中国人黏多糖贮积症ⅣA型家系GALNS基因新突变的鉴定

目的 研究黏多糖贮积症ⅣA型(mucopolysaccharidosis type ⅣA,MPS ⅣA)患者发病的分子遗传学机制,揭示其基因型与表现型的相互关系,为产前基因诊断等创造必要的前提条件.方法 采用尿糖胺聚糖(glycosaminoglycans,GAGs)定性检测法对疑似MPS ⅣA型的先证者进行初诊,然后采用PCR及扩增产物直接测序法对先证者及其家庭成员进行突变检测.在检出GALNS基因c.1567T＞G新突变后,先后建立XspⅠ酶切鉴定法和扩增阻碍突变系统(amplification refractory mutation system,ARMS)快速特异鉴定法,对随机采集的110名正常对照与先证者及其家庭成员的GALNS基因第14外显子进行序列分析,同时采用生物信息学方法对蛋白质二级、三级结构进行预测,以及直接测定患儿GALNS酶活性的方法,对该新突变进行致病性鉴定.结果 先证者尿检呈弱阳性GAGs(±),其GALNS基因第14外显子内存在杂合的c.1567T＞G终止密码突变,第4外显子存在杂合的c.374C＞T错义突变,为两种突变的复合杂合子.其妹突变类型与先证者完全相同,其母仅在第14外显子存在杂合的终止密码突变,为该病的携带者,其父仅在第4外显子存在杂合的错义突变,也为杂合子;第14外显子的PCR产物经XspⅠ酶切后,正常对照组切出28 bp、120 bp、399 bp 3条带,而患者和携带者的母亲均切出28 bp、120 bp、148 bp和399 bp 4条带;用ARMS特异引物扩增后,正常对照组均扩增阴性,而患者及携带者均扩增阳性;蛋白质二级、三级结构预测结果显示:c.1567T＞G变异导致终止密码(TAG)突变为谷氨酸(GAG),使多肽链延长了92个氨基酸残基,导致蛋白质二、三级结构发生明显改变,而正常对照无此变化.酶活性测定的结果显示:患者的GALNS酶活性仅为8.3 nmol/17 h/mg pr,明显低于正常值(正常参考值为41.9～92.1 nmol/17h/mg pr).结论 c.1567T＞G变异是一种新的致病性突变,是引起该家系患儿发病的根本原因.

Abstract：

Objective To study the molecular genetic mechanism of mucopolysaccharidosis type ⅣA(MPS ⅣA), and reveal the relationship between the genotype and phenotype, and provide a basis for prenatal gene diagnosis in the future. Methods A preliminary diagnosis was made by qualitative detection of urinary glycosaminoglycans of the suspected MPS ⅣA proband. Then, mutation detection was performed on the proband and her family members with PCR and direct sequencing of the PCR products. After a novel c.1567T＞G mutation was detected,XspⅠrestriction enzyme digestion and amplification refractory mutation system(ARMS) fast specific identification were established to analyze the sequences of exon 14 in GALNS gene, including 110 randomly selected healthy controls, the proband and other pedigree members. At the same time, bioinformatic approaches for protein secondary, tertiary structure prediction were applied to identify the novel pathologic mutation. Results The proband's urine GAGs test was a weak positive(±),and a c.1567T＞G heterozygous termination codon mutation in exon 14 and a c.374C＞T heterozygous missense mutation in exon 4 were found. The proband was compound heterozygous of the two mutations, so was her younger sister. Her mother was a carrier with only a c.1567T＞G heterozygous mutation in exon 14. Her father had a heterozygous mutation of c.374C>T in exon 4. After XspⅠrestriction enzyme digestion, healthy controls had three bands including 28 bp, 120 bp and 399 bp, while the proband and her mother had four bands consisting of 28 bp, 120 bp,148 bp and 399 bp. For amplification by ARMS specific primers, it was negative for the controls,while it was positive for the proband and the carrier. The results of protein secondary and tertiary structure prediction showed that the c.1567T＞G mutation located in the stop codon, resulted in stop codon (TAG) changing to glutamic acid (GAG), with the peptide chain extending 92 amino acid residues, and secondary and tertiary protein structure change, which were not found in the controls. The result of enzyme assay showed that the activity of GALNS enzyme in the affected child was 8.3 nmol/17h/mg pr, which was obviously lower than the normal value (the normal range is 41.9-92.1 nmol/17h/mg pr). Conclusion These results illustrate that the c.1567 T＞G is a novel pathologic mutation, which is the main cause of the disease in this family.     

双错配碱基ARMS结合RE法快速检测FGFR3基因的突变超热点

目的建立快速简便、特异灵敏的鉴定FGFR3基因c．1138G〉A突变超热点的基因诊断方法,为软骨发育不全（ACH）的产前基因诊断或植入前遗传学诊断（PGD）创造条件。方法针对突变率高达95％以上的FGFR3基因的突变热点c．1138G〉A,设计双错配碱基的ARMS特异引物,对已经临床确诊的先证者家系及正常对照和非c．1138G〉A突变的患者对照,分别用普通引物和特异引物进行PCR扩增和直接鉴定,然后对扩增产物进行DNA序列分析及sfcI酶切鉴定。结果用普通引物扩增,对照组和先证者均扩增阳性。sfcI酶切后,对照组仍为一条513bp的带,而患者切出205bp、308bp和513bp三条带;而用ARMS特异引物扩增,则先证者扩增阳性,而对照组扩增阴性,阳性者酶切后产生27bp和418bp两条带。这一切都与预期的结果完全吻合。结论该法快速、特异、准确性高,结合DNA序列分析和酶切鉴定（RE）,可用于ACH高危胎儿的快速产前诊断。     

单基因遗传病基因诊断技术研究进展

单基因遗传病(简称单基因病)种类、分型繁多,常规诊断难以确诊,而基因诊断技术在遗传病特别是单基因病的确诊、分型等方面都发挥着不可或缺的作用.近一、二十年来,基因诊断技术进展迅猛,各种检测方法层出不穷.本文重点围绕基因诊断技术的最新进展进行综述,以期对临床诊断和预防工作提供一些有益的启示.     

Maroteaux—Lamy综合征基因治疗的研究与应用

Maroteaux-Lamy综合征即粘多糖贮积症Ⅵ型（MPSⅥ），是由于溶酶体内N－乙酰半乳糖胺－4－硫酸酯酶（4S；E.C.3.1.6.1)缺乏而引起的一种常染色体隐性遗传病（AR）。本综述了近年来该病分子遗传学研究的最新成果，尤其是在基因治疗方面取得的进展。     

广东汉族群体粘多糖贮积症Ⅰ型KpnⅠ位点的PFLP

本文对８５例无亲缘关系的广东汉人的１７０例染色体进行ＩＤＵＡ基因ＫｐｎⅠ位点的ＲＦＬＰ研究，结果显示：等位基因Ａ１频率为０．１８，等位基因Ａ２频率为０．８２，杂合率为３２％。经ｘ＾２检验，证实这些结果与国外报宾无显著性差异。对３个家系１０位成员等位基因传递规律的研究结果表明：Ａ１、Ａ２在世代中的传递完全符合孟德尔遗传规律。     

遗传性侏儒及其鉴别诊断

遗传性侏儒常见的有软骨发育不全(achondrop lasia,ACH,M IM 100800)、软骨发育低下(hypochondrop lasia,HCH,M IM 146000)、致死性侏儒(thanatophoric dysp lasia,TD,M IM187600)、假性软骨发育不全(pseudoachondrop lasia,PSACH,M IM 177170)和多发性骨骺发育不全(mu ltip le     

Flash MX制作"线粒体遗传病"网络课件的研究

介绍了如何用Flash MX制作“线粒体遗传病”的网络课件，以及其中制作的技巧。     

Therapeutic Effect of Shenfu Injection(参附注射液)on Secondary Aplastic Anemia of Tumor Patients after Chemotherapy

Objective: To explore the effect of Shenfu Injection (参府注射液, SFI) on the secondary aplastic anemia (AA) of tumor patients after chemotherapy (CT). Methods: The 15 cases of SFI treated group, 10 cases of control group, 25 cases of SFI granulocyte-macrophage colony stimulating factor (GM-CSF) treated group and the group of 23 GM-CSF-treated tumor cases of secondary AA after CT were compared, with their increasing rate and the rebound speed of neutrophil, platelet, bone marrow nucleated RBC, granulocyte, and megakaryocyte all being investigated. Results: The increasing rate and rebound speed of granulocyte, platelet, and the increasing rate of bone marrow nucleated RBC, granulocyte, megakaryocyte were obviously higher than those of the control, and the clinical manifestations were also obviously improved. The increasing rate of platelet, bone marrow nucleated RBC, megakaryocyte of the SFI GM-CSF group were higher than those of the group which used GM-CSF alone, while the increasing rate of granulo     

一例抗维生素D 佝偻病的基因诊断和新突变的致病性鉴定

目的 研究低血磷抗维生素D 佝偻病即X 连锁低磷酸盐血症(XLH)患者发病的分子遗传学 机制,揭示其基因型与表现型的相互关系,为实施有效的对症治疗和今后的孕早期产前基因诊断创造必要的 前提条件.方法 在临床初诊的基础上,采用PCR 及扩增产物直接测序法对先证者的PHEX 基因进行全面 的突变检测;在检出PHEX 基因c.2197-2198insAACT 新突变后,采用PCR-DHPLC 筛检法对随机采集的108 例正常对照的相应外显子进行快速的突变筛查,以排除多态性变异的可能性,同时采用生物信息学方法对突 变部位氨基酸的保守性和突变蛋白的二、三级结构进行预测分析,从而对其致病性进行鉴定.结果 (1)先 证者PHEX 基因第22 外显子区域内存在一个新的杂合插入突变即c.2197-2198insAACT.(2)108 例正常对 照的DHPLC 筛检和序列分析中,均未检测到c.2197-2198insAACT 突变.(3)蛋白质二级、三级结构预测结 果显示:c.2197-2198insAACT 新突变引起密码子发生移位,导致肽链提前在第733 位遇上终止密码TAA,致 使肽链从正常的749 个氨基酸缩短至732 个,导致蛋白质二、三级结构发生明显改变,而正常对照无此变化.结论 PHEX 基因c.2197-2198insAACT 插入突变不是一般的多态性变异,而可能是一种新的致病性突变,它 极可能是引起先证者发病的根本内因.     

肾上腺皮质肿瘤与MEN1基因的相关性研究

目的 从分子水平上揭爪散发性肾上腺皮质肿瘤的发病与MEN1基因突变的相互关系,为今后开展症状前诊断和产前基因诊断创造前提条件.方法 在对先证者进行临床诊断的基础上,用微量快提法制备模板DNA,用自行设计的9对引物对先证者MEN1基因的所有编码区以及外显子与内含子的交界部位进行PCR扩增和DNA序列分析.结果 先证者MEN1基因第4内含子存在隐蔽性拼接位点ⅣS4as（-9）G〉A的杂合突变,而正常对照无此突变.结论 所用检测方法快速简便、微量经济,可用于肾上腺皮质肿瘤的快速确诊.所发现的MEN1基因的ⅣS4 as（-9）G〉A隐蔽性拼接位点突变为国内首报的病理性突变.