全基因组外显子测序技术在遗传性纤维蛋白原异常家系致病基因的克隆鉴定中的应用

目的研究采用全基因组外显子测序技术在遗传性纤维蛋白原异常家系致病基因鉴定中的应用价值。方法采集12例遗传性纤维蛋白原异常患者及其核心家庭成员外周血检测凝血指标,并提取其基因组DNA进行全基因组外显子测序,分析测序结果,探讨遗传性纤维蛋白原异常的分子机制。结果全基因组外显子测序结果显示:先证者A1、A4以及B2均为FGA基因g.1233GA突变,A1的妹妹、A4的父亲以及B2的母亲均携带有相同的突变;A2、A7、B4和B5均为FGG基因g.10819GA突变,家系成员中A2的母亲和外婆、A7的姐姐和女儿、B4的母亲和B5的母亲均携带有相同的突变;A3、A5、A6、B1和B3及其相关亲属共10例携带有FGB基因g.9692AG突变。先证者及家系主要成员中发生Fg基因突变的成员APTT、PT和TT均明显延长,但Fg活性显著降低。结论遗传性纤维蛋白原异常可由多种Fg基因外显子突变导致,FGB和FGG外显子突变较为常见。     

常染色体显性遗传先天性核性白内障家系的致病基因突变研究

目的 对一个常染色体显性遗传先天性核性白内障家系的致病基因突变进行研究,探索其潜在的分子遗传学缺陷.方法 选择一个确诊为常染色体显性遗传先天性核性白内障家系,随机选取家系中的6例白内障患者为实验组,6名有血缘关系的正常成员为对照组,常规采集外周血5 ml,提取基因组DNA,应用PCR直接测序法对实验组和对照组的基因片断进行先天性核性白内障候选基因的突变检测,并对测序结果进行分析.结果 实验组的GJA8基因出现了c.139G＞A的错义突变,对照组未出现该突变.结论 在中国家系的GJA8基因外显子上发现了新的突变点(D47N),为研究先天性核性白内障的分子发病机制、基因诊断和基因治疗提供了科学的理论依据.     

RET基因Cys634Arg突变致多发性内分泌腺瘤2A型2例家系分析

目的 总结2个多发性内分泌腺瘤2A型(MEN2A)家系的临床特征及RET基因的突变类型.方法 分析2例MEN2A先证者及其家系成员的临床表现;提取其外周血基因组DNA,应用PCR技术对RET基因的第10、11外显子进行扩增并双向测序.结果 2个家系的先证者及5名家庭成员均存在RET基因第11外显子的Cys634Arg点突变,且临床均表现为甲状腺髓样癌和嗜铬细胞瘤.其中,家系2合并有垂体无功能腺瘤.结论 该MEN2A家系的RET基因是Cys634Arg突变;其临床表现主要为甲状腺髓样癌及肾上腺嗜铬细胞瘤.     

颅骨锁骨发育不全家系基因多态性与表型关系的研究

目的研究家族性颅锁骨发育不全家系基因型与临床表型的关系。方法提取临床收集的一个先天性颅锁骨发育不全家系中患者和健康成员外周血基因组DNA,PCR扩增RUNX2/CBFA1基因7个编码外显子及其侧翼内含子序列,分别进行正反向测序,对发现的突变位点行酶切分析验证。结果家系中两位患者（先证者及其父亲）显示cDNA 346T→A的杂合突变,使编码的色氨酸变成精氨酸（W 116R）,属错义突变。酶切结果进一步证实了该错义突变。测序还发现先证者父亲cDNA 198G→A的杂合突变,致第66位氨基酸的密码子GCG被GCA取代,但二者均编码丙氨酸,属同义突变。先证者及家系健康成员中未见此改变。结论 RUNX2/CBFA1基因346T→A杂合突变是该家系发病的分子基础。     

促甲状腺素受体基因突变一个家系研究

目的：探讨1个甲状腺发育不良先天性甲状腺功能减退症(先天性甲减)患者家系促甲状腺素受体(TSHR)基因突变遗传规律。方法：调查包括先证者3代家系成员共计13人，检测血清甲状腺激素。用TKM法提取基因组DNA，PCR扩增TSHR基因第10外显子序列，对PCR产物正反向测序，测序结果同Genbank人TSHR基因序列对比。结果：先证者TSHR基因同时存在2个位点纯合子型点突变R450H／D727E，家系12人甲状腺功能均正常，6人为R450H／D727E杂合子型点突变，其中5人血清sTSH轻度升高。结论：R450H／D727E纯合子导致先证者甲状腺发育不良及先天性甲减，R450H／D727E杂合子甲状腺功能正常仅sTSH轻度升高。     

MTOR基因位点新突变致史密斯-金斯莫综合征患儿的遗传学分析

目的 分析哺乳动物雷帕霉素靶蛋白(MTOR)基因位点新突变致史密斯-金斯莫综合征(SKS)1例患儿的遗传学病因。方法 收集SKS患儿及父母外周血进行生化指标检测并抽提基因组DNA,应用全外显子测序(WES)进行基因突变位点检测,Sanger测序进一步验证突变位点真实性。结果 WES结合Sanger测序验证发现患儿MTOR基因在53号外显子发生c.7255G>A:p.E2419K错义突变。变异位点检测系统推测该突变形式为致病突变,与SKS相关。结论 先证者MTOR基因的外显子发生变异导致SKS。该突变c.7255G>A:p.E2419K是一种新型突变,突变位点的确认有助于患儿疾病诊断及父母再生育指导。     

肝豆状核变性患者ATP 7B基因外显子8、12、18的DNA测序突变

目的 对肝豆状核变性(WD)患者ATP7B基因外显子(exon)8、12、18进行PCR扩增测序,研究其突变特点.方法 对30例患者(WD组)和10例健康者(对照组)提取外周血基因组DNA,PCR扩增ATP7B基因外显子8、12、18相关片段,并对扩增产物进行测序和分析测序结果 .结果 对照组未见异常,WD组发现12例患者外显子8有突变,突变率为40.0%,测序发现有6例患者外显子Arg778leu呈复合杂合子突变;发现4例外显子12有突变,有2例患者存在双重突变,外显子18未检出突变.结论 WD的ATP7B基因外显子8、12为突变的热点区,18号外显子不是突变热点.     

一个常染色体显性遗传性先天性白内障家系分析

目的:对1个常染色体显性遗传性先天性白内障家系进行基因突变热点的筛查,以探索其潜在的遗传学缺陷.方法:经过详细的病史采集及完善的眼科学检查后,对1个家系共10名成员(包括患者,非患者)抽取外周血提取基因组DNA,针对国内外文献报道的与常染色体显性遗传先天性白内障发病相关的10个基因设计引物,PCR扩增后直接测序进行候选基因的突变检测.结果:被检查者的10个发病相关基因序列中,CRYBA4基因第四个外显子上c.281T＞C突变,并由此导致了βA4晶状体蛋白第94位的苯丙氨酸被丝氨酸取代,其余9个与GenBank发表的正常序列相同.结论:CRYBA4基因外显子上发现的已经报道过的杂合型突变位点,可能是这个家系的致病基因,其对先天性白内障的作用需进一步研究.     

全外显子测序检测5例青少年Gilbert综合征患者及家系验证分析

目的 探讨UGT1A1基因分子家系遗传在Gilbert综合征中的诊断价值.方法 分析5例因总胆红素异常增高,且治疗无明显改善的青少年患者全外显子测序结果,并对5例患者及其父母进行了筛选分析得出的致病基因UGT1A1突变位点验证.结果 5例患者经全外显子测序均发现在UGT1A1基因的c.-53_-52insTA和c.-3275T＞G位点发生突变;2例患者还在c.211G＞A(p.G71R)有突变,1例患者在c.686C＞A(p.P229Q)突变.全外显子测序还检测到5例患者其他基因突变,但并未发现患者间有其他共同的基因位点突变.针对所有突变对患者及父母进行Sanger测序验证,其结果与全外显子测序结果一致.结论 采用全外显子测序技术可准确锁定Gilbert综合征患者相关致病基因UGT1Al的突变位点,并且通过家系验证得以确定,有助于协助临床医生提高诊断率.     

病态窦房结综合征家系致病基因分析1例并文献复习

目的：通过全外显子测序（WES）探寻病态窦房结综合征（SSS）家系的致病基因,并探讨其基因突变型与临床表型的相关性。方法：收集在上海市儿童医院就诊的1例SSS病人及其家系成员的临床资料,采集病人及家系成员的外周血,抽提血液DNA,通过WES寻找致病基因,利用Sanger测序在家系中验证可能的致病基因突变,使用致病性预测软件预测基因型与表型关系。结果：将测序结果比对分析,多个生物数据库筛选、过滤,发现SCN5A基因c.999-1G> A位点发生杂合突变,是SSS的可能致病基因,此位点突变在汉族儿童中尚属首次报道。结论：SCN5A基因的c.999-1G> A位点突变是导致散发SSS的致病位点;通过对SSS相关致病基因的研究,对于临床医师精准判断此类病人以及未来尽早进行心脏生物起搏治疗具有重要的临床意义。     

Quantitative in vivo and in vitro sex differences in artemisinin metabolism in rat

1. The pharmacokinetics of the antimalarial compound artemisinin were compared in the male and female Sprague-Dawley rat after single dose i.v. (20 mg.kg) or i.p. (50 mg.kg) administration of an emulsion formulation. 2. Plasma clearance of artemisinin was 12.0 (95% confidence interval: 10.4, 13.0) l.h. kg in the male rat and 10.6 (95% CI: 7.5, 15.0) l.h. kg in the female rat suggesting high hepatic extraction in combination with erythrocyte uptake or clearance. Artemisinin half-life was 0.5 h after both routes of administration in both sexes. Values for plasma clearance and half-lives did not statistically differ between the sexes. 3. After i.p. administration artemisinin AUCs were 2-fold higher in the female compared with male rat (p 0.001). Artemisinin disappearance was 3.9-fold greater in microsomes from male compared with female livers and it was inhibited in male microsomes by goat or rabbit serum containing antibodies against CYP2C11 and CYP3A2 but not CYP2B1 or CYP2E1. 4. The unbound fraction of artemisinin in plasma was lower (p 0.001) in plasma obtained from the male (8.8 2.0%) compared with the female rat (11.7 2.2%). 5. The possibility of a marked sex difference, dependent on the route of administration, has to be taken into account in the design and interpretation of toxicological studies of artemisinin in this species.     

Quantitative in vivo and in vitro sex differences in artemisinin metabolism in rat

1. The pharmacokinetics of the antimalarial compound artemisinin were compared in the male and female Sprague-Dawley rat after single dose i.v. (20 mg x kg(-1)) or i.p. (50 mg x kg(-1)) administration of an emulsion formulation. 2. Plasma clearance of artemisinin was 12.0 (95% confidence interval: 10.4, 13.0) 1 x h(-1) x kg(-1) in the male rat and 10.6 (95% CI: 7.5, 15.0) 1 x h(-1) x kg(-1) in the female rat suggesting high hepatic extraction in combination with erythrocyte uptake or clearance. Artemisinin half-life was approximately 0.5 h after both routes of administration in both sexes. Values for plasma clearance and half-lives did not statistically differ between the sexes. 3. After i.p. administration artemisinin AUCs were 2-fold higher in the female compared with male rat (p < 0.001). Artemisinin disappearance was 3.9-fold greater in microsomes from male compared with female livers and it was inhibited in male microsomes by goat or rabbit serum containing antibodies against CYP2C11 and CYP3A2 but not CYP2B1 or CYP2E1. 4. The unbound fraction of artemisinin in plasma was lower (p < 0.001) in plasma obtained from the male (8.8 +/- 2.0%) compared with the female rat (11.7 +/- 2.2%). 5. The possibility of a marked sex difference, dependent on the route of administration, has to be taken into account in the design and interpretation of toxicological studies of artemisinin in this species.     

Interindividual variability in metabolic activation in humans in vivo

In assessing interindividual variability in metabolic activation, the toxic metabolite is often too unstable for conventional analysis. Possible alternatives include a stable product of the reactive metabolite e.g. cysteinyl derivatives of N-acetyl-4-benzoquinoneimine, the toxic metabolite of paracetamol, adducts with DNA or protein, and indirect measurement of the activity of the enzyme(s) producing the active metabolite. An example of the last approach is the use of furafylline, a highly specific inhibitor of human CYP1A2, to determine the extent of the metabolic activation of the cooked food mutagens PhIP and MeIQx. The extent of inhibition, determined from levels of unchanged amine in urine, is an indirect measure of the activity of the activation pathway. Further refinement of this approach, allied to improved measures of the biological process of interest should prove of value in evaluating interindividual variability and its role in the risk assessment process.     

Theophylline kinetics in peripheral tissues in vivo in humans

Several biochemical and cellular effects have been described for methylxanthines under in vitro conditions. However, it is unknown, whether threshold concentrations required to exert these effects are attained in target tissues in vivo. We therefore employed the microdialysis technique for measuring theophylline concentrations in peripheral tissues under in vivo conditions.Following in vitro and in vivo calibration, microdialysis probes were inserted into the medial vastus muscle and into the periumbilical subcutaneous adipose layer of healthy volunteers. Following single oral dose administration of 300 mg or i.v. infusion of 240 mg theophylline, in vivo time courses of theophylline concentrations were monitored in tissues and plasma. Major pharmacokinetic parameters (c_max, t_max, AUC) were calculated for plasma and tissue time courses. The mean AUC_tissue /AUC_plasma-ratio was 0.56 (p.o.) and 0.55 (i.v.) for muscle and 0.55 (p.o.) and 0.72 (i.v.) for subcutaneous adipose tissue.We conclude that microdialysis provides important information on the distribution and the tissue pharmacokinetics of theophylline.Abbreviations FPIA Fluorescence polarisation immuno assay - AUC Area under the curve - t_max Time to peak concentration - c_max Peak concentration     

休克时血中氧自由基的变化

本实验测定10名休克患者血浆和红细胞的丙二醛(MDA)、血浆总抗的氧化活性(AOA)的含量。结果表明:休克病人红细胞膜和血浆 MDA 含量(4.298±0.722;5.348±0.834)与对照组(3.235±0.682;4.356±1.081)比较明显增高(P<0.05);血浆 AOA(39.65±7.858)与对照组(48.21±10.81)比较明显降低(P<0.01)。提示:休克时,患者机体内自由基反应增强是引起组织细胞损伤的原因之一。     

Changes in angiopoietin expression in glomeruli involved in glomerulosclerosis in rats with daunorubicin-induced nephrosis

AIM: To study the potential pathological role of endogenous angiopoietins in daunorubicin-induced progressive glomerulosclerosis in rats. METHODS: Seventy male Wistar rats were allocated randomly into a daunorubicin group (DRB; n=40) or a control group (n=30). The rats in the DRB group were injected with DRB (15 mg/kg), in their tails. Subsequently, at intervals of 1, 2, 4, 6, 8, and 12 weeks, 5 male Wistar rats in each group were chosen randomly for 24 h urinary protein quantitative measurements (24 h UPQM), and determination of plasma tumor necrosis factor alpha (TNF-alpha), angiopoietin-1 (Ang1), and angiopoietin-2 (Ang2) levels. Kidney sections were examined by electron microscopy, Periodic Acid Schiff (PAS) staining, immunohistochemical staining and in situ hybridization histochemistry. RESULTS: As glomerulosclerosis progressed in the DRB group, expression of Ang1 mRNA and protein in glomeruli decreased and expression of TNF-alpha protein, Ang2 mRNA and protein in glomeruli increased. Expression of Ang1 mRNA and protein in glomeruli were negatively correlated with 24 h UPQM, Fn protein expression, and mean area of extracellular matrix (MAECM). In comparison, expression of Ang2 mRNA and protein in glomeruli were positively correlated with 24 h UPQM, Fn protein expression and MAECM; furthermore, there was a positive correlation between plasma Ang2 and 24 h UPQM. Plasma TNF-alpha and expression of TNF-alpha in glomeruli were positively correlated with expression of Ang2 mRNA and protein in glomeruli. There was a negative correlation between Ang1 protein expression and Ang2 protein expression in glomeruli. CONCLUSION: During DRB-induced glomerulosclerosis, podocyte injury led to a shift in the balance of Ang1 and Ang2 in glomeruli. Increased TNF-alpha in plasma and glomeruli may upregulate Ang2 expression in glomeruli. Elevated Ang2 in both plasma and glomeruli may mediate protein permeability through the glomerular filtration barrier. Moreover, local expression of Ang2 may facilitate the progress of glomerulosclerosis by upregulating a component expression of extracellular matrix.     

Trichinellosis in immigrants in Switzerland

We describe a case of trichinellosis diagnosed at the Division of Infectious Diseases, Hospital of Lugano, in January 2009. This case was associated with a cluster of cases and was traced to the consumption of contaminated meat after a wild boar hunt in Bosnia.