Nondisjunction of human acrocentric chromosomes: studies of 432 trisomic fetuses and liveborns

The present report summarizes molecular studies on the parent and meiotic stage of origin of the additional chromosome in 432 fetuses or liveborns with an additional chromosome 13, 14, 15, 21, or 22. Our studies suggest that there is little variation in the origin of nondisjunction among the five acrocentric trisomies and that there is no association between the origin of nondisjunction and the likelihood of survival to term of the trisomic conceptus. The proportion of cases of paternal origin was similar among the five trisomies: 12% for trisomy 13, 17% for trisomy 14, 12% for trisomy 15, 9% for trisomy 21, and 11% for trisomy 22. The stage of nondisjunction was also similar among the five trisomies, with the majority of cases of maternal origin being due to nondisjunction at meiosis I, whereas for paternally derived cases, nondisjuction occurred primarily at meiosis II.     

Genesis and systematization of cardiovascular anomalies and analysis of skeletal malformations in murine trisomy 16 and 19

Summary On account of genetic homologies, trisomy 16 in the mouse is generally regarded as a direct animal model of Down's syndrome. Mouse trisomy 19, on the other hand, can be seen as a general model of human trisomies. A detailed evaluation of the cardiovascular system and skeleton in 109 fetuses with trisomy 16 and 422 balanced siblings was carried out in order to systematize the cardiovascular anomalies and the pathogenetic mechanisms responsible for their formation according to (1) general retardation, (2) genetically determined impairment of neural-crest cell migration, and (3) direct gene action on organogenesis. Skeletal malformations in the form of a rib-vertebra syndrome encountered in Ts 16 are described here for the first time. In 108 fetuses and 219 neonates resulting from cross-breeding to induce trisomy 19, we found no significant increase in the frequency of the foregoing anomalies. These results are discussed with regard to a chromosome-specific genetic influence as opposed to a general effect of chromosome imbalance. The specificity of the Ts16 syndrome is compared with that of individual organ anomalies as can be induced by teratogenic agents. Our investigation shows that specific malformation patterns of a particular type can be produced by a variety of methods. However, the overall patterns of the two syndromes are highly chromosome-specific. On detailed examination, the malformation pattern of mouse trisomy 16 shows significant similarities with that of human trisomy 21.     

Mouse trisomy 16 as an animal model of human trisomy 21 (Down syndrome): production of viable trisomy 16 diploid mouse chimeras

We have previously proposed that mice trisomic for chromosome 16 will provide an animal model of human trisomy 21 (Down syndrome). However, the value of this model is limited to some extent because trisomy 16 mouse fetuses do not survive as live-born animals. Therefore, in an effort to produce viable mice with cells trisomic for chromosome 16, we have used an aggregation technique to generate trisomy 16 diploid (Ts 16 2n) chimeras. A total of 79 chimeric mice were produced, 11 of which were Ts 16 2n chimeras. Seven of these Ts 16 2n mice were analyzed as fetuses, just prior to birth, and 4 were analyzed as live-born animals. Unlike nonchimeric Ts 16 mouse fetuses which die shortly before birth with edema, congenital heart disease, and thymic and splenic hypoplasia, all but 1 of the Ts 16 2n animals were viable and phenotypically normal. The oldest of the live-born Ts 16 2n chimeras was 12 months old at the time of necropsy. Ts 16 cells, identified by coat color, enzyme marker, and/or karyotype analyses, comprised 50-60% of the brain, heart, lung, liver, and kidney in the 7 Ts 16 2n chimeric fetuses and 30-40% of these organs in the 4 live-born Ts 16 2n animals. Ts 16 cells comprised an average of 40% of the thymus and 80% of the spleen in the Ts 16 2n chimeras analyzed as fetuses, with no evidence of thymic or splenic hypoplasia. However, we observed a marked deficiency to Ts 16 cells in the blood, spleen, thymus, and bone marrow of live-born Ts 16 2n chimeras as compared to 2n 2n controls. These results demonstrate that although the Ts 16 2n chimeras were, with one exception, viable and phenotypically normal, each animal contained a significant proportion of trisomic cells in a variety of tissues, including the brain. Furthermore, our results suggest that although the abnormal development of Ts 16 thymus and spleen cells observed in Ts 16 fetuses is largely corrected in Ts 16 2n fetuses, Ts 16 erythroid and lymphoid cells have a severe proliferative disadvantage as compared to diploid cells in older live-born Ts 16 2n chimeras. Ts 16 2n chimeric mice will provide a valuable tool for studying the functional consequences of aneuploidy and may provide insight into the mechanisms by which trisomy 21 leads to developmental abnormalities in man.     

The induction of tail malformations in trisomy 16 mouse fetuses heterozygous for the curly tail recessive gene

The mouse mutant curly tail is thought to be inherited as an autosomal recessive (ct/ct) with incomplete penetrance so that approximately 60% of ct/ct individuals exhibit the curly tail (CT) phenotype. By outcrossing ct/ct with mouse stock carrying specific heterozygous combinations of Robertsonian (Rb) chromosomes, trisomy 16 (Ts16) and Ts19 mouse fetuses (and their chromosomally balanced littermates) were derived which were heterozygous for the ct gene. All of the Ts16 (ct/Rb;Rb) fetuses, studied between days 14-19 gestation had tail malformations, 86% of which were tail flexion defects (TFD) apparently very similar to the curly tail phenotype. Neither Ts19 nor any of the chromosomally balanced (ct/Rb) littermates from both experimental crosses showed any type of tail or other spinal malformation. At the 27-29 somite stage of development, Ts16 (ct/Rb;Rb) fetuses did not show any significant delay in the closure of the posterior neuropore (PNP) compared with their littermate controls, suggesting that the tail malformation observed in Ts16 (ct/Rb;Rb) occur as a result of mechanisms which differ significantly from those thought to be responsible to causing the curly tail malformation.     

Examination of the eyelid closure defect in trisomy 16 mice

A characteristic feature of trisomy 16 mouse conceptuses is a failure of their eyelids to close. This defect was investigated by examining ocular development in serially sectioned heads of trisomy 16 and normal littermate fetuses from 10 to 18 gestational days. Other heads were examined by using scanning electron microscopy. Between 10 and 15 days, trisomy 16 ocular structures were delayed, but there was no striking abnormal morphology. At 16 days, when the eyelids were closed and fused in normal mice, trisomic eyes had a large cell mass near the inner canthus that protruded between the open lids. The mass was covered by bulbar conjunctiva and cells of the mass were continuous with developing corneal tissue. The mass was not present in the eyes of normal mice on any gestational day and was not present in trisomic eyes at 17 and 18 days, when the lids began to show varying degrees of closure. Based on its positioning at the inner canthus, the mass may represent a transient hyperplasia of the developing semilunar fold which physically impedes lid closure in the trisomic conceptuses. Previously, the defect has been attributed to the trisomy 16 conceptus's overall pattern of growth retardation and delayed development. Masses such as those seen in the trisomic eyes have not been observed in other murine lid-gap defects that have been investigated. A second finding in this study is that trisomic eyes are positioned more superiorly in the head than normal eyes. This variation may be related to alterations in cranial base morphology that are associated with trisomy 16.     

Overexpression of esterase D in kidney from trisomy 13 fetuses.

Human trisomy 13 (Patau syndrome) occurs in approximately 1 in 5,000 live births. It is compatible with life, but prolonged survival is rare. Anomalies often involve the urogenital, cardiac, craniofacial, and central nervous systems. It is possible that these abnormalities may be due to the overexpression of developmentally important genes on chromosome 13. The expression of esterase D (localized to chromosome 13q14.11) has been investigated in both muscle and kidney from trisomy 13 fetuses and has been compared with normal age- and sex-matched fetal tissues, by using northern analysis. More than a twofold increase in expression of esterase D was found in the kidney of two trisomy 13 fetuses, with normal levels in a third. Overexpression was not seen in the muscle tissues from these fetuses.     

Altered calcium currents in cultured sensory neurons of normal and trisomy 16 mouse fetuses, an animal model for human trisomy 21 (Down syndrome)

Down syndrome is determined by the presence of an extra copy of autosome 21 and is expressed by multiple abnormalities, with mental retardation being the most striking feature. The condition results in altered electrical membrane properties of fetal dorsal root ganglia (DRG) neurons, as in the trisomy 16 fetal mouse, an animal model of the human condition. Cultured trisomic DRG neurons from human and mouse fetuses present faster rates of depolarization and repolarization in the action potential compared to normal controls and a shorter spike duration. Also, trisomy 16 brain and spinal cord tissue exhibit reduced acetylcholine secretion. Therefore, we decided to study Ca2+ currents in cultured DRG neurons from trisomy 16 and age-matched control mice, using the whole-cell patch-clamp technique. Trisomic neurons exhibited a 62% reduction in Ca2+ current amplitude and reduced voltage dependence of current activation at -30 and -20 mV levels. Also, trisomic neurons showed slower activation kinetics for Ca2+ currents, with up to 80% increase in time constant values. Kinetics of the inactivation phase were similar in both conditions. The results indicate that murine trisomy 16 alter Ca2+ currents, which may contribute to impaired cell function, including neurotransmitter release. These abnormalities also may alter neural development.     

Influence of mouse trisomy 16 on expression of specific genes

We examined developmental changes in the relative activities of three different isozyme systems: aldolase, enolase and phosphoglycerate mutase, in tissues of fetal mice with trisomy 16 and of fetal euploid littermates. We wanted to determine whether morphological abnormalities such as reduced weight and size, which are generally observed in murine trisomy, are reflected at the molecular level. Following electrophoretic separation and subsequent measurement of relative activities of enolase isozymes in brain and phosphoglycerate mutase isozymes in heart, we found no significant differences between trisomy 16 fetuses and their euploid littermates. Synthesis of liver-specific aldolase was, however, delayed in trisomy 16 fetuses.     

Altered placental morphology associated with murine trisomy 16 and murine trisomy 19

The morphology of placentas from trisomy 16 and trisomy 19 mouse conceptuses aged 12 to 18 gestational days was studied at the light microscopic level. Comparisons were made with placentas from normal littermate animals. Trisomy 16 placentas showed marked changes from normal: 1) the junctional zone showed little indication of normal morphologic differentiation throughout gestation; 2) clusters of germinal trophoblast cells persisted in the labyrinth throughout gestation, whereas these cells disappeared by gestational day 16 in the normal littermate placentas; 3) the labyrinth was reduced in size in the trisomic placentas, and the differentiation of the interhemal membranes was delayed. The size of the labyrinths from trisomy 19 placentas appeared to be decreased, but otherwise the placentas appeared to have normal morphology. These observations and others from the literature show that placental development is affected by the presence of a trisomic genome, and that different trisomies influence the development of the placenta differently. For trisomy 16, we propose that the striking changes of the junctional zone may be associated with the trisomy 16-related gene dosage effect for alpha- and beta-interferon cell surface receptors. Because of the homology for this and other genes on mouse chromosome 16 with genes on human chromosome 21, findings related to the altered development of the trisomy 16 mouse may be relevant to understanding some of the phenotypic variations associated with human trisomy 21, the Down syndrome.     

Maternal serum cell-free fetal DNA levels are increased in cases of trisomy 13 but not trisomy 18

Cell-free fetal DNA in the maternal circulation is a potential noninvasive marker for fetal aneuploidies. In previous studies with Y DNA as a fetal-specific marker, levels of circulating fetal DNA were shown to be elevated in women carrying trisomy 21 fetuses. The goal of this study was to determine whether cell-free fetal DNA levels in the serum of pregnant women carrying fetuses with trisomies 13 or 18 are also elevated. Archived maternal serum samples from five cases of male trisomy 13 and five cases of male trisomy 18 were studied. Each case was matched for fetal gender, gestational age, and duration of freezer storage to four or five control serum samples presumed to be euploid after newborn medical record review. Real-time quantitative polymerase chain reaction amplification of DYS1 was performed to measure the amount of male fetal DNA present. Unadjusted median serum fetal DNA concentrations were 97.5 GE/ml (genomic equivalents per milliliter; 29.2-187.0) for the trisomy 13 cases, 31.5 GE/ml (18.6-77.6) for the trisomy 18 cases, and 40.3 GE/ml (3.7-127.4) for the controls. Fetal DNA levels in trisomy 13 cases were significantly elevated ( P=0.016) by analysis of variance of the ranks of values within each matched set. In contrast, fetal DNA levels in trisomy 18 cases were no different from the controls ( P=0.244). Second trimester maternal serum analytes currently used in screening do not identify fetuses at high risk for trisomy 13. Fetal DNA may facilitate noninvasive screening for trisomy 13 provided that a gender-independent fetal DNA marker can be developed.     

Erythropoiesis in the fetal trisomy 19 mouse. I. Characterization of erythrocyte populations in peripheral blood

Changes in the type, size, and relative percentage of different erythrocyte populations in the peripheral blood of individual trisomy 19 and normal littermate mouse fetuses were studied from 12 gestational days to term. Large nucleated erythrocytes of yolk-sac origin comprise the first population of cells and are gradually diluted out of the circulation by nonnucleated erythrocytes of hepatic origin. This transition occurs between 12 and 16 gestational days. The rate of decline of the nucleated erythrocytes in the trisomic animals lagged by approximately 1 day behind the normal littermates, so that they did not completely disappear from the peripheral circulation until day 17. A slight decrease in size of the nonnucleated erythrocytes which occurs with increasing gestational age was also delayed by approximately 1 day in the trisomic fetuses. These observations are consistent with an hypothesis that one effect of the murine trisomy 19 genome is to retard by 1 day the growth and development of the affected animal.     

A cytogenetic study of human spontaneous abortions using banding techniques

Summary The karyotypes of 941 singleton and 42 twin abortuses and 4 cystic placentae were determined. 30.5% of the singletons were chromosomally abnormal; 49.8% of these were trisomic, 23.7% X-monosomics and 17.4% polyploid. 143 trisomies were identified by banding; over a third had an extra chromosome 16, more than 10% and extra 21 or 22 and about 5% an extra 2, 18 or 15. Examples of trisomy 3, 4, 8, 9, 10, 13, 14 and 20 were also encountered. Using the data from two other published studies, the prevalence of different trisomies was estimated and an attempt was made to relate the karyotype of the conceptus to its subsequent development.     

Defects of skeletal morphology, density, and structure in mouse fetuses with trisomy 16

Skeletal anomalies present in trisomy 16 in the mouse--an animal model of human trisomy 21--are described. Altogether 27 fetuses with trisomy 16 and 118 chromosomally balanced siblings were examined radiographically and by alizarin staining on day 20 of gestation; the radiographs were analyzed by computer-aided densitometry and structural differentiation. Extensive asymmetry or abnormal fusion of the vertebral centers and alterations of the vertebral arches were observed along with rib malformations (rib-vertebra syndrome). The skull primarily exhibited anomalies of the occipital bone. Ossification of the humerus, femur, and tibia was characterized by reduced mineralization. Typical, fracture-like alterations affecting only the tibia were also observed. Measurement of the lengths of the humeri of fetuses of comparable weight revealed a growth retardation not correlatable with the degree of mineralization. The significance of these skeletal abnormalities with regard to the trisomy 21 syndrome is discussed.     

Phenotypic characterization by high-resolution three-dimensional magnetic resonance imaging evidences differential effects of embryo genotype on intrauterine growth retardation in NOS3-deficient mice

The Nos3-knockout mouse, deficient for endothelial constitutive nitric oxide synthase (NOS3), is affected by a reduction in the number and weight of the embryos and constitutes a good model for some features of preeclampsia and intrauterine growth retardation (IUGR). Deficiencies in conceptus growth and survival may result from factors inherent in the embryo itself or from deficiencies in uterine function. In the current study, we aimed to determine the effects of embryonic genotype independently of maternal genotype, which can affect uterine environment. Therefore, by using magnetic resonance imaging (MRI), we characterized the phenotypes of NOS3-defective (Nos3(-/-); n = 6), normal wild-type (Nos3(+/+); n = 5), and heterozygous (Nos3(+/-); n = 16) mouse fetuses. All of them were littermates obtained by breeding heterozygous mice (Nos3(+/-)); therefore, the maternal genotype was the same for all the fetuses. At Day 13.5 (i.e., Theiler stage TS 21-22), females were anesthetized and scanned with three-dimensional MRI. Analysis of the different measurements of the embryos and the gestational annexes showed no significant differences between Nos3(+/+) and Nos3(+/-); however, there was a trend toward larger sizes in Nos3(+/+), and values in Nos3(-/-) were significantly smaller than in Nos3(+/+) and Nos3(+/-). The reduction in the crown-rump length of Nos3(-/-) reached 12% when compared to Nos3(+/+) (P < 0.05); the effect was higher for head measurements (16% for occipito-snout length and biparietal diameter, P < 0.05 for both) and trunk diameter (17%, P < 0.05). Overall, the maximum area of fetuses in longitudinal planes decreased 27% (P < 0.05) when comparing Nos3(-/-) to wild-type Nos3(+/+). Finally, Nos3(-/-) showed a reduction of 29% in the maximum thickness of the placenta, which may be related to the appearance of IUGR due to compromised nutritional delivery to the fetus.     

Neuronal apoptosis in mouse trisomy 16: mediation by caspases

Hippocampal neurons from the trisomy 16 (Ts16) mouse, a potential animal model of Down's syndrome (trisomy 21) and neurodegenerative disorders such as Alzheimer's disease (AD), die at an accelerated rate in vitro. Here, we present evidence that the accelerated neuronal death in Ts16 occurs by apoptosis, as has been reported for neurons in AD. First, the nuclei of dying Ts16 neurons are pyknotic and undergo DNA fragmentation, as revealed by terminal transferase-mediated dUTP nick end-labeling. Second, the accelerated death of Ts16 neurons is prevented by inhibitors of the caspase family of proteases, which are thought to act at a late, obligatory step in the apoptosis pathway. In the presence of maximally effective concentrations of caspase inhibitors, Ts16 neuron survival was indistinguishable from that of control neurons. These results suggest that overexpression of one or more genes on mouse chromosome 16 leads to caspase-mediated apoptosis in Ts16 neurons.     

FISH studies of the sperm of fathers of paternally derived cases of trisomy 21: no evidence for an increase in aneuploidy

Paternal nondisjunction accounts for approximately 5% of cases of trisomy 21. To test the hypothesis that, in some such cases, the fathers might be predisposed to meiotic nondisjunction, we utilized fluorescence in situ hybridization (FISH) to screen for aneuploidy in sperm. We analyzed sperm samples from ten males with a trisomy 21 offspring of paternal origin. Among these individuals, the overall frequency of disomy 21 was 0.15%, comparable to estimates of disomy 21 in the general male population. Furthermore, none of the ten fathers of trisomy 21 individuals had significantly elevated levels of disomic sperm. Thus, our results provide no evidence that the occurrence of a trisomy 21 conceptus of paternal origin imparts an increased risk of trisomy in subsequent pregnancies. Received: 9 September 1998 / Accepted: 30 September 1998     

Decreased HLA heterogeneity in parents of children with Down syndrome.

HLA-A and B antigens were determined in a study of 37 couples and their children with trisomy 21 Down syndrome (DS), using a standard microlymphocytotoxicity test. The comparison groups included 76 couples and their healthy children. All individuals were Caucasians from the same geographical area, and there was no history of consanguinity. The parents of children with DS did not show an association with a specific HLA antigen or haplotype. Sixteen of the 37 couples (43.24%) having children with DS share two or more antigens at the A and/or B locus. This was significantly higher than the proportion in the control group (6/76, or 7.88%). Of the 16 couples having children with DS and sharing two or more antigens, eight had a haplotype in common, in contrast with only two couples in the control group. The data suggest that sharing of parental HLA-A and B antigens may be related either to the occurrence of trisomy 21 zygotes or to prenatal survival of affected embryos and fetuses.     

Relationship between corpora lutea or fetal number and plasma concentrations of progesterone and testosterone in mice

Blastocysts (1-14) were transferred unilaterally into 63 pseudopregnant mice which were killed on Day 17. Plasma progesterone concentrations were significantly (P less than 0.05) lower in animals with one fetus than in those with 2-5 or 9-14 fetuses. Plasma testosterone concentrations were correlated with fetal number in mice with 1-13 fetuses (P less than 0.001). The total placental content of chorionic gonadotrophin in 13 litters varied directly with the number in the litter (1-6), and was 1.67 +/- 0.15 ng/placenta. The number of corpora lutea per mouse was negatively correlated with mean CL volume per mouse (P less than 0.001), and the number of conceptuses was positively correlated with mean CL volume per mouse (P less than 0.001). The effect of conceptuses on the ovary was systemic. The relationship between plasma testosterone concentration and conceptus number may be due to gonadotrophins acting on the ovary, or androgens produced by the placenta or fetus.     

Robertsonian translocations: mechanisms of formation,aneuploidy, and uniparental disomy and diagnostic considerations

Robertsonian translocations (ROBs) are rearrangements of the acrocentric chromosomes 13-15 and 21-22. Cytologically, ROBs between homologous chromosomes cannot be distinguished from isochromosomes that originate through duplication of a single homologue. Both types of rearrangements can be involved in aneuploidy. A conceptus with a trisomy or a monosomy can be rescued, and in a proportion of cases, a uniparental disomy (UPD) would result. If there are regions of genome imprinting on a uniparental chromosome pair, phenotypic consequences can result. Chromosomes 14 and 15 are imprinted, and UPD of these are known to result in abnormalities. Thus, prenatal testing should be considered in all pregnancies when one of the parents is a balanced carrier of a ROB because of the risk for aneuploidy, and UPD testing should be considered in fetuses found to carry a balanced ROB or isochromosome that involves chromosomes 14 or 15. Additionally, infants or children with congenital anomalies who carry a ROB should also be considered for UPD testing.     

Skewed X-Chromosome Inactivation Is Common in Fetuses or Newborns Associated with Confined Placental Mosaicism

The inactivation of one X chromosome in females is normally random with regard to which X is inactivated. However, exclusive or almost-exclusive inactivation of one X may be observed in association with some X-autosomal rearrangements, mutations of the XIST gene, certain X-linked diseases, and MZ twinning. In the present study, a methylation difference near a polymorphism in the X-linked androgen-receptor gene was used to investigate the possibility that nonrandom X inactivation is increases in fetuses and newborns that are associated with confined placental mosaicism (CPM) involving an autosomal trisomy. Extreme skewing was observed in 7 (58%) of 12 cases with a meiotic origin of the trisomy, but in none of 10 cases examined with a somatic origin of the trisomy, and in only 1 (4%) of 27 control adult females. In addition, an extremely skewed X-inactivation pattern was observed in 3 of 10 informative cases of female uniparental disomy (UPD) of chromosome 15. This may reflect the fact that a proportion of UPD cases arise by "rescue" of a chromosomally abnormal conceptus and are therefore associated with CPM. A skewed pattern of X inactivation in CPM cases is hypothesized to result from a reduction in the size of the early-embryonic cell pool, because of either poor early growth or subsequent selection against the trisomic cells. Since approximately 2% of pregnancies detected by chorionic villus sampling are associated with CPM, this is likely a significant contributor to both skewed X inactivation observed in the newborn population and the expression of recessive X-linked diseases in females.