FGFR3基因突变分析鉴别软骨发育不全及类似遗传性侏儒

目的 了解中国人软骨发育不全患者（ａｃｈｏｎｄｒｏｐｌａｓｉａ,ＡＣＨ）的基因突变情况,建立一种快速简便的从分子水平鉴别ＡＣＨ及类似遗传性侏儒的方法。方法 对２１例ＡＣＨ患者及６例颖似ＡＣＨ患者的干血滤纸片进行成纤维细胞成长因子受体３（ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ ｒｅｃｅｐｔｏｒ３,ＦＧＦＲ３）基因跨膜区特异性扩增,通过限性内切酶分析、单链构象多态和变性梯度凝胶电泳检测基因突     

Pfeiffer综合征的遗传异质性研究

目的为揭示Ｐｆｅｉｆｅｒ综合征的分子病理缺欠。方法采用ＳＳＣＰ－ＤＮＡ直接测序及ＰＣＲ－限制性内切酶酶切技术，对４个Ｐｆｅｉｆｅｒ综合征家系的外周血ＤＮＡ进行了分析。结果２个家系是由ＦＧＦＲ２基因突变所致：１例发生在ＦＧＦＲ２基因第８内含子３′剪切位点部位Ａ→Ｇ突变；１例为ＦＧＦＲ２基因第９外显子Ａｓｐ３２１Ａｌａ突变。另外１个家系是由ＦＧＦＲ１基因第５外显子Ｐｒｏ２５２Ａｒｇ突变所致。结论该项研究结果表明了Ｐｆｅｉｆｅｒ综合征的遗传异质性，为该病的病因学研究和临床研究提供了有用的资料     

先天性肾上腺皮质增生症患者21-羟化酶基因启动子区域突变的初步研究

目的研究先天性肾上腺皮质增生症（ｃｏｎｇｅｎｉｔａｌａｄｒｅｎａｌｈｙｐｅｒｐｌａｓｉａ,ＣＡＨ）患者２１－羟化酶基因启动子区域的突变。方法用ＰＣＲ、ＳＳＣＰ、内切酶酶谱分析及测序分析方法对１２例ＣＡＨ患者的２１－羟化酶基因启动子区域进行研究。结果１２例患者中有６例出现异常ＳＳＣＰ条带,其中１例在ＣＫ－２（－１０１）结合区域内的ＫｐｎⅠ内切酶识别位点及其－２０１处的ＴａｑⅠ内切酶识别位点存在突变,并经测序证实。结论在ＣＡＨ患者－２１羟化酶基因启动子区域存在突变,可能为ＣＡＨ发病机理之一。     

中国人血友病甲患者FⅧ基因突变研究

为研究血友病甲发病的分子机理，应用聚合酶链反应（ＰＣＲ）结合限制性内切酶ＴａｑⅠ酶切分析和ＰＣＲ结合变性梯度凝胶电泳（ＰＣＲ－ＤＧＧＥ），分别研究了７４名中国血友病甲患者ＦⅧ基因外显子１８、２２～２４、２６和外显子８与１４的３′端的基因突变情况，结果检测到一例基因突变，该突变位于第２４号外显子２２０９位密码子，ＣＧＡ－ＴＧＡ，导致了终止密码子的产生。ＰＣＲ－ＤＧＧＥ发现２例基因突变，分别位于外显子８的３４９位密码子，ＧＡＴ－ＧＡＧ，导致Ａｓｐ３４９Ｇｌｕ和外显子１４的１６８９位密码子，ＣＧＣ－ＴＧＣ，使Ａｒｇ１６８９Ｃｙｓ。血友病甲基因突变的研究将有助于开展遗传咨询和产前诊断工作。     

天津地区G6PD缺陷患者常见基因突变分析

目的研究北方地区葡萄糖－６－磷酸脱氢酶（ｇｌｕｃｏｓｅ－６－ｐｈｏｓｐｈａｔｅｄｅｈｙｄｒｏｇｅｎａｓｅ，Ｇ６ＰＤ）基因突变类型、基因突变与人口变迁。方法采用“错配引物”介导的聚合酶链反应／限制性酶切分析法和双脱氧核苷酸指纹印迹检测法，对天津地区２２例Ｇ６ＰＤ缺陷患者进行３种中国南方人群常见的Ｇ６ＰＤ基因突变的分析。结果８例患者（８／２２，３６．４％）有Ｒ４５９Ｌ（１３７６Ｇ→Ｔ）突变；７例患者（７／２２，３１．８％）有Ｒ４６３Ｈ（１３８８Ｇ→Ａ）突变；７例（７／２２）无１３７６和１３８８位点突变病例中，有３例做了Ｈ３２Ｒ（９５Ａ→Ｇ）突变分析，提示１例有该位点突变。结论北方地区也存在中国南方人群常见的３种Ｇ６ＰＤ基因突变，大多数中国北方Ｇ６ＰＤ缺陷患者是由于中国南方移民迁移造成的。     

一种N端缺失的rhGM—CSF（7～127）在大肠杆菌中的表…

在ｈＧＭ－ＣＳＦ结构与功能研究的基础之上，通过应用ＰＣＲ介导的缺失与突变和基因重组等技术，构建了ｒｈＧＭ－ＣＳＦ（７～１２７）的三种原核表达载体ｐＢＶ２２０／ＧＭ－ＴＧＡ，ｐＢＶ２２０／ＧＭ－ＴＡＡ和ｐＢＶ２２０／ＧＭ－３′ＵＴＲ。在三种载体内，ｈＧＭ－ＣＳＦ（７～１２７）ｃＤＮＡ的５′端块缺失６个氨基酸，３′端则分别为天然终止密码ＴＧＡ，突变终止密码ＴＡＡ和ＴＧＡ加３′ＵＴＲ。ＳＤＳ－ＰＡＧＥ表     

先天性肾上腺皮质增生症患者21—羟化酶基因启动子区域突变？…

目的 研究先天性肾上腺皮质增生症（ＣＡＨ）患者２１－羟化酶基因启动子区域的突变。方法 用ＰＣＲ、ＳＳＣＰ、内切酶酶谱分析及测序分析方法对１２例ＣＡＨ患者的２１－羟化酶基因启动子区域进行研究。结果 １２例患者中有６例出现异常ＳＳＣＰ条带，其中１例在ＣＫ－２（－１０１）结合区域内的ＫｐｎⅠ内切酶识别位点及其－２０１处的ＴａｑⅠ内切酶识别位点存在突变，并经测序证实。结论 在ＣＡＨ患者－２１羟化酶基因启动     

造血生长因子促进RV介导LacZ-NeoR基因在人骨髓造血细胞中转染的机制

目的：本文探索了不同组合的造血生长因子ＳＣＦ、ＩＬ３及ＩＬ６对逆转录病毒（ＲＶ）介导的ＬａｃＺ－ＮｅｏＲ双标志基因转染人骨髓造血细胞转染效率、表达水平的影响及其相关机理。方法：采用ＲＶ转染人骨髓非粘附造血细胞（ＮＡＢＭＣ）及经ＳＣＦ、ＩＬ３及ＩＬ６不同组合预激４８ｈ后的ＮＡＢＭＣ。经荧光素二－β－Ｄ－半乳糖呋喃苷脂（ＦＤＧ）标记的半乳糖苷酶、Ｇ４１８ＲＣＦＵ－ＧＭ及ＰＣＲ／Ｓｏｕｒｔｈｅｒｎ－ｂｌｏｔ检测ＮｅｏＲ和ＬａｃＺ基因的表达。结果：早期造血生长因子（ＨＧＦｓ）的预激明显改善了ＲＶ介导的ＬａｃＺ－ＮｅｏＲ基因在人骨髓造血细胞中的转染效率与表达水平,ＳＣＦ＋ＩＬ３＋ＩＬ６＞ＳＣＦ＋ＩＬ３＞ＩＬ３＋ＩＬ６＞ＳＣＦ＋ＩＬ６。氚标记脱氧胸苷（３Ｈ－ＴｄＲ）自杀及５－溴脱氧尿苷－碘化丙啶（ＢｒｄＵ－ＰＩ）双标流式细胞仪（ＦＣＭ）检测显示,ＨＧＦｓ预激后人骨髓造血细胞及ＣＦＵ－ＧＭ的Ｓ期比例显著提高。结论：ＳＣＦ＋ＩＬ３＋ＩＬ６的联合预激可显著改善ＲＶ介导的外源基因在人骨髓造血细胞中的转染效率与表达水平,这可能与ＨＧＦｓ预激后明显提高人骨髓造血细胞及ＣＦＵ－ＧＭ的Ｓ期比例密切相关     

一种N端缺失的rhGM-CSF(7～127)在大肠杆菌中的表达与调控

在ｈＧＭ－ＣＳＦ结构与功能研究的基础之上，通过应用ＰＣＲ介导的缺失与突变和基因重组等技术，构建了表达ｒｈＧＭ－ＣＳＦ（７～１２７）的三种原核表达载体ｐＢＶ２２０／ＧＭ－ＴＧＡ，ｐＢＶ２２０／ＧＭ－ＴＡＡ和ｐＢＶ２２０／ＧＭ－３′ＵＴＲ。在三种载体内，ｈＧＭ－ＣＳＦ（７～１２７）ｃＤＮＡ的５′端均缺失６个氨基酸，３′端则分别为天然终止密码ＴＧＡ，突变终止密码ＴＡＡ和ＴＧＡ加３′ＵＴＲ。ＳＤＳ－ＰＡＧＥ表明三种载体表达ｒｈＧＭ－ＣＳＦ（７～１２７）的水平分别为２１％，１８．８％和２５％。经过用ＰＣｇｅｎｅ软件和Ｚｕｌｃｅｒ算法分析ｈＧＭ－ＣＳＦ（７～１２７）－３′ＵＴＲｍＲＮＡ的二级结构，表明３′ＵＴＲ在终止密码ＴＧＡ附近形成两个茎－环结构，它可能与ｐＢＶ２２０／ＧＭ－３′ＵＴＲ载体高表达ｒｈＧＭ－ＣＳＦ（７～１２７）有关。表达产物ｒｈＧＭ－ＣＳＦ（７～１２７）经弱阴离子ＤＥＡＥ交换层析纯化后，纯度达到９２％，比活性为８×１０７Ｕ／ｍｇ。     

聚合酶链反应—单链构象多态性分析在家族性高胆固醇？…

目的 探讨聚合酶链反应－单链构象多态性分析（ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ－ｓｉｎｇｌｅ ｓｔｒａｉｎ ｃｏｎｆｏｒｍａｔｉｏｎ ｐｏｌｙｍｏｒｐｈｉｓｍ,ＰＣＲ－ＳＳＣＰ０在家族性高胆固醇胆固醇血症（ｆａｍｉｌｉａｌｈｙｐｅｒｃｈｏｌｅｓｔｅｒｏｌｅｍｉａｃ,ＦＨ）患者家系分析中的应用价值。方法 对于经ＰＣＲ－ＳＳＣＰ筛查,ＤＮＡ序列分析证实的４例ＦＨ患者（１例纯合子ＦＨ外     

Severe complications in a child with achondroplasia and two FGFR3 mutations on the same allele

We describe a unique case of achondroplasia with associated complications, including severe respiratory problems. Molecular analysis of the fibroblast growth factor receptor type 3 (FGFR3) gene in this patient showed the common p.G380R mutation and a second novel p.L377R mutation. An allele-specific PCR demonstrated that these mutations were on the same allele (cis). Both mutations were not present in the parents and appear to have occurred de novo. To our knowledge, this is the first report in the literature on an achondroplasia patient with two FGFR3 mutations on the same allele.     

A common FGFR3 gene mutation is present in achondroplasia but not in hypochondroplasia

Achondroplasia is the most common type of genetic dwarfism. It is characterized by disproportionate short stature and other skeletal anomalies resulting from a defect in the maturation of the chondrocytes in the growth plate of the cartilage. Recent studies mapped the achondroplasia gene on chromosome region 4p 16.3 and identified a common mutation in the gene encoding the fibroblast growth factor receptor 3 (FGFR3). In an analysis of 19 achondroplasia families from a variety of ethnic backgrounds we confirmed the presence of the G380R mutation in 21 of 23 achondroplasia chromosomes studied. In contrast, the G380R mutation was not found in any of the 8 hypochondroplasia chromosomes studied. Furthermore, linkage studies in a 3-generation family with hypochondroplasia show discordant segregation with markers in the 4p 16.3 region suggesting that at least some cases of hypochondroplasia are caused by mutations in a gene other than FGFR3. © 1995 Wiley-Liss, Inc.     

单细胞巢式PCR诊断软骨发育不全

目的 探讨单细胞水平诊断软骨发育不全(achondroplasia，ACH)的可靠性，为开展ACH的胚胎植入前遗传学诊断(preimplantation genetic diagnosis，PGD)打下基础。方法 采用巢式PCR扩增单淋巴细胞及单卵裂球的成纤维细胞生长因子受体3基因的高发突变位点G380R区域，用限制酶Bfm I消化PCR产物，10％聚丙烯酰胺凝胶电泳检测。结果 单淋巴细胞的PCR扩增成功率为90．4％，等位基因脱扣发生率为8．2％，诊断准确率为91．8％；单卵裂球的扩增成功率为75．4％。结论 单细胞巢式PCR诊断ACH是比较稳定、可靠的。     

软骨发育不全的产前基因诊断

目的 通过产前成纤维细胞生长因子受体3(fibroblast growth factor receptor 3,FGFR3)基因检测确诊胎儿软骨发育不全.方法 经脐带穿刺获取78例短肢发育异常胎儿脐血,常规行核型分析,并提取基因组DNA,扩增FGFR3基因第10外显子,应用限制酶Bfm I进行限制性片段长度多态性分析,并对其行DNA双向测序.同法分析阳性胎儿双亲FGFR3基因第10外显子.结果 短肢发育异常78例胎儿中,8例为G1138A杂合突变,诊断为软骨发育不全,其核型分析正常.余70例短肢发育异常胎儿,FGFR3基因第10外显子第1138位核苷酸检测结果正常,排除软骨发育不全.8例患病胎儿双亲中,1例父亲同为G1138A杂合突变,余检测结果正常.结论 对超声诊断的胎儿短肢发育异常,通过脐血FGFR3基因第10外显子的PCR-限制性片段长度多态分析和DNA双向测序,产前能明确诊断软骨发育不全.     

Mesomelic and rhizomelic short stature: The phenotype of combined Leri-Weill dyschondrosteosis and achondroplasia or hypochondroplasia

We studied two children with combined genetic skeletal disorders. Both had Leri-Weill dyschondrosteosis (LWD); one also had achondroplasia and the other had hypochondroplasia. Both had severe short stature and evidence of rhizomelia and mesomelia as well as other phenotypic features of their individual genetic disorders. Achondroplasia was due to the G380R FGF3R mutation and hypochondroplasia to a N540K mutation in the same gene. The patient with hypochondroplasia had a heterozygous SHOX deletion; no SHOX mutation was identified in the child with achondroplasia. The phenotypes of combined LWD and achondroplasia or hypochondroplasia appeared to be less than additive, suggesting that SHOX and FGFR3 act on overlapping pathways of bone growth and development.     

Novel fibroblast growth factor receptor 3 (FGFR3) mutations in bladder cancer previously identified in non-lethal skeletal disorders

Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are responsible for several autosomal dominant craniosynostosis syndromes and chondrodysplasias i.e. hypochondroplasia, achondroplasia, SADDAN and thanatophoric dysplasia--a neonatal lethal dwarfism syndrome. Recently, activating FGFR3 mutations have also been found to be present in cancer, i.e. at high frequency in carcinoma of the bladder and rarely in multiple myeloma and carcinoma of the cervix. Almost all reported mutations in carcinomas corresponded to the mutations identified in thanatophoric dysplasia. We here screened a series of 297 bladder tumours and found three FGFR3 somatic mutations (G380/382R; K650/652M and K650/652T) that were not previously identified in carcinomas or thanatophoric dysplasia. Another novel finding was the occurrence of two simultaneous FGFR3 mutations in four tumours. Two of the three new mutations in bladder cancer, the G380/382R and the K650/652M mutations, were previously reported in achondroplasia and SADDAN, respectively. These syndromes entail a longer life span than thanatophoric dysplasia. The K650/652T mutation has not previously been detected in patients with skeletal disorders, but affects a codon that has been shown to be affected in some cases of thanatophoric dysplasia, SADDAN and hypochondroplasia. From a clinical perspective, the patients with FGFR3-related, non-lethal skeletal disorders might be at a higher risk for development of bladder tumours than the general population.     

Predominance of the mutation at 1138 of the cDNA for the fibroblast growth factor receptor 3 in Japanese patients with achondroplasia

Summary Fibroblast growth factor receptor 3 (FGFR3) has recently been identified as a putative gene for achondroplasia. Since a guanine to adenine mutation at 1138 of the cDNA for FGFR3 had been identified in most of the patients in Western population, we examined 13 Japanese patients to see if they also share the same mutation. Specific endonuclease digestion of the amplified coding sequence for the transmembrane domain of the FGFR3 revealed that the 12 patients have the G to A change at 1138, while the other had the G to C substitution at the same point, both of which result in G380A substitution. As far as we studied, the homogeneity of the point mutation at 1138 is also authentic to Japanese patient as well as Western patients.     

Compound heterozygosity for the Achondroplasia-hypochondroplasia FGFR3 mutations: prenatal diagnosis and postnatal outcome.

We report on a male newborn infant, a compound carrier of heterozygous mutations in the FGFR3 gene causing achondroplasia and hypochondroplasia. The mother has achondroplasia and carries the common G1138 (G380R) mutation in the FGFR3 gene; the father has hypochondroplasia due to the C1620A (N540K) mutation in the same gene. The fetus was found to carry both mutations diagnosed prenatally by amniocentesis at 17.6 weeks of gestation, following maternal serum screening which showed an increased risk for Down syndrome (1:337). Detailed fetal ultrasound studies showed a large head, short limbs, and a small chest at 22 weeks of gestation. The changes were more severe than those of either achondroplasia or hypochondroplasia. The patient was born by cesarean section at 38 weeks of gestation and had rhizomelic shortness of the upper and lower limbs with excess skin folds, large head, enlarged fontanelles, frontal bossing, lumbar gibbus, trident position of the fingers, and a narrow chest with a horizontal line of demarcation at the narrowest area of the chest. Skeletal radiographs showed shortness of the long bones and flare of metaphyses. He had respiratory difficulties and was treated with nasal prongs. Seizures developed on day 2 of life and recurred on day 9 and responded to treatment with phenobarbital. Brain computed tomographic scan showed possible grey matter heterotopia, partial agenesis of the corpus callosum, and cortical dysplasia. To our knowledge, there are only two previously published cases of compound heterozygous achondroplasia-hypochondroplasia patients. The diagnosis was confirmed by DNA mutation analysis of the FGFR3 gene in both cases.     

Achondroplasia-hypochondroplasia complex in a newborn infant.

We describe the case of an 8-month-old girl with achondroplasia-hypochondroplasia complex. The diagnosis was suggested antenatally when obstetrical ultrasonography at 27 weeks of gestation showed short limbs, small chest, and macrocephaly. The father has achondroplasia due to the common G1138A (G380R) mutation in the fibroblast growth factor receptor 3 (FGFR3) gene, while the mother has hypochondroplasia due to the C1620G (N450K) mutation in the FGFR3 gene. Neither had had genetic counseling or molecular testing prior to the pregnancy. Antenatal ultrasound study at 29 weeks of gestation showed a large head, very short limbs, and a small chest; the findings were more severe than in achondroplasia or hypochondroplasia alone. The patient was born by cesarean section at 37 weeks of gestation and had rhizomelic shortness of limbs with excess skin creases, large head, and small chest, diagnostic of achondroplasia. Radiographs showed shortness of the long bones and flaring of the metaphyses. She had mild hypoplasia of lungs. Molecular testing showed both the G1138A and the C1620G mutations in FGFR3, confirming the diagnosis of achondroplasia-hypochondroplasia complex. At 8 months, she has disproportionate shortness of the long bones and a large head with frontal bossing and a depressed nasal bridge. Her chest remains small, and she is on home oxygen at times of respiratory stress. She has a large gibbus. She is delayed in her motor development and has significant head lag. To our knowledge, there is only one previously published report of achondroplasia-hypochondroplasia complex.