Evaluation of a fetus at risk for dihydropteridine reductase deficiency by direct mutation analysis using denaturing gradient gel electrophoresis

Dihydropteridine reductase (DHPR) is an enzyme involved in the recycling of tetrahydrobiopterin (BH₄), which is an obligate co-factor of the aromatic amino acid hydroxylases. DHPR deficiency is a rare, autosomal recessive disorder caused by mutations in the QDPR gene. DHPR-deficient patients are diagnosed by a lack of response to a low phenylalanine diet and by severe neurological symptoms. Final diagnosis is made by measurements of neurotransmitters and pterin metabolites in cerebrospinal fluid (CSF) and urine, in addition to DHPR enzyme activity, which can be assessed in whole red blood cells. Treatment of DHPR deficiency can be difficult and the outcome is not always satisfying, even if all treatment strategies are followed. Therefore prenatal diagnosis is of great importance in affected families. Prenatal diagnosis is possible by measuring DHPR activity in different cell types but this is time consuming. More than 25 different mutations have to date been identified in the QDPR gene and direct identification of a mutation in a fetus would be easy and rapid. We have developed a method based on denaturing gradient gel electrophoresis (DGGE) for the analysis of the QDPR gene. The method is useful for rapid and simultaneous scanning of all exons and flanking intronic sequences of the QDPR gene. We describe the first prenatal diagnosis conducted using this method. Copyright © 2001 John Wiley & Sons, Ltd.     

A synthetical methodology for identifying priority pollutants in reclaimed water based on meta-analysis

Wastewater reclamation and reuse is an increasing global project, while the reclamation treatment on wastewater does not completely remove all pollutants in water. The residual pollutants in reclaimed water would cause potential risk on human health and ecosystem safety during the long-term use. It is impossible to analyze and control all pollutants one by one in practice, therefore, identification and control of priority pollutants will be efficient strategy to ensure the safe use of reclaimed ...     

Genetic amniocentesis in biamniotic twin pregnancies by a single transabdominal insertion of the needle

We present a technique to aspirate amniotic fluid from both sacs in biamniotic twin pregnancies using a single abdominal insertion with a spinal needle. It was successful in 48 out of 55 cases of biamniotic twin pregnancies referred to our perinatal unit between 1985 and 1994. The single insertion technique was used when the inter-amniotic membrane was clearly evident and two separate free amniotic fluid pools could be reached by the operator with a single puncture. An adequate amount of amniotic fluid was sampled from both sacs to make a cytogenetic diagnosis in all cases. There were four fetuses with trisomy 21 in three twin pregnancies. In two cases, only one twin was affected whilst the co-twin was normal, so that a selective feticide was performed. No miscarriages due to genetic amniocentesis were reported. After 1990, all genetic amniocenteses in biamniotic twin pregnancies (except for one case due to late booking) were performed between 14 and 15 weeks of gestation and with all cases except one, it was possible to sample both twins by a single puncture. We suggest that early amniocentesis (14–15 weeks) by a single abdominal puncture could be a reliable and safe alternative to first-trimester chorionic villus sampling in twin pregnancies.     

Pure fetal blood samples obtained by cordocentesis: Technical aspects of 322 cases

Three hundred and twenty-two percutaneous umbilical blood samplings were performed over 4 years in our prenatal diagnostic centre. A 3·5 MHz sector ultrasound transducer was used to guide a 22·5-gauge needle under local anaesthesia. Sampling was performed fcir rapid fetal karyotyping (within 72 h) in 120 cases, for diagnosis of fetal toxoplasmosis in 133 cases, for determination of the severity of Rh immunization in 15 cases, and for diagnosis of congenital rubella in 4 cases. Pure fetal blood was obtained in 98·7 per cent of the cases after two attempts. The approach to the cord was either transamniotic or trans-piacental. Puncturing was preferentially done at the placental insertion of the cord (72·2 per cent of the cases) and the mean blood sample volume was 3·5 ml. The rate of fetal death in utero was 1 9 per cent, including two cases of amnionitis, one trisomy 18, and one severe bradycardia. The failures were due to sampling at an early stage of pregnancy (before gestation week 18), to maternal obesity, oligohydramnios, and the inexperience of the operator.     

The safety of fetoscopy: (I). Effect of umbilical vessel puncture on the fetal heart rate and cord histology

The fetal heart rate (FHR) was continuously monitored during 42 umbilical vessel punctures performed at the placental insertion of the cord in 24 diagnostic fetoscopies in which pure fetal blood was obtained. In only one patient did a deceleration first appear during puncture and aspiration of fetal blood. In two patients decelerations preceded fetoscopy and in two others they began during the fetoscopy but before puncture of an umbilical vessel. In 19 patients, the FHR did not change at all during the procedure. Fetal haemorrhage after sampling was either absent or minimal. Six pregnancies were terminated because a positive diagnosis had been made and 18 healthy babies were born. Umbilical cords were examined after 7 terminations of pregnancy and after 6 deliveries. In the former group the puncture could just be seen with the naked eye and the needle track was demonstrated histologically in 6. No traces of the puncture or other abnormalities were found in the cords after delivery. Fetal blood sampling from umbilical cord vessels, particularly at the placental insertion of the cord, is the technique of choice since pure fetal blood can be obtained without increasing the risk of fetoscopy.     

Prenatal ultrasound diagnosis of cloacal exstrophy associated with myelocystocele complex by the ‘elephant trunk–like’ image and review of the literature

A case of cloacal exstrophy (CE) was detected by ultrasound as early as 22 weeks of gestation in association with myelocystocele complex, an unusual form of occult spinal dysraphism often associated with such a disease. The ultrasonographic diagnosis was made through the detection of a wavy cord-like segment of soft tissue protruding from the anterior abdominal wall, just below the umbilical cord insertion, strongly resembling the trunk of an elephant. Our article enforces the suggestion that the ultrasound elephant trunk–like image should be added to the existing major criteria for making prenatal diagnosis of CE. Copyright © 2005 John Wiley & Sons, Ltd.     

Chorionic villus sampling: An analysis of the obstetric experience of 1000 cases

Chorionic villus sampling was performed between 7 and 12 weeks gestation in 1000 patients, 935 of whom intended to continue after fetal diagnosis. Transcervical and Transabdominal aspiration techniques were used providing a sampling success rate of 99 per cent. Anatomical and clinical contraindications to transcervical aspiration were pointed out, and the complementary role of the transabdominal approach evaluated. In the 615 concluded pregnancies an overall abortion rate of 4.1 per cent was observed. A significant association between fetal loss and number of catheter insertions was demonstrated. Bacterial inoculation by catheter insertion and colonization of uterine cavity was suspected as the cause of chorionamnionitis diagnosed in two cases (0.2 per cent) after CVS. Bleeding was the most frequent early complication (12.0 per cent) following chorionic aspiration, but was not significantly related to pregnancy wastage. Late complications, i.e. premature rupture of membranes (0.8 per cent), preterm delivery (6.3 per cent), perinatal losses (1.2 per cent), placental disorders (1.6 per cent), and congenital defects (2.6 per cent) did not exceed the expected values. Normal intrauterine growth patterns were ultrasonically estimated by cross-sectional and longitudinal studies, while the weight at birth was normally distributed in the range of the general population.     

The fetal intrahepatic umbilical vein as an alternative to cord needling for prenatal diagnosis and therapy

Seventy-one fetal blood samplings (FBS) were attempted from the intrahepatic portion of the umbilical vein (IHV) at 18–34 weeks; 54 were attempted primarily and 17 secondarily after a failed attempt at the placental cord insertion. Fetal blood was obtained in 89 per cent of the cases. Intravascular transfusion (IVT) was attempted on 31 occasions and successful in 24 (77 per cent). In all cases of failed sampling or transfusion via the IHV, prenatal diagnosis and/or therapy was accomplished using alternative procedures. On only one occasion was the procedure postponed. There were no losses or neonatal morbidity attributable to the procedure. FBS from the IHV may be considered as an alternative approach to sampling the placental cord insertion. It is recommended in cases where the approach to the placental cord insertion is difficult or hazardous.     

Prenatal diagnosis of carbamoyl-phosphate synthetase deficiency by fetal liver biopsy

In a pregnancy at risk for carbamoyl-phosphate synthetase (CPS) deficiency, prenatal diagnosis was attempted by fetal liver biopsy, performed at 18 weeks of gestation. CPS activity was absent and the diagnosis was confirmed after termination of the pregnancy. The technique employed for fetal liver biopsy is described together with an evaluation of its possible role in prenatal diagnosis.     

Prenatal diagnosis of chronic granulomatous disease (CGD) in four high risk male fetuses

Prenatal diagnosis of chronic granulomatous disease (CGD) was performed in four male high risk fetuses. The male sex was previously determined by an amniotic cell karyotype. Three kinds of test were performed on fetal blood obtained by umbilical venous puncture under fetoscopy at the 20th gestational week: nitroblue tetrazolium reduction (NBT) cytochemical test with phorbol myristate acetate (PMA) as activator; luminol enhanced chemiluminescence with activation by serum opsonized zymosan (STZ) or PMA; superoxide anion (0) production by measurement of the superoxide dismutase inhibitable reduction of cytochrome c with PMA as activator. Results were compared to those obtained in six fetuses investigated for other inherited diseases. In one case, absence of granulocyte defects was confirmed at birth. In three other cases, the tests showed deficient metabolic oxidative granulocytes. The pregnancy was terminated and the CGD diagnosis was confirmed on the products of abortion. The use of three different techniques performed on whole blood for CGD prenatal diagnosis is recommended instead of a single isolated test to ensure a higher confidence in the diagnosis.     

Rapid molecular prenatal diagnosis of ataxia-telangiectasia by direct mutational analysis

Mutations of the ataxia-telangiectasia-mutated (ATM) gene are responsible for the autosomal recessive disorder ataxia-telangiectasia (A-T). This study reports the first A-T prenatal diagnosis performed in Spain by direct molecular analysis. The pregnant woman had a previous child suffering from A-T due to a deletion in the ATM gene. The ATM coding region was sequenced in the A-T patient and her parents. Then, a specific polymerase chain reaction (PCR) to detect the deletion was performed for prenatal diagnosis. Additionally, polymorphic HLA loci were examined in order to exclude the possible contamination by maternal DNA. In this family of Gypsy origin, we carried out a rapid molecular diagnosis of A-T. Then, a prenatal diagnosis was carried out, identifying the deletion in the fetal DNA. Additionally, we performed a population study in unrelated Spanish Gypsies and in unrelated controls, showing that the deletion described could be a hotspot in the Spanish Gypsy population. The size of the coding region and the genomic structure, together with the absence of hotspots, make the mutation screening of the ATM gene difficult. The ability to identify ATM mutations provides a tool that can be applied in confirmatory diagnosis, genetic counselling, carrier prediction and prenatal diagnosis. Copyright © 2007 John Wiley & Sons, Ltd.     

Evaluation of prenatal diagnosis by a registry of congenital anomalies

Prenatal diagnosis performed by fetal karyotype and ultrasound scan is now a routine part of antenatal care in many countries. How many fetal anomalies are actually detected by these procedures? We have used our registry of congenital malformations to answer this question. In our region, prenatal diagnosis was performed in 23.1 per cent of fetuses with a chromosomal aberration and in 20.1 per cent of fetuses with non-chromosomal anomalies. Only 6.9 per cent of the pregnancies with fetuses with non-chromosomal anomalies were terminated. The sensitivity of prenatal diagnosis by ultrasonographic examination was much lower for isolated malformations (fetuses with only one anomaly) than for multiple malformed children, 15.3 and 48.3 per cent respectively, chromosomal anomalies excluded.     

Evaluation of routine prenatal diagnosis by a registry of congenital anomalies

Prenatal diagnosis performed by ultrasound scan is now a routine part of prenatal care in many countries. How many fetal anomalies are actually detected by these procedures? We have used our registry of congenital malformations to answer this question. In a previous study (Prenat. Diagn., 12 , 263–270, 1992), considering the period 1979–1988, we have shown that prenatal diagnosis was performed in 23.1 per cent of fetuses with a chromosomal aberration and in 20.1 per cent of fetuses with non-chromosomal anomalies. In 1991 and 1992, the percentatge of termination for Down syndrome was 44.4 and 41.9 per cent, respectively. From 1989 to 1992, the detection rate and the specificity of prenatal diagnosis by ultrasonographic examination were improved. The detection rate for isolated malformations (fetuses with only one anomaly) and for multiple malformed children was 26.2 and 66.0 per cent, respectively. The detection rate of congenital anomalies by ultrasonography was variable for the different categories of malformation. A high detection rate was observed for anencephaly (100 per cent) and urinary tract malformation. A low detection rate was seen for cleft lip (17.5 per cent) and limb reduction defects (18.2 per cent).     

First report of prenatal diagnosis of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency in a pregnancy at risk

Prenatal diagnosis of long-chain 3-hydroxyacyl-CoA dehydrogenase (3-HAD) deficiency was performed in a family at risk. The diagnosis of an affected fetus was carried out by enzyme assay in cultured chorionic villus cells.     

Prenatal diagnosis of pyruvate carboxylase deficiency

Prenatal diagnosis of pyruvate carboxylase (PC) deficiency was performed in a family at risk for the acute neonatal form of this disease which manifests secondary citrullinemia. The diagnosis of an affected child was confirmed by enzyme assay and ³H-biotin labelling of proteins in cultured fetal skin fibroblasts. Sufficient amniocytes were cultured in 3–4 weeks for enzyme analysis in two centres. Citrulline concentration in amniotic fluid (AF) was normal in the affected fetus.     

Evaluation of prenatal diagnosis of limb reduction defects by a registry of congenital anomalies

Prenatal diagnosis performed by ultrasound scan is now a routine part of antenatal care in our region. How many fetal anomalies are actually detected by this procedure? We have used our registry of congenital malformations to answer this question regarding limb reduction defects (LRDs). The mean time of detection of LRDs was 26 weeks of pregnancy (range 16–32 weeks). The sensitivity of prenatal diagnosis of LRDs by ultrasonographic examination was much lower for isolated malformations (fetuses with only one anomaly) than for multiply malformed children with LRDs, 4·0 and 18·2 per cent, respectively. For all cases of LRDs, the percentage of prenatal detection was 11·5. Termination of pregnancy was performed in 6·7 per cent of the cases.     

Metaphyseal chondrodysplasia McKusick type in a Chinese fetus,caused by novel compound heterozygosity 64T> A and 79G >T in RMRPgene

We present the first confirmed case by molecular analysis of a metaphyseal chondrodysplasia, McKusick type, in a 22-week fetus. Two novel compound heterozygous mutations, 64T> A and 79G > T, were found in the highly conserved regions of the RMRP gene. Twenty-two heterozygous g.1018 T> C mutations, two homozygous g.1018 T> C mutations, two heterozygous insertion mutations g.799_g.800insC and one heterozygous insertion mutation g.849_g.850insT were found among 100 normal controls. Careful radiological examination of the fetus for skeletal dysplasia allowed definitive diagnosis, proper genetic counselling and future prenatal diagnosis. Copyright © 2006 John Wiley & Sons, Ltd.     

Duplication of the long arm of chromosome 13 secondary to a recombination in a maternal intrachromosomal insertion (shift)

A rare chromosomal aberration consisting of a chromosomal shift was found in a woman who had prenatal diagnosis because she had previously had a malformed girl with phenotypic features compatible with the diagnosis of Patau syndrome. Chromosome analysis using G, C, and NOR banding showed a direct intrachromosomal insertion of bands 13q12 to 13q14 onto the short arm of chromosome 13 at band 13p13. We discuss this observation and compare it with other published reports of chromosomal shifts.     

Maternal contamination of amniotic fluid demonstrated by DNA analysis

DNA from 16 sets of samples comprising DNA from uncultured amniotic fluid cells, cultured amniotic fluid cells, fetal tissue, and maternal blood was analysed by the polymerase chain reaction (PCR) with AC-repeat primers. The analysis was performed to investigate the presence of contaminating maternal cells in amniotic fluid which would affect the reliability of DNA studies for prenatal diagnosis. In three sets, maternal contamination of uncultured amniotic fluid cells was detected. In one of the three sets, maternal contamination was present in both uncultured and cultured amniotic fluid cells. The use of amniotic fluid cells as a source of DNA for prenatal diagnosis should be limited to cases where the purity of the DNA can be demonstrated prior to the diagnostic test being performed. This limitation in the use of amniotic fluid DNA also extends to other forms of diagnosis relying on the purity of amniotic fluid samples, particularly the new in situ hybridization methods currently being developed.     

Prenatal diagnosis of pyruvate dehydrogenase E1α subunit deficiency

Pyruvate dehydrogenase (PDH) E1α subunit deficiency is an X-linked inborn error of metabolism affecting males and females with equal frequency. The diagnosis is usually based on determination of enzyme activity, although this may present difficulties in some females because of X-inactivation patterns favouring expression of the normal X chromosome. This is a particular problem for prenatal diagnosis using chorionic villus cells where normal enzyme assay results do not necessarily exclude the diagnosis and confirmatory X-inactivation analysis may be complicated by variable methylation of active and inactive X chromosomes. We describe prenatal diagnosis in two pregnancies in a family following diagnosis of a PDH E1α deficient male. The first prenatal diagnosis was performed by enzyme assay, but by the time of the subsequent pregnancy, the underlying mutation in the affected male had been identified and direct gene analysis was possible. This study highlights the limitations of diagnosis of PDH E1α deficiency based on measurement of the gene product and illustrates the need for mutation analysis in affected individuals.