一例Crigler-Najjar综合征Ⅱ型患儿的基因突变分析北大核心CSCD

目的对1例Crigler-Najjar综合征Ⅱ型患儿的尿苷二磷酸葡萄糖醛酸转移酶1A1（UGT1A1）基因进行突变分析,探讨其分子发病机制,为家系的遗传咨询及产前基因诊断提供依据。方法抽取患儿及其父母的外周静脉血提取基因组DNA,应用聚合酶链反应扩增UGT1A1基因的相应片段,经DNA测序法对UGT1A1基因进行突变检测,并随访患儿至3岁6个月。结果患儿为持续高未结合胆红素血症,DNA测序结果显示患儿UGT1A1基因的第1外显子存在c．610A〉G（p．Met 204 Val）杂合突变,第4外显子存在c．1091C〉T（p．Pro 364 Leu）杂合错义突变,患儿为UGT1A1基因复合突变杂合子。患儿父亲UGT1A1基因的第4外显子存在C．1091C〉T（p．Pro 364 Leu）的杂合错义突变,患儿母亲UGT1A1基因的第1外显子存在c．610A〉G（p．Met204 Val）杂合错义突变。结论UGT1A1基因第1外显子c．610A〉G、第4外显子c．1091C〉A复合杂合突变为该患儿的发病原因,为其家系的遗传咨询和产前基因诊断提供了依据。     

遗传性非综合征型耳聋患者GJB2基因编码序列分析

目的 对6个遗传性非综合征型耳聋家系成员的GJB2基因编码序列进行分析,寻找耳聋患者的致病基因突变,探讨GJB2基因突变致病的遗传模式.方法 提取患者及家系成员的外周血基因组DNA,扩增GJB2基因的编码序列,然后对扩增产物进行DNA测序,对出现重叠峰形的扩增产物进行TA克隆后再测序,确定基因突变是否存在于同一拷贝.结果 6个遗传性非综合征型耳聋家系中,4个家系是GJB2基因突变所致.患者的GJB2基因突变包括235delC、299-300delAT、79G→A+341A→G和109G→A.非致聋突变79G→A与341A→G组合具有致聋效应,109G→A和235delC的杂合突变可能也有致聋效应.结论 GJB2基因突变致聋具有明显异质性,非致聋突变并非完全不致聋,环境因素或其它基因可能参与GJB2基因突变所致耳聋.     

128例Ⅱ型糖尿病患者线粒体ND1基因突变位点的研究

目的通过对128例Ⅱ型糖尿病(Type 2 diabetes mellitus,T2DM)患者线粒体DNA ND1基因进行突变位点筛查,探索ND1基因点突变与山西人群T2DM的相关性。方法 PCR扩增患者ND1基因所在区段,PCR产物直接测序分析。结果128例患者共有38例患者检测到基因点突变,突变检出率为29.7%。38例患者共筛出22个突变位点,2种突变类型。其中31例存在单个位点突变,5例存在2个位点突变,2例存在3个位点突变。22个突变位点中,3552(T→A)突变频率最高,为40.9%(9/22);3394(T→C)、3435(C→T)、3497(C→T)、3316(G→A)、3571(C→T)、3537(A→G)的突变频率分别为22.7%(5/22)、22.7%(5/22)、18.2%(4/22)、13.6%(3/22)、13.6%(3/22)和10.1%(2/22);其余突变位点的突变率均为4.5%(1/22)。在所有突变位点中,除3688(G→C)为异质性突变外,其余突变均为同质性。此外,22个突变位点中存在一个新的突变位点3499(A→T),属首次报道。结论 3552(T→A)和3394(T→C)突变频率最高,可能与山西地区T2DM发病相关;新发现的突变位点3499A→T(Thr→Ser),其致病性需要进一步研究。     

EXT1基因1633-26(C→A)突变可能致多发性外生性骨疣

目的研究1例散发多发性外生性骨疣患者的基因突变情况，确定其致病基因。方法采用聚合酶链反应结合DNA直接测序法检测EXT1以及EXT2基因的突变热点区域；并应用错配引物PCR扩增引入酶切位点结合限制性片段长度多态性方法检测和鉴定突变。结果经测序证实在患者EXT1基因的第7内含子3’剪接位点上游26bp处发现一杂合突变，此杂合突变不存在于其表型正常的父母双亲中，是一个新生突变；错配引物扩增与限制性片段长度多态性分析结果表明在150名家系外正常对照者中没有此突变。结论EXT1基因1633-26（C→A）突变可能是导致这个患者发生多发性外生性骨疣的致病突变。     

一个琥珀酸半醛脱氢酶缺陷病家系ALDH5A1基因突变分析

目的 对1例临床怀疑且经尿筛查诊断为琥珀酸半醛脱氢酶缺陷病的患儿及家系进行ALDH5A1基因突变分析,以进一步明确诊断和辅助遗传咨询.方法 应用聚合酶链反应及DNA直接测序技术对1例琥珀酸半醛脱氢酶缺陷病患儿及其父母的ALDH5A1基因进行突变位点检测,同时检测100名健康对照者的ALDH5A1基因以排除多态性变异.结果 患儿携带有ALDH5A1基因编码区序列第3外显子c.527G＞A(p.Gly176Glu)和第4外显子c.691G＞A(p.Glu231Lys)两种杂合突变,其母亲为c.527G＞A杂合突变携带者,父亲为c.691G＞A杂合突变携带者.另外患儿还存在两种已报道的核酸多态性改变:c.545C＞T杂合突变和c.538C＞T纯合突变.患儿的c.545C＞T突变来源于父亲,c.538C＞T突变分别来源于父母.100名健康对照者中未检测到c.527G＞A和c.691G＞A突变.结论 c.527G＞A(p.Gly176Glu)和c.691G＞A(p.Glu231 Lys)错义突变可能是该患儿的致病突变.     

四川攀枝花地区非综合征性耳聋患者常见耳聋基因突变分析

目的分析四川攀枝花地区非综合性耳聋患者常见耳聋基因突变,初步了解该地区耳聋患者发病的分子机制。方法收集攀枝花地区74例非综合性耳聋患者,用遗传性耳聋基因芯片对4个常见耳聋相关基因(GJB2、SLC26A4、线粒体12Sr RNA及GJB3基因)的9个位点进行检测。结果 74例耳聋患者中共检出22例带有耳聋基因突变,检出阳性率为29.73%,其中GJB2基因235del C纯合突变8例,杂合突变7例,299del AT杂合突变1例,176del16纯合突变1例,SLC26A4基因IVS7-2AG纯合突变3例,杂合突变2例。结论攀枝花地区非综合性耳聋患者耳聋基因以GJB2基因235del C突变、SLC26A4基因IVS7-2AG突变为主,对该地区耳聋患者及高危人群进行耳聋基因突变的筛查,采用正确的康复干预措施,可有效减少该地区耳聋的发生。     

四例非综合征型白化病家系的产前基因检测

目的探讨非综合征型白化病产前基因诊断的临床价值和意义。方法本研究应用Sanger测序法分别对四个非综合征型白化病家系进行常见致病基因检测。结果家系1患者在OCA2基因发现致病突变位点c.1441AG和c.727CT,胎儿与患者检测结果一致。家系2患者在SLC45A2基因存在c.478GC纯合突变,胎儿发现该位点杂合突变,为携带者。家系3患者在TYR基因存在致病突变位点c.655GA和c.929ins C,胎儿发现c.655GA杂合突变,为携带者。家系4患者在TYR基因上存在突变位点c.346CT和c.929ins C,但胎儿在TYR基因上未见异常。结论明确患者的致病基因可明显缩短胎儿产前基因诊断的时间,为白化病家庭提供积极且准确的优生指导。     

糖原累积病I b型1例的临床和分子遗传分析

目的 初步探讨1例糖原累积病I b型(GSD I b)患者的临床特点和致病基因,分析该疾病发生的分子遗传机制.方法 收集患者临床资料,抽提患者外周血白细胞基因组DNA,通过多聚酶链反应扩增葡萄糖-6-磷酸酶转位酶基因SLC37A4的9个外显子,用DNA直接测序法确定其突变位点.结果 患者临床表现及实验室检查完全符合GSD I b.经PCR测序发现SLC37A4基因第3外显子572位碱基C→T纯合突变(c.572 C>T),造成第191位的脯氨酸被亮氨酸替代(P.P191L),导致葡萄糖-6-磷酸转位酶活性下降.结论 SLC37A4基因突变导致的葡萄糖-6-磷酸转位酶结构改变是该GSD I b患者临床表现的分子遗传基础.P191L纯合突变在中国大陆的报道尚属首次.相信不久DNA突变分析将会成为糖原累积病I b型的主要确诊方法.     

一例巨颅伴皮层下海绵样囊肿性脑白质病患儿的MLC1基因突变分析北大核心CSCD

目的探讨一例巨颅伴皮层下海绵样囊肿性脑白质病（megalencephalic leukoencephalopathy with subcorti calcysts,MLC）患儿临床特征与MLC1基因突变情况,为其家庭提供遗传咨询。方法收集患儿及其父母临床资料并提取外周血DNA,对MLC1基因编码区及侧翼序列进行聚合酶链反应扩增后测序。结果患儿生后出现头围异常增大,运动认知落后,头颅MRI示双侧大脑半球白质弥漫异常信号,测序发现MLC1第2外显子c．177-c180delC纯合型缺失突变。患儿父母为相同位点杂合型突变、表型正常携带者。结论该MLC患儿存在MLC1基因纯合型突变,突变基因来源于父母。     

3家系成骨不全基因突变及临床表型分析

目的对3家系成骨不全(OI)测序及其临床表型和突变分析,提高对成骨不全的诊断和认识。方法收集临床资料,提取患者及家属血标本;高通量测序,一代测序验证和结果分析。结果先证者1,FKBP10,EXON6,c.1016GA,p.R339Q,纯合突变,父、母分别为此位点杂合突变。先证者2,COL1A1,EXON9,c.671GA,p.G224D,杂合突变,父亲为此位点杂合突变,母亲基因无变异。先证者3,COL1A1,EXON30,c.2010del T,p.P670fs,杂合突变,母亲此位点杂合突变,父亲基因无突变。结论 FKBP10新位点突变可能导致了XI型成骨不全及其新表型的发生;证实了COL1A1两位点突变和成骨不全之间的关系。     

HLA-DRB1, -DQA1, -DQB1, -DPA1 and -DPB1 genes in Japanese multiple sclerosis patients

Japanese MS patients and controls were examined for the distribution of HLA-DRB1, -DQA1, -DQB1, -DPA1 and -DPB1 alleles using in vitro amplification of genomic DNA and probing with sequence-specific oligonucleotides. No significant difference in frequency of the examined alleles was observed among the two groups. This is in contrast to Norwegian MS patients, where an association to a combination of certain DQA1 and DQB1 alleles has previously been demonstrated.     

NDE1 and NDEL1: Multimerisation,alternate splicing and DISC1 interaction

Nuclear Distribution Factor E Homolog 1 (NDE1) and NDE-Like 1 (NDEL1) are highly homologous mammalian proteins. However, whereas NDEL1 is well studied, there is remarkably little known about NDE1. We demonstrate the presence of multiple isoforms of both NDE1 and NDEL1 in the brain, showing that NDE1 binds directly to multiple isoforms of Disrupted in Schizophrenia 1 (DISC1), and to itself. We also show that NDE1 can complex with NDEL1. Together these results predict a high degree of complexity of DISC1-mediated regulation of neuronal activity.     

韩国人CYP450 1A1、CYP450 2E1和GSTM1、GSTT1、GSTP1基因座遗传多态性分析

目的 调查代谢相关的CYP4501A1、CYP4502E1和GSTM1、GSIT1、GSTP1基因座在韩国人群中的遗传多态性分布状况。方法 采用多重聚合酶链式反应、聚合酶链式反应-限制性片段长度多态性技术，分析300名韩国健康大学生的CYP1A1基因3′端限制性内切酶Msp Ⅰ位点、CYP2E1基因5′端转录调节区Pst Ⅰ位点和GSTM1、GSTT1缺失与存在、GSTP1基因第5外显子BsmA Ⅰ位点的基因型，计算基因型和基因频率。结果 CYP1A1基因型频率为ml／ml型39．7％、ml／m2型49．7％、m2／m2型10．7％，基因频率为ml 0．645、m2 0．355。CYP2E1基因型频率为cl／cl型66．7％、cl／c2型30％、c2／c2型3．3％，基因频率为C1 0．818、C2 0．182。GSTM1基因缺失型频率为53．3％。GSTT1基因缺失型频率为54．7％。GSTP1基因型频率为Ile／Ile型62％、Ile／Val型34．3％、VaL／Val型3．7％，基因频率为Ile 0．792、Val 0．208。基因分布符合Hardy-Weirtberg平衡定律。结论 韩国人CYP1A1、CYP2E1、GSTM1、GSTT1基因分布与我国人群较为相近，半数以上人缺乏GSTM1和GSTT1基因，纯合缺失型频率超过印度人的3倍。     

Maternal genetic polymorphisms in CYP1A1, GSTM1 and GSTT1 and the risk of hypospadias

Hypospadias is one of the most common congenital anomalies. Increased exposure to environmental factors (endocrine-disrupting chemicals and smoking) or maternal endogenous estrogen may cause hypospadias because male sexual differentiation is dependent on normal androgen homeostasis. Moreover, interactions between genetic factors and cigarette smoking and other chemicals have been suggested. It has been demonstrated that the CYP1A1 metabolizes not only environmental chemicals but also estrogens, and glutathione-S-transferases (GSTs) are detoxification enzymes that protect cells from toxicants by conjugation with glutathione. In this study, to investigate the association of CYP1A1 (MspI), GSTM1 and GSTT1 polymorphisms with hypospadias, a case-control study of 31 case mothers who had boys with hypospadias and 64 control mothers was performed in Japan. These polymorphisms were investigated by PCR-based methods using DNA from peripheral lymphocytes. We found that the heterozygous CYP1A1 and heterozygous and homozygous CYP1A1 were less frequent in the case mothers than in the control mothers [adjusted odds ratio (OR)=0.17, 95% confidence interval (CI)=0.04-0.74, OR = 0.28, 95% CI = 0.08-0.97, respectively]. We found no effect of maternal smoking on the hypospadias risks among the gene polymorphisms. The results suggest that mothers with the CYP1A1 MspI variant allele may have a decreased risk for hypospadias.     

Circulatinglong non-coding RNA FEZF1-AS1 and AFAP1-AS1 serve as potential diagnostic biomarkers for gastric cancer

BackgroundGrowing evidence indicates that two long non-coding RNAs (lncRNAs), FEZ family zinc finger 1 antisense RNA 1 (FEZF1-AS1) and Actin filament associated protein 1 antisenseRNA1 (AFAP1-AS1), are highly expressed in different cancers, including gastric cancer (GC). However, the expression pattern and clinical utility of these two lncRNAs are still unknown.MethodsSerum expression levels of FEZF1-AS1 andAFAP1-AS1 were measured by quantitative real-time polymerase chain reaction (qRT-PCR). CEA and CA19-9 were detected by ARCHITET I2000 SR. Analyses were all performed using SPSS software version 20.0 (SPSS Inc., Chicago, USA). P < 0.05 was considered statistically significant.ResultsDetection of serum FEZF1-AS1 and AFAP1-AS1 showed both of them were up-regulated in GC patients compared with the normal controls (p < 0.0001), and high serum expression levels were correlated with tumor size, tumor-node-metastasis (TNM) stage and lymph node metastasis. Besides, the area under the ROC curve (AUC) demonstrated the two lncRNAs had higher diagnostic utility than CEA and CA19-9. Furthermore, when combined the two lncRNAs as a model, it yielded an AUC of 0.866, and the combination of the model, CEA and CA19-9 could observably improve diagnostic sensitivity to 95.5 %. What’s more, circulating FEZF1-AS1 and AFAP1-AS1 were significantly decreased after the GC patients underwent the operation (both p < 0.001).ConclusionOur study indicated that serum FEZF1-AS1 and AFAP1-AS1 had better sensitivity and efficiency for the diagnosis of GC and the combination of the two lncRNAs might be used as a potential prognostic indicator in GC.     

Rb1cc1 is critical for myoblast differentiation through Rb1 regulation

Rb1-inducible coiled-coil 1 (Rb1cc1) expressed at high levels is associated with the maturation of human embryonic musculoskeletal cells. To clarify the molecular role of Rb1cc1 in muscular differentiation, we investigated the expression of Rb1cc1 and other genes that regulate differentiation in murine embryonic tissues and in C2C12 myoblasts. We also evaluated the effects of RNA interference (RNAi)-mediated Rb1cc1 knockdown on C2C12 myoblast differentiation. After Rb1cc1, Rb1 and myosin heavy chain (Myhc) were expressed in mouse embryonic muscles. The synchronous expression of Rb1cc1 and Rb1 predicted Myhc expression during C2C12 myoblast differentiation. RNAi-mediated knockdown of Rb1cc1 led to Rb1 suppression, and C2C12 myoblasts failed to differentiate. These results indicated that Rb1cc1 is a potent regulator of the Rb1 pathway and a novel mediator that plays a crucial role in muscular differentiation. Rb1cc1 expression is, thus, a prerequisite for myogenic differentiation.     

HIV-1 Nef stabilizes AP-1 on membranes without inducing ARF1-independent de novo attachment

HIV-1 Nef affects the trafficking of numerous cellular proteins to optimize viral replication and evade host defenses. The adaptor protein (AP) complexes, which form part of the cytoplasmic coat of endosomal vesicles, are key cellular co-factors for Nef. Nef binds these complexes and alters their physiologic cycle of attachment and release from membranes. Specifically, while AP-1 normally becomes cytosolic when attachment events are blocked by inhibition of the GTPase cycle of ADP-ribosylation factor-1 (ARF1), the complex remains membrane-associated in Nef-expressing cells. To investigate the mechanism of this effect, we used a permeabilized cell system to detect the de novo attachment of exogenous AP-1 to endosomal membranes. Nef did not mediate de novo attachment independently of ARF1, despite its ability to maintain the association of AP-1 with endosomal membranes when the activity of ARF1 was blocked. We conclude that Nef stabilizes AP complexes on endosomal membranes after ARF1-dependent attachment. This stabilization may facilitate coat formation and stimulate the trafficking of multiple cellular proteins.     

神经系统RNA结合蛋白Musashi1 RRM1的初步结晶

目的 对Musashi1发挥功能的 RRM1结构域进行结晶,得到可用来衍射的蛋白晶体,为之后的结构解析打基础。方法 通过构建Musashi1RRM1的原核表达载体,并在BL21中表达、纯化高纯度的蛋白质,通过筛选结晶体条件得到蛋白晶体。结果 通过系统筛选和优化晶体生长条件得到了蛋白晶体。结论 Musashi1 RRM1的蛋白晶体质量较好,满足蛋白晶体衍射和数据收集的要求。     

IL—1受体相关激酶—1和—2协同调控IL—1诱导的AP—1的活化

目的研究白介素 - 1受体相关激酶 - 1(IRAK- 1)和 IRAK- 2在白介素 - 1(IL - 1)诱导 AP- 1活化中的作用。方法L ipofectin介导反义 IRAK- 1寡核苷酸和反义 IRAK- 2寡核苷酸转染 Hep G2细胞。用逆转录 PCR法检测 IRAK - 1和 IRAK- 2m RNA表达水平 ;Western blot分析 IRAK- 1和 IRAK - 2蛋白表达水平。以 Sandwich EL ISA法检测 AP- 1的活化。结果反义IRAK- 1寡核苷酸和反义 IRAK- 2寡核苷酸通过抑制各自靶基因 m RNA和蛋白表达抑制 IL- 1诱导的 AP- 1活化 ;反义 IRAK-1寡核苷酸与反义 IRAK- 2寡核苷酸共转染 Hep G2细胞对 AP- 1的抑制作用较两者单独转染明显增强。结论 IRAK- 1和 I-RAK- 2在调控白介素 - 1诱导的 AP- 1活化时协同作用。     

Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not involved in the risk of recurrent pregnancy loss

The etiology of recurrent pregnancy loss (RPL) remains unclear, but it may be related to a possible genetic predisposition together with involvement of environmental factors. We examined the relation between RPL and polymorphisms in four genes, human aryl hydrocarbon (Ah) receptor, cytochrome P450 (CYP) 1A1, CYP1A2 and CYP1B1, which are involved in the metabolism of a wide range of environmental toxins and carcinogens. All cases and controls were women resident in Sapporo, Japan and the surrounding area. The Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotypes were assessed in 113 Japanese women with recurrent pregnancy loss (RPL) and 203 ethnically matched women experiencing at least one live birth and no spontaneous abortion (control). No significant differences in Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotype frequencies were found between the women with RPL and the controls [Ah receptor: Arg/Arg (reference); Arg/Lys and Lys/Lys, odds ratio (OR)=0.67; 95% confidence interval (CI)=0.40-1.11, CYP1A1: m1m1 (reference); m1m2 and m2m2, OR = 0.86; 95% CI = 0.53-1.40, CYP1A2: C/C and C/A (reference); A/A, OR = 1.16; 95% CI = 0.71-1.88, CYP1B1: Leu/Leu (reference); Leu/Val and Val/Val, OR = 1.18; 95% CI = 0.68-2.02]. The present study suggests that the Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not major genetic regulators in RPL.