Prenatal detection of a cystic fibrosis mutation in fetal DNA from maternal plasma

OBJECTIVES: Maternal plasma and serum are being used to detect fetal DNA by PCR in order to determine certain conditions such as fetal gender and RhD without invasive procedures. Because of the presence of maternal DNA in plasma, these approaches are limited to paternally inherited disorders or those de novo present in the fetus. We have assessed the possibility of performing the detection of a single-gene disorder such as a fetal paternally inherited Cystic Fibrosis mutation (Q890X) in maternal plasma. METHODS: The analysis was performed at 13 weeks of gestation using DNA extracted from maternal plasma. We used a PCR amplification of the Q890X mutation and a posterior restriction analysis of the PCR product. RESULTS: We were able to detect the presence of the mutation and thus the fetal condition of being a carrier of the paternal mutation. CONCLUSIONS: We have made evident the possibility of detecting an inherited paternal mutation in a non-invasive way at the 13t(hr) weeks of pregnancy. This methodology could be very useful in cases of paternally inherited dominant disorders. The technical improvements in fetal DNA detection and analysis might lead to the development of new applications in the non-invasive prenatal diagnosis field.     

Prenatal diagnosis of glycogen storage disease type IV using PCR-based DNA mutation analysis.

Deficiency of glycogen branching enzyme activity causes glycogen storage disease type IV (GSD-IV). Clinically, GSD-IV has variable clinical presentations ranging from a fatal neonatal neuromuscular disease, to a progressive liver cirrhosis form, and to a milder liver disease without progression. Current methods for prenatal and postnatal diagnosis are based on an indirect method of measuring the enzyme activity, which has a limited sensitivity and cannot be used to distinguish patients with these variable clinical phenotypes. In this study, a GSD-IV family with a non-progressive hepatic form of the disease requested prenatal diagnosis. Determination of the branching enzyme activity in cultivated amniocytes showed 20 per cent residual activity overlapping with the level detected in the heterozygotes. Mutation analysis revealed that the fetus carried two mutant alleles, L224P and Y329S, the same as the proband of this family. The fetus was predicted to be affected and postnatally his clinical presentation is consistent with the diagnosis. We conclude that DNA mutation analysis should be used in the prenatal diagnosis of GSD-IV, especially in the situation of high residual enzyme activity.     

Late onset adrenal hyperplasia: mutation at codon 282 of the functional 21-hydroxylase gene is not ubiquitous

Ten patients affected with 21-hydroxylase (21-OH) deficient late-onset adrenal hyperplasia were studied to determine the prevalence of a mutation at codon 281 of the functional 21-OH gene (CYP21B) that results in a valine to leucine amino acid shift. This mutation has been reported in eight unrelated late-onset adrenal hyperplasia patients of Ashkenazi Jewish descent possessing the human leukocyte antigen-B14,DR1 haplotype. Normally, there are two 21-OH genes; a pseudogene (CYP21A) and a functional gene (CYP21B). The aberrant codon 281 sequence is normally present only in CYP21A. In all of our late-onset adrenal hyperplasia patients, hybridization of an oligonucleotide probe specific for this mutation was demonstrated to CYP21A but not to CYP21B. The mutation at codon 281 of CYP21B does not appear to be a ubiquitous genetic marker for 21-OH deficient late-onset adrenal hyperplasia, suggesting that this disorder may demonstrate the same molecular heterogeneity as congenital adrenal hyperplasia.     

Prenatal diagnosis of autosomal dominant hereditary spastic paraplegia (SPG4) using direct mutation detection

OBJECTIVE: To present a report on prenatal diagnosis using direct SPG4 gene analysis in a family with autosomal dominant hereditary spastic paraplegia (AD-HSP). METHODS: Genetic linkage and haplotype analysis were previously carried out with chromosome 2p markers. DNA was obtained from affected individuals, the affected father, the mother, and fetal DNA from an ongoing pregnancy by chorionic villus sampling (CVS) in the first trimester. The spastin gene (SPG4) was completely sequenced. RESULTS: A novel 832insGdelAA frameshift mutation, predicted to cause loss of functional protein, was identified in the affected father and in the fetal DNA. CONCLUSIONS: This is the first report on direct prenatal diagnosis of chromosome 2p-linked AD-HSP (SPG4). In addition, we report a novel SPG4-combined small insertion/deletion mutation in exon 5, which may be the first SPG4 mutational hot spot.     

Prenatal diagnosis for a novel homozygous mutation in PKLR gene in an Indian family

OBJECTIVE: To provide prenatal diagnosis of pyruvate kinase deficiency by direct DNA analysis in an Indian family. MATERIALS AND METHOD: This case report describes diagnosis of a novel homozygous mutation in PKLR gene that subsequently helped the family in the next pregnancy. RESULTS: Advancement in molecular genetics has resulted in the prenatal diagnosis of relatively uncommon genetic disorders like pyruvate kinase deficiency. CONCLUSION: This case reiterates the importance of application of molecular genetics in clinical practice and prenatal diagnosis especially for rare, incurable genetic disorders.     

Prenatal diagnosis of factor X deficiency using a combination of direct mutation detection and linkage analysis with an intragenic single nucleotide polymorphism

OBJECTIVE: To present a family in which it was possible to perform prenatal diagnosis for recessively inherited factor X deficiency using both direct mutation detection as well as linkage analysis. METHODS: In a family where both parents were known to be carriers of factor X deficiency, fetal DNA was obtained from an ongoing pregnancy by CVS in the first trimester. Direct DNA sequencing was used to detect a previously identified factor X mutation, and linkage analysis using a single nucleotide polymorphism (SNP) was used to follow the segregation of the other parent's factor X alleles. RESULTS: Our studies predicted the fetus in question to be a heterozygote carrier of factor X deficiency, and this was demonstrated to be correct at birth. CONCLUSIONS: This is the first reported case of prenatal diagnosis of factor X deficiency. In addition, this case demonstrates the remarkable utility of SNPs in linkage analysis of rare genetic disorders.     

Prenatal diagnosis of X-linked hyper-IGM syndrome by direct detection of mutation Q220X in the CD40L gene using PCR-mediated site directed mutagenesis

We present the first report of prenatal diagnosis of X-linked hyper-IgM syndrome by PCR-mediated site directed mutagenesis (PSM) in a woman known to carry the Q220X mutation in the CD40L gene. Using the simple PSM assay, the Q220X mutation was identified by chorionic villous sampling (CVS) at 11 weeks' gestation and the pregnancy was terminated.     

Prenatal diagnosis of Roberts syndrome and detection of an ESCO2 frameshift mutation in a Pakistani family

OBJECTIVES: We report two siblings with Roberts syndrome (RBS), and an attempt to delineate the underlying molecular mechanism leading to familial recurrence. METHODS: Cytogenetic studies and direct sequencing of the ESCO2 gene were carried out in the second affected fetus and the parents. Fetal DNA was obtained from amniocytes after amniocentesis. Parental DNA was obtained from peripheral blood samples. RESULTS: Cytogenetic analysis of amniocytes revealed a normal male karyotype in 20 analyzed metaphases and chromosomal aneuploidies in 10 metaphases. All metaphases displayed premature separation of centromeres and puffing of heterochromatic regions near the centromere. A homozygous mutation leading to a frameshift in ESCO2 was identified in the fetal DNA sample. Both parents are heterozygous carriers of the same mutation. CONCLUSION: The present case demonstrates the prenatal diagnosis of RBS associated with a frameshift mutation in ESCO2.     

Prenatal diagnosis of a rhodopsin mutation using chemical cleavage of the mismatch

OBJECTIVE: Mutations of the rhodopsin gene are responsible for autosomal dominant or recessive retinitis pigmentosa (RP). The present study reports the first prenatal diagnosis performed on chorionic villi biopsy of a pregnant woman affected by a severe form of autosomal dominant transmitted RP, due to the Arg135Trp substitution. METHODS: The rhodopsin gene was analysed by automated direct sequencing and, for the first time, by fluorescence-assisted mismatch analysis (FAMA). The latter is an inexpensive, rapid and particularly sensitive method, based on the chemical cleavage of the mismatch in heteroduplex DNA molecules marked with strand-specific fluorophores. RESULTS: FAMA is a feasible procedure for prenatal molecular diagnosis of rhodopsin mutations. The redundancy of signals obtained by FAMA and its sensitivity make it suitable for identifying exactly the position of the mutation and the nucleotide substitution. CONCLUSIONS: An association is proposed between FAMA and automated direct sequencing procedures, in order to achieve optimal results in terms of reliability for prenatal diagnosis of rhodopsin mutations.     

米非司酮不敏感妇女孕激素受体基因点突变的研究

目的了解米非司酮不敏感妇女中是否存在孕激素受体基因点突变。方法以１１例药物流产后继续妊娠和５例胚胎停育者为研究对象,随机选择１０例药物完全流产的妇女为对照组,采集其蜕膜组织,提取ＲＮＡ,进行逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）、单链构型多态性分析（ＳＳＣＰ）和限制性内切酶ＨｉｎｆＩ的酶切反应。并对所有研究对象进行临床随访。结果对照组除１例有痕迹量的孕激素受体基因点突变外,其余９例均无点突变存在。继续妊娠的１１例中,７例存在孕激素受体基因点突变,其中２例在黄体中期给予口服米非司酮后无二次阴道流血;另４例的孕激素受体基因无点突变存在,其中３例分别在６个月内再次妊娠,药物流产成功,１例于黄体中期单次口服米非司酮出现二次阴道流血。胚胎停育的５例中,孕激素受体基因均无点突变存在。结论米非司酮不敏感妇女中存在孕激素受体基因点突变     

Prenatal diagnosis of cystic fibrosis using linked DNA probes

This paper presents data collected in Europe on 107 prenatal diagnoses of cystic fibrosis (CF) using linked DNA markers. To date, 38 children have been born without CF, as predicted, demonstrating the present rapid move from research to clinical genetic service.     

Prenatal detection of fetal hemoglobin E gene from maternal plasma

In order to provide a noninvasive prenatal diagnosis of the hemoglobin E (Hb E) related disorder, we have evaluated the possibility of identifying the fetal beta(E)-globin gene in maternal plasma. The analysis was performed during 8 to 18 weeks of gestation using DNA extracted from 200 micro L of plasma from pregnant women whose husbands carried Hb E. The beta(E)-globin mutation in maternal plasma was detected by a nested PCR amplification followed by the Mnl I restriction analysis. The result was compared with that of routine analysis of the CVS specimens. Among the five pregnant women examined, the fetal beta(E)-globin gene was identified in maternal plasma in three of them and the result was completely concordant with the conventional CVS analysis. This simple noninvasive prenatal detection of the fetal beta(E)-globin gene should prove useful in a prevention and control program of Hb E/beta-thalassemia in countries where the beta(E)-globin gene is prevalent.     

hTERC基因表达在宫颈癌筛查中的意义

目的:探讨宫颈病变脱落细胞中人类染色体端粒酶基因(hTERC)的表达及hTERC基因检测在宫颈癌筛查中的临床意义。方法:收集宫颈病变患者的宫颈脱落细胞标本129例,依据组织病理学结果分为3组:宫颈上皮内瘤变低度病变组(CINⅠ)、高度病变组(CINⅡ/Ⅲ)、宫颈癌组(SCC);同时选择健康妇女的宫颈脱落细胞标本20例作为对照组建立实验室阈值;应用荧光原位杂交技术(FISH)检测两组标本hTERC基因的扩增情况,最终以病理学诊断为金标准,比较FISH检测结果。结果:研究各组中CINⅠ,CINⅡ/Ⅲ,SCC的hTERC基因阳性表达率分别为7.31%,56.25%,75.00%,hTERC基因扩增比例随病变进展而增加;研究组与对照组hTERC基因阳性表达率差异有统计学意义(P0.05);研究各组两两比较,hTERC基因阳性表达率低度病变组与高度病变组、宫颈癌组差异有统计学意义(P0.05),而高度病变组与宫颈癌组的差异无统计学意义(P0.05)。结论:FISH方法检测hTERC基因可用于筛查宫颈高度病变及宫颈癌,其阳性扩增信号多倍体型可能是宫颈病变进展的预测性指标。     

Prenatal exclusion of Leigh syndrome due to T8993C mutation in the mitochondrial DNA

Leigh syndrome (LS) is a mitochondrial encephalopathy that is caused by a mutation either in the mitochondrial DNA (mtDNA) or in the nuclear encoded genes of the mitochondrial proteins. Prenatal diagnosis of defects in the mtDNA is usually problematic because of mtDNA heteroplasmy and tissue specificity. However, the mutations T8993 G/C in the ATP synthase subunit 6 gene of the mtDNA show a more even tissue distribution and do not appear to change significantly over time. There are only few reports of prenatal diagnosis of the T8993G mutation in Leigh disease. Here we describe the first prenatal genetic testing of T8993C in a fetus of a mother whose previous child had died of Leigh syndrome due to the T8993C mutation. Mutant load in the chorionic villus sample (CVS) as well as in amniocytes was undetectable, thus predicting a very high likelihood of an unaffected outcome, indicative of a healthy baby. The diagnosis was confirmed after birth. Gathering data on the prenatal diagnosis of mtDNA mutations is of great importance so that prenatal diagnosis of both T8993G and T8993C mutations can be offered routinely.     

学语前性耳聋疾病相关基因的产前诊断

目的确定耳聋疾病的相关基因。方法从1例曾生育过一先天性耳聋男孩和一正常男孩、现孕21周的孕妇及其家庭成员的外周血和胎儿的羊水细胞中抽提基因组DNA,先对此家系中的耳聋先证者（为此孕妇的第1个儿子）进行GJB2和SLC26A4基因的突变检测,然后对家庭中其他成员（包括胎儿）的相关位点进行分析。结果先证者为SLC26A4基因的IVS7—2A〉G突变纯合子,GJB2基因的79G〉A（V29I）、341A〉G（E114G）、608T〉C（1203T）3种杂合改变;夫妇二人和其第2个儿子为IVS7-2A〉G突变杂合子;胎儿为IVS7—2A〉G野生型纯合子。结论GJB2和SLC26A4基因突变是先天性耳聋疾病的相关基因,通过产前诊断的方法可以预防先天性耳聋患儿的出生。     

Prenatal detection of complex chromosomal aberrations using advanced molecular cytogenetic techniques

OBJECTIVE: This study aimed to identify a marker chromosome and characterize the short arm of a derivative chromosome 5 in a foetus with the following karyotype: mos 47,XX,del(5)(p?),+i(5)(p10)[50]/48,XX,del(5)(p?),+i(5)(p10),+mar[25]. METHOD: Amniocentesis was performed in the 26th week of pregnancy because of ultrasound abnormalities (polyhydramnion and decreased amount of gastric filling). All classic banding techniques were performed. FISH and microdissection combined with reverse painting were used to reveal the exact origin of the marker and any extra material on the deleted chromosome 5p. The parents decided to continue the pregnancy and we compared the clinical features of the child born in week 34 with data from the literature on trisomy 5p. The possible contribution of trisomy of the centromeric region of chromosome 8 and trisomy 8p23.3-->8pter to this clinical picture was evaluated. RESULTS: GTG banding showed one normal and two aberrant chromosomes 5 [del(5)(p?) and i(5)(p10)] in all the cells examined. Furthermore, a supernumerary marker chromosome was present in approximately 30% of the cells. The marker was CBG positive and positive with the pancentromere probe, but dystamicinA/DAPI negative. It did not contain NOR-positive satellites. FISH proved this marker to be derived from the centromeric region of chromosome 8. MicroFISH disclosed the aberrant chromosome 5 as der(5)t(5;8)(p10;p23.3). The parent's karyotypes were normal. The baby showed the characteristic features of trisomy 5p syndrome. She died at the age of 15 days after cardiorespiratory arrest. CONCLUSION: The karyotype was interpreted as mos 47,XX,add(5)(p10).rev ish der(5)t(5;8)(p10;p23.3),+i(5)(p10) (WCP5+,D5S23+)[50]/48,XX,add(5)(p10).rev ish der(5)t(5;8)(p10;p23.3),+i(5)(p10)(WCP5+,D5S23+),+mar.ish 8(p10q10)(D8Z2+,WCP8-)[25]. Therefore, the baby had complete trisomy 5p, with trisomy of the distal part of 8p and of the centromeric region of chromosome 8. The clinical significance of de novo marker chromosomes is a major problem in prenatal counselling. Molecular cytogenetic tools such as FISH and microFISH are indispensable for characterizing markers and determining the breakpoints more precisely in deleted chromosomes.