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A female in her 60's presented with a left-sided breast mass. A core needle biopsy specimen showed diffuse proliferation of a round cell tumor, which was positive for vimentin, NKX2.2, BCOR, and focal CD99 on immunohistochemistry (IHC). No fusion genes of the Ewing family sarcomas were detected. With a tentative diagnosis of primary breast sarcoma (PBS), total mastectomy was performed after chemotherapy. The resected tissues showed proliferation of round or spindle-shaped tumor cells with a high nuclear-to-cytoplasmic ratio, exhibiting solid and fascicular arrangements but no epithelial component or organoid pattern. While IHC indicated no particular histological diagnosis, genomic examination revealed gene alterations in MED12 p.G44D, MLL2 (KMT2D) p.T1496fs*27, and EGFR variant III (vIII). Moreover, a retrospective IHC study showed overexpression of EGFRvIII. A malignant phyllodes tumor (PT) with extensive sarcomatous overgrowth was indicated as an integrative diagnosis. This is a rare case of a malignant PT harboring EGFRvIII. The present case provides an importance of accurate diagnosis and genomic analysis of rare breast tumors, as malignant PT and PBS are different in its treatment strategy and prognosis., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2024. Published by Elsevier GmbH.)

Oligodendroglioma, IDH-mutant and 1p/19q-codeleted is known for their relative chemosensitivity and indolent clinical course among diffuse gliomas of adult type. Based on the data from phase 3 clinical trials, the standard of post-surgical care for those tumors is considered to be initial chemoradiotherapy regardless of histopathological grade, particularly with PCV. However, partly due to its renewed definition in late years, prognostic factors in patients with those tumors are not well established. Moreover, the survival rate declines over 15 years, with only a 37% OS rate at 20 years for grade 3 tumors, even with the current standard of care. Given that most of this disease occurs in young or middle-aged adults, further improvements in treatment and management are necessary. Here, we discuss prognostic factors, standard of care and chemotherapy, and future perspectives with neoadjuvant strategy in those tumors., (© 2024. The Author(s), under exclusive licence to The Japan Society of Brain Tumor Pathology.)

DNA methylation at CG sites is important for gene regulation and embryonic development. In mouse oocytes, de novo CG methylation requires preceding transcription-coupled histone mark H3K36me3 and is mediated by a DNA methyltransferase DNMT3A. DNMT3A has a PWWP domain, which recognizes H3K36me2/3, and heterozygous mutations in this domain, including D329A substitution, cause aberrant CG hypermethylation of regions marked by H3K27me3 in somatic cells, leading to a dwarfism phenotype. We herein demonstrate that D329A homozygous mice show greater CG hypermethylation and severer dwarfism. In oocytes, D329A substitution did not affect CG methylation of H3K36me2/3-marked regions, including maternally methylated imprinting control regions; rather, it caused aberrant hypermethylation in regions lacking H3K36me2/3, including H3K27me3-marked regions. Thus, the role of the PWWP domain in CG methylation seems similar in somatic cells and oocytes; however, there were cell-type-specific differences in affected regions. The major satellite repeat was also hypermethylated in mutant oocytes. Contrary to the CA hypomethylation in somatic cells, the mutation caused hypermethylation at CH sites, including CA sites. Surprisingly, oocytes expressing only the mutated protein could support embryonic and postnatal development. Our study reveals that the DNMT3A PWWP domain is important for suppressing aberrant CG hypermethylation in both somatic cells and oocytes but that D329A mutation has little impact on the developmental potential of oocytes., Competing Interests: The authors have declared that no competing interests exist.

We report two cases of transthyretin familial amyloid polyneuropathy (ATTR-FAP) from non-endemic areas. Both cases showed chronic progressive distal limb numbness and weakness. Due to nonspecific symptoms, they were not diagnosed for a long period of time. A nerve conduction study revealed axonal neuropathy in the lower limbs and carpal tunnel syndrome. An echo test showed thickness of the left ventricle, one of the red flag symptom clusters of ATTR-FAP. Genetic analysis revealed a mutation in the transthyretin gene. In cases with chronic progressive neuropathy, it is important to consider a differential diagnosis of ATTR-FAP.

A 42-year-old female complaining of fever and night sweats was diagnosed with acute megakaryoblastic blast phase chronic myeloid leukemia (CML-BP). She had massive splenomegaly, left pleural effusion with leukemia infiltration, and moderate myelofibrosis. She received dasatinib monotherapy (140 mg/day) as for induction, after which her pleural effusion rapidly resolved and hematological remission was achieved. However, CML relapsed 4 months after starting dasatinib due to increased BCR-ABL fusion signals in the peripheral blood. The T315I mutation was also detected at the recurrence of CML. As a salvage treatment, ponatinib monotherapy (45 mg/day) was started immediately. After 5 months, BCR-ABL fusion signals decreased to 5%, and myelofibrosis improved from MF Grade 2 to 1; she then underwent allogeneic bone marrow transplantation from an unrelated donor. However, the graft failed, and cord blood transplantation (CBT) was performed. Ponatinib (15 mg/day) was continued after CBT as a maintenance treatment, with molecular complete response continuing for more than 1 year with no severe adverse events, including cardiovascular events. There is limited evidence regarding the optimal dose and schedule of ponatinib before and after allogeneic hematopoietic stem cell transplantation, especially in patients with CML-BP having T315I mutation; thus, well-designed clinical trials are warranted.

Mutations in CDCA7 and HELLS that respectively encode a CXXC-type zinc finger protein and an SNF2 family chromatin remodeler cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome types 3 and 4. Here, we demonstrate that the classical nonhomologous end joining (C-NHEJ) proteins Ku80 and Ku70, as well as HELLS, coimmunoprecipitated with CDCA7. The coimmunoprecipitation of the repair proteins was sensitive to nuclease treatment and an ICF3 mutation in CDCA7 that impairs its chromatin binding. The functional importance of these interactions was strongly suggested by the compromised C-NHEJ activity and significant delay in Ku80 accumulation at DNA damage sites in CDCA7- and HELLS-deficient HEK293 cells. Consistent with the repair defect, these cells displayed increased apoptosis, abnormal chromosome segregation, aneuploidy, centrosome amplification, and significant accumulation of γH2AX signals. Although less prominent, cells with mutations in the other ICF genes DNMT3B and ZBTB24 (responsible for ICF types 1 and 2, respectively) showed similar defects. Importantly, lymphoblastoid cells from ICF patients shared the same changes detected in the mutant HEK293 cells to varying degrees. Although the C-NHEJ defect alone did not cause CG hypomethylation, CDCA7 and HELLS are involved in maintaining CG methylation at centromeric and pericentromeric repeats. The defect in C-NHEJ may account for some common features of ICF cells, including centromeric instability, abnormal chromosome segregation, and apoptosis.

Objectives: Actin, alpha-2, smooth muscle, aorta (ACTA2) mutations are one of the major causes of familial thoracic aortic aneurysms and dissections. The aim of this study was to review our clinical results of young adult patients with aortic disease caused by ACTA2 mutations., Methods: We reviewed the medical records of 251 patients (<50 years old) who underwent surgery for thoracic aortic diseases between 2004 and 2014. Among them, nine patients (3.5%) had ACTA2 mutations. Their average age was 35 years (range 22-47) and two patients (22.2%) were males. No patients fulfilled the diagnostic criteria for Marfan syndrome. Preoperative diagnoses included annulo-aortic ectasia (n = 2), localized dissection of the sinus of Valsalva (n = 2), acute type B aortic dissection (n = 1), and chronic type B (n = 4). Eight patients (88.9%) had hypertension., Results: A thoracoabdominal aortic replacement was required in three patients who had descending replacement for residual chronic type B aortic dissection. A patient who had thoracic endovascular aortic repair for complicated acute type B aortic dissection showed no aortic dilatation for 7 years after TEVAR. Histological results revealed cystic medial necrosis (CMN) in most cases (7/8; 87.5%)., Conclusion: Surgical outcomes for patients with ACTA2 mutations were satisfactory. CMN was a major histological finding and family history of aortic event was detected in only half of the patients with ACTA2 mutations. Despite no characteristic physical findings besides hypertension, connective tissue disease including ACTA2 mutations should be considered for aortic dissection in young adult patients.

Spinocerebellar ataxia (SCA) is a group of dominantly inherited heterogeneous disorders in which 43 subtypes have been identified to date. Recently, Japanese and French families with SCA type 42 (SCA42) were found to have a missense mutation (c.5144G>A; R1715H) in CACNA1G. We performed genetic analysis of 84 unrelated families to find the prevalence of SCA42 in Japan. Two families were found to have the previously reported missense mutation. Clinical presentations of the affected members of these families were similar to those of the previously reported French and Japanese families. Our study demonstrates that SCA42 exists in small numbers in Japan, and further supports the idea that SCA42 is a slowly progressive, pure cerebellar ataxia.

Objectives: The T790M and C797S mutations of the epidermal growth factor receptor gene (EGFR) confer resistance to first- and third-generation EGFR tyrosine kinase inhibitors (TKIs), respectively, in patients with non-small cell lung cancer (NSCLC) harboring activating mutations of EGFR. C797S has been identified in cis or in trans with T790M in tumor specimens from patients who experienced treatment failure with first- and third-generation EGFR-TKIs. The allelic relation between T790M and activating mutations of EGFR has not been well characterized, however. We have now developed a digital polymerase chain reaction (dPCR)-based method for determination of the allelic relation between two types of EGFR mutation (T790M and either C797S or an activating mutation)., Materials and Methods: Seven clinical NSCLC specimens and two NSCLC cell lines harboring both an activating mutation and T790M were analyzed with this new method to identify the allelic relation between these EGFR mutations., Results: The median ratio of the number of alleles positive for both an activating mutation and T790M to the number of T790M-positive alleles was 97.1% (range, 90.0-100%). Confirmatory analysis by next-generation sequencing yielded a corresponding value of 96.7% (range, 89.1-99.5%). Our dPCR method thus reliably identifies the allelic relation between two EGFR mutations in a quantitative manner., Conclusions: Almost all T790M mutations were detected in cis with activating mutations of EGFR regardless of the de novo or acquired status of T790M, with cancer cells harboring T790M and activating mutations on the same allele appearing to be selected and enriched during EGFR-TKI treatment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Chordomas and chondrosarcomas are two major malignant bone neoplasms located at the skull base. These tumors are rarely metastatic, but can be locally invasive and resistant to conventional chemotherapies and radiotherapies. Accordingly, therapeutic approaches for the treatment of these tumors can be difficult. Additionally, their location at the skull base makes them problematic. Although accurate diagnosis of these tumors is important because of their distinct prognoses, distinguishing between these tumor types is difficult due to overlapping radiological and histopathological findings. However, recent accumulation of molecular and genetic studies, including extracranial location analysis, has provided us clues for accurate diagnosis. In this report, we review the genetic aberrations and molecular biology of these two tumor types. Among the abundant genetic features of these tumors, brachyury immunohistochemistry and direct sequencing of IDH1/2 are simple and useful techniques that can be used to distinguish between these tumors. Although it is still unclear why these tumors, which have such distinct genetic backgrounds, show similar histopathological findings, comparison of their genetic backgrounds could provide essential information.

Recent investigations revealed genetic analysis provides important information in management of gliomas, and we previously reported grade II-III gliomas could be classified into clinically relevant subgroups based on the DNA copy number aberrations (CNAs). To develop more precise genetic subgrouping, we investigated the correlation between CNAs and mutational status of the gene encoding isocitrate dehydrogenase (IDH) of those tumors. We analyzed the IDH status and CNAs of 174 adult supratentorial gliomas of astrocytic or oligodendroglial origin by PCR-based direct sequencing and comparative genomic hybridization, respectively. We analyzed the relationship between genetic subclassification and clinical features. We found the most frequent aberrations in IDH mutant tumors were the combined whole arm-loss of 1p and 19q (1p/19q codeletion) followed by gain on chromosome arm 7q (+7q). The gain of whole chromosome 7 (+7) and loss of 10q (-10q) were detected in IDH wild-type tumors. Kaplan-Meier estimates for progression-free survival showed that the tumors with mutant IDH, -1p/19q, or +7q (in the absence of +7p) survived longer than tumors with wild-type IDH, +7, or -10q. As tumors with +7 (IDH wild-type) showed a more aggressive clinical nature, they are probably not a subtype that developed from the slowly progressive tumors with +7q (IDH mutant). Thus, tumors with a gain on chromosome 7 (mostly astrocytic) comprise multiple lineages, and such differences in their biological nature should be taken into consideration during their clinical management., (© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Surgery was performed in a 65-year-old male patient with malignant gliomas at two locations in the left and right cerebral hemispheres that showed no apparent continuity in imaging studies. Slight differences in histopathological appearance were seen between the tumors, and multicentric malignant glioma was diagnosed. Detailed genetic examination showed both the left- and right-side tumors to be of the IDH-1 wild type with a p53 mutation at the same locus. Whole genome analysis by comparative genomic hybridization revealed many of the same mutations to be present in both tumors. The O(6)-methylguanine-methyltransferase promoter in both cases was unmethylated, and the genetic profiles of both showed them to be homologous tumors. They were therefore inferred to be multiple gliomas from the same clone. There have been occasional reports of multicentric gliomas classified by diagnostic imaging. This report discusses the need to examine tumor origin by genomic profiling.

Multiple mitochondrial DNA (mtDNA) deletions usually occur secondarily to a mutation in one of the enzymes involved in mtDNA maintenance, such as polymerase γ, which is encoded by the nuclear polymerase γ1 gene (POLG1) and POLG2. Patients with multiple mtDNA deletion disorders show clinical heterogeneity of symptoms, in addition to usually seen progressive external ophthalmoplegia (PEO). We conducted clinical, histological and genetic analyses of two affected sisters in a family with the autosomal dominant inheritance pattern of PEO. A 73-year-old woman (patient 1) with congenital hypogonadism and PEO developed L-dopa responsive parkinsonism about the age of 60. Neurological examination revealed mild proximal muscle weakness and polyneuropathy too. Her 69-year-old sister (patient 2) also showed PEO, parkinsonism and polyneuropathy. Histopathological studies of biopsied muscle specimens from patient 1 revealed numerous ragged red fibers as well as fibers with increased succinate dehydrogenase activity and decreased cytochrome c oxidase activity. Multiple mtDNA deletions were detected, both by Southern blot and long-range PCR assays of total DNA from the biopsied muscle specimens. A systemic mutational analysis in both sisters revealed a heterozygous p.Y955C (c.2864A>G) mutation in POLG1. This is the first Japanese family identified with this mutation. We reviewed cases with this mutation highlighting a wide phenotypic spectrum of this disorder.

Background: Progressive external ophthalmoplegia (PEO) and parkinsonism can be caused by genetic mutations that affect mitochondrial DNA (mtDNA) maintenance. We characterized parkinsonism in a family with dominantly inherited PEO., Methods: We conducted clinical, histological and genetic analyses on two affected members suffering from PEO and parkinsonism, and reviewed the cases in the literature. To clarify parkinsonism related to multiple mtDNA deletions, we used 3-T neuromelanin magnetic resonance imaging (MRI) to assess signal changes in the substantia nigra (SN) and locus ceruleus (LC) in our patients, and compared the results to those observed in idiopathic Parkinson's disease (iPD) (n = 35)., Results: We report the first case of a Japanese family harboring a heterozygous p.Y955C mutation in POLG1. The clinical features of parkinsonism related to the Y955C mutation in a total of 16 patients, including our two cases, are indistinguishable from iPD. However, neuromelanin MRI showed a distinct pattern in our cases compared to iPD. The neuromelanin imaging results were consistent with the neuropathological findings reported in cases of POLG1 mutations, in which neurons of the SN were profoundly affected while those in the LC were preserved., Conclusions: Our results suggest that 3-T neuromelanin MRI may be useful for differentiating POLG1 mutation-associated parkinsonism from iPD, and that POLG1 mutations may cause selective neuronal loss in the SN via a mechanism different from that of iPD., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Nuclear factor (erythroid derived 2)-like 2 (NRF2, gene name NFE2L2) gene mutations have been previously identified in lung cancers. The constitutive activation of NRF2 resulting from gene mutations has been correlated with the poor prognosis of patients with squamous cell lung cancer. However, DNA sequencing using PCR methods described to date is time-consuming and requires significant quantities of DNA. Thus, this existing approach is not suitable for a routine pre-therapeutic screening program. We genotyped the NRF2 gene mutation status in 262 surgically treated lung cancer cases using LightCycler analysis. The presence of the NRF2 gene mutation was confirmed by direct sequencing. We detected 6 cases (2.3%) with NRF2 gene mutations in our cohort, particularly smokers (P=0.04) with squamous histology (P=0.0001). NRF2 gene mutations were present in 10% (6/60) of the lung squamous cell carcinoma (SqCC) cases. The NRF2 gene mutation was exclusive of epidermal growth factor receptor mutations. The NRF2 gene mutation occurred with a tendency towards a higher frequency in male patients. Patients with the NRF2 gene mutation (n=22, 11 succumbed to disease) had a significantly worse prognosis when compared with the patients with the wild-type NRF2 gene (n=521, 98 succumbed to disease) from a larger cohort study (log-rank test, P<0.0001) even upon multivariate analysis. In our study, NRF2 gene mutations played a role in the prognosis of patients with SqCC of the lung.

Previous studies have identified several genetic loci associated with the development of familial inclusion body myopathy. However, there have been few genetic analyses of sporadic inclusion body myositis (sIBM). In order to explore the molecular basis of sIBM and to investigate genotype-phenotype correlations, we performed a clinicopathological analysis of 21 sIBM patients and screened for mutations in the Desmin, GNE, MYHC2A, VCP, and ZASP genes. All coding exons of the five genes were sequenced directly. Definite IBM was confirmed in 14 cases, probable IBM in three cases, and possible IBM in four cases. No cases showed missense mutations in the Desmin, GNE, or VCP genes. Three patients carried the missense mutation c.2542T>C (p.V805A) in the MYHC2A gene; immunohistochemical staining for MYHC isoforms in these three cases showed atrophy or loss of muscle fibers expressing MYHC IIa or IIx. One patient harbored the missense mutation c.1719G>A (p.V566M) in the ZASP gene; immunohistochemical studies of Z-band-associated proteins revealed Z-band abnormalities. Both of the novel heterogeneous mutations were located in highly evolutionarily conserved domains of their respective genes. Cumulatively, these findings have expanded our understanding of the molecular background of sIBM. However, we advocate further clinicopathology and investigation of additional candidate genes in a larger cohort.

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor belonging to the cap 'n' collar subfamily of the basic-leucine zipper (bZIP) family of transcription factors, which plays a significant role in adaptive responses to oxidative stress. Previously, we reported that NRF2 gene (NFE2L2) mutations correlate with poor prognosis of lung squamous cell carcinomas. We therefore hypothesized that an increased NRF2 gene copy number may correlate with clinicopathological features in lung cancer patients. In this study, the increased copy number of the NRF2 gene was analyzed by real-time polymerase chain reaction (real-time-PCR) amplifications in 90 surgically-treated non-small cell lung cancer (NSCLC) cases. In total, 16 NRF2 mutation cases were included. An increased NRF2 gene copy number was found in 7 (7.8%) lung squamous cell carcinoma patients. Increased NRF2 copy number status significantly correlated with mutation status (mutant, 31.25% vs. wild-type, 2.7%; p=0.0017). The mean NRF2 gene copy number was significantly higher in mutant (2.478 ± 0.668) compared to wild-type NRF2 (1.917 ± 0.737) (p=0.0048). However, the copy number did not correlate with smoking status (p=0.3741), gender (p=0.1545), age (≥65 vs. <65, p=0.1237) and pathological stage. Although an increased NRF2 copy number correlates with mutations in squamous cell carcinoma, the percentage of the increased copy number was low; therefore, another mechanism may exist for the activation of NRF2 mutations in cancer.

Objective: To determine whether the presence of Machado-Joseph disease (MJD, also spinocerebellar ataxia type 3 [SCA3]) among Australian aborigines was caused by a new mutational event or by the introduction of expanded alleles from other populations., Design: We sequenced a region of 4 kilobases (kb), encompassing the CAG repeat within the ATXN3 gene, in 2 affected Australian aboriginal families and compared them with the Joseph and Machado lineages described before. Full-extended haplotypes (including also more distant single-nucleotide polymorphisms and flanking short tandem repeats) were assessed by segregation and allele-specific amplification. A phylogenetic tree was inferred from genetic distances, and age of the Australasian Joseph-derived lineage was estimated., Setting: The aboriginal communities of Groote Eylandt and Yirrkala, in the Northern Territories, Australia (local ethics institutional permission was granted, and both community and individual informed consent was obtained)., Subjects: A convenience sample of 19 patients and unaffected relatives, from 2 Australian aboriginal families affected with MJD; 40 families with MJD of multiethnic origins and 50 unrelated Asian control subjects., Results: The 2 aboriginal families shared the same full haplotype, including 20 single-nucleotide polymorphisms:TTGATCGAGC-(CAG)(Exp)-CACCCAGCGC, that is, the Joseph lineage with a G variant in rs56268847.Among 33 families with the Joseph lineage, this derived haplotype was found only in 5 of 16 Taiwanese, all 3 Indian,and 1 of 3 Japanese families analyzed., Conclusion: A related-extended MJD haplotype shared by Australian aborigines and some Asian families (a Joseph-derived lineage) suggests a common ancestor for all, dating back more than 7000 years.

Glucose is the major source of energy for cells, and glucose transporter 1 (GLUT1) is the most common glucose transporter. GLUT1 has been found to be aberrantly expressed in several tumor types. From the results of the microarray and serial analysis of gene expression (SAGE), GLUT1 transcript expression was found to be higher in clones with mutant Kras alleles. We hypothesized that GLUT1 overexpression might be correlated with clinicopathological features of Japanese lung cancers. Immunohistochemistry for GLUT1 was performed in 283 surgically treated non-small cell lung cancer (NSCLC) cases from Nagoya City University Hospital. Thirty-six Kras mutant carcinoma cases were included. GLUT1 overexpression was found in 138 (48.8%) lung cancer patients. The GLUT1 overexpression status was significantly correlated with gender (women 31.9% vs. men 54.5%, P<0.0001), smoking status (never smoker 31.4% vs. smoker 59.4%, P<0.0001) and pathological subtypes (adenocarcinoma 36.4% vs. non‑adenocarcinoma 74.5%, P<0.0001). In addition, the GLUT1 overexpression status was significantly correlated with gene mutation status, including EGFR (mutation-positive 23.4% vs. -negative 58.3%, P<0.0001) and Kras (mutation-positive 66.7% vs. -negative 46.6%, P=0.038). The survival of patients with GLUT1 overexpression (n=137, 50 were deceased) was significantly worse when compared to the patients with normal expression of GLUT1 (n=142, 31 were deceased) (Log-rank test, P=0.0009). Thus, GLUT-1 overexpression correlates with an aggressive phenotype of lung carcinoma.

Stickler syndrome type I is caused by mutations in the type II collagen gene (COL2A1), which is specifically expressed in cartilage and vitreous humor. We developed a simple and noninvasive strategy for identifying the COL2A1 mutation using RNA from freshly isolated peripheral white blood cells and identified a new 3' splice site mutation in a Japanese family with Stickler syndrome. RNA was isolated from a patient's peripheral white blood cells that had been incubated with cycloheximide, an inhibitor of nonsense-mediated mRNA decay. COL2A1 cDNA fragments covering the entire coding region were obtained by RT-polymerase chain reaction cloning using a high-fidelity DNA polymerase and sequenced. Whole sequencing of the patient's cDNA resulted in identification of a 49-bp deletion in the region corresponding to exon 18. The deletion introduced a premature termination codon. Targeted genome sequencing identified a base substitution at the A (-2) position of the 3' splice acceptor site of intron 17. This mutation led to utilization of the cryptic splice site, which is located 49 bases downstream of the normal splice site and causes aberrant mRNA splicing, resulting in a 49-base deletion in the patient's mRNA. Our method is much easier than conventional genomic screening and provides a simple and noninvasive diagnostic test for patients with Stickler syndrome.

Purpose: Germline mutations of LKB1 (also known as SKT11; locus 19p13.3) cause the occurrence of autosomal dominant Peutz-Jeghers syndrome (PJS). Nearly half of the non-small cell lung cancer (NSCLC) cell lines and one-third of lung adenocarcinoma in Caucasian patients have an LKB1 mutation., Methods: This study examined the mutational hot spots of the LKB1 gene in surgical resectable lung adenocarcinoma. Exons 1, 6, and 7 of the LKB1 gene were sequenced in 174 Japanese patients with lung adenocarcinoma (including 157 men and 17 women)., Results: Only five patients had LKB1 gene alterations (2.9%). All of them were male smokers, and no LKB1 mutation was observed in any of the females. The details of LKB1 alterations were: one 5 bp deletion in intron 5, one Gly to Phe substitution at codon 279 of exon 6, and three Pro to Leu substitutions at codon 281 of exon 6. The P281L alteration and 5 bp deletion in the intron 5 were found to be germline polymorphisms. The G279F was confirmed to be a novel somatic mutation. None of the five patients with an LKB1 alteration showed either an EGFR or K-ras mutation., Conclusion: The LKB1 gene alteration is rare in Japanese patients with lung adenocarcinoma, and is generally limited to male smokers.

Introduction: Recent studies for the characterization of the lung cancer genome have suggested that Kras gene was frequently amplified and correlated with activating mutations of Kras, which occur in approximately 5 to 10% of Japanese lung cancers., Methods: We analyzed Kras mutation and Kras copy number in 172 Japanese non-small cell lung cancer (NSCLC) cases and their relation to the survival of patients. We also studied using fluorescence in situ hybridization to provide direct evidence of Kras amplification in 40 clinical specimens., Results: In 172 NSCLC cases, increased Kras copy number existed in 19 (11.0%) cases. Increased Kras gene copy number was correlated with Kras mutation. Nevertheless, Kras gene copy number gain was not correlated with gender, pathological subtypes, stages, and smoking status. Increased Kras copy number was not associated with overall survival in these 172 cases; however, patients with increased Kras copy number and Kras mutant had significantly worse prognosis, when compared with patients with Kras wild type and Kras not increased. From the fluorescence in situ hybridization analysis, Kras polysomy or amplified patients showed significantly worse prognosis, when compared with Kras disomy patients., Conclusion: Kras mutation plus increased copy number was a predictor of poor clinical outcome in patients with NSCLC.

Purpose: To examine an association between environmental tobacco smoke (ETS) and activating epidermal growth factor receptor (EGFR) mutations in never-smokers with non-small cell lung cancer (NSCLC)., Experimental Design: A total of 126 never-smokers with NSCLC were prospectively included in this study. Detailed ETS information was obtained through a standardized questionnaire including exposure period, place, and duration. Cumulative dose of ETS (CETS) was evaluated as a sum of the number of the exposure years at home and/or workplace. EGFR and K-ras mutations were determined using real-time PCR amplification., Results: A total of 124 patients (98.4%) had ETS exposure with median CETS of 50 years (range: 0-118). Activating EGFR mutations were detected in 62.7% of the 126 patients and K-ras in 2 of 114 patients. The incidence of activating EGFR mutations was significantly higher in females than in males (67.6% vs. 26.7%; P = 0.002), and increased in quintile groups separated on the basis of CETS (shortest group = 44.0%, longest = 84.6%; P = 0.0033). In the multivariate logistic regression model, including gender, CETS, age, and family history of cancer, both gender and CETS were significantly associated with an incidence of activating EGFR mutations; the odds ratio for the EGFR mutations were 5.13 [95% confidence interval, CI = 1.47-18.0; P = 0.0105] for females and 1.02 (95% CI = 1.00-1.04; P = 0.0193) for each 1-year increment in CETS., Conclusions: Females and increased ETS exposure are closely associated with EGFR mutations in never-smokers with NSCLC., (©2010 AACR.)

Introduction: Recently, the nuclear factor (erythroid derived 2)-like 2 (NFE2L2) gene mutations were identified in lung cancer. The constitutive activation of NFE2L2 in lung cancer cells promotes tumorigenicity. However, the correlation between NFE2L2 mutation status and clinicopathologic features of lung cancer has not been well characterized., Methods: We have investigated NFE2L2 gene mutation status in 263 surgically treated lung cancer cases at Nagoya City University Hospital. The NFE2L2 mutation was analyzed by direct sequencing of cDNA., Results: We detected 13 cases (5.1%) of NFE2L2 mutation in our cohort; all were male and all had a squamous histology. EGFR mutations were present in 78 patients (30.8%). The NFE2L2 mutation was exclusive with EGFR mutations. The NFE2L2 mutation tended to be more frequently found in patients with advanced stages. The patients with NFE2L2 mutation (n = 13, 8 were dead) had significantly worse prognosis than the patient with wild type NFE2L2 (n = 250, 72 were dead) (Log-rank test, p = 0.0032, Breslow-Gehan-Wilcoxon test, p = 0.0028)., Conclusion: NFE2L2 mutations might play a role in tumor prognosis of squamous cell carcinoma of the lung.

Background: Recently, to identify potential somatic mutations in genes of epidermal growth factor receptor (EGFR) signaling pathway, MEK1 gene mutation at exon 2 and mutation of the AKT2 (v-akt murine thymoma viral oncogene homologue 2) gene at kinase domain have been reported in non-small cell lung cancer., Methods: We investigated the MEK1 mutation (n = 280) and AKT2 mutation (n = 273) in surgically treated non-small cell lung cancer cases. The presence or absence of MEK1 mutation (exon 2) and AKT2 mutation at kinase domain was analyzed by direct sequences., Results: MEK1 mutation (K57K) was found from 1 of 280 patients with lung cancer (0.4%) and detected only one case (0.4%) of AKT2 mutation (R371H) in our cohort. MEK1 mutation was exclusive with EGFR, K-ras, and B-raf mutations at kinase domain. However, AKT2 mutation was coexisted with EGFR and PIK3CA mutations., Conclusion: This study demonstrated that mutation in the kinase domain of AKT2 and MEK1 exon 2 mutation occurred in a small fraction of Japanese lung cancers.

Approximately 20% of aortic aneurysm and/or dissection (AAD) cases result from inherited disorders, including several systemic and syndromatic connective-tissue disorders, such as Marfan syndrome, Ehlers-Danlos syndrome, and Loeys-Dietz syndrome, which are caused by mutations in the FBN1, COL3A1, and TGFBR1 and TGFBR2 genes, respectively. Nonsyndromatic AAD also has a familial background, and mutations of the ACTA2 gene were recently shown to cause familial AAD. In the present study, we conducted sequence analyses of the ACTA2 gene in 14 unrelated Japanese patients with familial thoracic AAD (TAAD), and in 26 with sporadic and young-onset TAAD. Our results identified three mutations of ACTA2, two novel [p.G152&#95;T205del (c.616+1G>T), p.R212Q] and one reported (p.R149C), in the 14 patients with familial TAAD, and a novel mutation (p.Y145C) of ACTA2 in the 26 sporadic and young-onset TAAD patients, each of which are considered to be causative for TAAD. Some of the clinical features of these patients were the same as previously reported, whereas others were different. These findings confirm that ACTA2 mutations are important in familial TAAD, while the first sporadic and young-onset TAAD case with an ACTA2 mutation was also identified.

Activity of proline uptake in Escherichia coli CSH4 was inhibited in the presence of 1 M NaCl, while it was recovered if the cells were incubated at 30 degrees C for 1 h in a moderate salinity stress (MSS) solution which consists of Davis minimal medium with 5 mM proline and 0.5 M NaCl. Then, an attempt was made to examine whether MSS treatment is also effective on the activity restoration of proline uptake and growth under high salinity for E. coli CSH4 mutants with different combinations of proP, putA, putP, and proU which are related to the transport and metabolization of proline. After MSS treatment, proline uptake was vigorously occurred for the mutants with proline transporter gene proP but not for its deficient ones. For the expression of proline uptake activities of these mutant strains after MSS treatment, PO(4)(3-) in MSS solution is more important than K(+). No growth of strain CSH4 and its mutants without MSS treatment was observed, when cultured in high osmotic medium G (0.8 M NaCl) consisting of 1 mM glycine betaine and Davis minimal medium without potassium phosphate supplemented. After MSS treatment, however, mutant strains lacking proP showed sufficient growth in medium G. Cell growth of proP(+) strains was recognized if MSS treatment was performed in the absence of proline. In conclusion, growth of mutant strains under high-salinity medium G depended on their amount of proline accumulated during MSS treatment, in which K(+) and PO(4)(3-) might play a key role to guarantee their sufficient growth.

Introduction: It has been reported that the R497K polymorphism of the epidermal growth factor receptor (EGFR) gene has attenuated functions in ligand binding, tyrosine kinase activation, and growth stimulation. On other hand, EGFR gene mutations at kinase domain in non-small cell lung cancer (NSCLC) have been examined for their ability to predict sensitivity to gefitinib or erlotinib., Materials and Methods: We investigated the EGFR mutations and/or R497K polymorphism statuses in 225 surgically treated NSCLC cases. 192 adenocarcinoma cases were included. The presence or absence of EGFR polymorphism of exon 13 was analyzed by PCR-RFLP method., Results: EGFR mutations at kinase domain were found from 95 of 225 lung cancer patients. In 86.2% of patients, homo- or heterozygous Lys497 allele was present. No correlation existed between R497K EGFR genotype and clinico-pathological features, such as gender, smoking status, and pathological subtypes., Conclusions: EGFR mutation status was not correlated with R497KEGFR genotype of lung cancers. In node-negative patients, R497KEGFR genotype was not correlated with disease outcome. In node-positive patients, however, R497K EGFR was significantly associated with better overall survival. This association was attributable to neo-adjuvant or adjuvant chemotherapy. In 46 total gefitinib treated NSCLC patients, the prognosis was not different between the EGFR wild type (GG) patients and AG+AA patients. R497KEGFR polymorphism might be associated with favorable prognosis of advanced lung cancers and correlated with chemosensitivity.

Catecholamines strongly promote lipolysis and thermogenesis, and play a central role in the regulation of body fat content. The beta1 adrenergic receptor (BAR-1) is a major mediator of catecholamine-induced lipolysis and thermogenesis. To explore whether mutations in the BAR-1 gene contribute to morbid obesity in Japanese, we scanned for mutations in the coding sequence of the gene in 50 morbid obese [body mass index (BMI)>==35.0kg/m(2); 99.7th percentile] Japanese subjects. Direct DNA sequencing was performed following polymerase chain reaction (PCR) amplification. Two common polymorphisms, Gly49Arg and Arg389Ser, were detected in these subjects. The frequencies of these polymorphisms, as determined by PCR-restriction fragment length polymorphism (RFLP) analysis, showed no significant difference between 180 severely obese subjects (BMI>==30.0kg/m(2); 97th percentile) and 132 control (BMI<25.0kg/m(2)) subjects. This study represents the first investigations of genetic variations of BAR-1 in relationship to morbid obesity and suggests mutations in the BAR-1 coding sequence is not likely a major cause of morbid obesity at least in Japanese.

Activating mutations of Ras gene families have been found in a variety of human malignancies, including lung cancer, suggesting their dominant role in tumorigenesis. Many studies have showed that the Kras gene is activated by point mutations in approximately 15-20% of non-small cell lung cancers (NSCLCs), however, there are only a few reports on Nras mutations in NSCLC. We have genotyped Nras mutation status (n=195) and Kras mutation status (n=190) in surgically treated lung adenocarcinoma cases. The presence or absence of Nras and Kras mutations was analyzed by real-time quantitative polymerase chain reaction (PCR) with mutation-specific sensor and anchor probes. EGFR mutation status at kinase domain has already been reported. Nras mutation was found in 1 of 195 patients. This mutation was a G-to-T transversion, involving the substitution of the normal glycine (GGT) with cystein (TGT) and thought to be a somatic mutation. The patient was male and a smoker. Kras mutant patients (11.1%; 21/190) had a significantly worse prognosis than wild-type patients (p=0.0013). Eighty-two EGFR mutations at kinase domain had exclusively Nras or Kras mutations. Although Nras gene mutation might be one of the mechanisms of oncogenesis of lung adenocarcinoma, this was a very rare event. Further studies are needed to confirm the mechanisms of Nras mutations for the sensitivity of molecular target therapy for lung cancer.

Background: Much evidence has accumulated that the epidermal growth factor receptor (EGFR) and its family members are strongly implicated in the development and progression of lung cancers. Recently, erbB4 kinase domain mutations were reported in Korean patients with lung cancer. We hypothesized that erbB4 mutations correlate with clinicopathologic features of lung cancers., Patients and Methods: The presence or absence of erbB4 kinase domain mutations was analyzed by reverse-transcription polymerase chain reaction amplification and direct sequencing in 105 surgically treated non-small-cell lung cancer cases from Nagoya City University Hospital. Sixty-three adenocarcinoma cases were included. The EGFR mutation status for these 105 samples were already reported. We have investigated erbB4 expression status by immunohistochemistry in 40 non-small cell lung cancer cases., Results: ErbB4 mutation was not found in 105 patients with lung cancer. The EGFR mutation status was significantly correlated with sex (women, 74.2% vs. men, 9.5%; P < 0.0001), smoking status (never-smokers, 68.8% vs. smokers, 11%; P < 0.0001), pathologic subtype (adenocarcinoma, 44.4% vs. non-adenocarcinoma, 4.8%; P < 0.0001), and differentiation status of the lung cancer (well-differentiated, 47.4% vs. others, 14.8%; P = 0.0004). We detected ErbB4 protein positivity in 20 samples. The ErbB4 protein status was not significantly correlated with sex (women, 28.6% vs. men, 54.5%; P = 0.4075), smoking status (never-smokers, 69.4% vs. smokers, 16.9%; P < 0.0001), pathologic subtype (adenocarcinoma, 46.2% vs. nonadenocarcinoma, 51.9%; P > 0.9999), or differentiation status of the lung cancer (well-differentiated, 54.5% vs. others, 48.3%; P > 0.9999)., Conclusion: Thus, erbB4 mutations are rare in Japanese people with lung cancer and of limited value for molecular-targeted therapy.

Systematic efforts are underway to decipher the genetic changes associated with tumor initiation and progression. However, widespread clinical application of this information is hampered by an inability to identify critical genetic events across the spectrum of human tumors with adequate sensitivity and scalability. Here, we have adapted high-throughput genotyping to query 238 known oncogene mutations across 1,000 human tumor samples. This approach established robust mutation distributions spanning 17 cancer types. Of 17 oncogenes analyzed, we found 14 to be mutated at least once, and 298 (30%) samples carried at least one mutation. Moreover, we identified previously unrecognized oncogene mutations in several tumor types and observed an unexpectedly high number of co-occurring mutations. These results offer a new dimension in tumor genetics, where mutations involving multiple cancer genes may be interrogated simultaneously and in 'real time' to guide cancer classification and rational therapeutic intervention.

Overexpression of the epidermal growth factor receptor (EGFR) is caused by EGFR gene amplification and is sometimes associated with expression of a variant EGFR (deletion exon 2-7 or EGFRvIII). EGFRvIII mutation has oncogenic potential and is investigated as a potential therapeutic target. We genotyped the EGFRvIII mutation status in 252 surgically treated lung cancer cases. The presence or absence of EGFRvIII mutation was analyzed by real-time quantitative polymerase chain reaction (PCR) with mutation specific sensor and anchor probes. EGFR copy number was evaluated with PCR-based assay. EGFR mutation status at kinase domain has been examined and reported. EGFRvIII mutation was found on 8 of 252 patients. All patients were male, smokers, and 7 had squamous cell carcinoma. The mutation status was significantly correlated with pathological subtypes (squamous cell carcinoma vs. adenocarcinoma, p=0.0114). Sixty EGFR mutations at kinase domain exclusively existed with EGFRvIII mutations. EGFR gene copy number was significantly higher in EGFRvIII mutant (4.711+/-4.968) than in non-EGFRvIII mutant (2.284+/-1.224) (p=0.0001). EGFRvIII gene mutation might be one of the mechanisms of increased EGFR copy number. Further studies are needed to confirm the mechanisms of EGFRvIII mutations for possible anti-EGFR therapy for lung cancer.

Somatic mutations of the epidermal growth factor receptor (EGFR) gene were found in about 25-40% of Japanese lung cancer patients. These mutations are associated with clinical and radiographic responses to EGFR tyrosine kinase inhibitors. Most common mutation are arginine for leucine substitution at amino acid 858 (L858R) and exon 19 deletions, especially deletion type 1 mutation. We investigated these EGFR mutation statuses in 575 surgically treated non-small cell lung cancer (NSCLC) cases. Three-hundred and sixty-two adenocarcinoma cases were included. The presence or absence of EGFR mutations of kinase domains was analyzed by genotyping analysis (TaqMan assay; n=386, and LightCycler assay; n=98) and sequences (n=91). EGFR mutations (CTG; CGG; L858R) were found from 63 of 575 lung cancer patients. We also detected the deletion 1a type mutations (2235-2249 del GGAATTAAGAGAAGC) from 39 patients and deletion 1b type mutations (2236-2250 del GAATTAAGAGAAGCA) from 15 patients in exon 19. These mutation statuses were significantly correlated with gender, smoking status (never smoker versus smoker), and pathological subtypes (adenocarcinoma versus non-adenocarcinoma). L858R mutation (p<0.0001), but not deletion 1 type mutation (p=0.0665), was correlated with differentiation status (well versus moderately or poorly) of lung cancers. L858R mutation ratio was significantly higher in non-smoker (p=0.0496) and adenocaicinoma (p=0.0136) when compared to deletion 1 type mutations. The EGFR mutation status, especially L858R mutation might be correlated with the clinico-pathological features related to good response to gefitinib, such as gender, smoking history, and pathological subtypes of Japanese lung cancers.

Several studies have suggested that epidermal growth factor receptor (EGFR) gene mutation, EGFR gene amplification, and some other biomarkers may be predictors of gefitinib sensitivity. We analyzed EGFR mutation and EGFR copy number in 22 gefitinib-treated non-small cell lung cancer (NSCLC) cases and their relation to the survival of patients. We also studied 143 gefitinib-naïve Japanese NSCLC cases. The erbB2 copy number was also studied in 59 gefitinib-naïve NSCLC cases. In gefitinib-treated patients, the presence of EGFR mutation was associated with a higher response rate to gefitinib and a longer overall survival, but the increased EGFR gene copy number was not. In gefitinib-naïve cases, EGFR mutation but not EGFR gene copy number was significantly correlated with gender, pathological subtypes, and smoking status. The erbB2 copy number was not significantly correlated with the EGFR mutation or EGFR copy number in 59 cases. In conclusion, EGFR mutation was a better predictor of clinical outcome in gefitinib-treated patients than the EGFR gene copy number.

The sensitivity of conventional DNA sequencing in tumor biopsies is limited by stromal contamination and by genetic heterogeneity within the cancer. Here, we show that microreactor-based pyrosequencing can detect rare cancer-associated sequence variations by independent and parallel sampling of multiple representatives of a given DNA fragment. This technology can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies.

Objective: The presence of epidermal growth factor receptor (EGFR) mutations has been reported to predict the response to gefitinib in pulmonary adenocarcinoma patients. A retrospective analysis was conducted to identify the correlation between computed tomographic findings of the nodules and EGFR status., Patients and Methods: Computed tomographic findings of 38 patients with peripheral pulmonary adenocarcinoma with EGFR mutations were reviewed and compared with those of 42 peripheral pulmonary adenocarcinoma patients with wild-type EGFR., Results: Mutations were found significantly more frequently among women (28 of 45 women versus 10 of 35 men) and among non-smokers (31 of 47 non-smokers and 7 of 33 smokers). The L858R mutation was found in 18 cases. Several types of deletion mutants in exon 19 were found in 18 cases. The nodules with EGFR mutations (2.5 +/- 1.0 cm) were significantly smaller in diameter than those in the wild-type group (3.1 +/- 1.9 cm). Ground glass opacity (GGO) was more often observed in the mutation group (28 of 38) than in the wild-type group (24 of 42), but the difference was not statistically significant. When mutations were analyzed with reference to both the tumor size and GGO ratio, patients with a tumor < or =3 cm and a GGO ratio > or =50% often had EGFR mutations, and most (10 of 12) were expressed in female patients. No male adenocarcinoma patients with a tumor larger than 4.0 cm had EGFR mutations., Conclusions: EGFR mutations were found most frequently in small peripheral adenocarcinomas with a GGO ratio > or =50%, especially among women. These factors may be useful in deciding therapeutic strategies for adenocarcinomas when resection or biopsy is not feasible.

Epidermal growth factor receptor (EGFR) gene mutations are frequently detected in lung cancer, especially in adenocarcinoma, in females, and non-smoking patients. EGFR mutations are closely associated with clinical response to EGFR tyrosine kinase inhibitor. Bronchioloalveolar carcinoma (BAC) appearance is a good predictor of response to this agent. Noguchi et al. subdivided small peripheral adenocarcinoma of the lung into two groups. One group was characterized with tumor cell growth replacing the normal alveolar cells with varying degree of fibrosis (types A-C), and the other shows non-replacing and destructive growth (types D-F). Using probes for the 13 mutations which have been previously described, we have genotyped the EGFR gene status in surgically resected atypical adenomatous hyperplasias (AAH) and small peripheral adenocarcinomas up to 2 cm in diameter using TaqMan PCR assay. In 95 small-sized adenocarcinomas, the EGFR mutations were detected in 37 patients (38.9%), and no mutations were found in five AAHs. In small peripheral adenocarcinomas, EGFR mutations were found 47.1% of types A, B, or C adenocarcinomas; it was less frequent (16%) in Noguchi's types D, E or F adenocarcinomas. These results suggest that type D, F adenocarcinomas are not derived from the less malignant types A-C adenocarcinomas; rather, they have arisen de novo by distinct mechanisms. Although types A and B adenocarcinomas are almost 100% cured by surgery, some type C adenocarcinoma show lymph node metastasis and relapse. EGFR mutation analysis may help identify patients who will respond to treatment with tyrosine kinase inhibitors, e.g., gefitinib.

Hereditary spastic paraplegia (HSP) is a group of genetically heterogeneous neurodegenerative disorders characterized by slowly progressive spasticity and weakness of the lower limbs. HSP is caused by failure of development or selective degeneration of the corticospinal tracts, which contain the longest axons in humans. The most common form of HSP is caused by mutations of the spastin gene (SPAST), located on chromosome 2p21-p22, which encodes spastin, one of the ATPases associated with diverse cellular activities (AAA). In this study, we detected four causative mutations of SPAST among 14 unrelated patients with spastic paraplegia. Two missense mutations (1447A-->G, 1207C-->G) and two deletion mutations (1465delT, 1475-1476delAA) were located in the AAA cassette region. Three of these four mutations were novel. Previous reports and our results suggest that the frequency of SPAST mutations is higher among Japanese patients with autosomal dominant HSP, although SPAST mutations are also observed in patients with sporadic spastic paraplegia.

Epidermal growth factor receptor (EGFR) gene mutations have been found in a subset of non-small cell lung cancer (NSCLC) with good clinical response to gefitinib therapy. A quick and sensitive method with large throughput is required to utilize the information to determine whether the molecular targeted therapy should be applied for the particular NSCLC patients. Using probes for the 13 different mutations including 11 that have already been reported, we have genotyped the EGFR mutation status in 94 NSCLC patients using the TaqMan PCR assay. We have also genotyped the EGFR mutations status in additional 182 NSCLC patients, as well as 63 gastric, 95 esophagus and 70 colon carcinoma patients. In 94 NSCLC samples, the result of the TaqMan PCR assay perfectly matched with that of the sequencing excluding one patient. In one sample in which no EGFR mutation was detected by direct sequencing, the TaqMan PCR assay detected a mutation. This patient was a gefitinib responder. In a serial dilution study, the assay could detect a mutant sample diluted in 1/10 with a wild-type sample. Of 182 NSCLC samples, 46 mutations were detected. EGFR mutation was significantly correlated with gender, smoking status, pathological subtypes, and differentiation of lung cancers. There was no mutation detected by the TaqMan PCR assay in gastric, esophagus and colon carcinomas. TaqMan PCR assay is a rapid and sensitive method of detection of EGFR mutations with high throughput, and may be useful to determine whether gefitinib should be offered for the treatment of NSCLC patients. The TaqMan PCR assay can offer us a complementary and confirmative test.

Betacellulin (BTC), a member of the epidermal growth factor (EGF) family, is an important factor in the growth and/or differentiation of pancreatic beta cells. In this point of view, we determined the transcriptional start site of the human BTC gene and screened the protein-coding region for mutations. The transcriptional start site was located 347 bp upstream from the translational initiation codon. After screening the protein coding exons (exons 1-5), we identified two novel missense mutations, Cys (TGC) to Gly (GGC) at codon 7 (C7G) and Leu (TTG) to Met (ATG) at codon 124 (L124M), and a single nucleotide substitution (-31c/t) in the intron 2. The C7G was located in the signal peptide and the L124M in the transmembrane domain and this Leu at codon 124 was conserved among human, bovine, rat, and mouse. The frequencies of these variants, however, were similar between type 2 diabetic patients (n = 228) and non-diabetic control subjects (n = 170). These data suggest that genetic variations in the protein-coding region of the human BTC gene are unlikely to be a major contributor to development of type 2 diabetes.

Background: Paired box gene 6 (PAX6) is a transcription factor involved in eye development. Mutations of PAX6 cause congenital eye anomalies, such as aniridia. PAX6 is also involved in the development of the endocrine pancreas, and reported to be a genetic factor common to aniridia and glucose intolerance, although the latter is usually mild. Here, we describe a case of PAX6 mutation with early-onset diabetes mellitus., Case Report: A 27-year-old woman was referred to our clinic. She was diagnosed having diabetes at the age of 15 with negative glutamic acid decarboxylase (GAD) antibody. Insulin treatment was started at age 24. Because she had aniridia, PAX6 gene mutation was investigated and a heterozygous 2-bp deletion (c.402del2) was identified. Her parents did not have aniridia and PAX6 mutations. Heterozygous PAX6 mutation may cause glucose intolerance. However, cases of early-onset diabetes mellitus have not been reported. Her parents did not have diabetes, but their insulinogenic indices were low (0.25 and 0.3, respectively). We thought her early-onset diabetes was partly as a result of PAX6 mutation and partly because of an unknown insulin secretory defect inherited from her parents. We could not find any mutations in HNF-1alpha, -1beta, -4alpha, IPF-1, ISL-1, BEAT2/NeuroD1, PAX4, and amylin genes., Conclusions: We report a case of PAX6 gene mutation with early-onset diabetes mellitus and aniridia. Low insulin secretory capacity in her parents suggested that her insulin secretory defect is as a result of not only PAX6 mutation but other genetic factors inherited from her parents.

Purpose: Recently, somatic mutations of the epidermal growth factor receptor (EGFR) gene were found in approximately 25% of Japanese lung cancer patients. These EGFR mutations are reported to be correlated with clinical response to gefitinib therapy. However, DNA sequencing using the PCR methods described to date is time-consuming and requires significant quantities of DNA; thus, this existing approach is not suitable for a routine pretherapeutic screening program., Experimental Design: We have genotyped EGFR mutation status in Japanese lung cancer patients, including 102 surgically treated lung cancer cases from Nagoya City University Hospital and 16 gefitinib-treated lung cancer cases from Kinki-chuo Chest Medical Center. The presence or absence of three common EGFR mutations were analyzed by real-time quantitative PCR with mutation-specific sensor and anchor probes., Results: In exon 21, EGFR mutations (CTG --> CGG; L858R) were found from 8 of 102 patients from Nagoya and 1 of 16 from Kinki. We also detected the deletion mutations in exon 19 from 7 of 102 patients from Nagoya (all were deletion type 1a) and 4 of 16 patients from Kinki (one was type 1a and three were type 1b). In exon 18, one example of G719S mutation was found from both Nagoya and Kinki. The L858R mutation was significantly correlated with gender (women versus men, P < 0.0001), Brinkman index (600 < or = versus 600, P = 0.001), pathologic subtypes (adenocarcinoma versus nonadenocarcinoma, P = 0.007), and differentiation status of the lung cancers (well versus moderately or poorly, P = 0.0439), whereas the deletion mutants were not. EGFR gene status, including the type of EGFR somatic mutation, was correlated with sensitivity to gefitinib therapy. For example, some of our gefitinib-responsive patients had L858R or deletion type 1a mutations. On the other hand, one of our gefitinib-resistant patients had a G719S mutation., Conclusions: Using the LightCycler PCR assay, the EGFR L858R mutation status might correlate with gender, pathologic subtypes, and gefitinib sensitivity of lung cancers. However, further genotyping studies are needed to confirm the mechanisms of EGFR mutations for the sensitivity or resistance of gefitinib therapy for the lung cancer.

Spinocerebellar ataxia 14 (SCA14) is associated with missense mutations in the protein kinase C gamma gene (PRKCG), rather than a nucleotide repeat expansion. In this large-scale study of PRKCG in patients with ataxia, two new missense mutations, an in-frame deletion, and a possible splice site mutation were found and can now be added to the four previously described missense mutations. The genotype/phenotype correlations in these families are described.

A complex of the transcription factors Gcr1p and Gcr2p coordinately regulates the expression of glycolytic genes in Saccharomyces cerevisiae. To understand the effects of gcr mutations on other metabolic pathways, genome-wide gene expression profiles in gcr1 and gcr2 mutants were examined. The biggest effects of gcr1 and gcr2 mutations were observed on the glycolytic genes and the expressions of most of the glycolytic genes were substantially decreased compared to those in the wild-type strain in both glucose and glycerol+lactate growth conditions. On the other hand, the expressions of genes encoding the TCA cycle and respiration were increased in gcr mutants when the cells were grown in glucose. RT-PCR analyses revealed that the expression of SIP4 and HAP5, which are known to affect the expression of some of the gluconeogenic, TCA cycle and respiratory genes, were also increased under this condition. The growth of gcr mutants on glucose was impaired by adding respiration inhibitor antimycin A, whereas the growth of the wild-type strain was not. The conversion of glucose to biomass was higher and, to the contrary, ethanol yield was lower in the gcr2 mutant compared to those in the wild-type strain. These results suggest the possibility that the gcr mutants, in which glycolytic activities are low, changed their metabolic patterns under glucose growth condition to enhance the expression of TCA cycle and respiratory genes to produce more energy., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

Purpose: Microarray-based comparative genomic hybridization analysis led us to detect a homozygous deletion at the cyclic AMP response element binding protein-binding protein (CBP) locus in a lung cancer cell line. Oncogenic roles of CBP had been suggested by functional and genetic studies; thus, involvement of CBP gene alterations in lung carcinogenesis was investigated by undertaking comprehensive analysis of genetic CBP alterations in human lung cancer., Experimental Design: Fifty-nine cell lines and 95 surgical specimens of lung cancer were analyzed for mutations, homozygous and hemizygous deletions, and expression of the CBP gene., Results: Homozygous CBP deletions, including two intragenic deletions, were detected in three (5.1%) lung cancer cell lines. CBP mutations, including missense, nonsense, and frame-shift mutations, were detected in six (10.2 %) cell lines and five (5.3%) surgical specimens of lung cancer. The wild-type CBP allele was retained in 9 of 11 cases with CBP mutations, and both the wild-type and mutant alleles were expressed in all the six cases with heterozygous CBP mutations examined. Three mutations with amino acid substitutions in the histone acetyltransferase domain caused significant reduction in transcription activation activity of CBP protein in vivo., Conclusions: A fraction of lung cancers carried mutations and/or deletions of the CBP gene, suggesting that genetic CBP alterations are involved in the genesis and/or progression of a subset of lung cancers.