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PubMed1995

Noninvasive screening for prenatal genetic diagnosis.

During the last two decades a number of methods of prenatal diagnosis have become available and have been used either in laboratory research or in routine genetic counselling. Despite the effectiveness of invasive sampling procedures in diagnosing genetic disorders, their use involves some risk. The advantage of noninvasive methods is that they provide an opportunity to make a genetic diagnosis without risk, and therefore are applicable for use in mass screening programmes. This article reviews three different approaches to noninvasive prenatal genetic diagnosis and offers conclusions and recommendations for their use. Maternal serum screening is a well-understood technique that should be universally offered to pregnant women, regardless of their risk status. Invasive tests can be used, as indicated, once serum testing results have been obtained. Although ultrasonography cannot be recommended for routine use, it can provide a useful adjunct to serum screening and deserves further investigation. Elaboration of fetal cells from maternal blood is a promising technique but can only be considered investigational on the basis of current research, and should not serve as the sole basis of clinical decision-making.

Female↗

PubMed2006

Noninvasive detection of fetal sex: the laboratory diagnostician's view.

Toward the end of the twentieth century it was discovered that cell-free fetal DNA sequences could be detected in maternal blood plasma. Initially, Y-chromosome sequences originating from male fetuses were targeted in cell-free DNA extracted from maternal plasma in order to demonstrate proof of this concept towards the development of noninvasive prenatal diagnosis methods. Clinical application of this approach is now possible. Fetal sex can be detected through a procedure that is noninvasive with respect to the fetus. Specifically, the presence of Y-chromosome sequences in maternal blood plasma indicates that the fetus is male, whereas lack of a signal will indicate that the fetus is female. Fetal sex can be detected very early, from at least the 7th week of pregnancy (and even earlier, according to several studies), about two months before this information is available through ultrasound scanning. Although the controversial issue of fetal sexing is not new, it is expected that with the availability of an accurate noninvasive test, public interest will rise. It is therefore imperative that an authorized committee of experts in each country generates an official policy regarding application of the test.

Attitude↗

PubMed1993

Fetal cells in the maternal circulation. Technical considerations for practical application to prenatal diagnosis.

Recent advances in cell separation technology and DNA analytic techniques leave little doubt as to the presence of fetal cells in the maternal circulation. The potential of using these cells for genetic analysis is compelling. The practical aspects of establishing a universal method utilizing the new capabilities in clinical practice have not been addressed to date. The major hurdles that still need to be traversed before this technology is universally adopted include the identification of appropriate sampling and separation methods yielding fetal cells amenable to genetic analysis by rapid DNA technologies, clinical studies of appropriate statistical power to validate and compare this approach to current genetic testing, and comparison of this approach to other noninvasive paradigms such as triple screening. Despite the tremendous value of noninvasive genetic screening, the rigorous course required to progress from description of scientific capability to validation of a clinical test must not be ignored or rushed for financial considerations.

DNA↗

PubMed2005

Diagnosis of human cytomegalovirus intrauterine infection using fetal cells from maternal blood.

OBJECTIVE: The sensitivity and specificity for the noninvasive prenatal diagnosis of human cytomegalovirus intrauterine infection were estimated by using isolating single fetal cells from maternal peripheral blood. METHODS: Micromanipulation techniques were employed to isolate single fetal nucleated erythroblasts from 273 maternal blood samples. SRY gene and HCMV-DNA in single fetal cells were detected by multiple primed in situ labeling (PRINS) from 76 HCMV-DNA positive samples of maternal peripheral blood. 273 samples of maternal peripheral blood were tested for SRY gene and HCMV-DNA in single fetal cells by primed extension preamplification (PEP) and polymerase chain reaction (PCR). RESULTS: The detection rate of fetal cells from maternal blood was 100% with micromanipulation techniques. The sensitivity of PRINS for SRY gene detection was 97.56% and its specificity was 100%. The sensitivity and specificity of PEP and PCR for SRY gene detection were 97.39% and 99.17%, respectively. The sensitivity of PRINS for HCMV-DNA detection was 92.68% and the specificity was 100%. The sensitivity and specificity of PEP and PCR for HCMV-DNA detection were 95.12%and 100%, respectively. CONCLUSION: The technique for noninvasive prenatal detection of intrauterine infection of HCMV using single fetal cells from maternal peripheral blood by using PRINS and PEP and PCR is more reliable than the CMV-DNA detection in peripheral maternal blood, amniocentesis or percutaneous umbilical blood sampling.

Cytomegalovirus↗

PubMed2003

Prenatal diagnosis of fetal trisomy 21 from maternal peripheral blood.

This study was undertaken to establish a noninvasive prenatal genetic diagnostic method for trisomy 21 using the fetal nRBCs that is rarely present in maternal circulation. Peripheral venous blood samples were collected from 30 women with an advanced maternal age, abnormal triple marker test results, or abnormal ultrasound findings such as an increased nuchal translucency. The blood samples were treated with heparin. The triple density gradient centrifugation, and MACS using CD45 and CD71 were used to isolate the fetal cells. FISH analysis using probe 21 was performed with GPA-immunostaining. The study population consisted of 30 patients from 13 to 25 weeks of gestation, and nRBCs were separated in all cases. In GPA-immuno FISH analysis using probe 21, 3 cases of trisomy 21 were diagnosed and these results were confirmed by the amniocentesis. In conclusion, a prenatal diagnosis of trisomy 21 through GPA- immuno fluorescence in situ hybridization (FISH) analysis using separated fetal nRBCs is a useful, innovative, accurate, rapid and non-invasive diagnostic method.

Adolescent↗

PubMed2006

MRI in fetal necropsy.

The fetal autopsy involves a series of investigations of the corpse, most of which are noninvasive and acceptable to the majority of parents and their physicians. The value of the perinatal autopsy is manyfold and well established, and the results can provide a basis for parental and family counseling, inform future obstetric management, and provide audit for prenatal care. Many techniques originally developed for diagnosis, such as histology, biochemical tests, photography, x-rays, and cytogenetic karyotyping, have become standard tools in perinatal autopsies. However, there has been an inexorable decline in the autopsy consent rate over the last 30 years due to social and cultural factors, and perhaps ignorance of the benefits to be derived from the examination. Growing evidence suggests that postmortem fetal MRI can assist the pathologist at autopsy, and in many cases can obviate the need for dissection or at least minimize and focus it. For the majority of cases in which no consent for surgical autopsy is given, MRI together with other noninvasive postmortem tests can provide a great deal of the information that was previously available only from autopsy.

Autopsy↗

PubMed1999

Urinary analyte screening: a noninvasive detection method for Down syndrome?

Prenatal screening for Down syndrome using maternal serum markers achieves detection rates of 60-80% with a 5% false positive rate. Improvement in the accuracy of screening, as well as its ease and safety, will increase the use of such tests. The most effective of the current serum markers is human chorionic gonadotropin (hCG). Studies on beta core fragment (beta CF), the major urinary metabolite of hCG, have indicated that screening with beta CF and other markers measured in maternal urine might improve the detection of Down syndrome and provide a less expensive and simpler test. However, recent results have been unusually variable. Although it has great potential, the true clinical value of maternal urine screening to detect Down syndrome still remains to be determined.

Chorionic Gonadotropin, beta Subunit, Human↗

PubMed1996

[Molecular genetic diagnosis and deletion analysis in Type I-III spinal muscular atrophy].

Autosomal recessive spinal muscular atrophy (SMA) is, after cystic fibrosis, the second most common fatal monogenic disorder. The disease is characterized by degeneration of anterior horn cells leading to progressive paralysis with muscular atrophy. Depending on the clinical type (Werdnig-Hoffmann = type I, intermediate form = type II, Kugelberg-Welander = type III), SMA causes early death or increasing disability in childhood. The SMA-critical region on the long arm of chromosome 5q13.1 contains many duplicated genes and polymorphisms. Recently, two presumptive SMA genes (survival motoneuron gene = SMN, and neuronal apoptosis inhibitory protein = NAIP) have been identified. Deletions involving critical regions of these genes are very often associated with SMA, and the extent of the deletions seems to correlate in part with disease severity. We have evaluated the diagnostic and prognostic value of molecular analysis in a large number of SMA patients. 57 patients and 78 healthy relatives were molecularly screened for deletions in the SMA critical region. We demonstrated homozygous deletions removing the SMN genes in over 90% of patients, whereas nearly 45% of patients exhibited NAIP gene deletions. Large deletions involving both genes on each chromosome are generally found in patients with severe SMA (Werdnig-Hoffman cases), while mildly affected Kugelberg-Welander cases frequently show only deleted SMN genes. Molecular classification based on combined deletion sizes, however, seems not to be exact, especially for the group with chronic SMA (type II and III). Direct DNA testing of patients in whom SMA is suspected is a highly reliable, fast, and noninvasive method. The ability to detect homozygous gene deletions in a high percentage of typical SMA patients will much improve genetic counselling and prenatal diagnosis in affected families.

Adult↗

PubMed2005

Fetal DNA detection in maternal plasma throughout gestation.

The presence of fetal DNA in maternal plasma may represent a source of genetic material which can be obtained noninvasively. We wanted to assess whether fetal DNA is detectable in all pregnant women, to define the range and distribution of fetal DNA concentration at different gestational ages, to identify the optimal period to obtain a maternal blood sample yielding an adequate amount of fetal DNA for prenatal diagnosis, and to evaluate accuracy and predictive values of this approach. This information is crucial to develop safe and reliable non-invasive genetic testing in early pregnancy and monitoring of pregnancy complications in late gestation. Fetal DNA quantification in maternal plasma was carried out by real-time PCR on the SRY gene in male-bearing pregnancies to distinguish between maternal and fetal DNA. A cohort of 1,837 pregnant women was investigated. Fetal DNA could be detected from the sixth week and could be retrieved at any gestational week. No false-positive results were obtained in 163 women with previous embryo loss or previous male babies. Fetal DNA analysis performed blindly on a subset of 464 women displayed 99.4, 97.8 and 100% accuracy in fetal gender determination during the first, second, and third trimester of pregnancy, respectively. No SRY amplification was obtained in seven out of the 246 (2.8%) male-bearing pregnancies. Fetal DNA from maternal plasma seems to be an adequate and reliable source of genetic material for a noninvasive prenatal diagnostic approach.

Cohort Studies↗

PubMed2006

Transrenal DNA testing: progress and perspectives.

Transrenal DNA (Tr-DNA) is a recently discovered class of extracellular urinary DNA that originates from cells dying throughout the body. Postapoptotic DNA is known to appear in the circulating plasma, but it is now recognized that a portion of these fragments cross the kidney barrier and appear in urine in the form of 150-200-bp fragments. Tr-DNA containing fetal sequences has been isolated from the urine of pregnant women, tumor-specific mutations have been detected in Tr-DNA from patients with colon and pancreatic tumors, and donor DNA has been found in Tr-DNA isolated from recipient urine. Furthermore, proviral HIV DNA, bacterial and parasite DNA sequences have been detected in Tr-DNA from infected patients. Potential applications of Tr-DNA-based tests cover a very broad area of molecular diagnostics and genetic testing, including prenatal detection of inherited diseases, tumor diagnostics and therapeutic monitoring and detection of infectious agents. The Tr-DNA test is expected to have utility in treatment monitoring, transplantation monitoring, drug development and broad public health screening, where a noninvasive, common-platform diagnostic technology has particular value. This review describes some of the highlights of Tr-DNA technology applications, advantages over existing technologies and potential problems anticipated in test development.

Animals↗

PubMed1991

Genetic analysis of the Duchenne muscular dystrophy gene.

Molecular biology techniques have changed the way in which we now consider a patient with Duchenne muscular dystrophy or Becker muscular dystrophy. Using cDNA probes, it has been shown that approximately 65% of the patients with Duchenne muscular dystrophy or Becker muscular dystrophy have gene deletions. The identification of a deletion allows the disease to be confirmed by noninvasive DNA testing. Furthermore, the identification of the gene defect can help distinguish Becker muscular dystrophy from other clinically similar neuromuscular disorders. Most importantly, the elucidation of the gene defects has resulted in the application of direct carrier and prenatal diagnostics.

DNA↗

PubMed2003

[Diagnostic methods for fetal malformations in the first half of pregnancy].

INTRODUCTION: Fetal abnormalities are the most common cause of perinatal and postnatal death and infant handicap. For this reason prenatal screening (for fetal malformation) has become a routine part of obstetric care in many countries. Most often used are biochemical tests and continuously developing ultrasound diagnostics which makes possible precise analysis of the fetal morphology. There is interesting to establish a noninvasive test for the early detection of fetal malformation in pregnancy which is based on ultrasound examination (NT measurement from the 10th to the 19th weeks, presence of nasal bone in the first trimester ultrasound), correlated with serum concentration of AFP, beta-HCG and oestriol in the second trimester of pregnancy (triple test). The main aim of the study was to establish a diagnostic schema for detection of fetal malformations based on NT measurement in the first and second trimester coupled with triple test performed in the second trimester. MATERIALS AND METHODS: A group of 775 pregnant women from the 10-th week of pregnancy until childbirth has been put under examination. Between the 10th and the 14th and than between 15th and 19th week of pregnancy ultrasound examination with fetal biometry and NT measurement was done. NT measurements have been performed in accordance with the standards worked out by professor K. Nicolaides. At the first ultrasound examination the presence of the nasal bone was observed. The next step was performing the triple test between the 15th and 19th week of pregnancy. On the same day as second ultrasound examination blood was taken to determine the results of the triple test (ELISA method). The obtained results have undergone statistical analysis. RESULTS: The age of women qualified for the examination oscillated between 15 and 45 (over 35 -9.4%). There were 8 fetal malformations recognized all connected with the chromosomal anomalies, namely, 4 Downs syndrome, 2 fetuses with trisomy of the 18th pair of chromosomes and 2 with triploidy. At all physiologic pregnancies nasal bone was seen during first ultrasound examination. The obtained results of nuchal fold measurements and concentrations for the parameters of the triple test have been the basis to calculate medians in the particular weeks of pregnancy. In all the cases of genetic malformations the widening of the nuchal fold above 99 percentile (MoM NT) has been observed. Fetal nasal bone were absent in 62.5% first trimester ultrasound examinations. The risk of the occurrence of a genetic malformation resulting from the mother's age combined with the risk connected with the NT measurements and the results of the triple test for the cut-off point 1:250 (which seems to be the best for this population) gave 100% sensitivity, 0.6% % of false positive results and the positive predictive value of 80%. The above mentioned results are better than the ones which were obtained within the triple test only, where for the previously fixed cut-off point 1:250 sensitivity reached 63%, positive predictive value 25% and 4.4% false positive rate. Performing the so-called integrated test in which the risk of the occurrence of any malformation is estimated on the basis of the NT measurement in the first and the second term of pregnancy seems to be far more useful. CONCLUSIONS: Diagnostic schema for detection of fetal malformations in the first half of pregnancy which is based on ultrasound examination (NT measurement from the 10th to the 19th weeks), correlated with serum concentration of AFP, beta-HCG and oestriol in the second trimester of pregnancy (triple test) is very sensitive and safe method of the prenatal diagnosis leading to significant decrease of the invasive procedures (amniocentesis).

Abnormalities, Multiple↗

PubMed2005

Detection of paternally inherited fetal point mutations for beta-thalassemia using size-fractionated cell-free DNA in maternal plasma.

CONTEXT: Currently, fetal point mutations cannot be reliably analyzed from circulatory fetal DNA in maternal plasma, due to the predominance of maternal DNA sequences. However, analysis of circulatory fetal DNA sequences in maternal plasma have been shown to selectively enrich for fetal DNA molecules on the basis of a smaller molecular size than maternal DNA. OBJECTIVE: To examine the prenatal analysis of 4 common beta-thalassemia point mutations: IVSI-1, IVSI-6, IVSI-110, and codon 39. DESIGN, SETTING, AND PATIENTS: A total of 32 maternal blood samples were collected at 10 to 12 weeks of gestation (mean, 10.7 weeks) between February 15, 2003, and February 25, 2004, in Bari, Italy, from women with risk for beta-thalassemia in their newborns immediately prior to chorionic villous sampling. Samples in which the father and mother did not carry the same mutation were examined. Circulatory DNA was size-fractionated by gel electrophoresis and polymerase chain reaction (PCR) amplified with a peptide-nucleic-acid clamp, which suppresses amplification of the normal maternal allele. Presence of the paternal mutant allele was detected by allele-specific real-time PCR. MAIN OUTCOME MEASURE: Detection of paternally inherited beta-globin gene point mutations. RESULTS: Presence or absence of the paternal mutant allele was correctly determined in 6 (86%) of 7 cases with the IVSI-1 mutation, 4 (100%) of 4 with the IVSI-6 mutation, 5 (100%) of 5 with the IVSI-110 mutation, and 13 (81%) of 16 with the codon 39 mutation. One false-positive test result was scored for the IVSI-1 mutation. Two cases with the codon 39 mutation were classified as uncertain and 1 case was excluded due to lack of a diagnostic test result at the time of analysis. These results yielded an overall sensitivity of 100% and specificity of 93.8%, with classified cases removed. CONCLUSION: Our recently described technique of the size-fractionation of circulatory DNA in maternal plasma may be potentially useful for the noninvasive prenatal determination of fetal point mutations.

Chorionic Villi Sampling↗

PubMed1993

[Systematic antepartum fetal evaluation ("Safe"). A concept for diagnosis of fetal function in threatened hypoxia].

TARGET: The significant aim of the study was to prove whether it makes sense to apply the classical approach of diagnostic procedures to the fetus. PATIENTS AND METHODS: In a group of a total of 534 singleton pregnancies we examined the value of various biochemical and biophysical tests for detecting the risk of perinatal hypoxia. The sequence and combination of the most decisive tests was considered as well as the different approach within the observation period. RESULTS: Maternal laboratory findings like HPL and E3 were significantly less predictive of growth disturbance and compromise than any biophysical test. A small fetal abdomen detected by ultrasound ("observation") is up to now the earliest and most reliable predictor of foetal growth disturbance, followed by Doppler measurements of the uteroplacental circulation ("examination of the chronic respiratory function"). Examination of the foetal heart-circulatory function with pulsed Doppler and the foetal cardiac frequency (CTG) are the procedures of choice to determine the risk of perinatal hypoxia following pre-selection of foetuses, which are too old or too thin. In these patients, additional stress tests have neither medical nor a diagnostic advantage compared to the published function tests. Amongst the variety of biophysical tests, the Apgar score ("fetal Apgar score") was superior to all other tests in predicting prenatal or perinatal hypoxia and might even prevent invasive procedures. CONCLUSION: The sequence from non-invasive biophysical tests, including the fetal Apgar score, to invasive procedures is recommended for screening and follow-up of preselected pregnancies at risk for perinatal hypoxia. This approach might be applied to other risk groups and will hopefully be supplemented by new noninvasive diagnostic tools allowing early prognosis and prevention of fetal and neonatal disease.

Asphyxia Neonatorum↗

PubMed1996

Rare event selection of fetal nucleated erythrocytes in maternal blood by flow cytometry.

A noninvasive method of prenatal genetic diagnosis requires fetal cell selection from the maternal circulation that allows efficient recovery for analysis by fluorescence in situ hybridization (FISH). We have solved several problems that negatively affect the isolation and FISH analysis of fetal nucleated red blood cells (nRBCs) in the maternal circulation. The use of glycophorin A (Gly A) antibodies (Abs) for selection is problematic because all five monoclonal antibodies (mAbs) tested caused agglutination of non-nRBCs, thereby changing both light scatter and fluorescence properties of cells by flow cytometry. Because the number of non-nRBCs is variable after Ficoll separation, isolation of nRBCs could be compromised severely by agglutination of nucleated cells with nonnucleated cells, causing them to shift light scatter and fluorescence properties. Several methods for the removal of unwanted maternal white blood cells with CD45 mAbs were also evaluated. Magnetic bead depletion was found to interfere with FISH detection because of residual bead debris after sorting. By contrast, removal of CD45+ cells by a panning technique eliminated this problem. Positive selection methods based on CD71, CD45, and LDS-751 staining and detection of fetal cells by gamma globin expression were also analyzed. Fetal cells were detected by FISH in 11 of 19 (CD71 selection) and in 13 of 15 (gamma selection) random pregnancies. These data support the possibility of a noninvasive method for isolation and analysis of fetal cells for prenatal diagnosis.

Antigens, CD↗

PubMed1994

Simultaneous immunophenotyping and FISH on fetal cells from maternal blood.

Current methods for the prenatal diagnosis of genetic disorders from fetal cells in the maternal circulation are restricted by maternal cell contamination and therefore must rely on the presence of features (such as Y-specific DNA sequences) absent from maternal cells. We have used density gradient centrifugation and magnetic-activated cell sorting to enrich maternal blood samples for fetal nucleated red cells. Mouse monoclonal antibodies for CD45 and CD32 were used to reduce the proportion of leukocytes. Unequivocal identification of fetal cells was achieved using an immunophenotypic test for fetal hemoglobin that allowed the simultaneous application of a diagnostic FISH analysis with chromosome-specific DNA probes. A positive diagnosis of female fetal sex (among other diagnoses) is thus possible even in the presence of an excess of maternal cells. The method, which appears to represent an advance over previous techniques, has considerable application in the development of noninvasive prenatal diagnosis from maternal blood.

Blood Cells↗

PubMed2006

Noninvasive prenatal diagnostic assay for the detection of beta-thalassemia.

The development of a noninvasive method for detection of beta-thalassemia in the population of Cyprus is based on the detection of paternally inherited single nucleotide polymorphisms (SNPs) as well as beta-thalassemia (beta-thal) mutations. We selected 11 informative SNPs for the Cypriot population linked to the beta-globin locus. Two different approaches were used: allele-specific polymerase chain reaction (AS-PCR) and the arrayed primer extension (APEX) method. The AS-PCR approach is being standardized, and the method was applied in two families. The paternally inherited allele was noninvasively detected with the AS-PCR approach on maternal plasma. Some preliminary tests were performed with the APEX method on genomic DNA of parents carrying the beta-thal mutation.

Alleles↗

PubMed2006

Noninvasive prenatal detection of fetal trisomy 18 by epigenetic allelic ratio analysis in maternal plasma: Theoretical and empirical considerations.

BACKGROUND: The discovery of cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis. However, the use of maternal plasma fetal DNA for the direct detection of fetal chromosomal aneuploidies has not been reported. We postulate that the aneuploidy status of a fetus could be revealed by an epigenetic allelic ratio approach, i.e., by analyzing the allelic ratio of a single-base variation present within DNA molecules exhibiting a placental-specific epigenetic signature in maternal plasma. METHODS: Placental-derived fetal-specific unmethylated maspin (SERPINB5) promoter sequences on human chromosome 18 were detectable in placental-maternal DNA mixtures and in maternal plasma by bisulfite modification followed by methylation-specific PCR (MSP) and primer extension. The ratios between the extension products of the 2 alleles were calculated for heterozygous placentas, placental-maternal blood cell DNA mixtures, and maternal plasma samples. The allelic ratios were compared between pregnancies carrying trisomy 18 and euploid fetuses. RESULTS: The epigenetic allelic ratios of all tested trisomy 18 samples deviated from the reference range obtained from euploid samples (placental DNA, 1.135 to 2.052; placental-maternal DNA mixtures, 1.170 to 1.985; maternal plasma, 0.330 to 3.044; without skew correction on the raw mass spectrometric data). A theoretical model was established and validated that predicted that a minimum of 200 copies of genomic DNA after bisulfite conversion were required for distinguishing euploid and aneuploid fetuses with confidence. CONCLUSION: Epigenetic allelic ratio analysis of maternal plasma DNA represents a promising approach for noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.

Chromosomes, Human, Pair 18↗