Alternative splicing implicated in immunity and prognosis of colon adenocarcinoma

Dysregulation of immune system is the hallmark of colon adenocarcinoma (COAD) patients. Aberrant alternative splicing (AS) is closely related to progression and immunotherapy of COAD. However, the intrinsic correlation of immune system with AS have not been elucidated. Here we identified 640 AS events related to immunescore by multi-omics data analysis. 7 key AS events were screened out and used to develop a riskscore model, the area under the ROC curve of riskscore model predicting 3-, 5-year survival probability was 0.750, 0.768. Also, the riskscore based on 7 key AS events is an independent prognostic factor. The AUC of the nomogram composed of riskscore and TMN grade reached to 0.872(3-year) and 0.841(5-year). Moreover, 11 AS events were identified to be associated with the infiltration of 8 types of immune cells. Interestingly, M1 macrophages and memory B cells had a higher infiltration in high-riskscore patients, and higher infiltration of M1 macrophages and memory B cells were significantly associated with worse prognosis. In conclusion, AS are closely related to immunescore, immunity stage and infiltrating immune cells. The riskscore is an effective diagnostic and prognostic indicator better than TMN grade, and AS events related to the immune system may be potential therapeutic targets for COAD.     

A study of exons 14, 15, and 24 of the ABCB11 gene in Egyptian children with normal GGT cholestasis

Background and study aimsProgressive familial intrahepatic cholestasis type 2 (PFIC2) is a rare inherited disorder caused by mutation in the ATP-binding cassette subfamily B member 11 gene (ABCB11) that encodes the bile salt export pump (BSEP), which is the main transporter of bile acids from hepatocytes to the canalicular lumen. Defects in BSEP synthesis and/or function lead to reduced bile salt secretion followed by accumulation of bile salts in hepatocytes and hepatocellular damage. This study aimed to detect variations in exons 14, 15, and 24 of the ABCB11 gene in patients with suspected PFIC2 among a group of Egyptian infants and children with normal gamma-glutamyl transpeptidase (GGT) cholestasis.Patients and methodsThis observational case-control study was conducted on 13 children with suspected PFIC2 and 13 healthy subjects as controls. Genotyping of the ABCB11 gene was performed via DNA extraction followed by PCR amplification, purification, and then sequencing analysis of exons 14, 15, and 24 of the ABCB11 gene.ResultsThe study detected two single nucleotide variations, c.1638+ 32T > C (rs2241340) in exon 14 and c.3084A > G (p.Ala1028 = ) (rs497692) in exon 24 of the ABCB11 gene. No variations were identified in exon 15.ConclusionThe study revealed two benign variants involving exons 14 and 24 of the ABCB11 gene. Exons 14, 15, and 24 are not hot spots for common mutations in Egyptian PFIC2 patients. Further study of other exons of the ABCB11 gene is necessary to confirm the diagnosis of PFIC2.     

SCN1A基因Exons7-21C〉T多态位点与全面性癫痫伴热性惊厥附加症相关性研究

目的研究SCN1A基因Exon7-21C〉T多态位点在全面性癫痫伴热性惊厥附加症患者中的分布。方法中国汉族人中收集155例全面性癫痫伴热性惊厥附加症患者和135例正常人标本,采用聚合酶链反应-变性高效液相色谱法检测SCN1A基因的第7编码外显子及与mRNA剪接有关的内含子进行筛查,对发现异常洗脱峰者进行测序并应用SPSS11.5分析结果。结果 135例正常人中,SCN1A Exon7-21C〉TCC、CT、TT基因型频率分别为：0.474、0.444、0.082,C、T等位基因频率分别为：0.696、0.304。155例全面性癫痫伴热性惊厥附加症患者中,SCN1AExon7-21C〉TCC、CT、TT基因型频率分别为：0.490、0.439、0.071,C、T等位基因频率分别为：0.710、0.290。实验组的基因型频率和等位基因频率与正常对照组比较,差异无统计学意义（P〉0.05）。结论 SCN1A基因单核苷酸多态位点Exon7-21C〉T与全面性癫痫伴热性惊厥附加症无相关性。     

人巨细胞病毒UL123基因外显子2,3诱饵质粒的构建与鉴定

目的构建和鉴定人巨细胞病毒(HCMV)UL123基因外显子2,3(ie1-exon2,3)的诱饵质粒,并评价其用于筛选胎儿脑文库的可行性。方法以重组质粒pTW IN1/ie1为模板扩增ie1-exon2,3,克隆入pBT质粒,转化E.coli XL1-B lue MRF'Kan,经PCR、限制性酶切和测序鉴定阳性重组子,提取阳性重组质粒转化入细菌双杂交系统报告菌株,诱导表达重组融合蛋白,用SDS-PAGE和W estern B lot对表达产物进行鉴定,并进一步鉴定重组子自身激活作用。结果在细菌双杂交系统中成功构建了细菌双杂交诱饵质粒pBT/ie1-exon2,3,并在报告菌株中表达了重组融合蛋白rIE1-N85/λC1,且pBT/ie1-exon2,3无自身激活作用。结论成功构建了无自激活作用的诱饵质粒pBT/ie1-exon2,3,可用于筛选胎儿脑文库。     

151个精神分裂症家系中多巴胺D3受体基因Ser9Gly多态的传递不平衡研究

目的 :探讨多巴胺D3 受体基因 (DRD3)第一外显子Ser9Gly多态性与精神分裂症的关系。 方法 :采集15 1个家系 ,每家有 2名或 2名以上符合ICD 10精神分裂症诊断标准的患病同胞父母存活且父母中至多只有一方有精神分裂症患病史。对DRD3基因的Ser9Gly多态性进行检测。结果 :(1)总体而言 ,DRD3基因的Ser9Gly等位基因频度和基因型频度在父母组、非患病同胞组和患病同胞组之间差异无显著性 ,在精神分裂症不同亚型之间基因型频度和等位基因频度的分布差异也没有统计学意义。 (2 )传递不平衡检验 (TDT)发现在患病同胞中存在传递不平衡 (χ2 =7.76 ,P =0 .0 0 5 3 ,OR =1.70 ,95 %置信区间为 1.15～ 2 .5 2 )。 (3)基因型与临床特征的相关分析显示 ,具有Ser9等位基因的患病同胞较多地出现阴性症状。结论 :DRD3基因的Ser9Gly多态与家族性精神分裂症相关联。研究支持DRD3作为精神分裂症的候选基因之一     

Ⅰ型神经纤维瘤一家系的基因检测及产前诊断

目的: 对一个Ⅰ型神经纤维瘤家系进行遗传学病因分析及产前诊断。方法: 应用目标捕获高通量测序和Sanger测序技术,对一个散发的Ⅰ型神经纤维瘤家系进行基因突变分析。提取先证者及其父母外周血淋巴细胞RNA,行RT-PCR及扩增产物测序分析。明确突变致病性后,抽取羊水标本对胎儿行产前基因诊断。结果: 先证者NF1基因存在c.1260+4A>T杂合剪接突变,为新发突变。RNA剪接分析提示先证者NF1基因发生转录时,11号外显子3'端发生相邻内含子区13个碱基的插入,理论上可导致蛋白编码提前终止,产生截短蛋白,影响蛋白功能。产前基因检测结果显示胎儿未携带该突变。结论: NF1：c.1260+4A>T杂合剪接突变是该Ⅰ型神经纤维瘤患者的致病原因,本研究结果为该家系遗传咨询和产前诊断提供了理论依据。     

elastin基因exon20、24、25的变异与盆底功能障碍性疾病的研究

目的检测elastin基因exon20、24、25在盆底功能障碍性疾病（pelvic floor dysfounction,PFD）女性患者中的突变情况,探讨基因变异与PFD疾病的关系。方法选择本院妇科因PFD住院手术的病人30例为实验组（A组）,同期非PFD患者20例为对照组（B组）;实验组患者按照美国盆腔器官脱垂定量分期法（pelvic organ prolaps quantitation,POP—Q）评分分为轻、重度（A1、A2）两组,均于术前采集静脉血,应用PCR、DNA直接测序法获得elastin基因exon20、24、25的序列,并与NCBIGENEBANK中标准序列比对,观察基因变异情况。结果（1）exon20检测到杂合错义点突变（c．114G｜A）,编码蛋白由甘氨酸变为丝氨酸,A2组与B组、A1组比较差异均有统计学意义（P〈0．05）。（2）exon24检测到杂合错义点突变（c．81C｜G）,编码蛋白由脯氨酸变为丙氨酸。各组之间比较差异均无统计学意义（P〉0．05）。（3）exon25A各组患者均未发现基因突变。（4）20—21内含子检测到杂交突变（c．17T｜c）,A2组与B组、A1组比较差异均有统计学意义（P〈0．05）。（5）24—25内含子检测到杂交突变（C．69A｜T）,各组之间比较差异均无统计学意义（P〉0．05）。结论PFD患者检测到elastin基因exon20、exon24的变异,以elastin基因exon20变异为主,其变异使编码蛋白发生改变,PFD患者发病可能与的elastin基因突变有关。     

中国早发性家族性阿尔茨海默病一家系淀粉样前体蛋白基因突变

目的 研究1个中国早发性家族性阿尔茨海默病(EOFAD)家系的临床表型及存在的基因突变.方法 收集1个EOFAD家系,分析2例患者的临床表现及辅助检查结果 .采集2例患者及其他家庭成员的血样,提取基因组DNA,采用直接测序法进行淀粉样前体蛋白(APP)基因第16、17外显子、早老素1基因和早老素2基因检测.结果 该家系中2例患者发病年龄较早,均缓慢起病,逐渐进展.首发症状为记忆力减退,Ⅱ3有发作性眨眼;Ⅱ5性格改变明显,病程中曾出现尿便障碍.2例患者的头部CT和MRI脑萎缩明显,以双侧颞叶为著.Ⅱ3的脑电图存在异常慢波.2例患者进行MMSE和蒙特利尔认知评估(MoCA)量表测试,提示为中重度智能减退.2例患者APP基因第17外显子中第2343位碱基发生突变(G→A),使编码APP的第715个密码子由GTG变为ATG,导致缬氨酸变为甲硫氨酸,发生V715M突变.Ⅱ1、Ⅱ3、Ⅱ5、Ⅲ1ApoE基因型为ε4/ε4,Ⅱ9 ApoE基因型为e2/ε4.结论 该家系APP基因发生V715M突变,证实在中国人群存在这一突变现象,并且导致发病,提示对可疑阿尔茨海默病患者或家系进行基因检测时不要遗漏该突变位点.

Abstract：

Objective To analyze the phenotype and genatics in a Chinese family with early-onset familial Alzheimer's disease(EOFAD). Methods Peripheral blood were collected in available members in the family and genomic DNA was extracted. PCR-sequencing of exon 16 and exon 17 of the amyloid precursor protein(APP) gene, presenilin 1 (PSEN1), and presenilin 2 (PSEN2) was performed. Results At age 40, two EOFAD patients (siblings) in the family developed an insidious onset of difficulties in memory. One ( Ⅱ3 in the pedigree) showed blinking. The other ( Ⅱ 5 ) showed irritability and bradykinesia.Progressive diffuse coritcal atrophy in bilateral temporal cortex was observed. Moderate diffuse cerebral dysfunction was observed in Ⅱ3 by the electroencephalogram study and neuropsychological assessments.Sequencing revealed that both patients were heterozygous for a mutation c. 2343 G ＞ A in exon 17 of APP,causing the amino acid substitution Val715Met. Four members ( Ⅱ1, Ⅱ 3, Ⅱ 5 and Ⅲ1 ) were homozygous for ApoE ε4 allele. Ⅱ9 was ε2/ε4. Conclusions This study identified a mutation, Val715Met in the APP gene in Chinese patients with EOFAD. We suggest screening for APP gene mutations in Chinese patients with EOFAD.     

载脂蛋白H基因7号外显子多态性与长沙地区汉族人脑卒中的相关性

目的 研究载脂蛋白H(apoH)基因7号外显子G817T(Leu247Val)多态性与长沙地区汉族人脑卒中的关系。方法 应用聚合酶链反应-单链构象多态(PCR-SSCP)技术和DNA序列测定法检测长沙地区汉族260例脑卒中患者(脑梗死组和脑出血组各130例)、20个脑卒中家系的成员(脑梗死组10个家系,142例成员,其中患病组25例;脑出血组10个家系,122例成员,其中患病组21例)、100名健康对照者的apoH基因7号外显子G817T(Leu247Val)多态;酶联免疫吸附法检测所有研究对象的血清抗磷脂抗体(APA)水平。结果 在脑梗死组,G817T(Leu247Val)多态T等位基因频率(0.338)明显高于对照组(0.220,P<0.05)。进一步研究发现,有家族史的脑梗死组T等位基因频率(0.370)明显高于对照组(0.220,P<0.05);APA阳性脑梗死组GT基因型频率明显高于对照组及APA阴性脑梗死组(P<0.01,P<0.05),T等位基因频率(0.439)明显高于对照组(0.220,P<0.01)。以脑梗死为主的家系中患病组与未患病组T等位基因频率(0.404,0.332)均明显高于对照组(0.220,P<0.05),未患病组GT基因型频率明显高于对照组(P<0.05)。apoH基因G817T(Leu247Val)多态性与脑出血无关。结论 长沙地区汉族人apoH基因G817T(Leu247Val)多态基因频率分布具有脑卒中家族聚集性。G817T(Leu247Val)多态GT基因型和T等位基     

单链构象多态性技术在家族性高胆固醇血症患者低密度脂蛋白受体基因13外显子点突变筛查中的应用

目的 探讨单链构象多态性(SSCP)技术在家族性高胆固醇血症患者低密度脂蛋白受体(LDLR)基因13外显子点突变筛查上的应用价值.方法 以16例临床诊断为家族性高胆固醇血症(FH)患者为研究对象,提取外周血DNA,扩增LDLR基因第13号外显子片段,组合最优条件进行SSCP电泳并银染.对异常条带进行DNA序列测定确定其突变性质和位置.结果 优化SSCP电泳条件为:不含甘油8%凝胶,在10℃条件下电泳;含5%甘油的8%凝胶,在常温条件下电泳(交联度均为49:1).凝胶厚度均不超过0.4 mm,电泳电压为5 V/cm,在此条件下进行SSCP电泳均可以得到满意电泳图谱.对发现的异常条带,结合DNA测序证实有4例患者LDLR基因第13外显子分别发生A606T,D601N,Y601D,或G636V错义突变,同时均存在1959碱基C→T同义突变.另外4例患者13外显子仅存在1959碱基C→T同义突变.结论 通过优化各种条件进行的PCR-SSCP银染方法,是高胆固醇血症病LDLR基因13外显子点突变初步筛查的有效手段.