TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade

An essential step in intricate visual processing is the segregation of visual signals into ON and OFF pathways by retinal bipolar cells (BCs). Glutamate released from photoreceptors modulates the photoresponse of ON BCs via metabotropic glutamate receptor 6 (mGluR6) and G protein (Go) that regulates a cation channel. However, the cation channel has not yet been unequivocally identified. Here, we report a mouse TRPM1 long form (TRPM1-L) as the cation channel. We found that TRPM1-L localization is developmentally restricted to the dendritic tips of ON BCs in colocalization with mGluR6. TRPM1 null mutant mice completely lose the photoresponse of ON BCs but not that of OFF BCs. In the TRPM1-L-expressing cells, TRPM1-L functions as a constitutively active nonselective cation channel and its activity is negatively regulated by Go in the mGluR6 cascade. These results demonstrate that TRPM1-L is a component of the ON BC transduction channel downstream of mGluR6 in ON BCs.     

Ankyrin gene mutations in japanese patients with hereditary spherocytosis

We studied mutations of the ankyrin-1 (ANK-1) gene of genomic DNA from Japanese patients with hereditary spherocytosis (HS). Forty-nine patients from 46 unrelated families were included in this study. Of these patients, 19 cases from 16 unrelated families had HS of autosomal-dominant inheritance, and 30 patients had non-autosomal-dominant HS. Fifteen mutations of the ANK-1 gene pathognomonic for HS were identified: 4 nonsense mutations, 7 frameshift mutations, and 4 abnormal splicing mutations. These 15 mutations have not been previously reported. The frameshift mutations were found from exon 1 to exon 26, corresponding particularly to the band 3-binding domain of ankyrin. The nonsense mutations, on the contrary, were present mostly at the 3'-terminal side, especially in the spectrin-binding domain and the regulatory domain. The patients with ankyrin gene mutations tended to be more anemic with a higher level of reticulocytosis than those without these mutations. Fifteen silent mutations of the ANK-1 gene, most of which have previously been detected in HS patients in Western populations, were also found. The allele frequency of these silent mutations in the HS patients was nearly identical to that in normal subjects. There was no difference between the Japanese and Western populations in the allele frequency of these gene polymorphisms in healthy subjects or HS patients.     

Identification of EGFR and KRAS mutations in Chinese patients with esophageal squamous cell carcinoma

The prognosis of esophageal squamous cell carcinoma (ESCC) is poor. It is urgent to improve this situation. Epidermal growth factor receptor (EGFR)‐targeted therapy possesses a promising clinical efficacy. Mutations of EGFR and V‐Ki‐ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) have been identified in esophageal carcinoma, but corresponding Chinese data are limited. So we investigated the mutation status of EGFR and KRAS in Chinese patients with ESCC, and explored their correlations with clinicopathological features. Formalin‐fixed paraffin‐embedded surgically resected tumor samples were obtained from 50 randomly selected Chinese patients with ESCC. EGFR mutations in exons 18–21 were detected by Scorpions amplification refractory mutation system technology. KRAS mutations in codons 12, 13 were detected by direct sequencing of polymerase chain reaction products. The correlations between clinicopathological features and the mutation status of EGFR and KRAS were analyzed using the Statistical Package for the Social Sciences. In the present study, EGFR mutations were found in 7 (14%) out of 50 patients, including G719X missense mutation (n= 1), in‐frame deletion (n= 2), and L858R missense mutation (n= 5). Six (12%) out of 50 patients had KRAS mutations in codon 12. Concurrent EGFR and KRAS mutations were detected in one sample. The presences of EGFR and KRAS mutations were not associated with gender, age, smoking history, cell differentiation, or cancer stage. In conclusion, the incidence of EGFR mutations in Chinese patients with ESCC was higher than that of previous reports, and the incidence of KRAS mutations was not low. EGFR and KRAS mutations were mainly located in exons 19 and 21 and codon 12, respectively. Unlike in NSCLC, concurrent EGFR and KRAS mutations existed.     

非小细胞肺癌胸水中癌细胞EGFR基因TK区突变研究

目的:检测非小细胞肺癌(NSCLC)患者胸水标本中癌细胞EGFR基因TK区突变。方法:收集21例NSCLC患者胸水标本,经密度梯度离心后,将胸水中细胞包埋,制成蜡块;巢式聚合酶链式反应(PCR)扩增EGFR基因18-21号外显子;应用PCR-LIS-SSCP及直接测序方法检测EGFR基因TK区突变。结果:21例NSCLC患者胸水癌细胞标本,19例为腺癌,1例为腺鳞癌,1例为大细胞癌;9例患者存在EGFR基因TK区突变,均为腺癌,突变率为42.9%(9/21),腺癌患者突变率为47.4%(9/19);9例患者中8例接受TKIs治疗,疗效评价为PR(部分缓解)或SD(稳定)。5例出现TKIs耐药,疾病进展TTP(疾病进展时间)为4～17个月不等。应用TKIs治疗前后对比胸水及癌组织中EGFR基因突变,2例出现获得性突变。结论:NSCLC胸水标本与文献报道的癌组织中EGFR基因TK区突变率基本相同;胸水标本中检测肿瘤细胞基因突变的方法可靠,值得在临床推广、应用。     

Toll-like receptor 9 gene mutations and polymorphisms in Japanese ulcerative colitis patients

Abnormal innate immune responses toward luminal bacteria play an important role in the pathogenesis of inflammatory bowel disease.It has been demonstrated that bacteria having CpG DNA ameliorate experimental colitis in mice,and Toll-like receptor 9 (TLR9) signaling mediates the anti-inflammatory effects in mouse colonic inflammation.A gene variation in NOD2/CARD15 has been reported in Crohn’s disease (CD) patients in Western countries,but this variation has not been identified in Japanese CD patients.Therefore,we hypothesized that TLR9 is a key factor in the development of ulcerative colitis (UC),and we investigated gene mutations and polymorphisms of TLR9 in Japanese UC patients.Three single nucleotide polymorphisms (SNPs) in TLR9 were identified in healthy controls,and were assessed in 48 UC patients and 47 healthy controls.Control subjects were matched for age,sex and date of blood sampling from among a subgroup of participants.We found that TLR9-1486CC,1174GG and 2848AA increase the risk of UC [odds ratio (OR) 2.64,95% confidence interval (95% CI):1.73-6.53,P=0.042],and TLR9-1486TT,1174AA and 2848GG decrease the risk of UC (OR 0.30,95% CI:0.10-0.94,P=0.039),although there were no correlations between SNPs and disease phenotype or TLR9 mRNA expression.These findings suggest that TLR9 polymorphisms are associated with increased susceptibility to UC.     

中国妊娠期血糖异常人群葡萄糖激酶基因突变初步筛查

目的 初步明确中国妊娠期血糖异常人群中葡萄糖激酶（GCK）基因突变情况.方法 回顾性选择2005年7月至2008年5月在北京协和医院进行妊娠期血糖筛查并进行了口服葡萄糖耐量试验（OGTT）和糖化血红蛋白（HbA1c）检测的妊娠妇女.以空腹血糖在5.5～ 10.0 mmol/L、OGTT试验2h与0h血糖差值小于4.6 mmol/L且HbA1c值小于8.0％作为筛选条件,对满足所有条件者进行GCK基因外显子区和启动子区-71G＞C的突变筛查.结果 共纳入577例受试者,符合GCK基因检测条件者30例,可获得标本数17例,发现1例GCK基因突变致青少年的成人起病型糖尿病2型（MODY2）患者和1处非编码区新变异.该MODY2患者6号外显子区c.626 C＞T（NM_000162.3）突变导致第209位编码氨基酸从苏氨酸变为甲硫氨酸（p.T209M,NP_000153.1）.推测中国妊娠期血糖异常人群GCK最小突变率为0.27％,估测中国总人群中MODY2的最小患病率为21/10万.结论 中国妊娠期血糖异常的人群中GCK基因突变并不常见.     

Diverse clinical outcomes of eosinophilic patients with T-cell receptor gene rearrangements: the emerging diagnostic importance of molecular genetics testing

Abnormal clones of clusters of differentiation (CD)3(-)CD4(+) or CD3(+)CD4(-)CD8(-) phenotypically abnormal lymphocytes have been identified in some patients who have the idiopathic hypereosinophilic syndrome. This report illustrates the disparate clinical courses of six eosinophilic patients with evidence of abnormal T-cell clones based on the finding of a T-cell receptor gene rearrangement. The data suggest that molecular genetics testing for T-cell receptor gene rearrangements should be included in the routine work-up of patients with idiopathic eosinophilia.     

Activation of the BMP-BMPR pathway conferred resistance to EGFR-TKIs in lung squamous cell carcinoma patients with EGFR mutations

The empirical criteria for defining a clinical subtype of lung cancer are gradually transiting from histopathology to genetic variations in driver genes. Targeting these driver mutations, such as sensitizing epidermal growth factor receptor (EGFR) mutations, has dramatically improved the prognosis of advanced non–small cell lung cancer (NSCLC). However, the clinical benefit of molecularly targeted therapy on NSCLC appears to be different between lung adenocarcinomas and squamous cell carcinomas (SqCCs). We report here that the resistance of lung SqCC harboring EGFR mutations to EGFR tyrosine kinase inhibitors (EGFR-TKIs) was due to the activation of BMP-BMPR-Smad1/5-p70S6K. The combined treatment of these tumor cells with EGFR-TKI, together with inhibitors specific to BMPR or downstream mTOR, effectively reversed the resistance to EGFR-TKI. Moreover, blocking the whole PI3K-AKT-mTOR pathway with the PI3K/mTOR dual inhibitor BEZ235 also showed efficacy in treating this subtype of lung SqCC. This study details the empirical basis for a feasible clinical solution for squamous cell carcinomas with EGFR mutations.Traditionally, the classification of lung cancer has been based primarily on histology and morphology (1, 2). With the identification of mutated driver oncogenes, methods from molecular pathology are gradually transforming the definition of the various types of lung cancers (3). Targeting gene aberrances such as epidermal growth factor receptor (EGFR) mutations (4–8) and anaplastic lymphoma kinase (ALK) fusion (9, 10) has significantly improved the prognosis of advanced non–small cell lung cancer (NSCLC). However, the clinical benefits brought by targeted therapies are mainly limited to nonsquamous NSCLC (11), while chemotherapy remains the major therapeutic choice for squamous cell carcinomas (SqCCs).Recent studies assessing somatic mutations and copy number alteration (CNA) profiles in SqCC performed by the Cancer Genome Atlas project and other investigators have disclosed specific gene mutations, including GRM8, BAI3, ERBB4, RUNX1T1, KEAP1, and FBXW7, and CNAs in 3q26, 24, 27, 32–34, and 8p12.35 in lung SqCC (12, 13). About half of all patients with lung SqCC carry multiple gene aberrances, indicating that complex genomic characterizations are more common in lung SqCC than in adenocarcinoma (ADC). Thus, successful therapeutic strategies that target a single driver gene in lung ADC might not be feasible for lung SqCC patients.Targeting EGFR mutations by EGFR tyrosine kinase inhibitors (TKIs) is one of the successful strategies in treating lung ADC. EGFR-TKIs obtained median progression-free survival (PFS) of 10–13 mo in EGFR-mutated lung ADC, but only ∼3 mo in lung SqCC with EGFR mutations (11, 14). Moreover, whether EGFR mutations exist in lung SqCC still remains controversial. There is a belief that EGFR mutations might not even occur in pure pulmonary SqCC, and that the occasional detection of these mutations in samples diagnosed as SqCC was due to mixed adenosquamous carcinoma and poorly differentiated ADC (15, 16). In current clinical practice, the utilization of EGFR-TKIs and the assessment of EGFR mutations are still routinely performed in lung SqCC, especially in nonsmokers. Therefore, it is critical to identify the subgroups of lung SqCC patients that are suitable for EGFR-TKI treatment. Exploring the mechanism of resistance to EGFR-TKIs in lung SqCC will deepen our understanding of the differences in tumorigenic profiling between lung SqCC and ADC, and should contribute to guiding clinical therapeutic decisions.In the present study, we demonstrated that lung SqCC patients with EGFR mutations indeed represent a subset of NSCLC. We further showed that lung SqCC cell lines were resistant to EGFR-TKIs both in vitro and in vivo. We also investigated the mechanisms of resistance to EGFR-TKI in this subset of patients and provided potential strategies for overcoming TKI resistance in these patients.     

Missense mutations in the gp91-phox gene encoding cytochrome b558 in patients with cytochrome b positive and negative X-linked chronic granulomatous disease

Chronic granulomatous disease (CGD) is a disorder of host defense due to genetic defects of the superoxide (O2-) generating NADPH oxidase in phagocytes. A membrane-bound cytochrome b558, a heterodimer consisting of gp91-phox and p22-phox, is a critical component of the oxidase. The X-linked form of the disease is due to defects in the gp91-phox gene. We report here biochemical and genetic analyses of patients with typical and atypical X-linked CGD. Immunoblots showed that neutrophils from one patient had small amounts of p22-phox and gp91-phox and a low level of O2- forming oxidase activity, in contrast to the complete absence of both subunits in two patients with typical CGD. Using polymerase chain reactions (PCR) on cDNA and genomic DNA, we found novel missense mutations of gp91-phox in the two typical patients and a point mutation in the variant CGD, a characteristic common to two other patients with similar variant CGD reported previously. Spectrophotometric analysis of the neutrophils from the variant patient provided evidence for the presence of heme of cytochrome b558. Recently, we reported another variant CGD with similar amounts of both subunits, but without oxidase activity or the heme spectrum. A predicted mutation at amino acid 101 in gp91-phox was also confirmed in this variant CGD by PCR of the genomic DNA. These results on four patients, including those with two variant CGD, are discussed with respect to the missense mutated sites and the heme binding ligands in gp91-phox.     

血氨基酸异常婴儿肝内胆汁淤积症患者SLC25A13基因的突变谱

目的 探讨血氨基酸异常婴儿肝内胆汁淤积症中是否存在SLC25A13基因突变及其突变谱特征.方法 对2003年6月-2007年6月就诊于复旦大学附属儿科医院的不明原因婴儿肝内胆汁淤积症患儿进行血氨基酸质谱分析,将检测有瓜氨酸、蛋氨酸至少一项2倍以上升高者共14例进行基因研究.全部入选对象先行SLC25A13基因的12种突变位点([Ⅰ]851de14、[Ⅱ]IVS11+1G>A、[Ⅲ]1638ins23、[Ⅳ]S225X、[Ⅴ]IVS13+1G>A、[Ⅵ]1800insl、[Ⅶ]R605X、[Ⅷ]E601X、[Ⅸ]E601K、[Ⅹ]IVS6+5G>A、[Ⅺ]R184X及[ⅪⅤ]IVS6+1G>C)的检测.对仅检出单个位点突变的研究对象,继续进行所有外显子区及其邻近序列分析.结果 检出SLC25A13基因突变8例,包括复合杂合突变851de14/1638ins23 2例,纯合突变851de14/851de14、复合杂合突变851de14/R184X及纯合突变IVS6+1G>A/IVS6+1G>各1例,杂合突变851de14 3例,其中纯合突变IVS6+1G>A/IVS6+1G>A是一种新型突变.结论 血氨基酸异常婴儿肝内胆汁淤积症中存在SLC25A13基因突变.[Ⅰ]851de14、[Ⅲ]1638ins23是中国SLC25A13基因的常见突变形式,新发现1例纯合突变IVS6+1G>A/IVS6+1G>A,和国外报道突变谱存在明显区别.     

非小细胞肺癌E19-Del、L858R突变临床特征分析

目的探讨非小细胞肺癌E19-Del、L858R突变临床特征。 方法纳入2012年7月至2018年7月在空军军医大学第二附属医院呼吸与危重症医学科基因检测中心行EGFR基因检测的498例非小细胞肺癌肺癌患者,回顾性分析其E19-Del、L858R突变临床特征及相互间突变的差异。 结果E19-Del在小于50岁、大于等于70岁、大于等于50岁而小于70岁时突变率逐渐降低(χ²＝5.984,P＝0.050),不吸烟组E19-Del突变率高于吸烟每天大于20支组(χ²＝4.328,P＝0.037),E19-Del在腺癌组、鳞癌组、其他类型非小细胞肺癌组突变率逐渐降低(χ²＝12.197,P＝0.000),E19-Del在Ⅳ期、Ⅲ期、Ⅰ+Ⅱ期突变率逐渐降低(χ²＝6.625,P＝0.036),以上均有统计学差异;L858R女性突变率高于男性(χ²＝13.755,P＝0.000),L858R在不吸烟组、吸烟每天小于或等于20支组、吸烟每天大于20支组突变率逐渐降低(χ²＝7.234,P＝0.027),L858R在腺癌组、其他类型非小细胞肺癌组突变率逐渐降低(χ²＝5.538,P＝0.021),胸膜转移组L858R突变率高于未发生胸膜转移组(χ²＝4.004,P＝0.045),以上均有统计学差异。 结论E19-Del在年轻非吸烟Ⅳ腺癌组突变率高;L858R在女性非吸烟腺癌并胸膜转移组突变率高;E19-Del、L858R在肿瘤发生部位,有无并发糖尿病、高血压、其它肿瘤或是否存在骨转移、头颅转移、淋巴结转移时突变率各有差异,但均无统计学差异。     

Clinical significance of elevated soluble interleukin-6 receptor levels in the sera of patients with plasma cell dyscrasias

Summary .We investigated the clinical significance of the serum soluble interleukin-6 receptor (sIL-6R) in 42 patients with plasma cell dyscrasias (27 with multiple myeloma (MM), 13 with monoclonal gammopathy of undetermined significance (MGUS), and two with plasma cell leukaemia (PCL)). Serum levels of sIL-6R in normal individuals were 77 ± 21 ng/ml (mean ± SD, n = 18); those in patients with MGUS and with MM were elevated (102 ± 33 ng/ml, mean ± SD, P < 0–05 and 126 ± 60ng/ml, mean ± SD, P < 0–01, respectively). Significant correlations were not found between the serum levels of sIL-6R and known prognostic factors (C-reactive protein, haemoglobin levels, calcium, creatinine, β₂-microglobulin, amounts of M-protein, or percentages of plasma cells in bone marrow). Elevated serum sIL-6R did not affect the survival of the patients with MM. Serial measurements of sIL-6R together with the clinical course of patients with plasma cell neoplasias revealed a good correlation between the sIL-6R level and disease activity. We conclude that sIL-6R can be used as a clinical factor correlated with the disease activity, at least in some patients with plasma cell neoplasias.     

Iron overload and HFE gene mutations in Polish patients with liver cirrhosis

BACKGROUND:Increased liver iron stores may contribute to the progression of liver injury and fibrosis,and are associated with a higher risk of hepatocellular carcinoma development.Pre-transplant symptoms of iron overload in patients with liver cirrhosis are associated with higher risk of infectious and malignant complications in liver transplant recipients.HFE gene mutations may be involved in the pathogenesis of liver iron overload and influence the progression of chronic liver diseases of different origin...     

Novel mutations in the STK11 gene in Thai patients with Peutz-Jeghers syndrome

Peutz-Jeghers syndrome (PJS), a rare autosomal dominant inherited disorder, is characterized by hamartomatous gastrointestinal polyps and mucocutaneous pigmentation. Patients with this syndrome have a predisposition to a variety of cancers in multiple organs. Mutations in the serine/threonine kinase 11 (STK11) gene have been identified as a major cause of PJS. Here we present the clinical and molecular findings of two unrelated Thai individuals with PJS. Mutation analysis by Polymerase Chain Reaction-sequencing of the entire coding region of STK11 revealed two potentially pathogenic mutations. One harbored a single nucleotide deletion (c.182delG) in exon 1 resulting in a frameshift leading to premature termination at codon 63 (p.Gly61AlafsX63). The other carried an in-frame 9-base-pair (bp) deletion in exon 7, c.907_915del9 (p.Ile303_Gln305del). Both deletions were de novo and have never been previously described. This study has expanded the genotypic spectrum of the STK11 gene.     

N-ras and p53 gene mutations in Japanese patients with myeloproliferative disorders

Alterations of the N-ras oncogene and p53 tumor suppressor gene have been demonstrated to play an important role in pathogenesis of hematological malignancies. We simultaneously investigated genetic lesions of both genes in bone marrow cells from 64 Japanese patients with myeloproliferative disorders (MPD), including polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis (MF), by direct sequencing analysis. No mutations of the N-ras gene were detected in any cases. Two patients, one with chronic neutrophilic leukemia derived from PV and one with acute mylogenous leukemia derived from ET, exhibited three mutations of the p53 gene. Among them, two were missense mutations in exon 5 or 7 and one was a deletion in exon 5. All samples in chronic phase or from MF were devoid of mutations in both genes. These data suggested that disruptions of both genes are extremely rare in MPD in chronic phase and that loss of functions in the p53 gene could be involved in progression of MPD such as PV and ET.     

Frequency and clinical expression of HFE gene mutations in a Spanish population of subjects with abnormal iron metabolism

Three HFE gene mutations (HFE 845 GA, 187 CG and 193 AT) are the most common mutations related to hereditary haemochromatosis (HH). The genotype for these mutations was analysed in 359 Spanish individuals with altered iron metabolism and iron overload. Various biochemical parameters were measured in serum samples from 96 of these individuals, and the effect of the genotype on these parameters was studied. Allele frequencies were 12.95% for the HFE C282Y variant, 28.97% for the HFE H63D variant and 0.69% for the HFE S65C variant, calculated in a total of 718 chromosomes. Multiple comparisons analysis showed very significant differences (p=0.001) in transferrin saturation index (TSI) between the HFE C282Y variant homozygous and control (ten healthy volunteers) groups. Highly significant (p=0.0001) and significant (p=0.005) differences in serum ferritin values were found between the HFE C282Y variant homozygous and control groups and between compound (HFE C282Y/H63D variant) heterozygous and control groups, respectively. Very significant differences (p=0.001) in serum iron values were observed between the HFE C282Y variant homozygous and control groups. TSI and serum ferritin values detected most HFE C282Y variant homozygotes and are recommended to facilitate the clinical diagnosis of HH.