一个遗传性胰腺炎家系中新发现的胰蛋白酶原基因突变

对1个遗传性胰腺炎(hereditary pancreatitis, HP)家系中6例成员和120例无亲缘关系健康人的胰蛋白酶原基因(protease serine 1, PRSS1)进行PCR扩增, 产物纯化后测序, 结合受检者的血清肿瘤标志物、糖尿病相关生化指标以及近亲属的一般临床资料进行分析。结果发现4例家系成员PRSS1基因3号外显子区136位碱基存在C→T杂合性突变, 他们的基因型表现为野生型与突变型杂合现象, 另外在先证者PRSS1基因的3号外显子区171位碱基还存在着一个同义突变点(C→T), 而对照组和家系其他成员中未发现此两种突变, 突变阳性患者表现为乳酸、糖基化血红蛋白和糖类肿瘤标志物(CA19-9、CA125)增高。因此, PRSS1基因3号外显子区136位碱基C→T杂合性突变与该家系遗传性胰腺炎有关, 是该家系中遗传性胰腺炎的遗传易感因素。     

改进的多片段重叠延伸PCR制作基因多位点突变

为在研究工作中提高制作目标基因多位点突变体的效率,对常规重叠延伸PCR进行适当改进:对于相距较近的两个突变位点,只需设计一对突变引物各自涵盖其中一个位点即可一次突变;对于相距较远的两个位点,可以采用三片段重叠延伸PCR的办法解决;两者结合则可一次性突变多个位点。以制作RBCT的6位点突变体PSM6为例,利用上述策略,设计两对突变引物;采用OE-PCR法,第一轮PCR扩增出三个片段,第二轮PCR同时利用三片段重叠延伸产物作为模板扩增出目标基因突变体,再按常规分子克隆方法将其连入质粒载体。经测序检测,发现得到了预期的目标基因多位点突变体。因此,采用灵活的引物设计策略,结合多片段重叠延伸PCR即可一次性制作基因的多位点突变体,此方案可解决研究工作中大多数多位点突变问题。     

枯草杆菌蛋白酶E基因多位点定点突变库的构建及其筛选

利用化学合成的15个寡聚核苷酸片段作为诱变引物,同时对枯草杆菌蛋白酶E(Subtilisin E或Apr E)基因进行体外突变,获得了合全部突变位点各种随机组合突变的突变库,通过点杂交法和DNA序列分析肯定了该突变库的可靠性.从突变库中选择一单点突变(Met222Ala)和3点突变(Asn76Asp/Asn109Ser/Ile205Cys)的基因进行克隆、表达和产物的酶学性质研究,发现其抗氧化性和热稳定性分别比野生型的有显著提高,与文献报道的一致.表明了该突变库在枯草杆菌蛋白酶工程研究中的应用价值.     

基孔肯雅病毒非结构蛋白基因合成中多位点缺失突变的校正方法

目的:建立一种PCR方法,以快速校正基孔肯雅病毒非结构蛋白基因合成过程中发生的多位点缺失突变。方法:用PCR方法合成基孔肯雅病毒非结构蛋白基因;对测序的克隆进行序列比对,分析不同克隆上缺失突变发生的位置,以保守区域互相重叠的寡核苷酸为上下游引物、以该区域测序正确的克隆为模板进行PCR扩增,得到所需片段,再将这些片段用PCR方法进一步组装成完整的基因序列并进行测序。结果:测序结果表明,经过2次PCR扩增,校正了基孔肯雅病毒非结构蛋白基因合成过程中发生的5个位点缺失突变。结论:得到序列正确的基孔肯雅病毒非结构蛋白基因。在进行基因合成过程中如发生多位点缺失突变,可利用该方法同时对以上突变进行校正,无须再合成引物,降低了实验操作难度,并提高了实验效率。     

Expression of human cationic trypsinogen (PRSS1) in murine acinar cells promotes pancreatitis and apoptotic cell death

Hereditary pancreatitis (HP) is an autosomal dominant disease that displays the features of both acute and chronic pancreatitis. Mutations in human cationic trypsinogen (PRSS1) are associated with HP and have provided some insight into the pathogenesis of pancreatitis, but mechanisms responsible for the initiation of pancreatitis have not been elucidated and the role of apoptosis and necrosis has been much debated. However, it has been generally accepted that trypsinogen, prematurely activated within the pancreatic acinar cell, has a major role in the initiation process. Functional studies of HP have been limited by the absence of an experimental system that authentically mimics disease development. We therefore developed a novel transgenic murine model system using wild-type (WT) human PRSS1 or two HP-associated mutants (R122H and N29I) to determine whether expression of human cationic trypsinogen in murine acinar cells promotes pancreatitis. The rat elastase promoter was used to target transgene expression to pancreatic acinar cells in three transgenic strains that were generated: Tg(Ela-PRSS1)NV, Tg(Ela-PRSS1*R122H)NV and Tg(Ela-PRSS1*N29I)NV. Mice were analysed histologically, immunohistochemically and biochemically. We found that transgene expression is restricted to pancreatic acinar cells and transgenic PRSS1 proteins are targeted to the pancreatic secretory pathway. Animals from all transgenic strains developed pancreatitis characterised by acinar cell vacuolisation, inflammatory infiltrates and fibrosis. Transgenic animals also developed more severe pancreatitis upon treatment with low-dose cerulein than controls, displaying significantly higher scores for oedema, inflammation and overall histopathology. Expression of PRSS1, WT or mutant, in acinar cells increased apoptosis in pancreatic tissues and isolated acinar cells. Moreover, studies of isolated acinar cells demonstrated that transgene expression promotes apoptosis rather than necrosis. We therefore conclude that expression of WT or mutant human PRSS1 in murine acinar cells induces apoptosis and is sufficient to promote spontaneous pancreatitis, which is enhanced in response to cellular insult.     

苯丙氨酸羟化酶（PAH）基因外显子7及其两侧内含子的突变研究

为探讨中国苯丙酮尿症（PKU）人群中苯丙氨酸羟化酶（PAH）基因外显子7的突变特征,对147例PKU患儿的294个PAH基因外显子7以及两侧部分内含子序列,应用PCR－单链构象多态性（SSCP）分析及基因序列分析的方法进行了筛查和确定。共发现13种突变基因：G239D、R241C、R241fs、R243Q、G247S、G247V、R252Q、L255S、R261Q、M276K、E280G、P281L、Ivs7+2T>A,其中7 种突变基因在中国PKU人群首次发现：G239D 、R241fs 、G247S 、E280G、L255S、R261Q、P281L,前4种在国际上尚未见到报道,并已提交到国际PAH突变数据库（www.pahdb.mcgill.ca）。突变基因的总频率为30.61%（90 /294）。突变涉及了错义、缺失、移码和剪接位点4种突变类型。结果明确了PAH基因外显子7的突变种类和分布等特征,表明外显子7是中国人PAH基因突变的热点区域。 Abstract: To study mutation in exon 7 of the gene for the phenylalanine hydroxylase（PAH）, the mutations in exon 7 and flanking sequence of PAH gene were detected by means of SSCP analysis and DNA sequencing, in 147 unrelated Chinese children with phynelketonuria and their parents. Thirteen different mutations, including 11 missense, 1 deletion and 1 splice mutation, were revealed in 90/294 mutant alleles (30.61%). The prevalent mutations were R243Q (22.8%) and Ivs7nt2t->a (2.38%). Seven novel mutations were identified: G239D, R241fsdelG, G247S, E280G, L255S, R261Q, P281L. These new mutations have not been described in Chinese PKU population and the first 4 mutants have not been reported and thus been submitted to www.pahdb,mcgill.ca. The missense was the most common type. The deletion and frameshift mutations were detected for the first time in Chinese PKU population. This study showed the mutation characteristics and their distribution in exon 7 of PAH gene and proved that the exon 7 was the hot region of PAH gene mutation in Chinese PKU population .     

The Frequency and Clinical Significance of IDH1 Mutations in Chinese Acute Myeloid Leukemia Patients

Objective

Mutations in the gene encoding isocitrate dehydrogenease 1 (IDH1) occur in various hematopoietic tumors including acute myeloid leukemia (AML), myeloproliferative neoplasms and myelodysplastic syndromes. IDH1 mutations are significant in both diagnosis and prognosis of these conditions. In the present study we determined the prevalence and clinical significance of IDH1 mutations in 349 samples from newly diagnosed AML patients.

Results

Of the 349 AML patient specimens analyzed, 35 (10.03%) were found to have IDH1 mutations including 4 IDH1 R132 mutations and 31 non-R132 mutations. IDH1 non-R132 mutations were largely concentrated within AML-M₁ (35.72%, p<0.01). We identified five IDH1 mutations that were novel to AML: (1) c.299 G>A, p.R100Q; (2) c.311G>T, p.G104V; (3) c.322T>C, p.F108L; (4) c.356G>A, p.R119Q; and (5) c.388A>G, p.I130V. In addition, we identified three IDH1 mutations that were previously described in AML. The frequency of IDH1 mutations in AML patients with normal karyotype was 9.9%. IDH1 non-R132 mutations were concurrent with mutations in FLT3-ITD (p<0.01), CEBPA (p<0.01), and NRAS (p<0.01), as well as the overexpression of MN1 (p<0.01) and WT1(p<0.01). The overall survival (OS) in the patients with IDH1 non-R132 mutations compared to patients without IDH1 mutations don''t reach statistically significance (median 521 days vs median: not reached; n.s.).

Conclusion

IDH1 non-R132 mutations occurred frequently in newly diagnosed adult Chinese AML patients, and these mutations were associated with genetic alterations. The OS was not influenced by IDH1 non-R132 mutations in the present study.     

Novel Mutations in the CLCN1 Gene of Myotonia Congenita: 2 Case Reports

Introduction: Myotonia Congenita is an inherited myotonia that isdue to a mutation in the skeletal muscle chloride channel CLCN1. These mutationslead to reduced sarcolemmal chloride conductance, causing delayed musclerelaxation that is evident as clinical and electrical myotonia.Methods: We report the clinical presentations of two individualswith Myotonia Congenita (MC).Results: Patient 1 has been diagnosed with the recessive form of MC,known as the Becker variant, and Patient 2 has been diagnosed with the dominantform of MC, known as the Thomsen variant. In both patients, the diagnosis wasmade based on the clinical presentation, EMG and CLCN1 gene sequencing. Patient1 also had a muscle biopsy.Conclusions: Genetic testing in both patients reveals previouslyunidentified mutations in the CLCN1 gene specific to Myotonia Congenita. Wereport the salient clinical features of each patient and discuss the effects andcommon types of CLCN1 mutations and review the literature.     

先天性甲状腺功能减退症伴发育不全患儿FOXE1基因突变的初步研究

目的：研究先天性甲状腺功能减退症(CH)伴甲状腺发育不全患儿转录因子2( FOXE )的基因突变。方法：选取90 例CH伴甲 状腺发育不全患儿及90 例正常儿童作为对照,提取外周静脉血基因组DNA,采用PCR扩增与直接测序技术,对基因外 显子进行突变筛查。结果：分别在1 例先天性甲状腺功能减退症伴甲状腺发育不全患者外显子测序中发现一杂合错义变体c. A3401G (p.K1134R),在1 例患者中发现1 个已知的单核苷酸多态性(single nucleotide polymorphisms,SNP)位点(rs755282859, c. 483G>C),在正常对照组中未发现以上变化。结论：在先天性甲状腺功能减退症(CH) 伴甲状腺发育不全患儿中发现新的关于FOXE1 杂合错义变体。     

乌司他丁治疗急性胰腺炎的临床疗效及对患者血清炎性因子水平的影响

目的:观察乌司他丁在急性胰腺炎临床治疗中的作用效果,分析其临床治疗价值。方法:选取我院治疗的急性胰腺炎患者178例,根据治疗方法不同分为观察组和对照组,每组89例。对照组患者采用奥曲肽治疗,观察组患者采用乌司他丁与奥曲肽联合治疗。观察并比较治疗前后患者APACHEⅡ评分和血清炎症因子变化及临床疗效。结果:入院时,两组血清CRP、IL-1、IL-6、TNF-α、APACHEⅡ评分无统计差异(P0.05),治疗后,组内CRP、IL-1、IL-6、TNF-α、APACHEⅡ评分明显下降(P0.05);观察组CRP、IL-1、IL-6、TNF-α、APACHEⅡ评分显著低于参照组(P0.05);观察组临床总有效率(86.52%)显著高于参照组(71.91%),观察组并发症(12.36%)明显低于参照组(24.72%),P0.05,组间有统计差异。结论:乌司他丁能够显著提升急性胰腺炎的临床疗效,促进患者康复,其作用机制可能与抑制炎症因子表达有关。     

SMARCA1基因组结构鉴定及其在Smith-Fineman-Myers综合征家系中的突变分析

为探讨SMARCA1基因在中国山东SFMS家系患者发生中的作用,采用计算机杂交结合DNA序列分析方法,首先确定了SMARCA1基因的基因组结构,发现该基因的基因组DNA全长超过71．7kb,含有24个外显子和23个内含子,所有外显子和内含子接头皆遵循GT-AG法则,基因组结构的阐明,为进行基因突变检测和分析其生物学功能奠定了基础。在以上分析的基础上,通过PCR扩增结合测序分析,对在山东省发现的1个SFMS家系患者的SMARCA1基因的全部外显子和外显子内含子接头序列进行了基因突变检测,未检测到导致疾病的突变,提示中国山东SFMS家系患者不是由于SMARCA1基因编码区域内基因突变所致。     

降钙素原与APACHEⅡ评分对急性胰腺炎病情严重程度及预后的评估价值

目的:探讨血清降钙素原(PCT)与急性生理学与慢性健康状况评分(APACHE Ⅱ)对急性胰腺炎(AP)患者病情严重程度及预后的评估价值。方法:选取本院2012年5月-2015年5月收治的急性胰腺炎患者280例为研究对象。根据急性胰腺炎患者病情严重程度将其分为低危组(83例)、中危组(102例)和高危组(95例);按患者临床结局将其分为存活组(248例)及死亡组(32例),采用酶联免疫吸附法(ELISA)检测各组血清PCT水平同时记录APACHE Ⅱ评分情况,分别比较PCT水平的差异以及与APACHE Ⅱ评分的相关性,评价血清PCT及APACHEⅡ评分对急性胰腺炎患者病情严重程度及预后的评估价值。结果:低危组、中危组及高危组间血清PCT水平和APACHE Ⅱ评分的差异具有统计学意义(P0.05)。其中,高危组血清PCT水平和APACHE Ⅱ评分最高,中危组次之,低危组最低(P0.05);死亡组PCT水平及APACHE Ⅱ评分显著高于存活组(P0.05)。相关性分析显示血清PCT水平与APACHE Ⅱ评分呈正相关(r=0.64,P0.01)。以PCT2.13 ng/m L为评估急性胰腺炎患者预后不佳界限时,其敏感性和特异性分别79.2%和91.3%;以APACHE Ⅱ评分18.1分为评估急性胰腺炎患者预后不佳界限时,其敏感性和特异性分别为82.7%和90.1%;两者指标串联评估敏感性及特异性分别为86.1%和92.9%,ROC曲线下面积为0.921(95%CI 0.824~0.938)。结论:急性胰腺炎患者血清PCT水平和APACHE Ⅱ评分具有较好的相关性,血清PCT水平越高,APACHEⅡ评分越高,患者病情越严重及预后也越差,二者联合可作为预测急性胰腺炎患者病情严重程度及预后的敏感指标,具有较好的临床应用价值。