A method to assess the clinical significance of unclassified variants in the BRCA1 and BRCA2 genes based on cancer family history

Introduction

Unclassified variants (UVs) in the BRCA1/BRCA2 genes are a frequent problem in counseling breast cancer and/or ovarian cancer families. Information about cancer family history is usually available, but has rarely been used to evaluate UVs. The aim of the present study was to identify which is the best combination of clinical parameters that can predict whether a UV is deleterious, to be used for the classification of UVs.

Methods

We developed logistic regression models with the best combination of clinical features that distinguished a positive control of BRCA pathogenic variants (115 families) from a negative control population of BRCA variants initially classified as UVs and later considered neutral (38 families).

Results

The models included a combination of BRCAPRO scores, Myriad scores, number of ovarian cancers in the family, the age at diagnosis, and the number of persons with ovarian tumors and/or breast tumors. The areas under the receiver operating characteristic curves were respectively 0.935 and 0.836 for the BRCA1 and BRCA2 models. For each model, the minimum receiver operating characteristic distance (respectively 90% and 78% specificity for BRCA1 and BRCA2) was chosen as the cutoff value to predict which UVs are deleterious from a study population of 12 UVs, present in 59 Dutch families. The p.S1655F, p.R1699W, and p.R1699Q variants in BRCA1 and the p.Y2660D, p.R2784Q, and p.R3052W variants in BRCA2 are classified as deleterious according to our models. The predictions of the p.L246V variant in BRCA1 and of the p.Y42C, p.E462G, p.R2888C, and p.R3052Q variants in BRCA2 are in agreement with published information of them being neutral. The p.R2784W variant in BRCA2 remains uncertain.

Conclusions

The present study shows that these developed models are useful to classify UVs in clinical genetic practice.     

香菇多糖对荷瘤小鼠IFNmRNA和SODmRNA的影响

目的：通过研究香菇多糖（LNT）对小鼠肝组织中干扰素（IFN）mRNA和超氧化物岐化酶（SOD）mRNA的影响,从分子水平探讨LNT的药理作用机制。方法：C57BL／6小鼠随机分面四组,其中三组左腋皮下接种S180肉瘤细胞,48h后,两组荷瘤小鼠分别腹腔注射5mg/kg和30mg/kgLNT,一天一次,另一组瘤荷小鼠及正常鼠等量注射生理盐水,6天后杀鼠取肝,以斑点杂交的方法检测肝组织中IFNmRNA和SODmRNA的表达量。结果：5mg/kgLNT对180肉瘤的生长有较明显的抑制作用;5mg/kg和30mg/kg的LNT均能提高IFNmRNA和SODmRNA的表达量,且以5mg/kg剂量的作用稍强,结论：LNT抗肿瘤和抗衰老的机理之一可能是促进了IFNmRNA和SOD mRNA的表达。     

Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation.

In the present study, we investigate the implication of the mitogen-activated protein kinases (MAPKs) Erk, p38, and JNK in mediating the effect of fetal calf serum (FCS) on the differentiation of MC3T3-E1 osteoblast-like cells. Erk is stimulated by FCS in proliferating, early-differentiating, as well as in mature cells. Activation of p38 by FCS is not detected in proliferating cells but is observed as the cells differentiate. JNK is activated in response to FCS throughout the entire differentiation process, but a maximal stimulation is observed in early differentiating cells. The roles of Erk and p38 pathways in mediating MC3T3-E1 cell differentiation was determined using specific inhibitors such as U0126 and SB203580, respectively. These experiments confirmed that the Erk pathway is essential for mediating cell proliferation in response to FCS, but indicated that this MAP kinase has little effect in regulating the differentiation of MC3T3-E1 cells. In contrast, p38 only marginally influenced proliferation, but appeared to be critical for the control of alkaline phosphatase (ALP) expression in differentiating cells. Finally, results obtained with high doses of SB203580, which also affected JNK activity, suggest that p38 and/or JNK are probably also involved in the control of type 1 collagen and osteocalcin expression in differentiating cells. The data indicate that MAPKs regulate different stages of MC3T3-E1 cell development in response to FCS. Distinct MAPK pathways seem to independently modulate osteoblastic cell proliferation and differentiation, with Erk playing an essential role in cell replication, whereas p38 is involved in the regulation of ALP expression during osteoblastic cell differentiation. JNK is also probably involved in the regulation of osteoblastic cell differentiation, but its precise role requires further investigation.     

构建大肠癌细胞相关基因Nrf3融合蛋白真核表达载体及其体外转染的初步功能分析

目的：应用基因芯片生物信息学分析思路筛查大肠癌细胞相关基因Nrf3，构建融合蛋白真核表达载体，检测其对体外转染癌细胞周期与凋亡的影响。方法：实验于2004—01／2006—07在南方医科大学病理解剖教研室及肿瘤研究所与中国农业大学农业生物技术国家重点实验室完成。①实验材料：大肠癌细胞组织及其配对的正常大肠黏膜各3例，由解放军总医院病理科提供：大肠癌LoVo细胞系由南方医科大学肿瘤研究所提供；肝癌SMMC7721细胞系由解放军军事医学科学院放射医学研究所提供；真核表达载体pEGFP-N1（Clontech公司）。②实验方法：分别应用Excel表、Affymetrix Microarray Suite Software5．0分析软件和STATA7．0分析软件，对基因芯片表达谱数据行交集补集分析、秩和检验及T检验，筛选“最重要的大肠癌细胞差异表达基因＆ESTs”，差异表达P〈0．05，差异倍数〉2。应用文献轮廓法进一步分析确定首选研究基因为Nrf3，提取组织样品总RNA，cDNA合成，PCR扩增产物经琼脂糖凝胶电泳分离后回收纯化，插入T载体中，SalⅠ和BamHⅠ双酶切后连接到pEGFP-N1载体．获得重组pEGFP-N1-Nrf3质粒。LoVo细胞在体外加入含体积分数为0．1小牛血清、100u／mL青链霉素的RPMI-1640培养基，置于37℃、体积分数为0．05的CO2饱和湿度培养箱中，待转染质粒pEGFP-N1-Nrf3与阴性对照质粒pEGFP-N1转染36h后，荧光显微镜观察候选基因亚细胞定位，FCM分选术观察其在体外对LoVo细胞周期和凋亡的影响。同法观察重组pEGFP-N1-Nrf3质粒体外转染对SMMC7721细胞周期和凋亡的影响。结果：①首选研究大肠癌细胞相关基因的确认：多种统计学方法分析获得最重要的大肠癌细胞相关基因17个，文献轮廓法进一步确认Nrf3为首选研究基因。②Nrf3基因表达：RT-PCR法检测Nrf3在3例大肠癌细胞组织均有表达，而在与其相配对的正常黏膜中表达较弱或不表达，与芯片检测结果基本一致；Nrf3在LoVo细胞中有表达。③Nrf3亚细胞定位：重组pEGFP-N1-Nrf3质粒转染的LoVo细胞其绿色荧光主要分布于细胞核内，提示Nrf3可能定位于细胞核内。（少Nrf3在体外对癌细胞周期与凋亡的影响：体外培养36h后与未转染LoVo细胞的空白对照比较，重组pEGFP-N1-Nrf3质粒转染的LoVo细胞S＋G2／M期所占比例明显减低，G2／M期下降尤为显著，G—G，期细胞所占比例明显增加。Nrf3转染作用于LoVo细胞后，未观察到明显“亚G1”峰（即凋亡峰）的出现。Nrf3体外转染的SMMC7721细胞周期及凋亡情况与LoVo细胞基本相似。结论：大肠癌细胞相关基因Nrf3在体外能够抑制LoVo细胞、SMMC7721细胞的DNA合成和有丝分裂，促使癌细胞阻滞于G0/G，期，抑制其生长增殖的同时不影响细胞凋亡，可作为大肠癌细胞候选分子标志物。     

On improvement in ejection fraction with iron chelation in thalassemia major and the risk of future heart failure

Background

Trials of iron chelator regimens have increased the treatment options for cardiac siderosis in beta-thalassemia major (TM) patients. Treatment effects with improved left ventricular (LV) ejection fraction (EF) have been observed in patients without overt heart failure, but it is unclear whether these changes are clinically meaningful.

Methods

This retrospective study of a UK database of TM patients modelled the change in EF between serial scans measured by cardiovascular magnetic resonance (CMR) to the relative risk (RR) of future development of heart failure over 1 year. Patients were divided into 2 strata by baseline LVEF of 56-62% (below normal for TM) and 63-70% (lower half of the normal range for TM).

Results

A total of 315 patients with 754 CMR scans were analyzed. A 1% absolute increase in EF from baseline was associated with a statistically significant reduction in the risk of future development of heart failure for both the lower EF stratum (EF 56-62%, RR 0.818, p < 0.001) and the higher EF stratum (EF 63-70%, RR 0.893 p = 0.001).

Conclusion

These data show that during treatment with iron chelators for cardiac siderosis, small increases in LVEF in TM patients are associated with a significantly reduced risk of the development of heart failure. Thus the iron chelator induced improvements in LVEF of 2.6% to 3.1% that have been observed in randomized controlled trials, are associated with risk reductions of 25.5% to 46.4% for the development of heart failure over 12 months, which is clinically meaningful. In cardiac iron overload, heart mitochondrial dysfunction and its relief by iron chelation may underlie the changes in LV function.     

血管内皮细胞生长因子及其受体在大鼠肺气肿模型中的表达

目的　探讨血管内皮细胞生长因子 (VEGF)及其受体 2 (VEGFR 2 )在大鼠肺气肿模型中的表达及意义。方法　经气管内注入脂多糖及熏香烟法建立大鼠肺气肿模型。免疫组化法检测VEGF在肺组织的表达部位 ,ELISA法检测支气管肺泡灌洗液 (BALF)中VEGF的含量 ,Westernblot检测VEGFR 2蛋白的表达。结果　VEGF在肺泡上皮细胞、支气管粘膜上皮细胞及血管内皮细胞表达 ,模型组BALF的VEGF含量 (110 2± 4 6 0 .1)pg/mL较对照组 (2 0 17± 84 0 .6 ) pg/mL明显降低 (P <0 .0 5 ) ;模型组VEGFR 2蛋白的表达低于对照组 (P <0 .0 5 )。结论　VEGF及VEGFR 2的减少可能参与了肺气肿的发生