Adult-onset GM2 gangliosidosis diagnosed in a fetus

Amniocentesis and subsequent tests are reported on a fetus conceived of a rare mating type: its mother has an intermediate level of β hexosaminidase A (HEX A), characteristic of carriers of Tay-Sachs disease (TSD), while the father suffers from an adult-onset GM₂ ganglio-sidosis (AOG) with severe HEX A deficiency. Activity of HEX A in the cultured fetal cells was very low when measured by the heat-inactivation method, thus showing the typical biochemical phenotype of TSD fetuses. However, upon separation of HEX isozymes by ion exchange chromatography, residual HEX A (17 per cent of total HEX) was demonstrated. Also in contrast to TSD fetuses, this fetus' fibroblasts were able to synthesize the precursor of a chains of HEX, and ultrastructural examination of its brain revealed few atypical lamellar bodies, unlike those found in TSD fetuses of the same gestational age. It is therefore concluded that the fetus was not affected with TSD, but rather with AOG.     

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

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

Differentiation in human chorionic villus cultures: hCG and hla expression

The present report describes methods to separate, culture, and study syncytio-cytotrophoblast and mesenchymal core of the first-trimester human chorionic villus. The cultured outer layer cells (syncytio-cytotrophoblast) are multinucleated, pleomorphic, and active in the formation of human chorionic gonadotrophin (hCG). The mesenchymal core cells are more fibroblast-like in appearance, do not show multinucleation, and have less hCG in their culture media. Both cultured cell types express HLA (ABC) Class I histocompatibility antigens but not HLA (DR) Class II antigens. These and previous studies from this laboratory postulate different embryonic origins: (1) Syncytio-cytotrophoblast cultures of chorionic villus derive from differentiated trophoblast and preserve multinucleation as well as hCG hormone function. (2) Cells cultured from the chorionic villus core originate from extraembryonic mesenchyme. (3) Amniocytes (AF cells) cultured from amniotic fluid resemble the multipotential and early-stage trophoblast, retaining pleomorphism, multinucleation, and lacunae formation as well as production of hCG, progesterone, oestrogen, basement membrane glycoprotein, and Type IV collagen. These cell types cultured from the chorionic villus and amniotic fluid provide a means for in vitro study of specific embryonic cell lineages.     

The cerebro-hepato-renal (Zellweger) syndrome: Prenatal detection based on impaired biosynthesis of plasmalogens

Prenatal diagnosis of the cerebro-hepato-renal (Zellweger) syndrome has been performed in 10 pregnancies at risk by measuring both the activity of acyl CoA: dihydroxyacetonephosphate acyltransferase (DHAP-AT) and the de novo plasmalogen biosynthesis, either in cultured amniotic fluid cells or in fibroblasts cultured from a chorionic villus biopsy. In 7 of the pregnancies both tests indicated no abnormality. All 7 continued to term and normal infants were delivered. However, in amniotic fluid cells from 2 fetuses affected by Zellweger syndrome unequivocal differences from control values were found. The activity of DHAP-AT was clearly deficient and the de novo plasmalogen biosynthesis was impaired. In one pregnancy at risk prenatal diagnosis was performed during the first trimester by measuring both the DHAP-AT activity and the de novo plasmalogen biosynthesis in fibroblasts cultured from a chorionic villi biopsy. From the deficient DHAP-AT activity and the impaired de novo plasmalogen biosynthesis it was concluded that the fetus was affected. This was confirmed biochemically after induced abortion. It can be concluded that measurement of the DHAP-AT activity and the de novo plasmalogen biosynthesis provides convenient methods for the early prenatal detection of Zellweger syndrome.     

First-trimester prenatal diagnosis of aspartylglucosaminuria

Fetal aspartylglucosaminuria (AGU) was studied during the first trimester of pregnancy in six at-risk pregnancies using chorionic villus samples. The activity of aspartylglucosaminidase (AGA) was high in five cases, indicating an unaffected fetus. This was confirmed through delivery of healthy newborns with a normal pattern of urinary oligosaccharides. Low enzyme activity in an uncultured biopsy specimen and in cultured amniotic fluid cells in one case demonstrated that the fetus was affected. The pregnancy was terminated and the prenatal diagnosis was confirmed by showing reduced AGA activity in cultured fibroblasts of the fetus.     

Cytogenetic analysis of trophoblasts by comparative genomic hybridization in embryo-fetal development anomalies

Cytogenetic studies of spontaneous abortions or intrauterine fetal death depend on conventional tissue culturing and karyotyping. This technique has limitations such as culture failure and selective growth of maternal cells. Fluorescent in situ hybridization (FISH) using specific probes permits diagnosis of aneuploidies but is limited to one or a few chromosomal regions. Comparative genomic hybridization (CGH) provides an overview of chromosomal gains and losses in a single hybridization directly from DNA samples. In a prospective study, we analyzed by CGH trophoblast cells from 21 fetuses in cases of spontaneous abortions, intrauterine fetal death or polymalformed syndrome. Six numerical chromosomal abnormalities including one trisomy 7, one trisomy 10, three trisomies 18, one trisomy 21 and one monosomy X have been correctly identified by CGH. One structural abnormality of the long arm of chromosome 1 has been characterized by CGH. One triploidy and two balanced pericentromeric inversions of chromosome 9 have not been identified by CGH. Sexual chromosomal constitutions were concordant by both classical cytogenetic technique and CGH. Contribution of trophoblast analysis by CGH in embryo-fetal development anomalies is discussed. Copyright © 2001 John Wiley & Sons, Ltd.     

Constraints on temperature-dependent sex determination in the leopard gecko (<Emphasis Type="Italic">Eublepharis macularius</Emphasis>): response to Kratochvil et al.

Kratochvil et al. (Naturwissenschaften 95:209–215, 2008) reported recently that in the leopard gecko (Eublepharis macularius) of the family Eublepharidae with temperature-dependent sex determination (TSD), clutches in which eggs were incubated at the same temperature produce only same-sex siblings. Interpreting this result in light of studies of sex steroid hormone involvement in sex determination, they suggested that maternally derived yolk steroid hormones could constrain sex-determining mechanisms in TSD reptiles. We have worked extensively with this species and have routinely incubated clutches at constant temperatures. To test the consistency of high frequency same-sex clutches across different incubation temperatures, we examined our records of clutches at the University of Texas at Austin from 1992 to 2001. We observed that clutches in which eggs were incubated at the same incubation temperature produced mixed-sex clutches as well as same-sex clutches. Furthermore, cases in which eggs within a clutch were separated and incubated at different temperatures produced the expected number of mixed-sex clutches. These results suggest that maternal influences on sex determination are secondary relative to incubation temperature effects.     

Does the mechanism of sex determination constrain the potential for sex manipulation? A test in geckos with contrasting sex-determining systems

The concentration of yolk steroids was suggested to influence offspring gender in oviparous animals subject to both temperature-dependent sex determination (TSD) and genotypic sex determination (GSD). However, the proposed mechanisms of steroid effects are thought to differ between TSD and GSD: a direct effect of oestrogens on gonad feminisation in TSD species vs a differential induction of male-producing or female-producing gametes in GSD species. Geckos offer an ideal opportunity for testing these suggested mechanisms. Closely related gecko species differ in their modes of sex determination. They lay clutches of two synchronously formed eggs; both eggs share equal steroid levels. If identical hormonal composition and environment during vitellogenesis, gravidity and incubation determine the sex of the progeny, siblings should share the same gender in both TSD and GSD geckos. We found strong support for this prediction in a TSD gecko species. Among clutches that were incubated at the temperature that produced both sexes, there were no clutches with siblings of the opposite sex. On the other hand, about half of the clutches yielded siblings of the opposite sex in four GSD species. These results suggest that sex-determining systems constrain the ability of the female to produce single-sex siblings and, hence, it seems that the GSD mechanism constrains the opportunities for sex ratio manipulation in geckos via yolk steroid manipulation.     

First trimester studies of A fetus at risk for triose phosphate isomerase deficiency

A first trimester prenatal diagnosis was offered to a mother whose child had died of haemolytic anaemia and multisystem disease caused by TPI deficiency. The deficiency state was characterized by greatly reduced TPI activity in both erythrocytes and peripheral lymphocytes. Specific activity of TPI in trophoblast homogenates from the index fetus was about 30 per cent less than in the controls, but the heat stability test showed overlap. These data were confirmed in uncultured and cultured amniotic cells, where glycolytic intermediate concentrations DHAP, GAP and FDP fell in the range of controls. These results suggested that the fetus was a TPI heterozygote. This prenatal prediction was confirmed by RBC and haematological studies at birth.     

An investigation of methods for enriching trophoblast from maternal blood

Trophoblast deportation is known to occur in normal human pregnancy, but it is not yet clear whether these cells routinely enter the maternal peripheral circulation and are available as a source of fetal DNA for non-invasive prenatal diagnosis of genetic disorders. To resolve this issue requires an efficient method of enriching trophoblast from maternal blood combined with a means to confirm its identity. Five different techniques were tested on ten retroplacental blood samples to determine the most sensitive and operator-efficient method. Lysis of red cells alone gave the best recovery of trophoblast but had to be discounted, together with Ficoll density gradient centrifugation, due to the very low purity and the excessive time required. Fluorescence-activated cell sorting (FACS) of pre-enriched trophoblast resulted in the lowest recovery rate (8 per cent) despite a 3250-fold enrichment and a very high purity. Immunomagnetic beads (Dynabeads) coated with anti-CD 16 antibody proved to be the best method for the subsequent immunocytochemical characterization of deported trophoblast. However, IO beads coated with anti-CD45 antibody may be more useful for isolating trophoblast for prenatal diagnosis due to the high purity, enrichment (32-fold), and recovery rate (78 per cent) obtained with this method.     

Prenatal diagnosis of pseudo arylsulphatase a deficiency

Prenatal diagnosis was performed on a pregnancy at risk for metachromatic leukodystrophy (MLD) in a family with the pseudo arylsulphatase A deficiency trait. Extracts of cultured amniotic fluid cells were deficient in arylsulphatase A indicating that the fetus was either affected with MLD or had the benign pseudodeficiency trait. In the cerebroside sulphate loading test, the at risk cells hydrolysed sulphatide like control cultured amniotic fluid cells implying that the fetus had pseudodeficiency. The pregnancy was carried to term and a male child was delivered. Placenta, urine and fibroblasts had very low activities of arysulphatase A. However, no sulphatide could be detected in urine and growing fibroblasts responded normally in the cerebroside sulphate loading test, suggesting pseudodeficiency. At 29 months, the infant is healthy and shows no stigmata of MLD. The prediction based on the results of the cerebroside sulphate loading test on cultured amniotic fluid cells appeared to be borne out.     

Trophoblast-like cells sorted from peripheral maternal blood using flow cytometry: A multiparametric study involving transmission electron microscopy and fetal dna amplification

Three monoclonal antibodies (MAbs) against trophoblast (GB17, GB21, and GB25) and flow cytometry were used to sort trophoblast-like cells (TLCs) from peripheral blood of pregnant women. Sorted TLCs were processed for electron microscopy and fetal DNA amplification of the Y-specific sequences from mothers carrying male fetuses. At the ultra-structural level, most of the nucleated cells had the morphology of leucocytes, suggesting maternal contaminants, and we did not find the characteristic features of the free inter-villous trophoblast cells. Nevertheless, polymerase chain reaction (PCR) analysis showed an amplification of Y-specific sequences in two out of three samples of sorted TLCs. These results suggest that besides the maternal leucocytes, sufficient trophoblast nucleated fetal cells can be obtained using cell enrichment by sorting. This sensitive method holds promise for non-invasive prenatal diagnosis of fetal sex and if sufficient Y(positive) nuclei are found, for the diagnosis of selected numerical chromosome abnormalities.     

Immediate prenatal diagnosis of Zellweger syndrome by direct measurement of very long chain fatty acids in chorionic villus cells

We report a gas chromatographic-mass spectrometric method which allows the very long chain fatty acids content of trophoblastic tissue to be directly measured in samples collected by biopsy between 8 and 11 weeks of gestation. This method has been successfully applied to the detection of fetal Zellweger syndrome in two pregnant women who had previously delivered affected infants. In one of them, increased concentrations of C26:0 (0.254 versus 0.108±0.035 μ/mg proteins) and C24:0 (1.32 versus 0.815±0.325 μ/mg proteins) in trophoblast indicated that the fetus had Zellweger syndrome, a diagnosis confirmed by pathological findings after abortion. In the second case, the pregnancy was allowed to proceed, on the basis of normal concentrations of very long chain fatty acids in trophoblastic tissue, and its outcome was actually a healthy newborn.     

A rare case of a false-negative finding in both direct and culture of a chorionic villus sample

A discrepancy is reported between the karyotype of both direct and cultured chorionic villus cells (46,XX) and a fetal skin biopsy (47,XX,+18). The significance of this result is discussed and compared with similar discordant findings reported in the literature.     

Circulating trophoblast in maternal blood

This review describes the status of circulating trophoblast, but is considered in the perspective that only a specific subset of trophoblast cells circulates in the maternal blood. The consequences for isolation, identification and clinical potential are described. Copyright © 2003 John Wiley & Sons, Ltd.     

Mosaicism for trisomy 12: Four cases with varying outcomes

Trisomy 12 observed in chorionic villus sampling (CVS) may reflect generalized mosaicism or indicate mosaicism confined to only the placenta. In this report, four cases of trisomy 12 observed in CVS or cultured placental biopsies with varying outcomes are presented. Seven dinucleotide repeat polymorphisms for chromosome 12 were used to determine the chromosome 12 origins in the fetus or child and to delineate the mechanism(s) that gave rise to the trisomy. In two cases (cases A and C), the mosaicism was confined to the placenta, resulting in normal liveborns. Although, in one case, the molecular results suggested an apparent duplication of one paternal chromosome 12 in the placenta, normal biparental inheritance was found in the diploid fetal cell line in both cases. In two other cases (cases B and D), trisomy 12 was observed in both extraembryonic and fetal tissues. In one of these pregnancies, a child was born by Caesarean section at 37 weeks because of intrauterine growth retardation and oligohydramnios, and resulted in neonatal death. Molecular markers and fluorescence in situ hybridization (FISH) revealed low-level trisomy 12 mosaicism in the spleen. In the fourth case, fetal abnormalities were detected on ultrasound and low-level trisomy 12 mosaicism was observed in amniotic fluid cells using conventional cytogenetics and FISH. Molecular markers revealed a maternal meiosis I non-disjunction of chromosome 12 in DNA from a cultured placental biopsy. Although predicting the outcomes of pregnancies involving confined placental mosaicism remains difficult, molecular techniques are valuable tools for distinguishing uniparental from biparental disomy and mechanisms of mosaicism.     

Prenatal diagnosis of the infantile type of neuronal ceroid lipofuscinosis by electron microscopic investigation of human chorionic villi

Chorionic villus biopsy specimens were studied electron microscopically in six pregnancies at risk of the infantile type of neuronal ceroid lipofuscinosis (INCL). The biopsy was performed in all cases in the first trimester of pregnancy (8–10 gestation weeks) by the transcervical route. In one case, the biopsy was repeated at 17 weeks by the transabdominal procedure. In two pregnancies, the endothelial cells and, to a lesser extent, the mesenchymal cells of the chorionic villi contained unit membrane-bound inclusions typical of INCL. In both cases, the pregnancy was terminated and in one of them identical inclusions were found in the brains and kidneys of the fetus at 20 weeks of gestational age. The children from the remaining four pregnancies are healthy and have shown no signs of the disease.     

First trimester prenatal diagnosis of Sanfilippo disease (MPSIII) type B

Two pregnancies of a family at risk for Sanfilippo disease type B were monitored in the first trimester. In one case an affected fetus was diagnosed on chorionic villi by the assay of N-acetyl-a-D -glucosaminidase and confirmed on cultured fibroblasts from the aborted fetus. Pathological findings are also reported and compared with changes observed later in life. The disease was excluded in the second pregnancy.     

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.