Prenatal diagnosis of β-thalassaemia by second-trimester chorionic villus sampling

In this study we evaluated the feasibility of second-trimester transabdominal chorionic villus sampling for prenatal diagnosis of β-thalassaemia in 80 pregnancies at risk presenting in the second trimester at the Antenatal Service. Sampling was carried out from 13 to 20 weeks and was successful in all cases. The amount of chorionic villi obtained varied from 10 to 40 mg, which was sufficient to make fetal diagnosis by oligonucleotide analysis within 10 days from sampling in all cases. No fetal losses occurred. From these results we conclude that transabdominal chorionic villus sampling is a useful procedure for prenatal diagnosis of β-thalassaemia in those couples presenting after the first trimester.     

Prenatal diagnosis of Huntington's disease (HD): Experiences with six cases and PCR

In the course of a 2-year predictive testing programme for Huntington's disease (HD), six couples from a total of 52 applicants requested prenatal testing. In each case, the pregnancy was in the first or second trimester when the couples were referred for DNA diagnosis. In five cases, exclusion testing was offered; in one case, a person at risk with an increased risk of being a gene carrier requested prenatal diagnosis. In all cases, informative markers for prenatal testing could be determined. Whenever possible, the newer technique of polymerase chain reaction (PCR) for D4S125 was applied to perform rapid prenatal diagnosis. Two couples withdrew before chorionic villus sampling was undertaken; prenatal diagnosis was completed in the remaining four cases. After exclusion testing, two pregnancies were determined to have an increased risk and two fetuses to have a low risk of being HD gene carriers.     

First-trimester fetal sex determination in maternal serum using real-time PCR

An Erratum has been published for this article in Prenatal Diagnosis 22(13) 2002, 1241. Fetal sex prediction can be achieved using PCR targeted at the SRY gene by analysing cell-free fetal DNA in maternal serum. Unfortunately, the results reported to date show a lack of sensitivity, especially during the first trimester of pregnancy. Therefore, determination of fetal sex by maternal serum analysis could not replace karyotype analysis following chorionic villus sampling. A new highly sensitive real-time PCR was developped to detect an SRY gene sequence in maternal serum. Analysis was performed on 121 pregnant women during the first trimester of pregnancy (mean gestational age: 11.8 weeks). Among them, 51 had at least one previous male-bearing pregnancy. Results were compared with fetal sex. SRY PCR analysis of maternal serum was in complete concordance with fetal sex. Among the 121 pregnant women, 61 were bearing a male fetus and 60 a female fetus. No false-negative results were observed. Furthermore, no false-positive results occurred, even though 27 women carrying a female fetus during the current pregnancy had at least one previous male-bearing pregnancy. This study demonstrates that a reliable, non-invasive sex determination can be achieved by PCR analysis of maternal serum during the first trimester of pregnancy. This non-invasive approach for fetal sex prediction should have great implications in the management of pregnant women who are carriers of an X-linked genetic disorder. Prenatal diagnosis might thus be performed for male fetuses only, avoiding invasive procedures and the risk of the loss of female fetuses. Copyright © 2001 John Wiley & Sons, Ltd.     

Discordant fetal sex on NIPT and ultrasound

Prenatal diagnosis of sex discordance is a relatively new phenomenon. Prior to cell-free DNA testing, the diagnosis of a disorder of sexual differentiation was serendipitous, either through identification of ambiguous genitalia at the midtrimester morphology ultrasound or discovery of genotype-phenotype discordance in cases where preimplantation genetic diagnosis or invasive prenatal testing had occurred. The widespread integration of cfDNA testing into modern antenatal screening has made sex chromosome assessment possible from 10 weeks of gestation, and discordant fetal sex is now more commonly diagnosed prenatally, with a prevalence of approximately 1 in 1500-2000 pregnancies. Early detection of phenotype-genotype sex discordance is important as it may indicate an underlying genetic, chromosomal or biochemical condition and it also allows for time-critical postnatal treatment. The aim of this article is to review cfDNA and ultrasound diagnosis of fetal sex, identify possible causes of phenotype-genotype discordance and provide a systematic approach for clinicians when counseling and managing couples in this circumstance.     

Transabdominal fine needle biopsy from chorionic villi in the first trimester

A technique for sampling first trimester chorionic villi for prenatal diagnosis by transabdominal fine needle biopsy is described. Specimens of chorionic villi were obtained from 49 out of 58 women, a success rate of 84.5 per cent. No fetal or maternal complications were demonstrated in the period before abortion. The procedure is useful for obtaining fetal tissue for culturing, DNA analysis and direct chromosome analysis.     

Microsatellite analysis provides efficient confirmation of fetal trophoblast isolation from maternal circulation

Fetal trophoblasts can be found in maternal circulation from an early stage of pregnancy and thus provide a potential source of DNA for non-invasive prenatal diagnosis. We have developed a two-step method for trophoblast isolation between the 8th and 12th week of pregnancy. Blood was sampled from 14 women undergoing termination of pregnancy or spontaneous abortion. Immunomagnetic beads precoated with HLA class I and II, and with anti-cytokeratin-18 monoclonal antibodies, were used to remove CD8+ and other maternal cells, and to select for fetal trophoblasts, respectively. Microsatellite analysis was performed on DNA extracted from the isolated, maternal, paternal and placental cells after PCR amplification. Recovery of the trophoblasts was confirmed in 13/14 cases (93%) by the identification of an identical microsatellite pattern for fetal and placental cells. Further evidence was the presence of heterozygous alleles of both maternal and paternal origin. The correct prediction of gender in all five male fetuses was an additional confirmation of trophoblast recovery. We conclude that trophoblasts can be effectively isolated from maternal blood in the first trimester, and by using polymorphic microsatellite markers to confirm sample purity, this method has potential future application in prenatal diagnosis. Copyright © 2001 John Wiley & Sons, Ltd.     

Krabbe's disease: First trimester diagnosis confirmed on cultured amniotic fluid cells and fetal tissues

Chorionic villi obtained during the first trimester from a pregnancy at risk for Krabbe's disease were shown to have reduced cerebroside-β-galactosidase (E.C. 3.2.1.46) activity using the artificial substrate trinitrophenylaminolauryl galactocerebroside (TNPAL-galactocerebroside). Assay of this enzyme in cultured amniotic fluid cells following amniocentesis, performed at the patient's request confirmed the diagnosis. Termination of pregnancy was performed and subsequent enzyme studies of the fetal tissues were consistent with the diagnosis of Krabbe's disease, thus confirming that chorionic villi can be used for first trimester diagnosis of this condition.     

β-glucuronidase deficiency: Identification of an affected fetus with simultaneous sampling of chorionic villus and amniotic fluid

Four pregnancies at risk for mucopolysaccharidosis VII were monitored by chorionic villus sampling obtained in the first or second trimester of gestation. One fetus showed reduced β-glucuronidase activity following simultaneous sampling of chorionic villus and amniotic fluid at 17 weeks of gestation. The pregnancy was terminated. Subsequent assay of β-glucuronidase activity in the fetal tissues was consistent with a diagnosis of mucopolysaccharidosis VII, thus confirming that chorionic villus samples provide useful information for diagnosis of this condition.     

DNA-based prenatal diagnosis of the infantile form of neuronal ceroid lipofuscinosis (INCL,CLN1)

Eleven fetuses at risk for the infantile form of neuronal ceroid lipofuscinosis (INCL, CLN1) were studied using DNA markers and the results were compared with the results of electron microscopy (EM) of chorionic villus specimens from pregnancies in the first or early second trimester of pregnancy. In four cases, the prenatal diagnosis was made independently with both methods, and in seven cases, the EM diagnosis was confirmed postnatally or from autopsy material using RFLP analysis. The two methods gave concordant results in all cases. The DNA analysis based on RFLP haplotypes also for the first time facilitates reliable carrier diagnostics. RFLP analysis based on polymorphic markers closely linked to the INCL locus is now available for prenatal diagnosis of this fatal brain disease, whose biochemical background is totally unknown and for which no treatment is available.     

Prenatal diagnosis for arginase deficiency by second-trimester fetal erythrocyte arginase assay and first-trimester ARG1 mutation analysis

Hyperargininemia is a progressive neurometabolic disorder caused by deficiency of hepatic cytosolic arginase I, resulting from mutations in the ARG1 gene. We diagnosed arginase deficiency in a three-year-old male child of first-cousin Palestinian Arab parents. Prenatal diagnosis of an unaffected fetus was achieved in the second trimester of a subsequent pregnancy by cordocentesis and analysis of arginase activity in fetal erythrocytes. ARG1 mutation analysis in the proband revealed homozygosity for a deletion of 10 753 bp extending from the first intron to beyond the poly (A) site of the gene. This is the first gross deletion in the ARG1 gene to be identified and the first mutation to be described in an arginase-deficient patient of this ethnic origin. The identification of the ARG1 deletion in this family enabled first-trimester prenatal diagnosis in a subsequent pregnancy by multiplex PCR analysis performed on chorionic villous DNA. Copyright © 2004 John Wiley & Sons, Ltd.     

First-trimester prenatal diagnosis in haemophilia A and B families—10 years experience from a centre in India

During the last 10 years (1995–2005) we have offered diagnosis in 438 families out of 502 families referred, by chorionic villus sampling procedure between 10 and 12 weeks of pregnancy in severe haemophilia A and B families. Sixty four families could not be offered a diagnosis in the first trimester either due to non-informativeness with the available techniques or due to the absence of affected members in the family and were subsequently offered diagnosis in the second trimester by direct analysis of factor VIII/IX clotting activity and antigen in the fetal blood samples. For first-trimester diagnosis in the chorionic villus samples, we have used both direct and indirect methods, that is, intron 22 and 1 inversions in the factor VIII gene, a multiplex PCR for the detection of gross deletions in the factor IX gene and RFLP analysis using a battery of markers within and outside the factor VIII/IX gene. The informativeness of all these techniques was found to be 92.21% in case of haemophilia A and 83.91% in case of haemophilia B. We followed up 122 children out of 326 (diagnosed unaffected prenatally) after birth and the diagnosis was adequately reconfirmed by both factor assays and by DNA analysis. Only one case of misdiagnosis was detected so far (0.22%), where the diagnosis was based only on the extragenic marker of the factor VIII gene. Copyright © 2006 John Wiley & Sons, Ltd.     

Comparative study of 15 lysosomal enzymes in chorionic villi and cultured amniotic fluid cells. Early prenatal diagnosis in seven pregnancies at risk for lysosomal storage diseases

A large number of chorionic villi samples obtained from women undergoing elective first trimester termination of pregnancy was analysed by enzyme assays similar to those applied to cultured amniotic cells. The levels of 15 lysosomal enzymes were compared to those observed in tissue cultures of amniotic cells obtained through amniocentesis at 16-18 weeks of pregnancy and the results were discussed in order to assess the usefulness of trophoblast biopsy for first trimester diagnosis of hereditary lysosomal diseases. The data suggest the applicability of this source of fetal cells for prenatal diagnosis of fifteen respective genetically determined enzyme deficiencies with the probable exception of α-L -iduronidase deficiency. Enzyme determinations were performed on chorionic villi samples of two pregnancies at risk for Tay-Sachs disease, three pregnancies for GM₁ gangliosidosis type 1, one for mucopolysaccharidosis type VI and one for Wolman's disease.     

A simple technique for obtaining high quality chromosome preparations from chorionic villus samples using FdU synchronization

A fluorordeoxyuridine (FdU) synchronization technique was applied to 30 chorionic villus samples (CVS) from patients undergoing first trimester fetal diagnosis. The villi were incubated for 15 h in the presence of FdU. The block in DNA synthesis was subsequently released using thymidine and after an additional 5 h of incubation the mitotic cells were arrested in metaphases using a high concentration of colcemid. This method results in improved morphology of the chromosomes and a high mitotic index. A diagnostic chromosome analysis could be obtained in each clinical case using at least 15 well-spread metaphases. G-banded karyotypes were prepared of four metaphases in each case. The diagnostic procedure was completed within 48 h from the time of CVS. Use of this technique significantly improves the success rate of ‘direct’ chromosome analyses from CVS in a busy cytogenetic laboratory.     

First-trimester prenatal diagnosis of severe alpha-thalassemia

By means of chorion biopsy together with restriction endonuclease analysis of fetal DNA, first trimester diagnoses were successfully made in 33 fetuses at risk for Bart's hydrops fetalis. Seven pregnancies with Hb H or hydrops fetalis were therapeutically terminated before 4 months of gestation. Of the 26 pregnancies intended to continue, 18 have come to term with normal deliveries; one with threatened abortion was terminated at the end of the first trimester and, seven are progresssing normally.     

Development of non-invasive fetal DNA diagnosis from maternal blood

Several attempts have been made to detect and retrieve fetal nucleated cells including nucleated erythrocytes (NRBCs), leukocytes, and trophoblasts in maternal blood. We have recently developed a new method for non-invasive fetal DNA diagnosis from maternal blood. Peripheral blood granulocytes including NRBCs were isolated by a discontinuous density gradient method using Percoll (Pharmasia). NRBCs were found and retrieved at a single cell level using a micromanipulator under a microscope. To determine whether the origin of the NRBCs was maternal or fetal, the NRBCs were analysed by polymerase chain reaction (PCR) amplification to determine the presence of a Y-chromosome-specific repeat sequence in mothers carrying male fetuses. We were successful in predicting fetal sex accurately in 10 out of 11 samples taken from maternal blood. This new technique opens up fetal DNA diagnosis from maternal blood during the first trimester of pregnancy to the whole population because there is no risk to the fetus or the mother.     

Prenatal diagnosis of a lethal form of Netherton syndrome by SPINK5 mutation analysis

Netherton syndrome (NS) is a severe autosomal recessive ichthyosis with no specific treatment or prenatal diagnosis available at present. The recent identification of SPINK5, which encodes a serine protease inhibitor, as the defective gene enables DNA-based prenatal diagnosis to be carried out. Here we report the first direct molecular prenatal diagnosis of a lethal form due to a recurrent SPINK5 mutation in three consanguineous Turkish families. XmnI restriction enzyme digestion and DNA sequencing demonstrated that each deceased affected child was homozygous for mutation 153delT inherited from each parent. Analysis of fetal DNA from amniotic fluid cells in Family 1 and from a chorionic villus sampling in Family 3 showed that the fetus was heterozygous for 153delT in both cases. The pregnancies were carried to term and the newborns were unaffected. In Family 2, fetal DNA analysis from chorionic villus biopsy showed in a first pregnancy that the fetus was homozygous for 153delT. The pregnancy was terminated at 13 weeks and DNA analysis of fetal keratinocytes confirmed the prenatal prediction. In a second pregnancy in Family 2, fetal DNA analysis showed heterozygosity for 153delT, and the pregnancy was continued. Direct SPINK5 mutation analysis in families at risk for NS represents the first early, rapid and reliable method for prenatal diagnosis of this life-threatening form of ichthyosis. Copyright © 2002 John Wiley & Sons, Ltd.     

Fetal toxoplasmosis and negative amniocentesis: necessity of an ultrasound follow-up

Prenatal diagnosis of congenital toxoplasmosis relies on the PCR test on amniotic fluid and ultrasound follow-up of the fetus. We report two cases of toxoplasma infection during the first trimester of gestation with a discrepant diagnosis of fetal infection. PCR performed more than four weeks after the estimated date of contamination was negative. Ultrasound follow-up was normal up to the third trimester when major hydrocephalus was detected, leading to pregnancy termination. In both cases, post-mortem examination revealed a diffuse infection with severe brain lesions. These observations confirm the necessity to continue a monthly ultrasound follow-up, even if amniocentesis is negative, in case of fetal toxoplasma infection in pregnancy. Copyright © 2003 John Wiley & Sons, Ltd.     

The time of appearance and disappearance of fetal DNA from the maternal circulation

A single copy Y-chromosome DNA sequence was amplified using the polymerase chain reaction (PCR) from the peripheral blood of 30 women who had achieved a pregnancy through an in vitro fertilization (IVF) programme. The time of conception was known precisely and was confirmed by serial ultrasound scans. Conceptions were dated as the number of weeks after fertilization plus 2, to give a time equivalent to the obstetric menstrual dating of the pregnancy (LMP). Y-chromosome-specific DNA was detected in all pregnancies with a male fetus (18/30). The earliest detection was at 4 weeks and 5 days, and the latest at 7 weeks and 1 day. Y-chromosome-specific sequences were no longer detected in any of the male pregnancies 8 weeks after delivery. No Y-chromosome sequences were detected in any of the pregnancies where only female babies were delivered. This demonstrates that fetal DNA appears in the maternal circulation early in the first trimester, that it can be identified in all pregnancies tested by 7 weeks, that it continues to be present throughout pregnancy, and that it has been cleared from the maternal circulation 2 months after parturition. Early non-invasive prenatal diagnosis for aneuploidies and inherited disorders will be possible in all pregnancies if fetal cells can be isolated free from maternal contamination (or identified accurately in the presence of maternal cells) without problems of contamination from previous pregnancies.     

Prenatal diagnosis of Apert syndrome: report of two cases

Two de novo cases with Apert Syndrome detected prenatally are presented herein. In the first, fetal ultrasound findings of syndactyly of the hands, craniosynostosis and proptosis resulted in a prenatal diagnosis in the nineteenth week of gestation. This is the earliest prenatal diagnosis of this syndrome in a not-at-risk case. Following counseling, this pregnancy was terminated and subsequent pathological examination and DNA analysis confirmed the diagnosis of Apert Syndrome and coarctation of the aorta. In the second case, fetal ultrasound at 21 weeks' gestation revealed a hypoplastic left heart and clover-leaf skull. Following counseling, this pregnancy was also terminated. Further examination of the fetus and DNA analysis led to a diagnosis of Apert Syndrome. These cases emphasize the need to complete a thorough fetal ultrasound in cases with potentially lethal cardiac abnormality and the importance of incorporating a fetal pathologist, as well as a medical geneticist, in the investigations performed after delivery or pregnancy termination when a fetal abnormality is detected on ultrasound. Copyright © 2003 John Wiley & Sons, Ltd.     

First trimester prenatal diagnosis of haemophilia A: Two years' experience

We evaluated the feasibility, reliability, and acceptability of prenatal diagnosis of haemophilia A by DNA analysis of chorionic villi. Twenty-two women at risk to transmit the abnormal gene were referred for prenatal diagnosis, two of them twice. Two of the 22 women appeared to be non-carriers by DNA analysis. In one of these women, the results were known only after chorionic villus sampling had been carried out. Thirteen of the twenty carriers were heterozygous for an intragenic (Bell or Xbal) marker; six women were only heterozygous for the extragenic DXS52 (Stl4) locus. One of the women was homozygous for all the presently known DNA markers within or closely linked with the factor VIII locus. Twelve of the 22 fetuses at risk were male, ten were female. Seven of the 12 male fetuses were shown to be affected and were subsequently aborted. Four male fetuses appeared to be not affected. In one case, the diagnosis was made by use of an extragenic marker. The woman rejected fetal blood sampling to confirm the diagnosis. After birth, a normal factor VIII level was found in three of the four cases. The fourth pregnancy is still continuing. In one of the 12 male fetuses, no diagnosis at the gene level was possible. DNA analysis is expected to provide maximum certainty as to the phenotype of the fetus for approximately 60 per cent of the women; for another 37 per cent a rate of misdiagnosis of 4–5 per cent applies. In only 3 per cent of the cases will no diagnosis at the gene level be possible as yet. The new possibility of a prenatal diagnosis in the first trimester of pregnancy enabled some of these women to have a family of their own and was appreciated in particular by the women who underwent fetoscopy in an earlier pregnancy.