Clinical aspects, prenatal diagnosis, and pathogenesis of trisomy 16 mosaicism.

INTRODUCTION: Analysis of data from cases of trisomy mosaicism can provide insight for genetic counselling after prenatal diagnosis and for the elucidation of the pathogenesis of trisomy during pregnancy. METHODS: Statistical analysis was carried out on data from 162 cases of pregnancies with prenatal diagnosis of trisomy 16 mosaicism. RESULTS: The majority of cases resulted in live birth (66%) with an average gestational age of 35.7 weeks and average birth weight of -1.93 standard deviations from the population mean. Among the live births 45% had at least one malformation, the most common being VSD, ASD, and hypospadias. The level of trisomy on direct CVS (cytotrophoblast) was associated with more severe intrauterine growth restriction (IUGR) and higher risk of malformation, while the level of trisomy on cultured CVS (chorionic villous stroma) was associated only with more severe IUGR. Similarly, the presence of trisomy on amniocentesis (amniotic fluid) was associated with both IUGR and malformation, while the presence of trisomy in the amniotic mesenchyme was associated only with IUGR. Surprisingly, the degree of trisomy in placental tissues appeared to be independent of the degree of trisomy in amniotic fluid and amniotic mesenchyme. The sex of the fetus was not associated with any outcome variables, although there was an excess of females (sex ratio = 0.45) that may be explained by selection against male mosaic trisomy 16 embryos before the time of CVS (approximately 9-12 weeks). CONCLUSION: The levels of trisomy in different fetal-placental tissues are significant predictors of some measures of outcome in mosaic trisomy 16 pregnancies.

Evidence for imprinting on chromosome 16: the effect of uniparental disomy on the outcome of mosaic trisomy 16 pregnancies.

Although a number of infants with maternal uniparental disomy of chromosome 16 (upd(16)mat) have been reported, the evidence for imprinting on chromosome 16 is not yet conclusive. To test the hypothesis that upd(16)mat has a distinct phenotype, which would support the existence of imprinted gene(s) on chromosome 16, statistical analysis was performed on a large series (n = 83) of mosaic trisomy 16 cases with molecular determination of uniparental disomy status. The incidence of upd(16)mat was 40%, which is consistent with the expected one third from random chromosome loss during trisomy rescue (P = 0.262). In pairwise comparisons, upd(16)mat was found to be associated with fetal growth restriction (P = 0.029) and with increased risk of major malformation (RR = 1.43; P = 0.053). Regression modeling showed that the effect of upd(16)mat on fetal/neonatal weight and malformation is independent of the degree of trisomy detected in the fetus. Regression modeling to control for the degree of trisomy detected in the placenta was not possible due to limited sample size. We conclude that upd(16)mat is associated with more severe growth restriction, and possibly, with higher risk of malformation. Our hypothesis is that imprinted gene(s) exist on chromosome 16 and that abnormal expression of these gene(s) in upd(16)mat cells during development results in decreased cell proliferation. Although we do not advocate prenatal testing for upd(16), studies on the long-term outcome of upd(16)mat neonates is necessary for counseling purposes.

Comparative genomic hybridization: a new approach to screening for intrauterine complete or mosaic aneuploidy.

In the practice of clinical genetics chromosomal aneuploidy in both mosaic and nonmosaic forms has long been recognized as a cause of abnormal prenatal and postnatal development. Traditionally, cytogenetic analysis of cultured lymphocytes has been used as a standard test for detection of constitutional aneuploidies. As lymphocytes represent only one lineage, chromosomal mosaicism expressed in other tissues often remains undetected. The purpose of this study was to assess the utilization of molecular cytogenetic analysis for detection of chromosomal aneuploidy in placental tissues. Using placentas from 100 pregnancies with viable nonmalformed livebirths, both trophoblast and chorionic stroma were analyzed using comparative genomic hybridization (CGH). In all cases with an indication of chromosomal imbalance by CGH, fluorescence in situ hybridization (FISH) analysis was performed to confirm the presence of aneuploidy. To differentiate between constitutional aneuploidy and confined placental mosaicism (CPM), amniotic membrane was analyzed by CGH and FISH techniques. Our results demonstrated five placentas with CPM for chromosomes 2, 4, 12, 13, and 18, respectively, and two constitutional nonmosaic aneuploidies (47,XXX and 47,XXY). Molecular cytogenetic studies of human placental tissues enables easy analysis of both embryonic (amnion) and extraembryonic (chorion) cell lineages. Detection at birth of chromosomal defects affecting intrauterine placental and fetal development is important because these chromosomal defects may continue to have an influence on postnatal development.

Trisomy 7 CVS mosaicism: pregnancy outcome, placental and DNA analysis in 14 cases.

Prenatal diagnosis by chorionic villus sampling (CVS) documents placental chromosomal mosaicism in approximately 2% of viable pregnancies at 9-12 weeks of gestation and can involve various chromosomes and placental cell lineages. Confined placental mosaicism (CPM) is the result of postzygotic mitotic errors occurring in either diploid or trisomic zygotes. With trisomic zygote rescue, depending on the parental origin of the chromosome which is lost, uniparental disomy (UPD) or biparental disomy (BPD) may arise [Kalousek et al., Am J Hum Genet 52: 8-16, 1993]. In this paper, we present 14 pregnancies which were diagnosed by CVS as mosaic trisomy 7. All follow-up amniocenteses showed a normal diploid karyotype. Using both classical cytogenetics and interphase analysis, studies of term placentae showed variable levels of trisomy 7. DNA analysis was performed in nine cases to determine whether the diploid fetus had BPD 7 or UPD 7. Fetal UPD 7 was present only in one case; in eight other cases biparental inheritance was demonstrated. DNA analysis to establish the origin of trisomy 7 in the placenta was fully informative in six cases. One trisomy resulted from a meiotic error and was associated with fetal UPD 7, while the rest were somatic in origin. It is difficult to compare the effect of CPM for trisomy 7 to other trisomies confined to the placenta, as for most chromosomes there are few available cases. It appears that intrauterine fetal growth is not greatly affected by the presence of a trisomy 7 cell line in the placenta. This finding is in contrast to the serious effect of high levels of trisomy 16 within the placenta on fetal intrauterine growth in a series of well-documented cases of CPM 16 [Kalousek et al. 1993].

Comparative genomic hybridization: a new tool for reproductive pathology.

OBJECTIVE: To demonstrate the effectiveness of comparative genomic hybridization (CGH) for analysis of reproductive pathology specimens in clinical cytogenetics laboratories. DESIGN: A total of 856 CGH analyses were performed on various placental and fetal tissues derived from 368 specimens of spontaneous abortions and on placentas from 219 pregnancies with live-born infants. The live-born infants were clinically evaluated as normally developed, with either a normal birth weight or with intrauterine growth restriction; some live-born infants had an abnormal prenatal triple screen with normal cytogenetic results on amniotic fluid cell cultures. RESULTS: Comparative genomic hybridization analysis was successfully performed on 856 samples from spontaneously aborted specimens and term placentas. Failure of analysis occurred in 1.6% of samples and was due to an insufficient amount of tissue for DNA extraction. Comparative genomic hybridization identified aneuploidy in 53% of spontaneous abortion samples and 3.1% of term placentas. CONCLUSIONS: Comparative genomic hybridization analysis is a useful clinical tool for detection of aneuploidy in placental and fetal tissues. It provides a genome-wide screen while eliminating tissue culture failures, culture artifacts, and maternal cell contamination. We present practical guidelines for interpreting CGH profiles derived from human reproductive specimens.

Placental mosaicism and intrauterine survival of trisomies 13 and 18.

Cytogenetic analysis of 14 placentas from live newborn infants or from terminated pregnancies with trisomies 13 and 18 revealed that all were mosaic. The mosaicism was confined to the cytotrophoblast and not detected in villous stroma, chorionic plate, or amnion. The percentage of cells with a normal karyotype varied from 12% to 100%, the average being 70%. No such confined mosaicism could be detected in 12 placentas of trisomy 21 fetuses. These findings suggest that a postzygotic loss of a trisomic chromosome in a progenitor cell of trophectoderm facilitates the intrauterine survival of trisomy-13 and -18 conceptuses. They also imply that it is placental function which determines the intrauterine survival and that the mother plays no active role in rejection of trisomic conceptions. The combination of both a pre- and post-zygotic cell division defect in viable trisomy-13 and -18 conceptions points to the possibility of a genetic predisposition to such events. The detection of only a diploid cell line in the cytotrophoblast of some pregnancies with trisomies 13 and 18 also suggests that direct preparation is unreliable for prenatal diagnosis of these trisomies on chorionic villi sampling and that long-term villous culture should be used.

Spontaneous abortion and confined chromosomal mosaicism.

Confined placental chorionic mosaicism is reported in 2% of viable pregnancies cytogenetically analyzed on chorionic villi samplings (CVS) at 9-12 weeks of gestation. In follow-up studies this mosaicism has been shown to be associated with increased frequency of second and third trimester pregnancy loss or intrauterine fetal growth retardation. We have studied 54 spontaneous abortions (SA) for the detection of confined placental mosaicism and found 11 of them to be mosaic. All mosaic cases were identified among first trimester spontaneous abortions, and the mosaicism was confined to specific placental or embryonic/fetal cell lineages. These results indicate that the previously reported mosaicism in SA represents both confined and generalized types of mosaicism and that its accepted frequency of 5%-10% in SA will likely be higher. Over the whole gestational period, the confined placental mosaicism is more common than the reported rate of 1%-2% seen in viable pregnancies at CVS, and a higher proportion of pregnancy complications than previously suspected may be associated with confined placental mosaicism.

Screening of human placentas for chromosomal mosaicism using comparative genomic hybridization.

Detection of confined placental mosaicism (CPM) in term placental tissues is usually accomplished by conventional cytogenetic analysis of cultured chorionic stroma and direct preparations from trophoblast or, more recently, by fluorescence in situ hybridization (FISH) on interphase nuclei. In this study, we describe the use of comparative genomic hybridization (CGH) for detection of chromosomal aneuploidy in term placentas and evaluate the sensitivity of this novel approach for CPM diagnosis in multiple placental samples acquired from five pregnancies prenatally diagnosed with CPM7 and CPM16. Each sample of placental villi was separated enzymatically into trophoblast and chorionic stroma, and the level of aneuploidy (three signals/nuclei) in each tissue was determined by FISH analysis, using centromeric DNA probes specific for chromosome 7 (D7Z1/Z2) or 16 (D16Z2). Aneuploidy levels ranged from 5.2-96.1% in the 11 tissues with CPM7 and 9.8-93% in the 29 tissues with CPM16. Subsequently, CGH analysis of DNA from the trophoblast and chorionic stroma of the same tissue sites detected the trisomic clone in all placental tissues with aneuploidy (16%, as determined by FISH analysis). Our results demonstrate the sensitivity of CGH analysis for detection of chromosomal aneuploidy mosaicism and support our contention that the CGH technique is the most effective cytogenetic method for screening term placentas for the presence of CPM.

Detection of confined placental mosaicism in trisomy 18 conceptions using interphase cytogenetic analysis.

Fluorescence in situ hybridization provides a rapid and accurate technique for detecting chromosomal aneuploidy. It is an excellent method for identifying mosaicism in placental tissues following prenatal diagnosis. Mosaicism, in the form of confined placental mosaicism, occurs im approximately 1%-2% of viable pregnancies studied by chorionic villus sampling at 9-11 weeks of gestation. It has been detected in pregnancies with both diploid and trisomic fetuses and appears to have an important effect on the intrauterine fetal survival. Using both standard cytogenetic analysis and fluorescence in situ hybridization, we have studied 12 placentas from pregnancies with trisomy 18 for the presence of chromosomal mosaicism. These included 2 that were spontaneously aborted, 5 that were terminated after prenatal diagnosis, and 4 that were delivered as either stillborn or liveborn. Significant levels of mosaicism, confined exclusively to cytotrophoblast, were detected in 7 pregnancies. This study demonstrates the usefulness of interphase cytogenetic analysis of uncultured tissues as an alternative method for the detection of mosaicism.

The utilization of interphase cytogenetic analysis for the detection of mosaicism.

This study describes a method for defining mosaic aneuploidy by interphase cytogenetics based on statistical limits established from control specimens. Fluorescence in situ hybridization (FISH) has been used to detect the number of copies of specific chromosomes in interphase nuclei from placental tissues of diploid controls and mosaic placentas. FISH was performed using probes D7Z1/D7Z2, D9Z1, D10Z1, and D18Z1, all purchased from Oncor, Inc. Statistical analysis of data obtained from diploid controls was used to determine the one-sided upper reference limit and corresponding 95% confidence interval for the proportion of cells with one and three signals for each of the probes used. The one-sided upper reference limits established the lower levels of monosomy and trisomy detectable using each of the four probes. These statistical parameters were then used to interpret the results obtained by FISH applied to the study of term placentas for the confirmation of prenatally diagnosed chromosomal mosaicism.

Interphase cytogenetic analysis of single cell suspensions prepared from previously formalin-fixed and paraffin-embedded tissues.

Fluorescence in situ hybridization (FISH) provides a rapid and accurate method for the detection of chromosomal aneuploidy. We have developed a technique for the use of FISH on single cell suspensions produced from either formalin-fixed or paraffin-embedded tissues. Preparation of such tissues involves sequential rehydration, enzymatic digestion to release single nuclei, and hybridization with a fluorescently labeled chromosome-specific centromeric probe. In a clinical setting formalin-fixed tissue from many tissue types is readily available for additional retrospective study. FISH on formalin-fixed tissues is especially beneficial in follow-up studies of cases involving termination after prenatal diagnosis or patients with a malignant disease where previous routine cytogenetics established the chromosomal aneuploidy. The use of this technique eliminates the biases of cytogenetic analysis due to clonal selection in tissue culture, the low number of cells analyzed, and the restriction to only dividing cell populations. We have demonstrated that this application of interphase cytogenetics to the study of various formalin-fixed tissues is amenable to the detection of chromosomal aneuploidies and has specific advantages over cytogenetic analysis.

Distribution of mosaicism in human placentae.

Traditional first trimester chorionic villus sampling (CVS) for prenatal diagnosis can be performed by cytogenetic analysis of cytotrophoblast or chorionic villous stroma. Approximately 2% of pregnancies studied by CVS show confined placental mosaicism (CPM) involving either cytotrophoblast, stroma or both. We present the results of a cytogenetic study of nine term placentae from pregnancies with prenatally diagnosed CPM. The aneuploid++ cell lines involved trisomies for chromosomes 7,9,16, and X. The cytotrophoblast and villous stroma from multiple biopsies of these placentae were examined using a combination of interphase and metaphase cytogenetic analysis. CPM was detected in all nine of the term placentae and both tissue-specific and site-specific patterns of mosaicism could be discerned. These results indicate that the analysis of villous stroma and cytotrophoblast from multiple placental biopsies is necessary to improve our understanding of the evolution of CPM during pregnancy and its effect on the fetus.

Confirmation of CVS mosaicism in term placentae and high frequency of intrauterine growth retardation association with confined placental mosaicism.

About 2 per cent of specimens from chorionic villus sampling (CVS) analysed either on direct preparation of cytotrophoblast cells or after culture of mesenchymal stroma reveal confined placental mosaicism (CPM), most commonly involving chromosomal trisomy. A significantly higher rate of prenatal loss (22 per cent) as well as the presence of intrauterine growth retardation (IUGR) has been reported among pregnancies with CPM. To evaluate more precisely the effect of these aneuploid cell lines confined to the placenta on intrauterine fetal growth and fetal survival, we have studied 34 term placentae from pregnancies with CPM diagnosed on CVS and confirmed identical mosaicism in 17 of these placentae. There was a direct correlation between a high number of aneuploid cells present at CVS and a high likelihood of their detection in term placenta. Also, the proportion of aneuploid cells in the mosaic term placentae correlated with that observed in CVS specimens. Among 17 gestations with confirmed CPM at delivery, there were six cases of IUGR identified, five in liveborns and one associated with intrauterine death.

Two cases of confined placental mosaicism for chromosome 4, including one with maternal uniparental disomy.

Two cases of trisomy 4 mosaicism are reported including one with molecularly confirmed uniparental disomy (UPD) of chromosome 4. Cytogenetic analysis of a chorionic villus sample (CVS) in Case 1 showed complete trisomy 4 in trophoblast and diploidy in chorionic stroma. Amniotic fluid analysis demonstrated a 46,XX complement. After intrauterine fetal death at 30 weeks, molecular analysis confirmed the presence of trisomy 4 of maternal meiotic origin, while fetal tissues showed maternal UPD for chromosome 4. Cultured CVS in Case 2 revealed trisomy 4 in 2/30 cells analyzed. This pregnancy resulted in a healthy livebirth with biparental inheritance of chromosome 4. Molecularly confirmed UPD4 has not been previously reported, and therefore, although the adverse outcome in Case 1 is likely due to the trisomy 4 in the placenta, an imprinting effect associated with UPD4 cannot be excluded.

Analysis of nine pregnancies with confined placental mosaicism for trisomy 2.

Karyotypically normal fetuses with completely trisomic or mosaic placentae may be at increased risk for intrauterine growth restriction (IUGR). Molecular and cytogenetic analyses on nine pregnancies with confined placental mosaicism (CPM) for trisomy 2 were performed at two collaborating centres. Seven cases were identified through prenatal testing of chorionic villi (CVS). Two of these seven cases demonstrated complete trisomy 2 while the remaining five cases showed various levels of trisomy 2 (33 per cent-75 per cent cells). Two cases identified after IUGR was observed in newborn infants demonstrated 65 per cent and 100 per cent trisomy 2 in cultured villi from term placentae. In all nine cases, blood chromosome analysis (n = 4), chromosome analysis of amniotic fluid cultures (n = 4), and cultured amnion (n = 5) were normal, failing to demonstrate any trisomic cells in tissues of fetal origin. Molecular studies on the fetal or newborn tissues using dinucleotide repeat polymorphisms on chromosome 2 revealed normal biparental inheritance of chromosome 2 in all nine cases. The parental origin studies of the extra chromosome 2 in the placenta showed that three cases were maternal in origin, at least two of which were consistent with a maternal meiotic non-disjunction giving rise to the trisomy 2, while in one case a paternal origin of the extra chromosome 2 was established.

Maternal uniparental disomy of chromosome 2 and confined placental mosaicism for trisomy 2 in a fetus with intrauterine growth restriction, hypospadias, and oligohydramnios.

We present a case of maternal uniparental heterodisomy for chromosome 2 (UPD 2) detected after trisomy 2 mosaicism was found on placental biopsy. This case presented prenatally with severe intrauterine growth restriction (IUGR) and oligohydramnios. The diploid newborn had hypospadias and features consistent with oligohydramnios sequence. He died shortly after birth of severe pulmonary hypoplasia. The term placenta had high levels of trisomy 2 in both the trophoblast and the stroma. A comparison of this case with others reported in the literature suggests that the IUGR and oligohydramnios are likely related to placental insufficiency due to the high levels of trisomy 2 present in the trophoblast of the term placenta and the presence of UPD 2 in the diploid placental line.

Meiotic origin of trisomy in confined placental mosaicism is correlated with presence of fetal uniparental disomy, high levels of trisomy in trophoblast, and increased risk of fetal intrauterine growth restriction.

Molecular studies were performed on 101 cases of confined placental mosaicism (CPM) involving autosomal trisomy. The origin of the trisomic cell line was determined in 54 cases (from 51 pregnancies), 47 of which were also analyzed for the presence of uniparental disomy (UPD) in the disomic cell line. An additional 47 cases were analyzed for parental origin in the disomic cell line only. A somatic (postmeiotic) origin of the trisomy was observed in 22 cases and included the majority of cases with CPM for trisomy 2, 7, 8, 10, and 12. Most cases of CPM involving trisomy 9, 16, and 22 were determined to be meiotic. Fetal maternal UPD was found in 17 of 94 informative CPM cases, involving trisomy 2 (1 case), 7 (1 case), 16 (13 cases), and 22 (2 cases). The placental trisomy was of meiotic origin in all 17 cases associated with fetal UPD (P = .00005). A meiotic origin also correlated with the levels of trisomy in cultured chorionic villi samples (CVS) (P = .0002) and trophoblast (P = .00005). Abnormal pregnancy outcome (usually IUGR) correlated with meiotic origin (P = .0003), the presence of fetal UPD (P = 4 x 10(-7)), and the level of trisomy in trophoblast (P = 3 x 10(-7)) but not with the level of trisomy in CVS or term chorion. The good fit of somatic errors with the expected results could have been observed only if few true meiotic errors were misclassified by these methods as a somatic error. These data indicate that molecular determination of origin is a useful predictor of pregnancy outcome, whereas the level of trisomy observed in cultured CVS is not. In addition, UPD for some chromosomes may affect prenatal, but not postnatal, development, possibly indicating that imprinting effects for these chromosomes are confined to placental tissues.

An association between skewed X-chromosome inactivation and abnormal outcome in mosaic trisomy 16 confined predominantly to the placenta.

Skewed X-chromosome inactivation (XCI) is frequently found in the diploid fetal tissues of individuals with mosaic trisomy that originated from a 'trisomic zygote rescue' event. This may result from a high number of trisomic cells in the embryonic cell pool at the time of XCI, which are subsequently eliminated by selection. We hypothesize that extremely skewed XCI in these mosaic cases will be associated with a poor fetal outcome due to failure to completely eliminate the trisomy from all fetal tissues. To test this hypothesis, XCI status was evaluated in 17 cases of prenatally detected trisomy 16 mosaicism. Ten of the 15 informative cases showed extreme XCI skewing ( > or = 90% inactivation of one allele) in blood or other diploid fetal tissues compared to six of the 111 controls (p < 0.001). Among these 10 'skewed' cases, 6 showed an abnormal outcome, defined as developmental abnormalities and/or intrauterine or neonatal death. In contrast, of the 5 cases without extreme skewing, none showed abnormal outcome, although outcome information was incomplete in 1 case. An additional 6 cases analyzed, involving trisomy mosaicism for other chromosomes, showed similar results. Further studies are warranted to determine if XCI status adds useful information to the prediction of pregnancy outcome in prenatally detected mosaic trisomy.