Reliable Method For Prenatal Diagnosis Research Articles

Prenatal Diagnosis of Clubfoot: A Review of Current Available Methodology

Clubfoot is one of the most common congenital limb deformities. Prenatal diagnosis of the condition is essential as it can help treat the malformation as early as possible. We reviewed the recent available literature concerning the current methods for prenatal diagnosis of clubfoot. The following databases were searched from 1966 to 2015: PubMed, OVID, Cochrane, CINAHL, Google scholar and Embase. Out of a total number of 197 retrieved articles, after abstract or title page evaluation, 158 articles not matching the inclusion criteria were excluded. The full text versions of the remaining 39 articles were obtained, and their reference lists screened, with the addition of another 5 full-text articles. Currently, ultrasonography is considered the most reliable method of prenatal diagnosis of clubfoot. Ultrasonographic diagnosis of clubfoot appears more likely between the 18th and the 24th week of pregnancy. Alternative imaging is not indicated. There is no agreement whether to propose foetal karyotyping when isolated clubfoot is diagnosed by prenatal ultrasonography. Early detection of clubfoot should prompt a careful surveillance during pregnancy in order to detect any possible additional abnormalities and, if any of these are detected, invasive testing should be offered.

Quantitative Fluorescent-PCR: Current Scenario and Future Approach for the Investigations of Spontaneous Miscarriages in Karachi

Chromosomal numerical aberration is one of the leading reasons behind spontaneous pregnancy loss. The probability of spontaneous miscarriage increases with advance parental age. After miscarriage patients and their families feel desperate, which consequently create psychological illness. The review evaluates current techniques being used in the diagnosis and focuses toward a recently introduced molecular technique Quantitative Fluorescent-PCR (QF-PCR) which based on the use of fluorescent primers (probes). Several published articles were selected for the review regarding the frequency of spontaneous miscarriages, its reasons, and outcomes. Studies that focus on QF-PCR, karyotyping and their comparison were included. Current situation of management of spontaneous miscarriages was also observed through surveys and consultant views in Karachi. On the basis of the above mentioned piece of information, QF-PCR would be a good addition in the field of prenatal diagnosis. However, karyotyping is also a reliable method of prenatal diagnosis with little limitations.

Comparison of Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) and Conventional Cytogenetic Analysis in Detection of Fetal Anueploidies

Objective: Cytogenetic analysis, the gold standard in the diagnosis of fetal aneuploidies, requires the time-consuming generation of primary cell cultures, which limits the success rate of this technique. In this study, we examined the efficiency and accuracy of quantitative fluorescent polymerase chain reaction (QF-PCR) for the rapid detection of fetal aneuploidy. Methods: Sixty pregnant women at high risk for fetal aneuploidies were recruited to the study group. Indications for invasive prenatal diagnosis were: advanced maternal age, positive biochemical screening, abnormal ultrasound findings and previous history of fetal abnormalities. All samples were tested by both QF-PCR and traditional karyotyping. Results: Twenty-six samples showed normal patterns. All normal samples were detected by QF-PCR without false positive or false negative results. All trisomies, including trisomies 21, 13, and 18, (n = 25, 2 and 2, respectively) were successfully detected by QF-PCR. Four Turner cases were identified by karyotyping, while only two were detected by QF-PCR. QF-PCR failed to accurately diagnose a balanced translocation. Conclusions: QF-PCR is a rapid and reliable method for prenatal diagnosis of the common chromosomal aneuploidies, especially trisomies 13, 18 and 21. This rapid and inexpensive technique may be a suitable prenatal screen in developing countries. Synopsis: QF-PCR is a rapid and reliable method for prenatal diagnosis of the common chromosomal aneuploidies. It may replace fluorescence in-situ hybridization as rapid prenatal screen.

Molecular diagnosis of Down syndrome using quantitative APEX‐2 microarrays

To develop a new rapid and high-throughput microarray-based prenatal diagnostic test for the detection of trisomy 21 (T21). The T21 arrayed primer extension-2 (APEX-2) assay discriminates between trisomy and euploid DNA samples by comparing the signal intensities of allelic fractions of heterozygous single nucleotide polymorphisms (SNPs) after APEX reaction. After preliminary validation using DNA samples from Down syndrome patients, we analyzed DNA samples from cultured and uncultured amniocytes and chorionic villus for 90 SNPs with high heterozygosity from the 21(q21.1q22.2) region. Differences in allelic ratios of heterozygous SNPs in normal and T21 individuals were verified by t-test. Analysis of the T21 APEX-2 assay results revealed that 90 SNPs were sufficient for reliable discrimination between T21 and euploid DNA samples (P≤0.05 for one or both strands). Using 134 clinical samples from cultured or uncultured fetal cells, both the sensitivity and the specificity of the assay were 100%. Our study provides a proof of principle demonstration of the use of the modified APEX-2 assay as a new, fast and reliable method for prenatal diagnosis of fetal T21.

Prenatal diagnosis of methylmalonic aciduria by analysis of organic acids and total homocysteine in amniotic fluid

Methylmalonic aciduria (MMA) is the most frequent disease of organic aciduria in China. Various biochemical strategies are followed for the prenatal diagnosis of MMA. However, since fetuses affected by MMA have decreased excretion of methylmalonic acid, the difficulties of prenatal biochemical diagnosis are obvious. Gas chromatography mass spectrometry (GC/MS) and tandem mass spectrometry (ESI/MS/MS) have allowed us to identify the disease in affected fetuses. The aim of this study was to determine the value of analysis of organic acids and total homocysteine in amniotic fluid in prenatal diagnosis of MMA. The clinical diagnoses and outcomes of nine probands with MMA and the prenatal diagnoses based on biochemical analysis of nine fetuses at risk for MMA were investigated. Amniotic fluid samples from pregnancies at risk for MMA and metabolically normal pregnancies were obtained at 16 - 24 weeks of gestation. Methylmalonic acid and methylcitric acid were measured by GC/MS, propionylcarnitine was analyzed by ESI/MS/MS, and total homocysteine was determined by fluorescence polarization immunoassay. In two pregnancies, high levels of methylmalonic acid, methylcitric acid, propionylcarnitine, and total homocysteine indicated combined MMA and homocysteinemia in the fetuses. One of the mothers continued pregnancy and received cobalamin supplement as prenatal treatment, and the other terminated her pregnancy. In one pregnancy, significantly elevated levels of methylmalonic acid, methylcitric acid, and propionylcarnitine, and normal level of total homocysteine was found indicating isolated MMA in the fetus; abortion was performed on this case. In the other six pregnancies, all the levels of the above mentioned metabolites were normal suggesting that the fetuses were not affected by MMA. The diagnoses were confirmed after delivery by testing urinary organic acids and plasma total homocysteine. The metabolic abnormalities of MMA occur early in gestation. The level of total homocysteine in amniotic fluid may be an additional indicator of fetal combined MMA and homocysteinemia. Determination of total homocysteine level in amniotic fluid may become a convenient and reliable method for prenatal diagnosis of the disease.

Prenatal diagnosis of Smith‐Lemli‐Opitz syndrome (SLOS) by DHCR7 mutation analysis

We review our experience using mutation analysis of the DHCR7 gene for prenatal diagnosis of Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive disorder of endogenous cholesterol biosynthesis caused by deficiency of 7-dehydrocholesterol reductase (DHCR7). Prenatal diagnosis of SLOS was conducted for 21 pregnancies involving 15 families. DNA was isolated directly from amniotic fluid cells or chorionic villus samples (CVS), and the DHCR7 gene was screened for the parental mutations using PCR-ARMS and nucleotide sequencing. Sixteen of the pregnancies were unaffected and five were affected. There have been no incidences of misdiagnosed pregnancies. DHCR7 mutation is a rapid and reliable method for prenatal diagnosis of SLOS, and provides an alternative to specialized biochemical tests for elevated 7DHC in amniotic fluid or CVS.

Prenatal diagnosis of Ullrich congenital muscular dystrophy using haplotype analysis and collagen VI immunocytochemistry.

Ullrich congenital muscular dystrophy (UCMD) is a recessively inherited condition characterised by proximal joint contractures, marked distal joint hyperextensibility, rigidity of the spine and early respiratory failure. Recently, mutations in the genes encoding the subunits of collagen VI have been identified in this disease. We undertook two prenatal diagnoses for UCMD in a consanguineous family where the disease was consistent with linkage to the COL6A3 locus and immunolabelling of collagen VI in the proband's skeletal muscle was severely reduced. Both haplotype analysis and collagen VI immunolabelling were used to determine the status of the fetuses. Haplotype analysis of DNA extracted from chorionic villus samples (CVS) from the initial at-risk pregnancy with markers encompassing COL6A3 demonstrated that this fetus had inherited the same haplotypes as the affected child, and immunolabelling of the at-risk CVS demonstrated the virtual absence of collagen VI. A second latter fetus inherited neither of the at-risk haplotypes and collagen VI expression in the CVS was normal. During the second pregnancy, a homozygous G > A change in the last nucleotide of exon 27 of COL6A3 was identified in the proband, substantiating the results obtained from haplotype analysis and collagen VI immunolabelling. These findings demonstrate that haplotype analysis in combination with immunocytochemistry is a rapid and reliable method for prenatal diagnosis of UCMD, provided the family is genetically informative and reduced collagen VI expression in the proband has been demonstrated.

Prenatal molecular diagnosis of glutaric aciduria type I by direct mutation analysis

Various biochemical strategies are followed for the prenatal diagnosis of glutaric aciduria type I (GA I). However, since the description of patients with normal excretion of glutarate and significant residual activity, the difficulties of prenatal biochemical diagnosis are obvious. The characterization of the glutaryl-CoA dehydrogenase (GCDH) gene has allowed us to develop a single strand conformation polymorphism (SSCP) screening method, followed by direct sequencing, to identify the disease causing mutations in patients with GA I. Here we report the first prenatal diagnoses based on DNA analysis in chorionic villi biopsy or cultured amniotic fluid cells in three families at risk for GA I. Our results show that this strategy provides a fast and reliable method for prenatal diagnosis. In addition we report two new mutations (1209-1210ins G and R161W) in the GCDH gene that occurred at hypermutable loci.

Prenatal diagnosis of haemophilia.

Genotype assessment based on direct identification of the pathogenic mutation in a chorionic villi sample obtained in the 11-12th gestational week is the most reliable method for prenatal diagnosis and should be used if available. Genetic linkage studies of polymorphisms should be the second choice in the assessment of carriers and in prenatal diagnosis. Carriers of haemophilia should be offered adequate psychosocial support before, during and after the prenatal diagnostic procedures.

Prenatal diagnosis of glycogen storage disease type Ia by restriction enzyme digestion

Glycogen storage disease type Ia (GSD Ia) is an autosomal recessive condition, caused by a deficiency of hepatic glucose-6-phosphatase (G6Pase) activity. In a consanguineous family originating from northern Africa whose first daughter was affected with GSD Ia, we were able to identify the disease-causing mutation, a cytosine to thymine substitution at nucleotide 326 in exon 2 of the G6Pase gene (R83C). This mutation causes the disappearance of an HgaI site, and is thus easily detectable by restriction enzyme digestion. Both parents were heterozygous for this mutation. During the third pregnancy, fetal genomic DNA was extracted from a chorionic villus biopsy sampled at the 24th week of gestation. Exons 2 of the G6Pase gene were amplified by the polymerase chain reaction followed by HgaI digestion. Fetal DNA analysis indicated that the fetus had received both normal G6Pase alleles. This result was confirmed after birth. DNA analysis is the only reliable method for prenatal diagnosis of GSD Ia. © 1998 John Wiley & Sons, Ltd.

Prenatal diagnosis of glycogen storage disease type Ia by restriction enzyme digestion

Glycogen storage disease type Ia (GSD Ia) is an autosomal recessive condition, caused by a deficiency of hepatic glucose-6-phosphatase (G6Pase) activity. In a consanguineous family originating from northern Africa whose first daughter was affected with GSD Ia, we were able to identify the disease-causing mutation, a cytosine to thymine substitution at nucleotide 326 in exon 2 of the G6Pase gene (R83C). This mutation causes the disappearance of an HgaI site, and is thus easily detectable by restriction enzyme digestion. Both parents were heterozygous for this mutation. During the third pregnancy, fetal genomic DNA was extracted from a chorionic villus biopsy sampled at the 24th week of gestation. Exons 2 of the G6Pase gene were amplified by the polymerase chain reaction followed by HgaI digestion. Fetal DNA analysis indicated that the fetus had received both normal G6Pase alleles. This result was confirmed after birth. DNA analysis is the only reliable method for prenatal diagnosis of GSD Ia.

MUTATION-BASED PRENATAL DIAGNOSIS OF HERLITZ JUNCTIONAL EPIDERMOLYSIS BULLOSA

Epidermolysis bullosa (EB) is a group of heritable diseases which manifest with blistering and erosions of the skin and mucous membranes. Due of life-threatening complications and significant long-term morbidity associated with the severe, neonatal lethal (Herlitz) form of junctional EB (H-JEB), there has been a demand for prenatal diagnosis from families at risk for recurrence. Previously, the only reliable method of prenatal diagnosis of EB was a fetal skin biopsy performed at 16-20 weeks' gestation and analysed by electron microscopy. Recently, the genes LAMA3, LAMB3, and LAMC2, encoding the polypeptide subunits of laminin 5, an anchoring filament protein, have been shown to contain mutations in H-JEB. In this study, direct detection of pathogenetic mutations in the laminin 5 genes was used to perform polymerase chain reaction (PCR)-based prenatal testing. DNA was obtained by chorionic villus sampling (CVS) at 10-15 weeks or amniocentesis at 12-19 weeks' gestation in 15 families at risk for recurrence of JEB. In 13 cases, the fetus was predicted to be either genetically normal or a clinically unaffected carrier of a mutation in one allele. These predictions have been validated in all cases by the birth of a healthy child. In two cases, an affected fetus was predicted, and the diagnosis was confirmed by subsequent fetal skin biopsy. These results demonstrate that DNA-based prenatal testing offers an early, expedient, and accurate method of prenatal diagnosis or an exclusion of Herlitz JEB.

Diagnosis of fetal rubella infection with reverse transcription and nested polymerase chain reaction: A study of 34 cases diagnosed in fetuses

OBJECTIVE: Our purpose was to develop a reliable method for prenatal diagnosis of fetal rubella infection through the detection of viral ribonucleic acid extracted from the chorionic villi, amniotic fluid, or fetal blood in pregnant women. STUDY DESIGN: Double amplification of rubella viral ribonucleic acid by nested polymerase chain reaction after reverse transcription was applied to samples from 34 women suspected of having rubella. The results were compared with those of serum antibody and levels of rubella virus - specific immunoglobulin M antibodies in fetal blood. RESULTS: Viral ribonucleic acid was revealed in 8 of 34 cases (23.5%). In the remaining 26 cases, healthy babies were born in 24, 1 was electively aborted, and 1 died in the thirty-sixth week of pregnancy of unknown causes. CONCLUSIONS: This method allowed very early detection of fetal rubella infection by sampling of chorionic villi and amniotic fluid compared with evaluation of the maternal symptoms and serum antibody levels. Fetal blood was also more useful for making a diagnosis up to the twentieth week of pregnancy than was measuring rubella virus - specific immunoglobulin M antibodies. (AM J OBSTET GYNECOL 1996;174:578-82.)

Consequences of varicella and herpes zoster in pregnancy: prospective study of 1739 cases

In a joint prospective study in Germany and the United Kingdom between 1980 and 1993, 1373 women who had varicella and 366 who had herpes zoster during the first 36 weeks of gestation were followed up. 9 cases of congenital varicella syndrome were identified, all occurring after maternal varicella during the first 20 weeks of gestation. The highest risk (2 0%) was observed between 13-20 weeks gestation, with 7 affected infants identified among 351 pregnancies (95% Cl of risk 0·8-4·1%). Only 2 cases of congenital varicella syndrome were identified among 472 pregnancies in which maternal varicella occurred before 13 weeks (observed risk 0·4%, 95% Cl 0·05-1 5%). Herpes zoster in infancy was reported in 10 children whose mothers had had varicella in pregnancy. No infants with clinical evidence of intrauterine infection were born to the 366 women with herpes zoster in pregnancy (upper 95% confidence limit of estimated risk 1 0%). Varicella-zoster-specific IgM antibody was found at birth in 4 of 16 (25%) infants with clinical manifestations of intrauterine infection and persistent specific IgG antibody in 5 of 7 infants tested. The corresponding rates in asymptomatic infants whose mothers had varicella were 12% (76/615) and 7% (22/335) respectively. No serological evidence of intrauterine infection was found in infants who mothers had herpes zoster in pregnancy. In 97 pregnant women, varicella occurred after post-exposure prophylaxis with anti-varicella-zoster immunoglobulin. No cases of congenital varicella syndrome or zoster in infancy occurred in this group. Our estimates provide a sound basis for counselling women with varicella in pregnancy. Although the risk of congenital varicella syndrome is small, the outcome for the affected infant is so serious that a reliable method of prenatal diagnosis would be valuable. In the long term, prevention of maternal varicella would be an option if a safe and effective vaccine were to become routinely available.

Prenatal detection of Canavan disease (aspartoacylase deficiency) by DNA analysis.

Amniocentesis was performed in four pregnancies at risk for Canavan disease (CD). In all families both parents were of Ashkenazi-Jewish origin and harboured the C854 mutation in the cDNA of the aspartoacylase gene. Using DNA analysis of the amniotic cells, three fetuses were predicted to be non-affected and one fetus was predicted to be affected. The concentration of N-acetylaspartic acid (NAA) in the amniotic fluid was in agreement with these results. In urine samples of the three newborns predicted to be non-affected, the concentration of NAA was normal. Tissues of the aborted fetus were not available. We conclude that DNA analysis is probably a reliable method for prenatal diagnosis of CD.

Prenatal diagnosis of beta thalassaemia by fetal red cell enrichment with NH4-Cl-NH4HCO3 differential lysis of maternal cells.

Prenatal diagnosis with globin chain synthesis analysis on fetal red blood cells concentrated by NH4Cl-NH4HCO3 differential lysis of maternal cells (Orskov lysis) was carried out in 27 pregnancies at risk for beta thalassaemia and one at risk for sickle cell beta0 thalassaemia. The beta/gamma globin chain synthesis ratio was also determined after anti-i-differential agglutination (12 cases), in almost pure fetal samples (sic cases) and by extrapolation (one case). Differential lysis permitted the study of samples drawn by placental aspiration containing as little as 3.2% fetal red blood cells. There was no consistent difference between the beta/gamma ratios observed after differentail lysis and those determined after the use of the other approaches. A presumptive diagnosis of homozygous beta thalassaemia was made in nine cases. All but one of these pregnancies was terminated. The absence of beta chain synthesis was confirmed by the study of fetal blood after abortion in four cases with suitable samples. Of the remaining pregnancies, six proceeded to term and non-homozygous infants were delivered. The others are still in progress. No fetal loss occurred. Orskov lysis seems to be a very reliable method for prenatal diagnosis of beta chain abnormalities. Moreover it can minimize the number and duration of placental aspirations required and thus the risk to the fetus.