribution of mutations in a large cohort of familial HCM in Japan.

Amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS/PDC) is a distinct neurodegenerative disorder characterized by ALS pathology with neurofibrillary tangles (NFTs) in the spinal cord and brain. Recent clinical studies have revealed a high incidence and a high familial occurrence of ALS/PDC in both Guam and the Kii peninsula of Japan, suggesting a strong genetic predisposition to this disorder. The T1482I variant (rs8042919) of TRPM7 gene which is suggested to play roles in regulating the cellular homeostasis of Ca(2+), Mg(2+), and trace metals, has recently been reported to be associated with Guamanian patients with ALS/PDC. To investigate whether TRPM7 is associated with Kii ALS/PDC, we conducted parametric linkage analyses of the TRPM7 locus in a large extended family with ALS/PDC. Linkage analysis did not reveal any evidence supporting the linkage to the TRPM7 locus. Resequencing of the entire coding region of TRPM7 did not reveal any pathogenic mutations in an affected individual in this family. The allele frequencies of the T1482I in affected individuals in this family or in those from other families are not significantly different from those in regional controls or those in HapMap-JPT samples. These results indicate that TRPM7 is not associated with ALS/PDC in the Kii peninsula of Japan., ((c) 2009 Wiley-Liss, Inc.)

Objectives: To investigate the prevalence of anti-ribosomal P protein (anti-P) antibodies in Japanese patients with connective tissue diseases (CTDs) using enzyme-linked immunosorbent assays (ELISAs) and western blotting (WB) and to evaluate the indirect immunofluorescence (IIF) staining patterns of anti-P-positive sera., Methods: Anti-P antibodies were measured by two different commercially available ELISA kits and WB in 239 outpatients, 99 with systemic sclerosis (SSc), 73 with systemic lupus erythematosus (SLE), 45 with dermatomyositis (DM), and 22 with Sjögren's syndrome (SjS). Sera positive for anti-P antibodies by WB were analysed by IIF., Results: The frequency of positive WB findings in SLE (18/73, 25%) was higher than in other diseases. ELISA kits A and B for anti-P antibodies showed 21% and 43% sensitivity, and 93% and 88% specificity, respectively, for diagnosing SLE, based on the manufacturer's cut-off values. Receiver operating characteristic (ROC) curve analysis, based on positive WB findings, determined a new cut-off threshold but revealed that both ELISA kits still had good diagnostic characteristics. In IIF assays on anti-P antibody positive sera, typical anti-P antibody cytoplasmic staining patterns (n=8) were seen less frequently than other staining patterns (n=17)., Conclusions: Routine screening for anti-P antibodies by IIF has low sensitivity. ELISAs using cut-off values established by individual facilities are suitable for detecting anti-P antibodies and provide a tool with good diagnostic characteristics, on a parity with WB.

The authors report a Japanese patient with hereditary sensory and autonomic neuropathy type 2 (HSAN2) who has a new mutation of the HSN2 gene. The pathologic findings of the patient matched those of Canadian patients. They identified a homozygous 1134-1135 ins T mutation, resulting in a frameshift, and the subsequent premature stop codon at residue 378. These observations support the hypothesis that HSN2 is a causative gene for HSAN2.

Objective: Polo-like kinase 1 (PLK1) is one of the serine-threonine kinases that contributes to cell mitosis and is regarded as a marker of cellular proliferation. However, its protein expression in human carcinoma has not been studied in depth. In this study, we investigated PLK1 expression in medullary thyroid carcinoma by means of immunohistochemistry., Methods: We immunohistochemically investigated PLK1 expression in 67 cases of medullary thyroid carcinoma., Results: The PLK1 expression level was elevated in 43 of the 67 cases (64.1%). Furthermore, the expression level was directly linked to lymph node metastasis, advanced stage and male sex. All patients who were negative for PLK1 expression are currently alive without tumor recurrence, while 6 of the 43 PLK1-positive patients showed recurrence and 3 have already died of this disease., Conclusions: These findings suggest that PLK1 expression significantly reflects aggressive characteristics of medullary thyroid carcinoma., (Copyright (c) 2005 S. Karger AG, Basel.)

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is known to act as a lipid phosphatase hydrolyzing phosphatidylinositol (PI)(3,4,5)P(3) to PI(4,5)P(2). Since the PI3-kinase product, PI(3,4,5)P(3), is an important second messenger leading to the metabolic action of insulin, PTEN functions as a potent negative regulator of insulin signaling and its gene is one of the possible candidates involved in susceptibility to the development of type 2 (non-insulin-dependent) diabetes. In the present study, we investigated the polymorphisms of the PTEN gene in Japanese patients with type 2 diabetes and non-diabetic control subjects. We identified three mutations of the gene in the type 2 diabetes patients. Among these mutations, the frequency of the substitution of C with G at position -9 (-9C-->G) (SNP1), located in the untranslated region of exon 1, was significantly higher in type 2 diabetic patients than in control subjects. In addition, transfection of the PTEN gene with SNP1 resulted in a significantly higher expression level of PTEN protein compared with that of the wild-type PTEN gene in Cos1 and Rat1 cells. Furthermore, insulin-induced phosphorylation of Akt in HIRc cells was decreased more greatly by transfection of SNP1 PTEN gene than that of wild-type PTEN gene. These findings suggest that the change of C to G at position -9 of the PTEN gene is associated with the insulin resistance of type 2 diabetes due possibly to a potentiated hydrolysis of the PI3-kinase product.

Objective: This study aims to reveal the morphological, histological, and immunohistochemical mechanism of pannus formation using resected pannus tissue from patients with prosthetic valve dysfunction., Method: Eleven patients with prosthetic valve (St Jude Medical valve) dysfunction in the aortic position who underwent reoperation were studied. We used specimens of resected pannus for histological staining (hematoxylin and eosin, Grocott's, azan, elastica van Gieson) and immunohistochemical staining (transforming growth factor-beta, transforming growth factor-beta receptor 1, alpha-smooth muscle actin, desmin, epithelial membrane antigen, CD34, factor VIII, CD68KP1, matrix metalloproteinase-1, matrix metalloproteinase-3, and matrix metalloproteinase-9)., Results: Pannus without thrombus was observed at the periannulus of the left ventricular septal side; it extended into the pivot guard, interfering with the movement of the straight edge of the leaflet. The histological staining demonstrated that the specimens were mainly constituted with collagen and elastic fibrous tissue accompanied by endothelial cells, chronic inflammatory cells infiltration, and myofibroblasts. The immunohistochemical findings showed significant expression of transforming growth factor-beta, transforming growth factor-beta receptor 1, CD34, and factor VIII in the endothelial cells of the lumen layer; strong transforming growth factor-beta receptor 1, alpha-smooth muscle actin, desmin, and epithelial membrane antigen in the myofibroblasts of the media layer; and transforming growth factor-beta, transforming growth factor-beta receptor 1, and CD68KP1 in macrophages of the stump lesion., Conclusions: Pannus appeared to originate in the neointima in the periannulus of the left ventricular septum. The structure of the pannus consisted of myofibroblasts and an extracellular matrix such as collagen fiber. The pannus formation after prosthetic valve replacement may be associated with a process of periannular tissue healing via the expression of transforming growth factor-beta.

To evaluate the genetic factors of familial predisposition to gastric cancer, genetic alterations in the surgically resected stomach samples from gastric-cancer-prone families were investigated. Familial gastric cancer (FGC) was defined as gastric cancer occurring in a family with 3 or more gastric cancer patients over at least two successive generations. We examined replication error (RER) of six microsatellite markers and screened mutations of the 10-(A) repeat sequence in the transforming growth factor-beta receptor type II (TGF-betaRII) gene in individuals from seven unrelated FGC families. Three cases showed RER at one of the six (CA)n microsatellite markers but the other 4 cases showed no RER at any of these loci. No mutation was found in the 10-(A) repeat of the TGF-betaRII gene. Additionally, no germline mutation was found by polymerase chain reaction-single strand conformation polymorphism in exons 1-16 of E-cadherin, exons 5-8 of p53 and in the mutation cluster region of APC. These results indicate that disorders in the DNA mismatch repair system, E-cadherin, p53 and APC may be infrequently involved in the carcinogenesis of Japanese FGC., (Copyright 2001 S. Karger AG, Basel)

Background: Microsatellite instability occurs frequently in hereditary nonpolyposis colorectal carcinoma, in sporadic gastrointestinal carcinoma, and in other tumors. In these tumors, slippage-related frameshift mutations have been detected at coding mononucleotide repeats in genes such as those for transforming growth factor-beta receptor type II (TGFbetaRII), mannose 6-phosphate/insulinlike growth factor II receptor (M6P/IGFIIR), hMSH3, hMSH6, and Bcl-2-associated X protein (BAX). Because these genes regulate cell growth or repair DNA mismatches, loss of their function is thought to promote tumor development. The authors screened for these frameshift mutations and investigated the incidence of microsatellite instability (MI) in hepatocellular carcinoma (HCC) in Japan., Methods: Fifty HCC samples were analyzed in this study. The authors used polymerase chain reactions to screen for frameshift mutation at the TGFbetaRII (A)(10) tract, the M6P/IGFIIR (G)(8) tract, the hMSH3 (A)(8) tract, the hMSH6 (C)(8) tract, and the BAX (G)(8) tract. For MI analysis, matched tumor and nontumor liver DNA were investigated with respect to 10 microsatellite loci., Results: No frameshift mutation was detected in any case, and only 4% of these cancers exhibited MI in comparisons between tumor and nontumor liver specimens., Conclusions: This study suggests that frameshift mutation at coding mononucleotide repeats within TGFbetaRII, M6P/IGFIIR, hMSH3, hMSH6, and BAX genes did not seem to be involved in hepatocarcinogenesis in the Japanese population studied., (Copyright 2000 American Cancer Society.)

Somatically acquired mutations in several genes have been reported as playing an important role during colorectal tumorigenesis. Two alternative groups of carcinomas, termed LOH+ and RER+, have been defined on the basis of their genetic anomalies, a biallelic inactivation of the APC or the TGF-betaRII genes, occurring as an alternative, in LOH+ or RER+ tumors. It is a generally accepted hypothesis that most of colorectal cancers (CRC) develop from a pre-existing adenomatous polyp. Such benign lesions are usually exophytic polyps, a small proportion of adenomas having been described as flat lesions. The latter histological category has thus been proposed to bear specific genetic alterations. In order to examine this hypothesis, we have characterized a series of 44 flat colorectal neoplasias for their RER status and for somatic APC, KRAS and TGF-betaRII genes mutations. Flat colorectal neoplasias were found to be of the RER+ subtype in 22% of cases, all of them exhibiting a TGF-betaRII mutation. A mutation of the APC and KRAS genes has been found in 42% and 4% of tumors, respectively, none of these tumors being of the RER+ subtype. With the exception of a low KRAS mutation rate, flat adenomas appear to follow tumorigenesis pathways very similar to those identified in exophytic adenomas and carcinomas.

The ATM (A-T, mutated) gene on human chromosome 11q22.3 has recently been identified as the gene responsible for the human recessive disease ataxia-telangiectasia (A-T). In order to define the types of disease-causing ATM mutations in Japanese A-T patients as well as to look for possible mutational hotspots, reverse-transcribed RNA derived from ten patients belonging to eight unrelated Japanese A-T families was analyzed for mutations by the restriction endonuclease fingerprinting method. As has been reported by others, mutations that lead to exon skipping or premature protein truncation were also predominant in our mutants. Six different mutations were identified on 12 of the 16 alleles examined. Four were deletions involving a loss of a single exon: exon 7, exon 16, exon 33 or exon 35. The others were minute deletions, 4649delA in exon 33 and 7883del5 in exon 55. The mutations 4612del165 and 7883del5 were found in more than two unrelated families; 44% (7 of 16) of the mutant alleles had one of the two mutations. The 4612del165 mutations in three different families were all ascribed to the same T-->A substitution at the splice donor site in intron 33. Microsatellite genotyping around the ATM locus also indicated that a common haplotype was shared by the mutant alleles in both mutations. This suggests that these two founder mutations may be predominant among Japanese ATM mutant alleles.

We have studied linkage disequilibrium between CTG repeats and an Alu insertion/deletion polymorphism at the myotonin protein kinase gene (DMPK) in 102 Japanese families, of which 93 were affected with myotonic dystrophy (DM). All of the affected chromosomes are in complete linkage disequilibrium with the Alu insertion allele. Among the normal chromosomes, alleles of CTG repeats 5 and > or = 17 are exclusively associated with the insertion allele. On the other hand, intermediate alleles of 11-16 repeats show a significantly greater association with the deletion allele. A strikingly similar pattern of linkage disequilibrium observed in European populations suggests a common origin of the DM mutation in the Japanese and European populations.

To determine the relation between the mutation of the TGF-beta type II receptor gene and genomic instability in the tumorigenesis of hereditary nonpolyposis colorectal cancer (HNPCC), we screened genomic DNA of 38 tumors from 25 HNPCC patients, 15 colorectal cancers from familial adenomatous polyposis patients, and 8 sporadic endometrial cancers, in two areas containing a (A)10 repeat or a (GT)3 repeat of the gene. Seventeen of the 24 (71%) genomic instability-positive HNPCC tumors carried one or two A deletions in the (A)10 repeat, while none of the 14 genomic instability-negative tumors did. These deletions inactivate the receptor through a frameshift mutation and the resultant protein truncation. No mutation was detected in the (GT)3 repeat sequence, but we found a missense mutation of codon 537 in the same area in one tumor. One A deletion was also detected in a genomic instability-positive sporadic endometrial cancer, but none in familial adenomatous polyposis tumors. No mutations were detected in the corresponding normal cells of these cases, indicating a somatic mutation. These data suggest that the TGF-beta type II receptor gene is a major target of genomic instability in HNPCC tumorigenesis.