遗传性蛋白S缺陷症家系基因突变的研究

Objective To identify the clinical phenotypic diagnosis and gene mutation detection of two kindreds with PS deficiency. MethodsPS: A was measured by chromogenic substrate method;TPS:Ag, FPS: Ag levels were measured by ELISA method; PS gene(PROS1 gene)was detected by amplifying 15 exons and flanking intron sequences from the propositus with PCR method. PCR products were purified and directly sequenced. Results For propositus 1,PS: A was 48.6% ,TPS: Ag was 136 mg/L, FPS : Ag was 41 mg/L, PROSI gene exon 2 was in c. Heterozygous base substitutions was detected in C121T locus, which led to Arg-1Cys (R-1C) heterozygous roissense mutation encoded in PS proteins. For propositus 2, PS: A was 29.2%, TPS: Ag was 83 mg/L, FPS: Ag was 26 mg/L, PROSI gene exon 14 was in c. Heterozygous base substitutions was identified in CI687T locus, in which Gln.522Stop heterozygous nonsense mutation was encoded in PS proteins. Conclusions c. C121T is a novel mutation locus detected in PROS1 gene. This heterozygous mutation could lead to type Ⅱ PS hereditary deficiency, while c. C1687T heterozygous mutation could bring about type Ⅰ PS hereditary deficiency.     

遗传性蛋白S缺陷症家系基因突变的研究

目的 对2个遗传性PS缺陷症家系进行临床表型诊断和基因突变检测.方法 PS:A测定采用发色底物法;TPS:Ag、FPS;Ag测定采用ELISA法;PS基因(PROS1基因)检测采用PCR方法 对先证者PROS1基因的15个外显子及其侧翼内含子序列进行扩增,PCR产物纯化后直接测序.结果 先证者1的PS:A为48.6%,FPS:Ag为41 mg/L,TPS:Ag为136 mg/L,PROS1基因2号外显子在c.C121T位点发生杂合碱基替换,导致编码的PS蛋白存在Arg-1 Cys(R-1C)杂合错义突变.先证者2的PS:A为29.2%,FPS;Ag为26 mg/L,TPS:Ag为83 mg/L,PROSl基因14号外显子在c.C1687T位点发生杂合碱基替换,导致编码的PS蛋白存在Gln522Stop杂合无义突变.结论 c.C121T是PROS1基因的一个新的突变位点,该杂合突变可以导致Ⅱ型遗传性PS缺陷症;c.C1687T杂合突变导致Ⅰ型遗传性PS缺陷症.     

X-连锁迟发性脊髓骨骺发育不良家系的基因诊断

ObjectiveTo investigate the molecular pathogenesis of a pedigree of X-linked spondyloepiphyseal dysplasia atarda (SEDL) and to establish methods of gene diagnosis. Methods Clinical diagnosis was made based on height measurement, radiological examination and pedigree analysis. Peripheral blood samples of relevant family members were collected. After genomic DNA extraction, single strand conformation polymorphism (SSCP) followed with DNA sequencing was used to detect SEDL gene exons 36. Microsatellite marker DXS16 was selected for linkage analysis. Results The abnormal electrophoretic bands were detected in exon 4 of probands by PCR-SSCP. A c. 218C ＞ T mutation in exon 4 of SEDL gene was found in three probands, which resulted in a change in amino acid sequence S37L. The heterozygous exon 4 mutation was identified in three carriers, but not in healthy individuals, and no mutations were detect in exon 3, 5 and 6 of probands. Three unmarried young females (Ⅲ10, Ⅳ6 and Ⅳ7) were found to harbor the mutation by DNA sequencing analysis. ConclusionsA c. 218C ＞ T missense mutation in exon 4 of SEDL gene is the cause of molecular pathogenesis of the pedigree. SSCP and DNA sequencing can be used for prenatal gene diagnosis.     

X-连锁迟发性脊髓骨骺发育不良家系的基因诊断

ObjectiveTo investigate the molecular pathogenesis of a pedigree of X-linked spondyloepiphyseal dysplasia atarda (SEDL) and to establish methods of gene diagnosis. Methods Clinical diagnosis was made based on height measurement, radiological examination and pedigree analysis. Peripheral blood samples of relevant family members were collected. After genomic DNA extraction, single strand conformation polymorphism (SSCP) followed with DNA sequencing was used to detect SEDL gene exons 36. Microsatellite marker DXS16 was selected for linkage analysis. Results The abnormal electrophoretic bands were detected in exon 4 of probands by PCR-SSCP. A c. 218C ＞ T mutation in exon 4 of SEDL gene was found in three probands, which resulted in a change in amino acid sequence S37L. The heterozygous exon 4 mutation was identified in three carriers, but not in healthy individuals, and no mutations were detect in exon 3, 5 and 6 of probands. Three unmarried young females (Ⅲ10, Ⅳ6 and Ⅳ7) were found to harbor the mutation by DNA sequencing analysis. ConclusionsA c. 218C ＞ T missense mutation in exon 4 of SEDL gene is the cause of molecular pathogenesis of the pedigree. SSCP and DNA sequencing can be used for prenatal gene diagnosis.     

家族性高胆固醇血症患者二例的LDLR基因复合杂合新突变分析

目的 探讨2例临床确诊的湖北籍FH患者的LDLR基因突变状况,为FH的基因诊断提供依据.方法 收集2例临床确诊的FH患者及其父母血脂检测指标等临床资料,通过PCR扩增LDLR基因的1～18个外显子和内含子区域,再将扩增产物进行正、反双向核苷酸序列分析,并与GenBank中LDLR基因的正常序列对比找出突变后,结合FH先证者的临床表型证实致病突变的类型.结果 氧化酶法测定1号、2号FH先证者血浆TC,分别为12.79、11.98 mmol/L;经核苷酸序列分析,其ApoB100基因涵盖的3 500～3 531区域均未见突变;LDLR基因均为复合杂合突变,1号FH先证者LDLR基因第4外显子的665位碱基G>T为杂合错义突变,且该突变为新的点突变,第9内含子的1 358+32位碱基C>T突变也为新的点突变,并均由其父母遗传.2号先证者第9外显子1 257位碱基C>A突变导致终止密码子提前出现,但其核苷酸改变与比利时报道的C>G不同,第13外显子检测到1 879位碱基G>A杂合错义突变,且分别来源于其父母.结论 2例FH先证者均存在LDLR基因复合杂合突变,1号FH先证者的第4外显子665位碱基G>T和第9内含子1 358+32位碱基C>T、2号FH先证者的第9外显子1 257位碱基C>A突变均为新突变,这可能是导致FH的分子机制.

Abstract：

Objective To determine LDLR gene mutation in 2 clinically diagnosed FH patients from Hubei province and provide basis for gene diagnosis of FH.Methods Clinical data of 2 FH patients and their parents were collected.The promoter region and exon 1 to exon 18 region of LDLR gene were amplified through PCR and the amplified products were analyzed by forward and reverse DNA sequencing.The mutations were identified after comparison with LDLR gene sequence in GenBank.The pathogenic gene mutations were confirmed according to both genotype and phenotype of FH probands.Results The levels of plasma TC of two probands were 12.79 and 11.98 mmol/L.respectively.No gene mutations were detected in region 3 500 to 3 531 of ApoB100. The mutations of LDLR gene were compound heterozygous mutations. The novel mutation 665G > T detected in the exon 4 of No. 1 proband's LDLR gene was heterozygous missense mutation. The novel mutation 1 358 +32C > T was detected in the exon 9 of No. 1 proband's LDLR gene.The mutations 665G > T ( paternal origin) and 1 358 + 32C > T ( maternal origin) were inherited from the parents. A novel mutation 1 257 C > A was detected in the exon 9 of No. 2 proband's LDLR gene, resulting the presence of a premature termination codon, which was different from 1 257 C > G reported in Belgium.Another heterozygous missense mutation 1 879 G > A was detected in exon 13. They were derived from paternal origin and maternal origin, respectively. Conclusions There are three novel gene mutations:665G >T, 1 358 +32C > T, 1 257C > A found in two probands with compound heterozygous mutations in LDLR respectively. They maybe play a potential role in FH pathogensis.     

遗传性凝血因子Ⅶ缺乏症的分子发病机制研究

目的 对一个遗传性FⅦ缺乏症家系进行F7基因突变检测,探讨其分子发病机制.方法 采用ELISA法检测先证者及家系成员FⅦ∶Ag,采用一期凝固法检测先证者及家系成员PT、FⅦ∶C等凝血指标进行实验表型诊断.基因检测采用DNA直接测序法分析先证者及家系成员F7基因的全部外显子及侧翼、5'和3'非翻译区,发现突变位点用反向测序加以证实;用CLC Protein Workbench软件分析基因突变位点的物种保守性和蛋白质二级结构改变.选择100名健康对照者以排除基因多态性.结果 先证者及其妹妹的PT、FⅦ∶C、FⅦ∶Ag明显异常,分别为36.3 s、5.0%、40.7%和33.4 s、5.0%、37.4%;先证者的父亲、母亲、女儿、外甥女的PT稍延长,分别为14.9 s、14.6 s、15.5 s、14.6 s;其FⅦ:C稍减低,分别为70%、85%、59%、79%.先证者F7基因8号外显子c.784T＞C杂合突变导致Ser269Pro,8号外显子c.964T＞G杂合突变导致Cys329Gly;先证者妹妹为c.784T＞C和c.964T＞G双重杂合突变,母亲为c.784T＞C杂合子,父亲、女儿、外甥女均为c.964T＞G杂合子.蛋白质生物学特性分析发现,Cys329Gly导致蛋白质空间构型发生改变,Ser269Pro导致氨基酸极性及疏水性发生改变.结论 F7基因Ser269Pro和Cys329Gly双重杂合突变是导致该例遗传性FⅦ缺乏症的分子发病机制.

Abstract：

Objective To identify gene mutations and explore the molecular mechanism of a pedigree with inherited coagulation F Ⅶ deficiency. Methods The levels of F Ⅶ: Ag in the proband and other family members were measured by ELISA assay. The values of PT, F Ⅶ: C and other coagulant parameters were determined by one-stage clotting for laboratory phenotype diagnosis. All the exons,exon-intron boundaries and 5',3' untranslated sequences of F7 gene were amplified by direct sequencing. The detected mutations were further confirmed by sequencing the other stand. The CLC Protein Workbench software was used to analyze the species conservation of the mutated site and the protein secondary structure. 100 healthy individuals were selected to exclude gene polymorphism. Results PT, FⅦ∶C and FⅦ: Ag in the proband and his sister were abnormal, which were 36. 3 s, 5.0%, 40. 7% and 33.4 s,5. 0%, 37.4%, respectively. Both PT and FⅦ∶C in the proband's father, mother, daughter and niece were slightly abnormal, which were 14.9 s, 14. 6 s, 15.5 s, 14. 6 s and 70%, 85%, 59%, 79%, respectively.The heterozygous mutations c. 784T ＞ C and c. 964T ＞ G in exon 8 of F7 gene were found in the proband,resulting in the substitutions of Ser269Pro and Cys329Gly respectively. Compound heterozygous mutations c. 784T ＞ C and c. 964T ＞ G were found in the proband's sister. The proband's mother was heterozygous for c. 784T ＞ C. His father, daughter and niece were heterozygous for c. 964T ＞ G. The protein biological characteristics analysis revealed that the Cys329Gly caused the change of spatial configuration, and Ser269Pro led to the change of amino acid polarity and hydrophobicity. Conclusion Compound heterozygous mutations of Cys329Gly and Ser269 Pro in F7 gene may be the underlying molecule mechanism of FⅦ deficiency in this pedigree.     

Clinical characteristics on manifestation and gene mutation of a transient neonatal cyanosis: A case report

BACKGROUND We analyzed the main features of an infant diagnosed with temporary neonatal cyanosis in order to strengthen our understanding of the disease.CASE SUMMARY We report a patient diagnosed with temporary neonatal cyanosis.The main clinical characteristics,gene mutation and treatment are discussed and a review of related literature was conducted.The neonate aged 1 d and 5 h was admitted to hospital due to cyanosis after birth.The main clinical manifestation was cyanosis,which was not improved by auxiliary ventilation and the patient showed no obvious shortness of breath or methemoglobinemia.Gene mutation analysis showed a heterozygous c.190C>T mutation in the HBG2 gene associated with transient neonatal cyanosis,which was derived from his mother.Symptomatic supportive treatment was given for 2 mo.The neonate was discharged and gradually improved with oral administration of vitamin C and vitamin B2 for 2 wk.CONCLUSION There is no special treatment for temporary neonatal cyanosis caused by heterozygous mutation of the HBG2 gene.     

酶切富集联合DHPLC检测肿瘤患者外周血EGFR和KRAS基因突变及其临床应用

Objective To establish a REDE-DHPLC method for detecting the EGFR and KRAS mutations in plasma DNA from tumor patients, and investigate its clinical significance. Methods Restriction endonucleases Mse Ⅰ , Msc Ⅰ , BstN Ⅰ and Bgl Ⅰ were used to digest the wild type fragments of exon 19,exon 21 of EGFR gene and coden 12, 13 of KRAS gene for enriching the mutation fragments, and REDE-DHPLC method was established to detect EGFR and KRAS mutations. The sensitivities of REDE-DHPLC and conventional DHPLC were analyzed by using a series of plasmids containing 50%, 10%, 5%, 1% and 0. 1% mutation genes. Then, Plasma samples and paraffin-embedded tissue samples of 120 NSCLC patients and 120 colorectal cancer patients were detected by REDE-DHPLC. Compared with conventional DHPLC and sequencing, the diagnostic efficiency of REDE-DHPLC method was evaluated by detecting the mutation status of 2 genes in plasma of NSCLC and colorectal cancer patients. Results The sensitivity values of REDE-DHPLC and conventional DHPLC for detecting mutations in 4 loci were 0. 1% and 1%respectively. Plasmid DNA containing 0.1% mutation gene was detected to be positive continually for 2 to 3 times by REDE-DHPLC. EGFR mutation rates of 120 plasma from NSCLC patients detected by REDE-DHPLC, conventional DHPLC and sequencing methods were 27. 5%, 16. 7% and 12.5% respectively, and KRAS mutation rates of 120 plasma from colorectal cancer patients were 38. 3%, 25. 8% and 16. 7%,respectively. The positive rates of EGFR and KRAS mutation detected by REDE-DHPLC were significantly higher than conventional DHPLC(x2 = 4. 092, 4. 301, all P ＜ 0. 05 ) and sequencing method (x2= 8. 438,14. 127,all P ＜ 0. 05 ). In comparison with conventional DHPLC, the sensitivities of REDE-DHPLC for detecting EGFR and KRAS mutation were 100% (20/20,31/31), the specificities were 87. 0% (87/100)and 83. 2% (74/89). In comparison with sequencing method, the sensitivities of REDE-DHPLC were 100%( 15/15,20/20), the specificities were 82.9% (87/105)and 74. 0% (74/100). The coincidence rate of the two methods for detecting EGFR and KRAS mutation were 89. 2% ( 107/120, Kappa = 0. 690, P ＜ 0. 05 ) and 87.5% ( 105/120, Kappa= 0. 718, P ＜ 0. 05 ). The Consistency of EGFR and KRAS mutation status in plasma and tissues detected by REDE-DHPLC were 91.7% (33/36, Kappa =0. 939,P ＜0. 05)and 90. 2 %(46/51, Kappa = 0. 914, P ＜ 0. 05 ), respectively. Conclusions The REDE-DHPLC method is highly sensitive and specific for detecting EGFR and KRAS mutations in plasma DNA from tumor patients. The results are easy to be interpreted without missing homozygous point mutation, which indicate that the detection of EGFR and KRAS mutations in plasma DNA by REDE-DHPLC could therefore extend to be usedin clinical laboratory.     

酶切富集联合DHPLC检测肿瘤患者外周血EGFR和KRAS基因突变及其临床应用

Objective To establish a REDE-DHPLC method for detecting the EGFR and KRAS mutations in plasma DNA from tumor patients, and investigate its clinical significance. Methods Restriction endonucleases Mse Ⅰ , Msc Ⅰ , BstN Ⅰ and Bgl Ⅰ were used to digest the wild type fragments of exon 19,exon 21 of EGFR gene and coden 12, 13 of KRAS gene for enriching the mutation fragments, and REDE-DHPLC method was established to detect EGFR and KRAS mutations. The sensitivities of REDE-DHPLC and conventional DHPLC were analyzed by using a series of plasmids containing 50%, 10%, 5%, 1% and 0. 1% mutation genes. Then, Plasma samples and paraffin-embedded tissue samples of 120 NSCLC patients and 120 colorectal cancer patients were detected by REDE-DHPLC. Compared with conventional DHPLC and sequencing, the diagnostic efficiency of REDE-DHPLC method was evaluated by detecting the mutation status of 2 genes in plasma of NSCLC and colorectal cancer patients. Results The sensitivity values of REDE-DHPLC and conventional DHPLC for detecting mutations in 4 loci were 0. 1% and 1%respectively. Plasmid DNA containing 0.1% mutation gene was detected to be positive continually for 2 to 3 times by REDE-DHPLC. EGFR mutation rates of 120 plasma from NSCLC patients detected by REDE-DHPLC, conventional DHPLC and sequencing methods were 27. 5%, 16. 7% and 12.5% respectively, and KRAS mutation rates of 120 plasma from colorectal cancer patients were 38. 3%, 25. 8% and 16. 7%,respectively. The positive rates of EGFR and KRAS mutation detected by REDE-DHPLC were significantly higher than conventional DHPLC(x2 = 4. 092, 4. 301, all P ＜ 0. 05 ) and sequencing method (x2= 8. 438,14. 127,all P ＜ 0. 05 ). In comparison with conventional DHPLC, the sensitivities of REDE-DHPLC for detecting EGFR and KRAS mutation were 100% (20/20,31/31), the specificities were 87. 0% (87/100)and 83. 2% (74/89). In comparison with sequencing method, the sensitivities of REDE-DHPLC were 100%( 15/15,20/20), the specificities were 82.9% (87/105)and 74. 0% (74/100). The coincidence rate of the two methods for detecting EGFR and KRAS mutation were 89. 2% ( 107/120, Kappa = 0. 690, P ＜ 0. 05 ) and 87.5% ( 105/120, Kappa= 0. 718, P ＜ 0. 05 ). The Consistency of EGFR and KRAS mutation status in plasma and tissues detected by REDE-DHPLC were 91.7% (33/36, Kappa =0. 939,P ＜0. 05)and 90. 2 %(46/51, Kappa = 0. 914, P ＜ 0. 05 ), respectively. Conclusions The REDE-DHPLC method is highly sensitive and specific for detecting EGFR and KRAS mutations in plasma DNA from tumor patients. The results are easy to be interpreted without missing homozygous point mutation, which indicate that the detection of EGFR and KRAS mutations in plasma DNA by REDE-DHPLC could therefore extend to be usedin clinical laboratory.     

遗传性蛋白C缺陷症四个家系的分子发病机制研究

目的 对4个遗传性PC缺陷症家系进行临床表型诊断和基因型分析.方法 分别用发色底物法和凝固法测定4个家系先证者及家系成员的血浆PC:A、TPS:A和FPS:A;PC:Ag和FPS:Ag的测定采用ELISA法.采用凝血酶生成试验检测患者凝血功能的变化.PCR法扩增先证者PC和PS基因的全部外显子及其侧翼序列,PCR产物纯化后直接测序,检测其基因突变.结果 先证者1的PC:A为36%,PC:Ag 为57%,TPS:A为48%,FPS:A为18%,FPS:Ag为23.7%,基因分析显示其PC基因2号、7号和8号外显子分别存在L-34P、K150del和A209V 的杂合突变,其中L-34P和A209V来自父亲,K150del来自母亲;先证者2的PC:A为46%,PC:Ag为64.4%,TPS:A为36%,FPS:A为19.5%,FPS:Ag为20.9%,其PC基因的2号和7号外显子分别存在T66C的多态性和R147W的杂合突变,PS基因的14号外显子存在Tyr519stop的杂合突变,其中PC突变来自母亲,PS突变来自父亲.凝血酶生成试验显示先证者2及其父母的抗凝功能减弱,其中PS缺陷的先证者和其父亲的外源性活化蛋白C抑制凝血酶生成的能力下降,而其母亲没有明显变化.先证者3的PC:A为33%,PC:Ag为48.42%,其PC基因同时存在R147W和R178W突变;先证者4的PC:A为21%,PC:Ag为18.36%,基因检测结果显示先证者4是R178W和D255H的双杂合子.结论 遗传性PC缺陷症或PC、PS联合缺陷症是导致4例先证者出现静脉血栓的原因.杂合PC基因突变(L-34P、A209V、R147W、R178W和D255H)是导致4例先证者遗传性PC缺陷症的原因.