Molecular prenatal diagnosis of ataxia telangiectasia heterozygosity by direct mutational assays.

Ataxia telangiectasia (AT) is a severe autosomal recessive disease, rare but not infrequent in Italy. Owing to the seriousness of the disease, prenatal diagnosis has been attempted in the past by means of cytogenetic, biochemical, radio-biological and indirect molecular analyses. We performed the first direct molecular prenatal diagnosis of AT on a chorionic villi sample from a 37-year-old woman at the 10th week of pregnancy. She had two previous children suffering AT and two induced abortions. At molecular analysis her affected children were compound heterozygotes for mutations 7792C-->T in exon 55 (from the mother) and 8283delTC in exon 59 (from the father). The prenatal diagnosis was performed by two different operators in double-blind form. Mutation 7792C-->T was studied by restriction enzyme analysis using TaqI. Mutation 8283delTC was screened by heteroduplex analysis. The fetus was heterozygous for the mutation 7792C-->T (confirmed by sequencing). In order to verify the possible contamination by maternal DNA, polymorphic loci HLA-DRB1 and HLA-DQA1, together with microsatellite markers D6S259, D11S2000, D11S29, D11S1778 and D11S2179, were examined. All these loci were informative, showing that the fetus received only one allele from each parent. The heterozygosity for ATM mutation 7792C-->T was confirmed by molecular studies after the birth of a healthy male baby.     

Prenatal molecular diagnosis of Wiskott-Aldrich syndrome by direct mutation analysis

We have performed prenatal diagnosis for Wiskott Aldrich syndrome (WAS) in two unrelated families by direct gene analysis. Using a combined non-radioactive analysis of single-strand conformational polymorphism (SSCP) and heteroduplex formation (HD), followed by automated sequencing, we studied DNA from chorionic villus sampling (CVS), allowing the diagnosis of one affected and one healthy male at the 12th week of gestation.     

先天性软骨发育不全的产前快速基因诊断

目的 探讨先天性软骨发育不全(ACH)的产前快速基因诊断方法.方法 选择2007年5～11月南京大学医学院附属鼓楼医院妇产科产前诊断中心收治的3个ACH家系:其中家系1为年龄6个月的ACH患儿;家系2为妊娠18周的孕妇,待采集胎儿羊水细胞进行产前诊断;家系3为孕39周经超声诊断为ACH的胎儿,待采集脐带血进行诊断.同时采用限制性内切酶(Sfc Ⅰ和MspⅠ)酶切、变性高效液相色谱(DHPLC)分析及序列分析3种检测方法,对外周静脉血或脐血中的成纤维细胞生长因子受体3(FGFR3)基因进行突变检测.结果 (1)DHPLC分析:家系1患者的色谱分析结果为异源双链杂合峰,家系2患者和胎儿的结果均为异源双峰,家系3胎儿的结果为异源双峰,正常对照的结果为同源单峰.(2)FGFR3基因10号外显子PCR产物酶切前的聚丙烯酰胺凝胶电泳检测结果:家系1患者、家系2患者及胎儿的PCR产物经Sfc Ⅰ酶切后除有247 bp条带外,还产生162和85 bp两个酶切产物条带,但不能被Msp Ⅰ酶切,且只有247 bp 1条产物条带.家系3胎儿PCR产物不能被Sfc Ⅰ酶切,但经Msp Ⅰ酶切后可出现162和85 bp两个产物条带.正常对照PCR产物均不能被SfcⅠ和Msp Ⅰ酶切.(3)PCR产物测序结果:家系1患者PCR产物经测序发现FGFR3基因1138G→A杂合子突变,家系2患者及胎儿也均为FGFR3基因1138 G→A杂合子突变,家系3胎儿为FGFR3基因1138 G→C杂合子突变,正常对照在FGFR3基因1138位点为G纯合子.家系2胎儿以上各项检测结果均与家系2患者相同,是遗传母亲致病突变基因的结果.结论 DHPLC和限制性内切酶酶切分析均能准确检测致病突变的FGFR3基因,但DHPLC技术操作方便快捷、准确,而且敏感性更高,可在临床上推广用于ACH的产前快速基因诊断.     

Rapid interphase analysis for prenatal diagnosis of translocation carriers using subtelomeric probes

Interphase fluorescence in situ hybridization (FISH) has become an accepted laboratory technique for the rapid and preliminary prenatal assessment of chromosome aneuploidy. The introduction of subtelomeric FISH probes now allows for the molecular-cytogenetic analysis of terminal chromosome rearrangements. In a prospective study, we examined the prenatal use of subtelomeric probes on interphase cells to rapidly detect the carrier status of a fetus when a parent carried a known reciprocal or Robertsonian chromosome translocation. Three of the cases were identified as being abnormal. All cases were confirmed by routine cytogenetic analysis. These findings clearly demonstrated the utility of this technique and these probes to rapidly and correctly identify balanced and unbalanced chromosome anomalies of a fetus that could result from parental translocations.     

Status of prenatal diagnosis using direct interventions on the fetus

Prenatal diagnosis has developed into a large flourishing multidisciplinary branch of medicine. At present prenatal diagnosis is world-wide possible in the second trimester of pregnancy by analysis of both amniotic fluid obtained by amniocentesis or amniotic cell cultures. Ultrasound plays a central role in prenatal diagnosis, and has revolutionized obstetric practice. Without dealing with all aspects of prenatal diagnosis we have discussed the following invasive procedures, which give the prenatal diagnosis its forward thrust: fetal blood sampling including intravascular transfusion, fetal skin-, liver- and muscle biopsy, chorion biopsy, selective birth in twin pregnancies, and fetal therapy including fetal surgery. In utero surgery is in its earliest stages. Such simple procedures as decompression of the hydronephrotic kidney in obstructive uropathy, of the fetal lungs in pleural effusions caused by chylothorax, or of the hydrocephalic ventricle may be useful, but the possible success rate for such efforts remains still uncertain. The possibilities of first-trimester prenatal diagnosis especially by DNA-technology or direct chromosomal and biochemical analysis has stimulated the development of a multiplicity of methods for taking chorion biopsies. Compared with second trimester amniocentesis, the introduction of prenatal diagnosis by chorionic villi sampling would reduce the psychic trauma for patients waiting on results and allow earlier and safer termination of pregnancy.     

Cystic fibrosis prenatal diagnosis: confirmation of an equivocal microvillar enzyme result by direct analysis of the common gene mutation

A child was tentatively diagnosed as having cystic fibrosis, based on neonatal presentation with severe gastrointestinal complications; the diagnosis was not confirmed biochemically and no tissues were available for DNA analysis. The mother presented in her subsequent pregnancy, and microvillar enzyme analysis of cell-free amniotic fluid at both 18 and 20 weeks gestation gave equivocal results. The pregnancy was terminated voluntarily because of a trend towards abnormal enzyme assay results on the second amniocentesis. Retrospectively, fetal tissues were found to be homozygous for the most common mutation of the cystic fibrosis gene (delta F508), which confirmed the prenatal assessment and suggested that the first infant of the couple was probably also affected by the disease.     

荧光定量PCR产前快速诊断唐氏综合征可行性研究

目的建立快速高效的产前诊断唐氏综合征的分子生物学方法。方法分别在21号染色体上选取7个微卫星重复序列(SmallTandemRepeat,STR)(D21S1433,D21S1442,D21S1444,D21S1411,D21S1412,D21S1413,D21S1414)作为遗传标记,利用荧光定量PCR(QF-PCR)扩增技术及片段分析技术,对250例羊水标本进行检测,并与羊水标本染色体核型分析结果进行对比。结果250例羊水标本中,核型分析发现24例21三体,2例性染色体数目异常,24例唐氏综合征样本采用QF-PCR全部检出。224例正常羊水标本中,QF-PCR检出阴性标本223例,1例样本呈假阳性,假阳性率为0.4%,24例21三体标本中,七对引物同时检测诊断阳性率为100%。所有试验结果均在24h内得出。结论QF-PCR作为一种快速、准确、高效的分子生物学方法,对诊断唐氏综合征具有重要意义。     

家系分析在产前筛查诊断中的价值

家系分析有助于临床医生了解疾病的传递方式、分析检测结果,进而为家系其他成员提供检测建议。目前遗传学检测技术不断进步并应用于临床,可能发现大量“阳性”结果,更需要重视家系分析在致病性判断中的价值。     

Improved direct molecular diagnosis and rapid fetal sexing

Adaptations of the techniques of modern molecular biology to prenatal diagnosis has opened new avenues for the detection of genetic diseases. We have taken advantage of the rapid adhesion of colony forming cells in cultured amniotic fluid samples to develop an improved method for molecular diagnosis. By employing the cell adherence regime sickle cell diagnosis using Mst II can be undertaken directly. In addition, hybridization with a cloned repetitive sequence that is of Y origin and has limited autosomal homology permits rapid fetal sexing in 3 to 4 days without compromising conventional cytogenetic or biochemical analysis. This combination of techniques provides a useful adjunct to convential prenatal genetic diagnosis in the second trimester.     

Rapid method for targeted prenatal diagnosis of Duchenne muscular dystrophy in Vietnam

ObjectiveSince there is no effective curative treatment for Duchenne muscular dystrophy (DMD), prevention mostly depends on genetic counseling and prenatal diagnosis. About two-thirds of the affected patients have large deletions or duplications, which can be detected by multiplex ligation-dependent amplification (MLPA). The remaining cases include small mutations, which cannot be easily identified by routine techniques. In such cases, linkage analysis may be a useful tool for prenatal diagnosis. Here we compared results obtained from linkage using short tandem repeats (STRs) with those by MLPA and sequencing analysis.Materials and methodsEight Vietnamese pregnant women at risk of having a baby with DMD and requesting prenatal diagnosis were recruited in this study. MLPA and direct sequencing were applied to screen large rearrangements and point mutations in the dystrophin gene in the DMD probands and the fetal samples. STR linkage was also performed to analyze fetal mutation status.ResultsBy MLPA and sequencing analysis, five DMD patients showed deletions of the dystrophin gene, and no deletions of exons were detected in seven amniotic fluid cell samples; one patient harbored the out-of-frame small deletion of exon 43, which was also found in the fetal sample of this family. STR analysis revealed the transmission of a mutant allele inside each family.ConclusionOur results suggest that the combination of STR and MLPA could be a rapid, reliable, and affordable detection protocol for determination of the carrier's status and prenatal diagnosis of DMD in a developing country such as Vietnam.     

Prenatal diagnosis of Fabry's disease by direct analysis of chorionic villi

Six pregnancies of three carriers for X-linked Fabry's disease, were monitored by chromosome and enzyme analysis. Two affected male fetuses were detected by the demonstration of alpha-galactosidase deficiency in amniotic fluid cells and chorionic villi respectively. The use of chorionic villi enabled a diagnosis within a few hours after sampling in the ninth week of pregnancy whereas the use of amniotic fluid cells in the earlier case required two weeks of culturing after amniocentesis in the 16th week. Four female fetuses were found; heterozygosity was demonstrated in one by analysis of clones in the primary amniotic fluid cell culture.