多重等位基因特异多聚合酶链反应产前基因在诊断β-珠蛋白生成障碍性贫血中的应用

目的 探讨多重等位基因特异多聚合酶链反应（PCR）产前基因诊断β-珠蛋白生成障碍性贫血的临床应用价值。方法 在B超监视下对21名孕妇行羊膜腔穿刺，抽取羊水20ml，常规酚-氯仿法提取DNA，应用多重等位基因特异PCR检测其羊水的细胞的β-珠蛋白的5种基因突变类型（CD17，CD41-42，IVS-Ⅱ654，ht-28,nt-29）。结果 检测的21例中，4例双重杂合子及1例纯合子，均选择流产。16例为正常或单个突变的杂合子，胎儿出生后取脐验证并随访观察，现小儿9个月-3.2岁，均健康。结论 多重等位基因特异PCR可用于β-珠蛋白生成障碍性贫血高风险胎儿的产前基因诊断，在指导β-珠蛋白生成障碍性贫血阳性家系的选择性具有一定的临床意义。     

Molecular Genetic Techniques for Prenatal Diagnosis*

Abstract Recently, molecular genetic techniques have been rapidly incorporated into routine clinical as well as laboratory medicine, and genetic diseases including inborn errors of metabolism have come to be diagnostically viewed as changes in DNA. Molecular genetic techniques have the advantage that rapid and accurate analysis can be made with a small amount of samples in comparison with analysis at the protein level by conventional biochemical techniques. They are, therefore, clinically used today not only for the diagnosis of various genetic diseases but also for the detection of infection, including viral infections, in the outpatient clinic. They have also been applied to fetal diagnosis, and are bringing about major changes in prenatal medicine.     

Non-invasive prenatal diagnosis and determination of fetal Rh status

RhD blood group incompatibility between a pregnant woman and her fetus can result in maternal alloimmunization and consequent haemolytic disease of the newborn (HDN) in subsequent pregnancies. The D-negative blood group is found in 15% of whites, 3-5% of black Africans, and is rare in Asians. Recent technological advances in non-invasive prenatal determination of the fetal RHD status using cell-free fetal DNA (cffDNA) have opened new avenues for the management of D-negative pregnant women. In this review applications for the high risk women, as well as potential for routine screening will be discussed. The use of non-invasive prenatal diagnosis and the management of other blood incompatibilities will also be discussed.     

Prenatal diagnosis of homozygous α°-thalassaemia by direct DNA analysis of chorionic villi in Singapore

Abstract First-trimester prenatal diagnosis by DNA analysis was carried out for seven pregnancies at risk for homozygous α°-thalassaemia. Transabdominal placental biopsy was carried out at 10–12 weeks'gestation. The presence of α-globin genes in the fetal DNA was determined by restriction endonuclease mapping and hybridization with cloned α-globin probe. Homozygous α°-thalassaemia was detected in two fetuses and the pregnancies were interrupted. α°-thalassaemiat in both cases was confirmed by electrophoresis of the umbilical cord blood where only haemoglobin Bart's was detected. The remaining five fetuses were diagnosed as normal or as possessing α°-thalassaemia-1 trait and the pregnancies are being carried to term. The use of DNA analysis in prenatal diagnosis of fetuses at risk for homozygous α°-thalassaemia enables detection of the haemoglobinopathy at 10 weeks'gestation.     

Non-invasive prenatal diagnosis: implications for antenatal diagnosis and management of high-risk pregnancies

There has been a huge effort in the last 2-3 decades to develop non-invasive prenatal diagnosis to avoid the risks to the fetus caused by invasive procedures. Obtaining fetal nucleic material for molecular analysis without the need of invasive procedures has been a goal of prenatal diagnosis for many years; this is now been made possible by the use of non-cellular fetal nucleic acids circulating in maternal blood. The placenta is the primary source of these nucleic acids, raising the possibility that they could be a marker for pregnancy complications resulting from placental disease/dysfunction such as pre-eclampsia and fetal growth restriction. If so, these markers might be able to identify cases at risk, predict disease and/or its severity or allow early diagnosis. This has the potential to allow improvements in the management of complicated pregnancies.     

肠道病毒感染的病原诊断

肠道病毒(EV)感染临床常见,但病原诊断一直是困扰临床医师的一个难题。随着病毒学研究的不断深入,EV感染的诊断方法也逐步完善。传统的EV分离和血清学鉴定技术繁杂费时,从临床标本中检测到EV的阳性率也较低,无法满足病毒感染暴发流行期间同时处理大量样本的需求。RT-PCR技术克服了以上缺点,已成为EV感染快速诊断的重要手段。     

Rapid,sensitive and specific diagnosis of Bordetella pertussis using the polymerase chain reaction

Use of a repetitive DNA sequence of Bordetella pertussis allowed successful detection of the organism by the polymerase chain reaction (PCR). The method was highly sensitive, being able to detect B. pertussis in specimens containing only a few cells. It was also highly specific, with no amplification of specimens containing other organisms, for example Haemophilus influenzae or Neisseria, being observed. A diagnosis could be made within 1 day. The PCR assay was also evaluated in clinical specimens. Among 47 nasopharyngeal specimens obtained from 24 patients with laboratory-confirmed pertussis, 27 were positive by PCR and 19 by culture. In particular, all three bronchial aspirates from one patient with pertussis were positive by PCR, but only one showed positive on culture. Eleven specimens from parapertussis patients and 65 specimens from patients without pertussis tested negative. It was concluded that this newly developed PCR method for the diagnosis of pertussis was more rapid and sensitive than the usual culture method. Polymerase chain reaction could have a major impact on the treatment and control of this infection and would be a useful tool for studying the pathogenesis of B. pertussis infection.     

产前诊断先天性胆总管囊肿37例临床分析

目的探讨产前诊断先天性胆总管囊肿患儿的临床治疗。方法将2006年9月至2013年2月收治的产前诊断为先天性胆总管囊肿患儿37例,按手术时年龄分为A组（0~3个月,20例）和B组（>3个月,17例）,回顾性分析两组患儿的临床特点、手术前后肝功能指标、术后并发症及肝脏组织病理检查结果等。结果 A组5例患儿出生后有黄疸,B组2例黄疸;两组丙氨酸转氨酶（ALT）和天门冬氨酸转氨酶（AST）差异无统计学意义（P均>0.05）,A组手术前、后总胆红素（TBIL）和直接胆红素（DBIL）水平均高于B组,差异有统计学意义（P均<0.05）;A组2例出现吻合口狭窄,1例胆漏,B组无术后并发症。肝脏活检提示胆汁性肝硬化改变11例,其中A组4例（36.36%）,B组7例（63.64%）,但两组肝硬化发生率差异无统计学意义（P=0.160）。结论对产前诊断为先天性胆总管囊肿患儿,应密切观察,发现黄疸、白便、ALT和AST升高明显、超声提示囊肿短期增大明显者应尽快手术治疗,以减轻肝功能损害,减少肝硬化发生。[临床儿科杂志,2013,31(9)：858-861]     

云南苯丙氨酸羟化酶基因内(TCTA)n多态性及其在苯丙酮尿症诊断中的应用

目的应用苯丙氨酸羟化酶（ＰＡＨ）基因内（ＴＣＴＡ）ｎ多态性连锁分析进行经典型苯丙酮尿症（ＰＫＵ）的基因诊断和产前诊断。方法应用聚合酶链反应扩增片段长度多态性（ＰＣＲＡｍｐＦＬＰ）方法,分析云南省１３个家系苯丙氨酸羟化酶（ＰＡＨ）基因内（ＴＣＴＡ）ｎ多态性。结果在１３个家系中检测到２２４～２５２ｂｐ的８种等位片段,其ＰＩＣ为０．６９８,杂合频率是５１％。可诊断率为１００％和５０％的家系各６个,１个家系因双亲带型为纯合型而未能诊断,可诊断率为６９％。完成１例产前基因诊断和２例回顾性的基因诊断。结论（ＴＣＴＡ）ｎ的ＰＣＲＡｍｐＦＬＰ分析可作为经典型ＰＫＵ基因诊断和产前诊断的一种简便有效的方法。     

Prenatal diagnosis of Gaucher disease using next‐generation sequencing

In the prenatal diagnosis of Gaucher disease (GD), glucocerebrosidase (GBA) activity is measured with fetal cells, and gene analysis is performed when pathogenic mutations in GBA are identified in advance. Herein is described prenatal diagnosis in a family in which two children had GD. Although prior genetic information for this GD family was not obtained, next‐generation sequencing (NGS) was carried out for this family because immediate prenatal diagnosis was necessary. Three mutations were identified in this GD family. The father had one mutation in intron 3 (IVS2 + 1), the mother had two mutations in exons 3 (I[‐20]V) and 5 (M85T), and child 1 had all three of these mutations; child 3 had none of these mutations. On NGS the present fetus (child 3) was not a carrier of GD‐related mutations. NGS may facilitate early detection and treatment before disease onset.     

Prenatal Diagnosis of Duchenne Muscular Dystrophy (DMD) by the Polymerase Chain Reaction (PCR)

The diagnosis of Duchenne muscular dystrophy (DMD) has been drastically improved by recent advances in DNA analysis. The Southern blot hybridization using the cDNA 8 probe and the restriction enzyme Hind III was conducted in a gravida and her family in blood samples. The diagnosis revealed partial gene deletions in both the gravida and the DMD-affected second child. The prenatal diagnosis was performed by studying the PCR (polymerase chain reaction) for target DNAs of exons 48 and 51 that correspond with cDNA 8 probe. In the affected child, the 506 bp band at exon 48 was detected but 388 bp at exon 51 was missing. On the other hand, both the 506 bp band at exon 48 and the 388 bp band at exon 51 were detected in the cultured amniotic cells. Thus, the fetus was determined to be not affected.     

Clinical applications of real-time PCR for diagnosis and treatment of human cytomegalovirus infection in children

There are many methods of detecting human cytomegalovirus (HCMV) infection. So far, the quantitative polymerase chain reaction (PCR) has been very useful not only in aiding in the diagnosis of HCMV but also in determining the severity and predicting HCMV infection. However, it is time-consuming and labor intensive. Real-time PCR (RT-PCR) is an exception, for it allows rapid quantification of HCMV DNA load. Our group used this method for detecting and monitoring HCMV and compared it with the diagnostic criterion recommended by the Pediatric Branch of Chinese Medical Association, in 45 children suspected of having HCMV infection. The response to two types of antiviral treatment on HCMV DNA load was also monitored in HCMV hepatitis cases. RT-PCR was positive in 30 cases while the diagnostic criterion, which includes enzyme-linked immunosorbent assay (ELISA) and/or conventional PCR, was positive in 32 cases. The decrease in the HCMV DNA load was achieved earlier in the modified treatment group compared with the conventional treatment group. A 10(3) copies/ml of HCMV DNA load of is a useful cut-off value in predicting patients who will have symptoms of the disease. RT-PCR can be used not only in detecting HCMV but also in monitoring response to antiviral treatment and risk of having symptoms of the disease.     

Congenital chloride diarrhoea: a prenatal differential diagnosis of small bowel atresia

We compared ultrasound findings and pre- and postnatal clinical signs in 8 patients with congenital chloride diarrhoea and 14 with small bowel atresia diagnosed in 1977–1991 in order to evaluate the possibility of a prenatal distinguishing diagnostic sign. In the patients with congenital chloride diarrhoea the pregnancy was complicated by marked polyhydramnios, the symphysis-fundus distance exceeded +2 SD before gestational week 31 and the fetus displayed normal peristalsis in extensively dilated intestines and the "frog position". In the patients with small bowel atresia the symphysis-fundus distance was normal before gestational week 31 and the fetus displayed increased peristalsis in a few dilated intestinal loops.     

Detection of congenital cytomegalovirus infection in Chinese newborn infants using polymerase chain reaction

Polymerase chain reaction (PCR) amplification was used to detect cytomegalovirus (CMV) in 1000 urine specimens from Chinese newborns for defining the incidence of congenital CMV infection in the Chinese population. The major immediate-early and the late antigen genes of CMV were amplified and detected by gel electrophoresis. There were 18 congenitally infected infants found when tests were performed with one or both primer pairs. Comparing with tissue culture, PCR of both primer sets provided a sensitivity of 94%, a specificity of 100% and a predictive value of positive result of 100%.     

利用孕妇外周血浆小片段游离胎儿DNA检测Y染色体性别决定区基因

目的探索从孕妇外周血浆中提取小片段游离胎儿DNA（cffDNA）提高检测胎儿Y染色体性别决定区（SRY）基因准确率的方法,评估利用小片段cffDNA进行无创性产前诊断可行性。方法收集117例孕妇外周血,利用柱吸收方法提取其血浆cffDNA,经琼脂糖凝胶电泳分离富集小片段cffDNA,使用二重PCR反应（dulex-PCR）检测SRY基因和磷酸甘油醛脱氢酶（GAPDH）基因。结果来源于66例孕男胎孕妇血浆标本小片段cffDNA均检出SRY和GAPDH基因,来源于50例孕女胎孕妇血浆标本小片段cffDNA仅检出GAPDH基因。与绒毛/羊水标本检测结果相符。特异性和敏感性均为100%。结论利用琼脂糖凝胶电泳,切胶回收,可选择性富集孕妇外周血小片段cffDNA,相对提高胎儿DNA水平,结合二重PCR扩增SRY基因技术可用于无创性产前性连锁遗传疾病和单基因突变疾病产前诊断。     

Acrania/encephalocele sequence (exencephaly) associated with 92,XXXX karyotype: Early prenatal diagnosis at 9+5 weeks by 3D transvaginal ultrasound and coelocentesis

A 27-year-old pregnant woman was diagnosed by 3D transvaginal ultrasound as carrying a fetus of 9⁺⁵ weeks gestation affected by acrania/encephalocele (exencephaly) sequence. A 2D transvaginal ultrasound-guided aspiration of 5 mL of extra-coelomic fluid was performed under cervical block before uterine suction. Conventional cytogenetic analysis demonstrated a 92,XXXX karyotype. Transvaginal 2D ultrasound-guided coelocentesis for rapid karyotyping can be proposed to women who are near to miscarriage or in cases where a prenatal ultrasound diagnosis of congenital anomaly is performed at an early stage of development. Genetic analysis can be performed using traditional cytogenetic analysis or can be aided by fluorescence in situ hybridization (FISH). Coelocentesis may become an integral part of first trimester armamentarium and may be clinically useful in the understanding of the pathogenesis of early prenatally diagnosed congenital anomalies.     

Genetic analysis of cystic fibrosis in Denmark. Implications for genetic counselling, carrier diagnosis and prenatal diagnosis

Cystic fibrosis is the most common, severe, inherited disease in the Caucasian population. As a consequence, the demand for genetic counselling of patients with cystic fibrosis and their families is large. In Denmark the incidence of cystic fibrosis is 1:4700, which is quite low compared to other European countries. We have investigated 268 Danish cystic fibrosis patients with respect to DNA markers (haplotypes) and the most common mutation delta F508. The delta F508 mutation is found on 88% of all cystic fibrosis chromosomes, the highest frequency reported so far. This had had an important impact on genetic counselling, prenatal diagnosis and eventually population screening. In the Danish population 78% of all couples at risk will be informative for delta F508 and will be identifiable by simple screening methods.     

Letter to the editor: Ifosfamide nephrotoxicity in children

An 11-year-old boy with prior bone marrow and testicular relapses of his acute lymphoblastic leukemia (ALL) developed an isolated metatarsal bone relapse during complete hematologic remission 10 months after completion of chemotherapy. Although there was no radiographic or histologic evidence of additional occult leukemia, the polymerase chain reaction (PCR) technique detected a leukemic clone in both his bone marrow and metatarsal. A literature survey revealed only 10 reported cases of isolated bone relapse occurring during complete bone marrow remission in childhood ALL. Most of these patients had prior bone marrow or extramedullary relapses. The majority experienced subsequent relapses after their isolated bone recurrence. We report a case of isolated bone recurrence, review all previously reported cases, and suggest that PCR elucidation of clonal disease may provide a better understanding of these extremely rare extramedullary events. © 1994 Wiley-Liss, Inc.