Rapid prenatal testing for human beta-glucuronidase deficiency (MPS VII)

Prenatal diagnosis for the lysosomal storage disorders is typically achieved by enzymatic analysis of the relevant lysosomal enzyme in cultured amniocytes or chorionic villi. While prenatal diagnosis of some genetic diseases can be done by analysis of pertinent metabolites in amniotic fluid, there are few data regarding prenatal diagnosis of lysosomal disorders by enzyme analysis of amniotic fluid. Prenatal diagnosis by enzyme analysis of amniotic fluid has the potential advantage of providing a more rapid prenatal test result. In this study we describe an assay for the prenatal diagnosis of the mucopolysaccharidosis beta-glucuronidase deficiency (MPS VII; MIM #253220) using amniotic fluid and we confirm its reliability in detecting an affected fetus in an at-risk pregnancy by enzyme analysis of cultured amniocytes and fetal fibroblasts. Because MPS VII is rare and few instances of prenatal diagnosis for this and nearly all other lysosomal disorders have been accomplished by enzyme analysis of amniotic fluid, confirmation of results obtained from enzyme analysis of amniotic fluid should be carried out by enzyme or mutation analysis using cultured amniocytes or chorionic villus specimens.     

Prenatal diagnosis of Sanfilippo disease type B

Summary The prenatal diagnosis of a fetus affected with Sanfilippo disease type B is described. The deficiency of -N-acetylglucosaminidase in the cultured amniotic fluid cells was shown by a microassay enabling early prenatal diagnosis. In addition an increased level of heparan sulphate was demonstrated in the amniotic fluid by two-dimensional electrophoresis of glycosaminoglycans. The latter result confirmed the value of this test as an adjunctive method in the prenatal diagnosis. The pregnancy was terminated and the prenatal diagnosis was confirmed by enzyme analysis of cultured fetal fibroblasts and fetal liver.     

Prenatal diagnosis of hypophosphatasia; genetic, biochemical, and clinical studies.

This report has considered three approaches to the prenatal diagnosis of the severe, early onset form of hypophosphatasia. Two of these approaches, ultrasonography and the determination of the bone/liver isozymes of alkaline phosphatase (ALP) in cultured amniotic fluid cells, have proven useful diagnostically. The third method, assay of the bone/liver isozyme activity or total activity in supernatant amniotic fluid, was not informative for the affected fetus we studies. Failure to visualize a well-defined fetal skull after 16 weeks of pregnancy when the level of alpha-fetoprotein in the amniotic fluid is normal should arouse the suspicion of hypophosphatasia. Because the disease is known to manifest clinical variabiltiy, studies to detect both the biochemical defect as well as the structural manifestations should be considered. The combined use of ultrasonography, analysis of amniotic fluid alpha-fetoprotein, and the measurement of the bone/liver ALP in cultured amniotic fluid cells would appear to be the best approach to the prenatal diagnosis.     

Genotyping and prenatal assessment of collagen lysyl hydroxylase deficiency in a family with Ehlers-Danlos syndrome type VI.

Collagen lysyl and prolyl hydroxylase activities were measured in cultured fibroblasts from a child with clinical features of Ehlers-Danlos syndrome. Lysyl-to-prolyl hydroxylase activity ratios in cells from the proband, mother, father, and control were .24, .86, .52, and 1.00, respectively, providing a biochemical diagnosis of Ehlers-Danlos syndrome type VI and indicating an autosomal recessive mode of inheritance in this family. Prenatal assessment of lysyl hydroxylase deficiency was requested and accomplished for the first time during a subsequent pregnancy in the family. A series of control cultures established lysyl hydroxylase activity to be similar in cultured amniotic fluid cells (AF and F cells) and in cultured dermal fibroblasts. Cultured F and AF cells from the monitored pregnancy had enzyme activity similar to controls, indicating that the fetus should not be affected by lysyl hydroxylase deficiency. This finding was confirmed by demonstration of normal lysyl hydroxylase activity in fibroblasts cultured from the newborn baby. These studies show that cells cultured from second trimester amniotic fluid have collagen lysyl hydroxylase activity similar to that in dermal fibroblasts, making prenatal diagnosis of lysyl hydroxylase deficiency possible.     

Argininosuccinic aciduria: prenatal studies in a family at risk.

We have monitored two successive pregnancies in a family which we found to be at risk for argininosuccinic aciduria. We measured argininosuccinic acid (ASA) concentrations in amniotic fluid and utilized an indirect assay of ASA lyase activity in cultured amniotic fluid cells. The assay procedure is based on the uptake of 14C from [14C]citrulline and of [3H]leucine into protein. ASA was easily measured in amniotic fluid from the first fetus at risk, whereas none was detectable in control fluids. Amniotic fluid cells cultured from this fetus had only 5.5% of control ASA lyase activity. The pregnancy was terminated, and hepatic ASA lyase activity in the fetus was shown to be about 1.3% of control values. In addition, eight fetal tissues were analyzed for ASA, and all had significant accumulation. ASA was not detected in amniotic fluid from the second fetus at risk, and ASA lyase activity in cultured cells was 80% of control activity. Enzymatic analysis of erythrocyte lysate confirmed the diagnosis of an unaffected child (ASA lyase = 46% of control) and indicated heterozygosity. Thus, we provide further evidence that argininosuccinic aciduria can be diagnosed successfully in utero by indirect assay of ASA lyase activity in cultured amniotic fluid cells. In addition, high amniotic fluid ASA concentrations provide strong adjunctive evidence for such a prenatal determination, and may prove to be sufficient for diagnosis.     

The use of DNA probes in preimplantation and prenatal diagnosis

DNA probes are now widely used for prenatal diagnosis, but the prospect of preimplantation diagnosis of genetic disorders requires the development of sensitive genetic tests that can be performed on small numbers of cells removed from a preimplantation-stage pre-embryo. The sensitivity of molecular tests can now be increased by specifically amplifying the target DNA with the polymerase chain reaction. In situ hybridisation with chromosome-specific DNA probes to repeated sequences also permits the detection of particular numerical chromosome aberrations or the distinction of male and female pre-embryos when only a few interphase nuclei are available. We have used in situ hybridisation to a Y chromosome-specific DNA probe to sex preimplantation-stage pre-embryos and to sex fetuses from samples of chorionic villus cells, amniotic fluid cells, and fetal blood. These two approaches (amplification of target DNA and in situ hybridisation) provide suitable tests for improving prenatal diagnosis particularly when few cells are available and they offer the possibility of tests suitable for preimplantation diagnosis.     

Prenatal diagnosis of familial hypercholesterolemia: importance of DNA analysis in the high-risk South African population

In this report on the outcome of the first prenatal diagnosis performed for familial hypercholesterolemia (FH) in a South African family, we aim to demonstrate the value of a population-directed screening strategy to identify FH patients in populations with an enrichment for certain low-density lipoprotein receptor (LDLR) gene mutations. Prenatal diagnosis was offered to an Afrikaner couple, both partners heterozygous for the FH mutation D206E, whose first child was diagnosed with heterozygous FH and the second with homozygous FH. Genomic DNA isolated from parental peripheral blood and subsequently amniotic fluid was amplified by the polymerase chain reaction (PCR) and subjected to mutation analysis. Heterozygosity for mutation D206E was confirmed in both parents, whilst this mutation was not detected in DNA directly amplified from amniotic fluid. To exclude the possibility of a false-negative result due to the limited number of cells in the uncultured amniotic fluid sample, cells were also cultured in vitro, and the DNA extracted and subjected to a second round of analysis. This confirmed the absence of mutation D206E in the fetus. This case illustrates the application of a DNA-based mutation detection technique as a simple and rapid diagnostic aid that can be carried out at a relatively early gestational stage. Prenatal diagnosis of FH, aimed at the detection of homozygous cases, is particularly feasible in populations and families with molecularly defined LDLR gene mutations.     

Heterogeneity for adenosine deaminase deficiency: Expression of the enzyme in cultured skin fibroblasts and amniotic fluid cells.

Adenosine deaminase (ADA) could be quantitated and the isozyme pattern characterized in cultured amniotic fluid cells. In 20 amniotic fluid cell cultures the mean specific activity was 14.3 U/g protein +/- 6.7 (SD) and compared favorably with values of 14.6 U/g protein +/- 6.8 (SD) observed in 26 cultures of skin fibroblasts. In cultures of skin fibroblasts established from two obligate heterozygotes for ADA deficiency, the specific activity of ADA was 7.0 and 7.7 U/g protein. The ADA isozyme pattern that existed in cultures of amniotic fluid cells was the same as that observed in cultured skin fibroblasts. This identification of the same apparent enzyme may permit the prenatal diagnosis of that form of combined immunodeficiency disease caused by ADA deficiency. Residual enzyme activity of less than 1% and 10% of the mean of normal fibroblasts could be measured in cultured fibroblasts from two unrelated children with ADA deficiency and combined immunodeficiency disease. The tissue-specific enzyme from cultured skin fibroblasts from the child with 10% residual activity had a faster electrophoretic mobility and greater heat stability than normal ADA. This enzymatic evidence indicates that at least two mutant alleles exist at the locus for ADA which predispose to combined immunodeficiency disease when present in the homozygous state.     

Citrullinemia: prenatal diagnosis of an affected fetus.

We monitored a pregnancy in a family at risk for citrullinemia due to argininosuccinic acid (ASA) synthetase deficiency. ASA synthetase activity in cultured epithelioid amniotic fluid cells from the fetus at risk was less than 2% of control epithelioid amniotic fluid cell activity. An increased concentration of citrulline was found in the at-risk amniotic fluid (0.14 mumol/ml) as compared with fluid from six controls and one at-risk but unaffected pregnancy (trace). The pregnancy was terminated, and the in utero diagnosis was confirmed by assay of ASA synthetase activity in cultured fetal skin fibroblasts (4.4% of control activity). In addition, all five fetal tissues studied had significant accumulation of citrulline, whereas control fetal tissues had none. These data provide evidence that, if precise control is maintained over tissue culture variables, citrullinemia can be diagnosed successfully in utero by microassay of ASA synthetase activity in cultured amniotic fluid cells. They also suggest that amniotic fluid citrulline concentrations provide strong adjunctive evidence for this prenatal diagnosis.     

First trimester prenatal diagnosis of 21-hydroxylase deficiency by linkage analysis to HLA-DNA probes and by 17-hydroxyprogesterone determination

Summary The close genetic linkage between the gene for congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency and HLA genes allowed us to use the polymorphism of this system as a marker of the disease. HLA genotyping can be performed by using restriction enzyme fragments hybridized with specific probes instead of serologic methods. In seven pregnancies at risk for 21-OH deficiency, a first trimester prenatal diagnosis has been performed by determining the fetal genotype by linkage analysis of DNA from chorionic villi using HLA class I and class II probes. In four of these pregnancies, determination of 17-OH progesterone in first trimester amniotic fluid afforded a complementary approach to the diagnosis.     

Cultivation of chorionic cells in studying the fetal karyotype in the 1st trimester of pregnancy

Primary culture of chorion cells obtained during the first trimester of pregnancy (7-9 weeks) through artificial abortion or by transcervical biopsy of the chorion tissue is an object for cytogenetic examination of fetus cells. The application of this cell culture for prenatal diagnosis of human hereditary diseases has a considerable advantage in time as compared with a routine method of using amniotic fluid cell culture during the second trimester of pregnancy.     

Fetal tissue sampling--indications,techniques, complications,and experience with sampling of fetal skin,liver, and muscle.

Invasive prenatal testing has become an important way to evaluate fetuses at increased risk for hereditary disorders. In utero sampling of fetal skin, liver, and muscle may be required to diagnose before-birth disorders that cannot be diagnosed by analysis using chorionic villi or amniotic fluid. In the next few years, many of these conditions will be detected by DNA analysis, and the need for these procedures may decrease dramatically. First performed by fetoscopy, fetal tissue sampling is now most frequently done by inserting a biopsy needle under continuous ultrasonographic guidance. We describe the indications, techniques, complications, and experience with obtaining fetal skin, liver, and muscle biopsy specimens.     

Zytogenetische Methoden in der Pr?nataldiagnostik

Cytogenetic analysis of amniotic fluid cells or chorionic villi are standard methods in invasive prenatal diagnosis. In certain cases, analyzing fetal blood cells or fetal cells of other origin represents an excellent supplementary investigation to disclose a fetal chromosomal aberration. At the microscopic level, chromosome analysis allows an examination of the complete genome. In the case of molecular analysis of monogenic disorders, native chorionic villi are the preferred tissue for targeted examination. Rapid advances in molecular non-invasive prenatal diagnosis will broaden parents?? options to exclude certain relevant genetic changes and will have an important impact on the field of invasive prenatal diagnosis.     

Perspective on the Technical Challenges Involved in the Implementation of Array-CGH in Prenatal Diagnostic Testing

Our aim was to construct a streamlined technical workflow to facilitate a prospective, multi-centre evaluation of array comparative genomic hybridisation (array-CGH) in the prenatal diagnostic context. A collection of commercially available DNA extraction and quantification techniques were evaluated and compared using minimal quantities of amniotic fluid, chorionic villi and cultured cells. When prenatal DNA of suitable quality and quantity was obtained, array-CGH was performed using Oxford Gene Technology’s (OGT, Oxford, UK) CytoSure? ISCA 8 × 60 K oligo array platform. With starting quantities of 2–4 ml amniotic fluid, 2–5 mg chorionic villi or under 150,000 cultured cells the following optimised technical workflow was identified: DNA extraction using the iGENatal? kit (igenbiotech, Madrid, Spain) and quantification by the Qubit^® 2.0 Fluorometer with the Qubit^® dsDNA BR assay kit (Invitrogen?, Eugene, OR, USA). In addition, it was elucidated that array-CGH can be successfully performed with as little as 125 ng DNA in the experiment using the OGT CytoSure? ISCA 8 × 60 K oligo array platform. Amidst an on-going debate on whether array-CGH should be applied in the prenatal diagnostic setting, by following the technical recommendations described here genetics laboratories can now gain exposure to prenatal array-CGH testing without compromising the conventional karyotype result.     

Properties of fetal and adult red blood cell arginase: a possible prenatal diagnostic test for arginase deficiency.

Prenatal diagnosis of inborn errors of metabolism has been possible only if the enzyme affected is expressed in amniotic fluid cells grown in culture. Arginase is essentially undetectable in normal human fibroblasts, amniotic fluid, and amniotic fluid cells but is present in high amounts in red blood cells. It is absent in the red blood cells of patients with liver arginase deficiency. The properties of the enzyme in the red cells of healthy children and adults were compared to those of the enzyme obtained from cord blood red cells of 13--20-week fetuses obtained at hysterotomy. The activities, heavy metal requirements, heat stability, pH optimum, kinetic properties, and reaction with anti-arginase antibody were examined. Both enzyme species were either identical or substantially similar by all criteria. The adult and fetal enzymes are, therefore, probably determined by the same structural gene. Fetal red cells obtained during amniocentesis and amnioscopy should then be a suitable tissue to use to make the prenatal diagnosis of arginase deficiency.     

Cryopreservation does not alter karyotype, multipotency, or NANOG/SOX2 gene expression of amniotic fluid mesenchymal stem cells

Cryopreservation of mesenchymal stem cells from amniotic fluid is of clinical importance, as these cells can be harvested during the prenatal period and stored for use in treatments. We examined the behavior of mesenchymal stem cells from human amniotic fluid in culture that had been subjected to cryopreservation. We assessed chromosomal stability through karyotype analysis, determined whether multipotent capacity (differentiation into adipogenic, chondrogenic, and osteogenic cells) is maintained, and analyzed SOX2 and NANOG expression after thawing. Five amniotic fluid samples were cryopreserved for 150 days. No chromosomal aberrations were observed. The expression levels of NANOG and SOX2 also were quite similar before and after cryopreservation. Capacity for differentiation into adipogenic, chondrogenic, and osteogenic tissues also remained the same. We conclude that cryopreservation of amniotic fluid does not alter karyotype, NANOG/SOX2 gene expression, or multipotent capacity of stem cells that have been collected from amniotic fluid during pregnancy.     

Transport and storage of amniotic fluid samples for prenatal diagnosis of metabolic diseases

Summary 1. A method for freezing uncultured amniotic fluid cells is presented, which allows their use as pregnancy-age matched controls in prenatal diagnosis of metabolic diseases. 2. Amniotic fluid cells were successfully cultured after up to 7 days in transport, which makes prenatal diagnosis available to parents living a long way from specialized centers.

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Zusammenfassung 1. Eine Einfriermethode für nichtkultivierte Fruchtwasserzellen wird beschrieben, wodurch man diese wie Kontrollmuster anwenden kann bei pr?nataler Diagnostik von Stoffwechselkrankheiten. 2. Fruchtwasserzellen wurden erfolgreich kultiviert nach langzeitigem Transport (7 Tage); dieser Befund bringt eine pr?natale Diagnostik im Bereich entfernt von spezialisierten Zentren lebender Eltern.

     

The use of quantitative cytochemical analyses in rapid prenatal detection and somatic cell genetic studies of metabolic diseases

Synopsis The biochemical analysis of enzyme activities in cultured human cells has become an increasingly important tool in the (prenatal) diagnosis of inborn errors of metabolism and in fundamental studies of somatic cell genetics and metabolic interaction of normal and mutant human cells. In all these fields the use of microchemical analyses offers new possibilities in the assay of enzymes in small numbers or even single cultured cells.When cell cultivation is carried out in dishes with a thin plastic foil at the bottom, groups of a few hundred cells and single cells can be sampled after quick freezing and freeze-drying of the culture. Incubation of this material in (sub)microlitre volumes ofp-nitrophenyl or methylumbelliferyl substrates, followed by extinction or fluorescence measurements in microdroplets using a microspectrofluorometer enables lysosomal enzyme activities in the range of 10^–11–10^–14 mol/hr/cell to be detected.Application of these techniques in assays of -1,4-glucosidase and -galactosidase on groups of 100–300 freeze-dried cultured amniotic fluid cells have enabled the prenatal diagnosis of glycogenosis-II and Fabry's disease respectively within 10–12 days after amniocentesis in the 14–15th week of pregnancy. This is a considerable reduction in the time interval hitherto required, 4–6 weeks, for the prenatal diagnosis of metabolic diseases.Examples of application of enzyme assays on single cultured cells are presented for studies on the interaction between enzyme-deficient and normal cultured cells and for genetic complementation studies after hybridization of two human cell strains. By using Lowry's principle of NADP(H) cycling, glucose-6-phosphate dehydrogenase (G-6-PD) activity could be reliably measured in single binuclear hybrid cells after fusion of normal and G-6-PD deficient human cell strains. With the same technique we were also able to detect two cell populations (normal and G-6-PD-deficient) within one culture of a heterozygous carrier for G-6-PD deficiency without the need of elaborate cell cloning techniques.Finally, a microfluorometric assay procedure was developed to enable certain lysosomal enzyme activities to be measured in single (hybrid) human cells. An example of this application is presented for -galactosidase activity measurements in single cells from a mixed culture of normal fibroblasts and cells from a patient with GM1-gangliosidosis (-galactosidase-deficient). In none of our experiments was any evidence obtained for the metabolic correction of enzyme-deficient cells by the uptake of enzyme from normal cells.     

Variation in lysosomal enzyme activity during growth in culture of human fibroblasts and amniotic fluid cells

1. 1. The specific activity of the lysosomal hydrolases in cultured skin fibroblasts varies according to the phase of growth in culture.

2. 2. Diagnosis of heterozygous genotypes for lysosomal enzyme deficiency diseases is unreliable with cultured fibroblasts, at least partly because of the growth curve-associated variations in specific activity.

3. 3. Fluctuations in specific activity during the beginning of the growth curve in vitro can be avoided by initiating cultures with cells which are in the early log phase of growth.

4. 4. Primary amniotic fluid cell cultures show no relationship between length of time in culture and lysosomal enzyme specific activity.

5. 5. Secondary amniotic fluid cell cultures exhibit growth curve-related variations in lysosomal enzyme specific activity as they assume fibroblast-like growth kinetics.

6. 6. Prenatal and postnatal diagnosis on cultured amniotic fluid cells and fibroblasts requires the use of appropriate controls which are matched for stage of growth and length of time after the last trypsinization.

     

Prenatal enzymatic diagnosis of Krabbe disease (globoid-cell leukodystrophy) using chorionic villi

Summary Sixteen pregnancies in families with children enzymatically diagnosed as having Krabbe disease (KD) were monitored for prenatal KD using the assay of galactosyl ceramide -galactosidase (GCG) in uncultured chorionic villi (CV), cultured CV, or cultured amniotic fluid cells (AFC). Prenatal KD diagnoses were made for 5 pregnancies on the basis of lower than 10% normal GCG activity in cultured CV or AFC. Uncultured CV were studied in 3 out of the 5 KD embryos, although the GCG activities of 14%–23% as compared with control villi were diagnostically inconclusive; the relatively high activities were considered to be caused by maternal GCG contamination of these very small villus samples. Although the villi from 6 of the other pregnancies yielded more conclusive results, the use of uncultured CV alone is not recommended for prenatal KD diagnosis, this material being subject to possible uncontrolled contamination with maternal enzyme.