造血器官及血液疾病

940299散发性血友病A家系的分子遗传学研究/李秀玲…刀中国实用儿科杂志一1993,8(4)一231一232应用SOuthbem印迹杂交和PCR技术对6个散发性血友病A家系进行了限制性片段长度多态性分析。结果表明,家系1,2中11个成员外同血进行了SOuthem印迹杂交,未能提供信息。家系1,3,4,5,6成员PCR扩增,其中家系3均出现142bp带,家系4各成员均出现99bp带,此两家系也未提供PCR扩增信息。而凝血资料表明家系1,2,3,4,中的母亲或外祖母,其中之一为女性携带者。而一部分肯定携带者从凝血资料检查中仍不能确定。家系5中先证者母亲F物:C/VWF正常,但从基因水…     

扩增片段多态性结合银染法对广西汉族人FⅧ基因内(CA)n的研究

目的研究广西汉族人群在FⅧ基因内含子13（CA）n及内含子22（GT）n（AG）n两位点的基因频率分布,并用于血友病甲的家系分析。方法采用聚合酶链反应（PCR）扩增FⅧ基因内含子13（CA）n及22（GT）n（AG）n的多态性片段,银染色法显示扩增结果;任选一标本,测其扩增片段长度获得其i13（CA）n及i22（GT）n（AG）n的具体重复数,从而推算出所有标本两位点的二核苷酸重复数。结果在91名广西汉族人135条X染色体中,共检出6种i13（CA）n和4种i22（GT）n（AG）n,i13（CA）n位点基因频率在0．0002～0．5408间,多态信息量（polymorphism information content,PXC）为0．5899,实际杂合度为0．6364（28／44）;i22（GT）n（AG）n位点基因频率为0．0444～0．4963,PIC为0．5359,实际杂合度为0．5227（23／44）。13个有阳性家族史的血友病家系,用该方法进行家系分析,9个家系可作出诊断,可诊断率为69％。结论（1）i13（CA）n、i22（GT）n（AG）n在广西汉族人中PIC高,是血友病甲家系分析中的有用位点;（2）相比FⅧ相关的某些单个限制性片段长度多态性位点（restrictive fragment length polymorphism,RFLP）,i22（GT）n（AG）n是一个较为有用的位点。     

血友病乙患儿一家系基因突变分析

目的探讨凝血因子Ⅸ(FⅨ)基因突变致血友病乙的分子机制。方法回顾分析1例确诊血友病乙并经家系基因检测患儿的临床资料。结果先证者男,2岁4个月,反复皮肤瘀斑、鼻衄。查活化部分凝血活酶时间118. 3 s,凝血酶原时间15 s,血小板287×10~9/L,FⅨ2. 3%。明确诊断血友病乙型。收集其家系临床资料,提取家系DNA,运用PCR扩增及Sanger双脱氧链终止法对其FⅨ基因8个外显子及其侧翼序列进行测序,先证者FⅨ基因突变为c. 129 delGp.Pro 44 Glnfs*60,突变位于2号外显子,家系中5例女性为该位点的杂合突变,先证者大姨的胎儿经产前诊断提示未携带该致病基因。结论 c.129delG p.Pro44Glnfs*60是该家系FⅨ基因突变类型,基因检测有助于产前诊断。     

4个甲基丙二酸尿症的家系基因突变分析及其胎儿产前诊断

目的 分析4 个甲基丙二酸尿症（MMA）的家系基因突变情况,阐明开展MMA 基因突变分析及产前诊断的意义。方法 对诊断为MMA 的先证者或其父母行相关基因的高通量测序,确定基因突变位点,并采用聚合酶链反应和直接测序法对家系行一代测序验证。家系1、3、4 于先证者母亲再次妊娠11~13 周时超声引导下行绒毛活检,进行早期产前诊断。结果 家系1 先证者父亲检出MUT 基因c.656A > T 杂合突变,先证者母亲检出c.729-730insTT 杂合突变;早期产前诊断发现胎儿为c.656A > T 和c.729-730insTT 双重杂合突变,终止妊娠。家系2 先证者检出MUT 基因c.1106G > A 和c.755-756insA 双重杂合突变,c.1106G > A 来自父亲,c.755-756insA 来自母亲。家系3 先证者检出MMACHC 基因c.217C > T 和c.609G > A 双重杂合突变,c.217C > T 来自父亲,c.609G > A 来自母亲;产前诊断提示胎儿携带c.609G > A 杂合突变,胎儿出生时脐血检测结果与产前诊断一致。家系4 先证者检出MMACHC 基因c.609G > A 和c.567dupT 双重杂合突变,c.609G > A 来自父亲,c.567dupT 来自母亲;产前诊断提示胎儿携带c.567dupT 杂合突变,胎儿顺利出生,脐血检测结果与产前诊断一致。结论 明确基因突变有助于MMA 家系行产前诊断,避免缺陷患儿出生。     

Duchenne肌营养不良家系临床表型及遗传学分析

目的 分析Duchenne肌营养不良（DMD）患儿家系临床表型与遗传学特点。方法 选择基因检测明确诊断为DMD的7例患儿（6个家系）为研究对象，总结家系临床特征、遗传学特点。结果 6个家系中新发突变2个，母源遗传性4个。家系1先证者同时存在DMD基因1处点突变和1处插入突变，均为新发突变；3个家系为DMD基因点突变，1个家系为DMD基因外显子大片段缺失，1个家系DMD基因外显子大片段重复。所有先证者均具有骨骼肌运动障碍和肌酶改变，最小发病年龄为6个月，临床表型轻重不一；所有先证者心脏彩超检查结果均正常；3例先证者伴有轻度智力障碍。仅家系6先证者母亲具有轻度临床表型。结论 基因检测可作为DMD确诊方法，智力障碍亦是DMD较多伴发的临床表型，早期心肌受累症状不显著，女性携带者可有轻度临床症状。     

7个血友病A家系基因型与临床表型分析

目的 确定各家系凝血因子Ⅷ(F Ⅷ)基因突变类型,了解基因突变与临床表型的关系。方法 对7 个家系患者进行活化部分凝血酶原时间(APTT)及凝血因子Ⅷ凝结活性(F Ⅷ :C)检测,再采用PCR 方法对7 个血友病A家系患者进行内含子22、内含子1 倒位检测,对检测结果阴性患者采用直接测序法确定基因突变类型,并对家系相关成员进行相应位点的扩增与测序。结果 8 例血友病A患者APTT 为91.6~131 s,F Ⅷ :C 为0.8%~2%,基因检测结果显示8 例患者中未检出内含子22 倒位,仅检出1 例内含子1 倒位。余7 例血友病A 患者中,共检出5 种基因型,其中6例位于外显子14,1例位于外显子23,均为单碱基突变;有1 例检出基因型为p.His1202LeufsX16(c.3666delA),经检索为新突变。结论 F Ⅷ突变热点区域位于外显子14,新突变p.His1202LeufsX16 的发现丰富了F Ⅷ基因突变数据库。     

6-丙酮酰-四氢蝶呤合成酶缺陷症的产前诊断

目前 对6－丙酮酰－四氢喋呤合成酶（6－myruvoyl tetrahydropterin synthase,PTPS)缺陷所致非经典型苯酮尿症家系先证母亲现孕的第3胎进行产前DNA突变分析,以确诊其是否受累。方法 用PCR和DNA测序在先证者及其父母身上找到PTPS基因突变。分离胎儿羊水细胞DNA,以PCR－限制酶识别突变位点的方法进行基因突变分析;同时以辅以高压液相（HPLC）测定羊水中的生物蝶呤／新喋呤＋生物蝶蛉比例（B％）,对照该家系的尿生物蝶呤水平进行生化检查。结果 该家系先证PTPS－对等位基因各有－286G→A和226G→A和226C→T的突变,分别来自父母;先证者姨母也是226C→T突变携带者,待测胎儿不含这两种突变。结论 对PTPS基因而言,该家系的待测胎儿为完全正常。     

散发性血友病A家系的分子遗传学研究

本文应有用Southern印迹杂交和PCR技术对6个散发性血友病A家系进行了限制性片段长度多态性分析,并结合凝血资料,确定了其中两个家系中的母亲和1名姐姐为女性携带者,另外4个家系亦与遗传具有相关性,这一结果为遗传咨询提供了信息。     

中国南方汉族人家族性激素耐药型肾病综合征家系NPHS2基因突变

目的 分析中国南方汉族人家族性激素耐药型肾病综合征(SRNS)家系NPHS2基因突变及其特点.方法 研究对象为A、B、C 3个南方汉族人SRNS家系先证者及其姐和父母,50例尿检正常的南方汉族成年人作为对照人群.取所有研究对象外周静脉血3 ml,提取基因组DNA,PCR扩增NPHS2全部8个外显子及其周围的部分内含子和启动子全长序列,对PCR产物直接进行DNA序列测定.结果 对3个南方汉族人SRNS家系先证者NPHS2全部8个外显子及其周围的部分内含子进行突变分析,未发现NPHS2突变,仅在外显子8上检测到1个NPHS2基因多态性(954T＞C).在3个家系的先证者及其姐和父母的NPHS2启动子上检测到6个变异:-1715A＞G、-1709G＞A、-1000A＞T、-670C＞T、-116C＞T和-51G＞T.其中5个变异(-1709G＞A、-1000A＞T、-670C＞T、-116C＞T和-51G＞T)在100条正常染色体中也有检出,它们在SRNS患者中的等位基因频率分别与在对照人群中的等位基因频率比较差异均无统计学意义(P＞0.05);另1个变异(-1715A＞G)在家系C的先证者及其母亲(尿检正常)中检出,为杂合变异,而在100条正常染色体中未发现.-1000A＞T为新发现的NPHS2基因多态性,-1715A＞G为新发现的NPHS2变异.结论 NPHS2基因突变不是本研究3个南方汉族人家族性SRNS家系的主要致病原因.     

与ATP7B基因紧密连锁的微卫星DNA单体型在汉族Wilson病的多态性分析

目的：了解与ATP7B基因紧密连锁的微卫星DNA(D13S314,D13S301和D13S316)单体型在正常人、Wilson病(WD)患者及其杂合子的分布特点及意义。方法：采用荧光标记3个短串联重复标记(D13S314,D13S301和D13S316),测序定位和Genotype TM 软件技术分析71例WD患者和123位父母的单体型。结果：在D13S314,D13S301和D13S316位点分析中得到71例WD患者和123位携带者及54例正常个体等位基因片段;片段大小分别为134～157 bp,128～156 bp 和136～154 bp;获得等位基因数分别为19,20和15个;3个位点的杂合率分别是 0.79,0.82 和 0.23。D13S314,D13S301和D13S316位点的等位基因分布在WD患者和正常人群之间明显不同,其中D13S314和D13S301位点显示各有9个等位基因片段存在明显差异(P<0.05),D13S316位点显示有4个等位基因片段存在明显差异(P<0.01);显示的81种单体型中以 12-6-5,15-10-5,6-10-5和6-14-5最多见,分别占 5.2%,4.5%,4.5% 和 3.7%,其次为12-8-5,12-9-5和6-16-5,各占 3.0%,13-10-8,6-13-5,6-14-13和6-9-5各占 2.2%。结论：单体型的类型较多可能和基因突变的类型多样化相关,D13S314-D13S301-D13S316的单体型分析对WD的诊断,尤其是症状前或产前诊断都有重要的意义,是有效及可行的方法之一。     

Cord blood analysis for prenatal diagnosis of thalassemia major and hemophilia A

Beta thalassemia and Hemophilia A are common genetic disorders for which prenatal diagnosis (PND) is an accepted option. Our aim was to evaluate cord blood analysis as a method for PND of these disorders. Cord blood samples at 18-26 weeks gestation from nine mothers with previous thalassemia major child and five families with previous hemophilia A were studied. In the former; HbF, HbA2 and HbF were determined by high performance liquid chromatography (HPLC) and in latter; Factor VIII and IX assays were done by one stage method. In HPLC studies for thalassemia, three out of nine fetuses were affected, five were carriers and one was normal. In hemophilia PND samples, 2 out of five fetuses were affected. Thus, HPLC and factor VIII assay in cord blood are feasible alternatives for PND in Beta thalassemia and hemophilia A respectively, especially when DNA analysis is uninformative or there are financial constraints.     

北京地区苯丙酮尿症基因突变构成及基因型与表型相关分析

目的 明确北京地区苯丙酮尿症(phenylketonuria,PKU)患儿苯丙氨酸羟化酶基因(phylalanine hydroxylase gene,PAH)突变图谱及其突变基因的微单体型(STR/VNTR)构成,探讨突变基因型与生化代谢表型的相关关系.方法 应用PCR/SSCP、序列分析和变性凝胶电泳等技术,对50例北京地区PKU患儿及其父母,进行PAH基因的全部外显子及两侧的内含子序列和STR与VNTR多态性分布的分析.依据由基因型预测生化代谢表型的方法进行基因型与生化代谢表型的相关关系分析.结果 (1)共检测到34种PAH突变基因,总的检出率为95%;较为常见的突变有R243Q(20%)、EX6-96A＞G(11%)、Y356X(9%)和V399V(7%),其次是R111x(5%)、R413P(5%)、R252Q(3%)和A434D(3%).(2)北京地区PKU患儿的STR杂合度较高,共检测出8种等位基因,以240 bp(34%)和244 bp(44%)最为常见;而VNTR的杂合度较低,只检测到3种等位基因,以VNTR3(83%)最为常见.(3)由基因型预测的生化代谢表型与患儿实际的生化代谢表型之间一致率为81.5%,在经典型PKU中一致率达到87.5%.结论 (1)北京地区共有34种PAH基因突变,检出率为95%,以R243Q、EX6-69A＞G、Y356X和V399V突变为常见突变.(2)PAH突变基因的微单位(STR/VNTR)构成以240/3和244/3最为常见.(3)PKU患儿基因型与生化代谢表型之间存在较好的相关关系,一致率达到80%以上.     

Specific gene deletion in patients with cystic fibrosis: pilot study of a small patient cohort

Identification and molecular cloning of the cystic fibrosis (CF)-gene was a major progress in genetic counseling of families with one or more affected children. In caucasian families about 70% of the CF-patients show a homozygous or heterozygous deletion of one amino acid, that is phenylalanine at position 508 (delta F508). In a pilot study we examined the DNA of 14 CF-patients for F508 deletions. DNA was amplified by PCR and hybridized with a oligonucleotide-probe specific for the mutation containing CF-gene. Surprisingly all of the patients had a deletion of F508 of at least one allele (10 for both alleles, 4 for one allele). This method is of great importance for carrier-diagnosis. The finding of these or other deletions within the CF-gene may represent a prognostic marker for this disease.     

X-连锁迟发性脊椎骨骺发育不良的基因诊断研究

目的 探讨应用连锁分析和基因测序方法对X-连锁迟发性脊椎骨骺发育不良(Xinked spondylepiphyseal dysplasia tarda,SEDL)进行基因诊断的可行性。方法 利用与SEDL基因毗邻的微卫星遗传标记DXSl6多态性对一个SEDL大家系的21名成员进行连锁分析,确定与该家系SEDL致病基因连锁的DXSl6等位基因型,对家系中的年轻女性和年幼男性进行基因诊断。为探讨连锁分析诊断的准确性,随后对SEDL基因编码外显子3—6进行PCR扩增产物双向直接测序以确定导致该家系发病的SEDL基因突变型,通过检测致病基因对待诊对象进行直接诊断。结果 21名家系成员的连锁分析结果显示与致病基因连锁的DXSl6是D2等位基因,6位待诊对象中IVt4、IVl9、IV21、V7各有一个DXSl6D2等位基因,表明她们是携带者;而IV23和V4不具有与致病基因连锁的DXSl6等位基因,表明她(他)们是正常人。对21名成员的DNA测序证实该家系的致病突变是首次发现的SEDL基因第2内含子剪接受体处IVS2-2A→C突变。待诊对象IVl4、IVl9、IV21、V7携带该突变,IV23和V4基因型正常。基因测序和连锁分析的诊断结果完全一致。结论 连锁分析用于SEDL基因诊断简便、快速、花费少、与基因测序方法一样准确,解决了长期以来SEDL症状前患者和潜在女性携带者无法诊断的难题,有重要的临床价值。     

Value of Dna Analysis with Multiple Dna Probes for the Detection of Hemophilia a Carriers

Detection of hemophilia carriers is an important issue and should be addressed with great care. The allelic frequencies of three intragenic probes (Bcl I for probe p1 14.12, Xba I for probe p482.6, and Bgl I for probe C) and ow linked probe (Bgl II for probe DX 13) are reported, together with their diagnostic yield singly and in combination. In this series, 725 individuals (405 females) an 156 families were analyzed for restriction fragment-length polymorphisms. A total of 255 females (63%) were found to be information for their carrier state with one or more probes. The most informative intragenic probe was p482.6 (useful in 49% of informative females). The most informative probe was DX 13 (useful in 59% of informative females), but this k a linked probe that carries a 5% risk of cross-over. By the use of probes p1 14.12, p482.6, and DX 13, almost 98% of all the informative females could be detected. In about 71% of families with a family history and a known carrier, prenatal diagnosis was feasible.     

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??Hemophilia A??HA?? and HB is a X-link recessive hereditary hemorrhagic disorder with genetic characteristics of male suffering and female carrying. In this paper??we will focus on three aspects of hemophilia??including the genetic diagnosis??inhibitor of factors and pharmacokinetics. The method of gene diagnosis for hemophilia included DNA sequencing??PCR??DGGE??SSCP??etc. At present??the F?? intron 22 inversion analysis can be used as a clinical screening test for severe HA. Prenatal diagnosis is helpful to reduce the birth rate of hemophilia. The factor inhibitor causes invalid replacement therapy for hemophilia. Individual hemophilia patients have different pharmacokinetics??PK??. In addition to considering the bleeding event??the PK parameters should be considered as an important indicator for adjustment of individual preventive treatment. The relevant PK parameters include the half-life??clearance rate??in vivo recovery rate??IVR????the area under the curve??AUC????peak concentration??etc. The influencing factors for PK include age??body weight??blood type and von Willebrand factor??vWF?? level. The above progress contributes to individual adjustment of treatment strategy for patients with hemophilia.     

SHOX intragenic microsatellite analysis in patients with short stature

BACKGROUND: SHOX haplo-insufficiency is considered the molecular basis of short stature in patients with Turner's syndrome, and gives rise to the short stature with mesomelic dysplasia and Madelung deformity of patients with Leri-Weill syndrome. OBJECTIVE: Analysis of the intragenic SHOX microsatellite to define its utility in detecting SHOX haplo-insufficiency in patients with short stature. PATIENTS AND METHODS: 207 patients with short stature (57 girls with Turner's syndrome [TS] [24 mosaicisms]; 73 children with isolated short stature [ISS]; 77 patients with short stature and skeletal disproportion) and 30 control subjects. DNA extraction and PCR amplification of the intragenic SHOX microsatellite, at the 5'-untranslated region. SSCP and partial sequencing of the SHOX gene in one patient with Madelung deformity and two SHOX alleles. DXS1055 (Xp) and DXS1192 (Xq) microsatellites were also analyzed, together with DXS233 and DXS234 at 0 and 2 cM of the pseudoautosomal region (PAR), in patients with one SHOX allele. RESULTS: 1. 93% of patients with TS had a single SHOX allele, and allele unbalance was detected in the remainder. 2. Patients with ISS were not different from the normal population with respect to SHOX heterozygosity (0.92 and 0.93, respectively; p = 0.997). 3. Patients with short stature and skeletal disproportion showed a higher frequency of SHOX homo/hemizygosity (0.27 vs 0.08; p = 0.027). 4. Five patients with short stature with SHOX haplo-insufficiency were detected: three had Madelung deformity (inherited Yq;Xp translocation, de novo PAR deletion, and SHOX microdeletion), and two had de novo/inherited Xp partial monosomy. CONCLUSIONS: The SHOX intragenic microsatellite might be a useful molecular marker to detect TS (including Xp distal deletions). SHOX haplo-insufficiency seems not to be an important contributor to ISS, but when skeletal disproportion is associated with short stature, a significant proportion of patients is found to have a single SHOX allele. Some of these patients were found to be SHOX haplo-insufficient upon molecular, cytogenetic and radiological examination.