Susceptible chiasmate configurations of chromosome 21 predispose to non-disjunction in both maternal meiosis I and meiosis II.

The cause of non-disjunction of chromosome 21 remains largely unknown. Advanced maternal age is associated with both maternal meiosis I (MI) and meiosis II (MII) non-disjunction events. While reduced genetic recombination has been demonstrated in maternal MI errors, the basis for MII errors remains uncertain. We studied 133 trisomy 21 cases with maternal MII errors to test the hypothesis that segregation at MII may also be influenced by genetic recombination. Our data support a highly significant association: MII non-disjunction involves increased recombination that is largely restricted to proximal 21q. Thus, while absence of a proximal recombination appears to predispose to non-disjunction in MI, the presence of a proximal exchange predisposes to non-disjunction in MII. These findings profoundly affect our understanding of trisomy 21 as they suggest that virtually all maternal non-disjunction results from events occurring in meiosis I.

Cytological studies of recombination in rhesus males.

An immunofluorescence approach was used to directly examine meiotic recombination events in 483 pachytene spermatocytes from 11 male rhesus monkeys. Specifically, we examined the nuclear localization patterns of the DNA mismatch repair protein MLH1, known from analyses of other mammalian species to be a useful marker of meiotic cross-overs. Our results indicated that rhesus pachytene spermatocytes contain approximately 40 cross-overs per cell, corresponding to about one cross-over per chromosome. The chromosomal distribution of these exchanges was consistent with data from human and mouse males but, surprisingly, the overall number of foci was lower, and the number of 'exchangeless' bivalents higher, than reported for either humans or mice.

Trisomy in humans: incidence, origin and etiology.

Molecular studies conducted over the past year have demonstrated the importance of aberrant genetic recombination in the etiology of several human trisomies, and have begun to shed light on the basis of the association between advancing maternal age and trisomy. Preliminary studies of gametes using fluorescence in situ hybridization indicate that this will be a useful approach in the analysis of human non-disjunction.

Akt1/PKB upregulation leads to vascular smooth muscle cell hypertrophy and polyploidization.

Vascular smooth muscle cells (VSMCs) at capacitance arteries of hypertensive individuals and animals undergo marked age- and blood pressure-dependent polyploidization and hypertrophy. We show here that VSMCs at capacitance arteries of rat models of hypertension display high levels of Akt1/PKB protein and activity. Gene transfer of Akt1 to VSMCs isolated from a normotensive rat strain was sufficient to abrogate the activity of the mitotic spindle cell-cycle checkpoint, promoting polyploidization and hypertrophy. Furthermore, the hypertrophic agent angiotensin II induced VSMC polyploidization in an Akt1-dependent manner. These results demonstrate that Akt1 regulates ploidy levels in VSMCs and contributes to vascular smooth muscle polyploidization and hypertrophy during hypertension.

Effect of meiotic recombination on the production of aneuploid gametes in humans.

Within the last decade, aberrant meiotic recombination has been confirmed as a molecular risk factor for chromosome nondisjunction in humans. Recombination tethers homologous chromosomes, linking and guiding them through proper segregation at meiosis I. In model organisms, mutations that disturb the recombination pathway increase the frequency of chromosome malsegregation and alterations in both the amount and placement of meiotic recombination are associated with nondisjunction. This association has been established for humans as well. Significant alterations in recombination have been found for all meiosis I-derived trisomies studied to date and a subset of so called "meiosis II" trisomy. Often exchange levels are reduced in a subset of cases where the nondisjoining chromosome fails to undergo recombination. For other trisomies, the placement of meiotic recombination has been altered. It appears that recombination too near the centromere or too far from the centromere imparts an increased risk for nondisjunction. Recent evidence from trisomy 21 also suggests an association may exist between recombination and maternal age, the most widely identified risk factor for aneuploidy. Among cases of maternal meiosis I-derived trisomy 21, increasing maternal age is associated with a decreasing frequency of recombination in the susceptible pericentromeric and telomeric regions. It is likely that multiple risk factors lead to nondisjunction, some age dependent and others age independent, some that act globally and others that are chromosome specific. Future studies are expected to shed new light on the timing and placement of recombination, providing additional clues to the link between altered recombination and chromosome nondisjunction.

A systematic search for uniparental disomy in carriers of chromosome translocations.

A systematic search was made for uniparental disomy in carriers of apparently balanced chromosome translocations who also had unexplained abnormalities of mental or physical development. Of 65 families studied, biparental origin of both translocated chromosomes was demonstrated in 64, and only 1 case of maternal uniparental disomy of chromosome 14 was detected in the carrier of a Robertsonian t(13q14q). We conclude that uniparental disomy is a rare occurrence in this population.

A reinvestigation of non-disjunction resulting in 47, XXY males of paternal origin.

We have used polymorphisms within the Xp/Yp pseudoautosomal region (PAR 1) to determine the frequency and location of recombination in 80 paternally derived 47, XXY males. Of 64 informative results, there were 10 single cross-overs, one double cross-over and 53 without a cross-over. Therefore 2/3 of 47, XXY males of paternal origin result from meiosis in which the X and Y chromosomes fail to recombine. This failure was not associated with the presence of an increase in recombination in the smaller Xq/Yq pseudoautosomal region (PAR 2) or with the presence of microdeletions within PAR 1.

Cytological studies of meiotic recombination in human males.

We combined immunostaining and fluorescence in situ hybridization (FISH) methodology to directly examine meiotic exchanges in over 2,000 pachytene stage spermatocytes from 25 individuals. Our results indicate that, on average, there are about 50 exchanges per cell and that, with the exception of the acrocentric chromosomes, all chromosome arms harbor at least one exchange. We also identified significant among-individual variation in the mean number of exchanges, with an approximate 20% difference between individuals with "low" and those with "high" exchange frequencies.

Systematic search for uniparental disomy in early fetal losses: the results and a review of the literature.

About 20% of all human conceptuses are estimated to be trisomic and trisomy of all chromosomes remains a common cause of early fetal loss. Uniparental disomy (UPD) has been reported for most human chromosomes and may be an underrecognized contributor to embryonic lethality. To investigate the contribution of UPD to spontaneous abortions, we devised a genome-based screening strategy to identify holochromosomic UPD in 18 fetal losses. No cases of UPD were identified using this approach. Based on our data, UPD does not appear to be a significant contributor to early embryonic lethality. The results of the study are presented along with a review of the cases of UPD reported in the literature by chromosome, parental origin, mode of ascertainment, and phenotypic consequences due to imprinting.

Paternal uniparental disomy in a child with a balanced 15;15 translocation and Angelman syndrome.

Chromosome 15 (15q11-q13) abnormalities cause two distinct conditions, Angelman syndrome (AS) and Prader-Willi syndrome (PWS). We present the first case of a child with a balanced 15;15 translocation and AS in whom molecular studies were crucial in confirming a diagnosis. DNA polymorphisms demonstrated paternal uniparental disomy for chromosome 15, consistent with the diagnosis of AS. The molecular studies also showed the patient to be homozygous at all loci for which the father was heterozygous, suggesting that the structural rearrangement was an isochromosome 15q and not a Robertsonian translocation.

Molecular and cytogenetic investigation of complex tissue-specific duplication and loss of chromosome 21 in a child with a monosomy 21 phenotype.

Several recent molecular studies have suggested that the clinical phenotype of Down syndrome may be due to triplication of 21q22 [McCormick et al., 1989] as initially suggested by Niebuhr [1974], and perhaps just 21q22.2 [Korenberg et al., 1989, 1990; Rahmani et al., 1989]. Recently, we studied a patient with a phenotype inconsistent with Down syndrome, whose lymphocyte karyotype on several occasions detected only 46,XX,-21, + dic(21)(qter----p11::p11----qter). Combined karyotype and molecular studies on both lymphocytes and fibroblasts allowed correct identification of the abnormality as a complex monosomy/trisomy 21 mosaicism involving a marker derived from idic (21) (p11), and probable assignment of a maternal origin for the error(s). The patient's phenotype was found to be most consistent with monosomy 21. Detailed study of our patient underscores (1) the need for confirmation that there is phenotype/karyotype correlation and (2) the usefulness of molecular analyses to complement the cytogenetic interpretation of marker chromosomes.

Early complete hydatidiform moles contain inner cell mass derivatives.

In four cases of early complete hydatidiform moles, confirmed to be androgenetic in origin by DNA studies, we have identified nonchorionic inner cell mass derived structures which are not commonly observed in specimens of later gestational age. These structures include nucleated red blood cells, endothelial cells, stromal macrophages, amnion and yolk sac. The latter four structures were confirmed by specific immunocytochemical stains. Recognition that such structures can accompany complete hydatidiform moles has both theoretical and practical significance. From a theoretical perspective, it demonstrates that the maternal genome is not required for the initiation of amniogenesis, development of the yolk sac, vasculogenesis, or hematopoiesis. From a practical perspective it emphasizes that complete hydatidiform moles, with their markedly increased risk of subsequent choriocarcinoma, cannot be excluded based on the finding of "fetal structures."

Cytogenetic analysis of spontaneous abortions: comparison of techniques and assessment of the incidence of confined placental mosaicism.

Cytogenetic studies on spontaneous abortions traditionally have used one of two methodologies, direct preparations or long-term culture, to determine the chromosome constitution of either the cytotrophoblast or villous stroma, respectively. Few studies have utilized both techniques simultaneously to compare the relative efficiencies of each method and to assess the contribution of confined placental mosaicism (CPM). The present report summarizes cytogenetic studies on 691 consecutive spontaneous abortions using long-term culture, direct preparations, or both. All 691 cases were analyzed by long-term culture and 177 cases were analyzed using both long-term culture and direct preparations. The results indicate that the two methods have similar success rates, 82% for long-term culture and 76% for direct preparation; however, the proportion of normal females was significantly increased in the culture method, presumably attributable to maternal contamination. In 107 cases, results were obtained from both methods with 22 discrepancies identified. However, most of these involved a 46,XX result in culture, consistent with maternal contamination in the cultured preparation. Therefore, to estimate the proportion of CPM we excluded cases with a 46,XX result in culture and found four (6.1%) of the remaining 65 cases to be consistent with CPM. These cases consisted of normal or mosaic aneuploid cytotrophoblast and non-mosaic aneuploid villous stroma. These studies suggest that each method has specific advantages in the analysis of spontaneous abortions. Direct preparations are less prone to maternal contamination, but certain chromosome abnormalities are more likely to be identified using long-term culture.

Population-based study of congenital heart defects in Down syndrome.

Mental retardation and hypotonia are found in virtually all Down syndrome (DS) individuals, whereas congenital heart defects (CHDs) are only present in a subset of cases. Although there have been numerous reports of the frequency of CHDs in DS, few of the studies have had complete ascertainment of DS in a defined geographic area. The Atlanta Down Syndrome Project, a population-based study of infants born with trisomy 21, provides such a resource. In the first 6.5 years of the study, 243 trisomy 21 livebirths were identified in the five-county Atlanta area (birth prevalence: 9.6/10,000). Cardiac diagnoses were available on 227 (93%) of the cases and 89% of these evaluations were made by echocardiography, cardiac catheterization, surgery, or autopsy. Of the 227 DS infants, 44% had CHDs including 45% atrioventricular septal defect (with or without other CHDs), 35% ventricular septal defect (with or without other CHDs), 8% isolated secundum atrial septal defect, 7%, isolated persistent patent ductus arteriosus, 4% isolated tetralogy of Fallot, and 1% other. This report is unique in that it contains the largest number of trisomy 21 infants ascertained in a population-based study where modern techniques for diagnosing cardiac abnormalities predominate.

Prevalence of developmental and inflammatory lesions in nonmolar first-trimester spontaneous abortions.

The management of patients with first-trimester spontaneous abortions is handicapped by two problems: difficulty in recognizing conceptions that abort because of abnormal karyotypes and an incomplete understanding of what causes abortions with normal karyotypes. Our goals in this study were to define features useful in distinguishing normal from abnormal karyotype and to identify pathological processes contributing to abortions with a normal karyotype. The study population consisted of 668 well-characterized first-trimester spontaneous abortions derived from a larger study of 1,054 consecutively karyotyped spontaneous abortions. Clinical factors increased in specimens with normal karyotype were maternal age younger than 20 years (P=.0003) and autoimmune markers (P=.0474). Developmental features associated with abnormal karyotype were developmental stage less than 6 weeks (P=.0017), hydropic villi greater than 1 mm (P=.0004), and villi with two or more dysmorphic features (P=.0001). Developmental stage greater than 11.5 weeks was increased with normal karyotype (P=.0001). Histological features increased in specimens with a normal karyotype were chronic intervillositis (P=.0003), increased perivillous fibrin deposition with intermediate trophoblast (P=.0006), decidual plasma cells (P=.0040), deciduitis without plasma cells (P=.0660), and chronic villitis (P=.1581). Overall, 19% of samples with a normal karyotype versus 8% with abnormal karyotype had one or more of these findings (P < .0001). Autoimmune markers, chronic intervillositis, and increased perivillous fibrin with intermediate trophoblast all had positive predictive values greater than 85% for normal karyotype, whereas dysmorphic villi had a positive predictive value of 90% for abnormal karyotype. Patients with recurrent spontaneous abortion and normal karyotype were more likely to have one or more of the histological features listed above (31%) than patients with normal karyotype and no prior abortions (13%) and patients with recurrent abortion and abnormal karyotype (11%).

Prevalence of the partial molar phenotype in triploidy of maternal and paternal origin.

Triploid partial moles are at risk for trophoblastic neoplasia, yet the prevalence, parent of origin, and evolution of the partial molar phenotype amongst all triploids remains controversial. We determined parental origin by polymerase chain reaction (PCR) analysis, stage of development by gross and histological criteria, and partial molar status according to strict diagnostic criteria for all triploids identified amongst 1,054 consecutively karyotyped spontaneous abortions. Triploidy was detected in 64 of 832 successfully karyotyped specimens. Complete data were collected in 59 cases. Diandric origin was found in 39 specimens, and 20 of these fulfilled all four criteria for partial mole (trophoblast hyperplasia, dimorphic population of large and small villi, villous hydrops greater than 0.5 mm, and irregular villous contour). We separated the 19 diandric triploids not fulfilling all criteria for partial mole into four groups: specimens of early developmental stage, which we believed represented developing ("early") partial moles (n = 3), cases of late developmental stage, which we believed represented involuting ("ancient") partial moles (n = 4), cases showing some but not all criteria for partial mole (n = 7), and specimens with few if any criteria suggestive of partial mole (n = 5). In triploids of digynic origin (n = 20), developmental stage was significantly lower, fetal tissue was more frequently identified, and all specimens showed well-preserved fetal red blood cells. Digynic triploids occasionally showed irregular contour, dimorphic villi, and a mild form of trophoblast hyperplasia but never showed hydropic degeneration and were never suspicious for partial mole.

Very early complete hydatidiform mole.

Recent trends toward early pregnancy ultrasound have led to evacuation of complete hydatidiform moles at a stage before the development of diffuse trophoblast hyperplasia and villous cavitation. Absence of these recognized diagnostic criteria can lead to misdiagnosis and subsequent trophoblastic neoplasia. The authors identified a case of very early complete hydatidiform mole (VECM) on review of a previous curettage specimen when the patient presented 4 weeks later with increasing human chorionic gonadotropin (HCC) titers and the typical histological features of complete mole on a subsequent curettage. DNA studies on this index case and three subsequent similar specimens confirmed the diagnosis of complete hydatidiform mole using polymerase chain reaction (PCR) amplification of eight microsatellite markers on microdissected maternal and villous tissue. VECM were compared with spontaneous abortions and elective terminations of a similar gestational age to develop diagnostic criteria. Five cardinal diagnostic features were identified: redundant bulbous terminal villi, hypercellular villous stroma, a labyrinthine network of vinous stromal canaliculi, focal cytotrophoblast and syncytiotrophoblast hyperplasia on both villi and the undersurface of the chorionic plate, and enlarged hyperchromatic implantation site trophoblast.

Etiology of nondisjunction in humans.

Aneuploidy is the most common class of chromosome abnormality in humans, occurring in at least 0.3% of newborns and approximately 50% of spontaneous abortions. Considered as a class, it is the most common known cause of mental retardation and the leading cause of pregnancy loss. Despite the high frequency of aneuploidy, its obvious clinical importance, its severe impact on human reproduction, and the 35 years of research since the first human chromosome abnormality was described, we still know very little about its causes, let alone the contribution of environmental exposures. Recently, however, with the advent of molecular and molecular cytogenetic techniques and advances in reproductive biology, a body of evidence has been generated that is beginning to shed light on the incidence, origin, and etiology of human aneuploid conditions. The bulk of this evidence comes from two sources: 1) studies of the incidence of aneuploidy in the cells of origin, namely oocytes and sperm; and 2) examinations of meiotic stage, parent of origin, and meiotic recombination in trisomic conceptuses, both liveborn and abortuses. Using a multicolor fluorescence in situ hybridization (FISH) approach, it is now possible to screen on extremely large number of human sperm to determine chromosome-specific rates of disomy. Likewise, because of the introduction in the past decade of in vitro fertilization technology, a population of human oocytes suitable for aneuploidy screening became available. The examination of the cells of origin of aneuploidy, the sperm and oocytes, has provided data on the incidence of chromosome aberrations and valuable insight into possible mechanisms of nondisjunction. Additionally, the recent identification of multiple, highly informative DNA polymorphisms on all human chromosomes has made the determination of parental origin and the analysis of recombination a straightforward matter. We now know that the vast majority of trisomic conceptuses are maternal in origin, that increased maternal age is associated with nondisjunction, and that the amount and position of recombination on nondisjoined chromosomes is altered. In this review we will restrict discussions to these recent developments and to new models of the mechanism(s) of human nondisjunction based on the molecular cytogenetic analyses. Additionally, we will discuss the direction of future epidemiological research made possible through the development of molecular and molecular cytogenetic techniques. These technological advances have now allowed for a systematic search for genetic and environmental components to human nondisjunction.

Aneuploidy in germ cells: etiologies and risk factors.

A 2 1/2-day workshop on germ cell aneuploidy was convened September 11-13, 1995 at the National Institute of Environmental Health Sciences in Research Triangle Park, North Carolina to discuss current understandings of the etiology and origin of human aneuploidy, especially in regard to potential environmental causes, and to identify gaps in our research knowledge. The workshop was designed to facilitate interactions among research experts conducting studies on the fundamental biology of chromosomal movement and segregation, on aneuploidy as a human clinical problem, and on toxicological aspects of aneuploidy induction. Overview presentations provided perspectives on aneuploidy as a human clinical problem, the genetics of aneuploidy, and the issues of concern in toxicological testing and regulatory risk assessment. The four chairs introduced the topics for each of their workgroups, setting the stage for subsequent, in-depth discussions on (1) chromosome mover components, (2) altered recombination, (3) parental age effects, and (4) differential chromosome susceptibility. From these discussions, gaps in our research knowledge related to the role of the environment in the etiology of aneuploidy and associated molecular, cellular, and genetic processes involved were identified, and will be used to establish a research agenda for filling those gaps.