应用长度多态性对非缺失型Hb H病进行产前基因诊断

利用ζ珠蛋白基因的限制性内切酶HindⅢ特异片段长度多态性分析,鉴别了一个非缺失型HbH病患儿及其父母的单体型,并对该家系正在妊娠中的重型HbH危险胎儿进行了早期产前诊断,确定其基因型为(αα/αα~T)。     

应用胎儿特异性抗体HbF（γ链）标记法无创性 产前基因诊断DMD

以孕8~26周孕妇外周血为材料,经过Percoll密度梯度离心初步富集,胎儿细胞特异性抗体—HbF标记、识别胎儿有核红细胞,母体和胎儿有核红细胞的精确区分是以胎儿和成人血红蛋白的组成差异为基础的。胎儿细胞胞浆黄染,而具有成人血红蛋白的母体细胞没有颜色。显微操作法获取全部阳性细胞后,以其全基因组扩增（PEP）的产物为模板,进行性别检测、DMD基因的多重PCR检测和STR连锁分析。结果,20名孕妇外周血中均发现与HbF呈阳性反应的胎儿NRBC。并完成7例DMD的产前基因诊断。HbF抗体标记法能有效识别胎儿有核红细胞,是无创性产前基因诊断中很有应用前景的标记方法。 Abstract： Maternal blood was obtained at 8-26weeks of gestation.After discontinuous density gradient centrifugation with Percoll ,HbF antibody was used to identify fetal NRBC.The precise differentiation between fetal and maternal NRBC is based on the constitutional difference between fetal and adult hemoglobin (Hb).Fetal cells appear yellow cytoplasmic staining,while adult cells colorless. NRBCs were collected by micromanipulation andwhole genome amplification was performed. DMD was prenatally diagnosed by using the combination of sex determination,multiplex PCR and linkage analysis of several STR sites of dystrophin. NRBCs stained with HbF were found in all of 20 maternal blood samples with gestations, and 7 fetuses with risk of DMD were diagnosed. We concluded that HbF antibody could identify fetal NRBC efficaciously,and this is a kind of more prospective application method.     

两个DMD cDNA片段在进行性肌营养不良症基因诊断中的应用

本文使用了缺失热点区的两个DMD cDNA片段1b-2a及8为探针检测Duc-henne型及Becker型肌营养不良(DMD/BMD)患者的基因缺失。在34例不相关患者中分别检测到5例及8例基因片段缺失,缺失检测率分别为14.7％及23.5％,总检出率为38.2％。结果表明,中国肌营养不良患者的基因缺失也不是随机分布的,主要集中于基因中心附近,其次在基因5′侧。     

Real-time PCR技术的应用研究进展

Real-time PCR(RT-PCR)是基于PCR,利用不同的荧光检测定量核酸的技术,广泛应用于基因分型,单核苷酸多态性,等位基因突变检测等方面.综述了RT-PCR作为一种检测技术,在医学、食品、环境微生物等不同领域的应用进展.     

改良的PEP方法在无创性产前基因诊断中的应用

应用显微操作技术获取孕妇外周血中的单个有核红细胞,改良的PEP方法扩增单个有核红细胞的全基因组DNA;在此基础上,应用荧光标记聚合酶链反应扩增9个微卫星片段,进行基因型分析判定单个有核红细胞来源。综合性别和DMD基因内的数个STR位点连锁分析进行DMD基因诊断,应用PCR-STR连锁分析进行PKU基因诊断。结果显示,对10例DMD高危胎儿中的6例成功地进行了无创性产前基因诊断。同时对1例PKU也成功地进行了无创性产前基因诊断。改良的PEP方法扩增单个细胞的全基因组可以满足基因诊断的要求,是无创性产前基因诊断中一种极有价值的全基因组扩增的方法。 Abstract：We investigated the feasibility of using improved primer extension preamplificat ion method to diagnose DMD and PKU. The fetal nucleated red blood cells from the peripheral blood of pregnant women were detected and individually retrieved into glass capillary pipettes using a micromanipulator under microscopic observation. The whole genome of a single cell was amplified by improved primer extension preamplification (PEP).Genotypes were analyzed by amplifying the 9 STR fragments using fluorescence?PCR technique and NRBC's(nucleated red blood cell) origin w as determined.We diagnosed DMD prenatally using sex determination and linkage an alysis of several STR sites of dystrophin,and we diagnosed PKU prenatally using PCR?STR linkage analysis.6 of 10 potential DMD patients were diagnosed,includin g 1 male fetal patient,1 potential PKU patient was also diagnosed.The improved P EP method is a very valuable method of amplifying the whole genome of single cel ls,and the products of amplification are enough to the requirements of DNA in no n-invasive prenatal diagnosis.     

基因诊断中测序技术的应用及优缺点

基因突变是当今生命科学研究的热点之一,其检测方法和诊断技术发展迅猛。测序技术在基因病的确诊、分型等方面发挥着不可或缺的作用。文章重点围绕第一~三代测序技术的研究进展、优缺点及其在基因诊断中的应用进行评述 ,并对未来的发展趋势进行了预测和展望。     

软骨发育不全（侏儒症）能产前基因诊断吗？

问：我是一名侏儒症患者,在医院被诊断为软骨发育不全,无家族史,兄弟姐妹无同样的病,我现在28岁,已怀孕3个月,想咨询是否可以通过基因诊断来判断我的胎儿是否有病？ 答：软骨发育不全是侏儒症中最常见的类型。患者主要表现为头颅巨大、前额突出、身材矮小、短肢畸形及与躯干不成比例、三又手畸形和典型的X线放射学特征。     

HCVRNA反转录PCR技术的建立及对血液样品的检测

实验建立了ＨＣＶ　ＲＮＡ的反转录和套式ＰＣＲ技术,扩增出２３２ｂｐ的核酸片段,经酶切电泳图谱和Ｓｏｕｔｈｅｒｎ杂交鉴定,来自ＨＣＶ基因５,端非编码区。实验从抗ＨＣＶ阳性的１８例血浆样品和１９例血清样品中分别检出７例和１３例ＨＣＶＲＮＡ阳性。     

肽核酸与核酸分子杂交的模式及其在基因诊断领域中的应用

在发明肽核酸(peptide nucleic acid,PNA)后的短短十年间,由于PNA分子独特的生物学性能,使之可以与DNA及RNA分子通过不同的机制形成稳定的复合物,导致PNA在基因诊断领域有得天独厚的优势。本文综述了PNA与核酸分子杂交的几种不同模式以及最近几年PNA在基因诊断领域的一些最新进展。     

缺失型Duchenne/Becker 肌营养不良家系女性携带者及新发突变的确认

应用多重PCR（multiple polymerase reaction／mPCR）技术,联合DMD基因内部及附近11个短串连重复序列（short tandem repeats,STRs）位点连锁分析,对缺失型Duchenne／Beeker肌营养不良（Duchenne／Becker Muscular Dystrophy,DMD／BMD）家系成员进行DMD基因分型,确定家系中女性成员是否携带者,并进行产前诊断。3个家系中的4名缺失型患者,其中2例为新发突变：4位女性成员中,1名为携带者。应用mPCR和11个STRs的连锁分析,能快速、准确、客观判断家系中女性成员是否携带者身份,适于DMD／BMD临床研究机构遗传咨询、基因诊断和产前诊断常规应用。但在mPCR分析过程中,发现45号外显子扩增产物在不同凝胶中电泳迁移率不同。聚丙烯酰胺凝胶电泳（Polyacrylamide Gels Electrophoresis／PAGE）对mPCR产物分析快速、清晰,但需要注意片段迁移率,以防止分析错误。     

脆性X综合征的基因诊断与产前诊断

为了探讨简便、快速、准确、价廉的脆性X综合征的诊断方法,对6个智能低下家系进行了细胞遗传学检查,以及PCR直接扩增FMR1 5^'端(CGG)_{n<\sub>重复序列、RT-PCR扩增FMR1基因的cDNA序列的分子遗传学检查。A家系先证者脆性X染色体高表达（35/273）,分子遗传学检查证实为脆性X综合征全突变患者;B家系先证者及其母亲无脆性X染色体表达,分子遗传学检查证实为非脆性X综合征患者;C家系的男性胎儿脆性X染色体表达（5/93）,先证者及其母亲未发现脆性X染色体,分子遗传学检查证实男性胎儿为脆性X综合征全突变患者,其母亲为前突变携带者,哥哥为嵌合体患者;D家系先证者脆性X染色体高表达17%,其姐姐脆性X染色体5%,分子遗传学检查证实先证者为脆性X综合征全突变患者,其姐姐为嵌合体患者;E家系先证者及其母亲,F家系先证者发现可疑脆性X染色体,分子遗传学检查证实为非脆性X综合征家系。结论： PCR直接扩增FMR1基因(CGG)n<\sub>重复序列联合RT-PCR扩增FMR1基因cDNA 序列简便、快速、价廉。可用于脆性X综合征的筛查、诊断及产前诊断,有推广应用价值。}     

Genetic Modifiers of Duchenne Muscular Dystrophy and Dilated Cardiomyopathy

Objective

Dilated cardiomyopathy (DCM) is a major complication and leading cause of death in Duchenne muscular dystrophy (DMD). DCM onset is variable, suggesting modifier effects of genetic or environmental factors. We aimed to determine if polymorphisms previously associated with age at loss of independent ambulation (LoA) in DMD (rs28357094 in the SPP1 promoter, rs10880 and the VTTT/IAAM haplotype in LTBP4) also modify DCM onset.

Methods

A multicentric cohort of 178 DMD patients was genotyped by TaqMan assays. We performed a time-to-event analysis of DCM onset, with age as time variable, and finding of left ventricular ejection fraction < 50% and/or end diastolic volume > 70 mL/m² as event (confirmed by a previous normal exam < 12 months prior); DCM-free patients were censored at the age of last echocardiographic follow-up.

Results

Patients were followed up to an average age of 15.9 ± 6.7 years. Seventy-one/178 patients developed DCM, and median age at onset was 20.0 years. Glucocorticoid corticosteroid treatment (n = 88 untreated; n = 75 treated; n = 15 unknown) did not have a significant independent effect on DCM onset. Cardiological medications were not administered before DCM onset in this population. We observed trends towards a protective effect of the dominant G allele at SPP1 rs28357094 and recessive T allele at LTBP4 rs10880, which was statistically significant in steroid-treated patients for LTBP4 rs10880 (< 50% T/T patients developing DCM during follow-up [n = 13]; median DCM onset 17.6 years for C/C-C/T, log-rank p = 0.027).

Conclusions

We report a putative protective effect of DMD genetic modifiers on the development of cardiac complications, that might aid in risk stratification if confirmed in independent cohorts.     

羊水直接PCR法快速诊断脊肌萎缩症的实验研究

目的：建立一种快速、简便的产前诊断脊肌萎缩症的检测方法。方法：基于运动神经元生存基因SMNc和SMNt的两个同源拷贝碱基的差异,对SMNt基因第7、8外显子进行缺失分析,抽取孕妇羊水后用作者自行研制的“HpH—Buffer”,以离心后羊水沉淀中的胎儿细胞为模板直接进行PCR扩增,扩增产物进行酶切限制性片断长度多态性分析,对2例有脊肌萎缩症患儿生育史的孕妇怀有的胎儿进行产前基因诊断。同时,为考察羊水直接扩增方法的效率,对羊水样本直接扩增和对羊水样本中提取的基因组DNA扩增进行了平行实验。结果：2例胎儿均有SMNt基因第7、8外显子缺失;以羊水为模板和以基因组DNA为模板进行扩增的效率相似。结论：应用“HpHBuffer”直接对羊水样本中SMN基因上外显子7、8进行扩增,结合限制性片断长度多态性分析SMNt上是否发生缺失,可缩短诊断时间,节约诊断成本,减少检测过程中可能出现的样本交叉污染,有望成为临床上产前诊断脊肌萎缩症的新方法。     

Mutation-independent Proteomic Signatures of Pathological Progression in Murine Models of Duchenne Muscular Dystrophy

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Highlights

• •Proteomics analysis was performed in two murine models of Duchenne muscular dystrophy (mdx and mdx52) at three ages (8, 16 and 80 weeks) and compared with wild-type controls.
• •High-resolution isoelectric focusing liquid chromatography-tandem mass spectrometry enabled the quantification of 4974 proteins in all samples.
• •This study has revealed protein signatures of dystrophin deficiency and the progression of dystrophic pathology.
• •In contrast, the proteomes of the mdx and mdx52 mice were highly similar.
• •Pathway analysis revealed crosstalk between inflammatory, metabolic and muscle growth processes in dystrophic muscle.