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.     

单基因遗传病基因诊断技术研究进展

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Rb基因功能片段重组质粒的构建

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Maroteaux—Lamy综合征基因治疗的研究与应用

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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

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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

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