Detection of mosaicism in lymphocytes of parents of free trisomy 21 offspring

Genetic selection assays were developed to measure rates of deletion of one or more (CAG).(CTG) repeats, or an entire repeat tract, in Escherichia coli. In-frame insertions of >or=25 repeats in the chloramphenicol acetyltransferase (CAT) gene of pBR325 resulted in a chloramphenicol-sensitive (Cm(s)) phenotype. When (CAG)25 comprised the leading template strand, deletion of one or more repeats resulted in a chloramphenicol resistant (Cm(r)) phenotype at a rate of 4 x 10(-2) revertants per cell per generation. The mutation rates for plasmids containing (CAG)43 or (CAG)79 decreased significantly. When (CTG)n comprised the leading template strand the Cm(r) mutation rates were 100-1000 lower than for the opposite orientation. As an initial application of this assay, the effects of mutations influencing mismatch repair and recombination were examined. The methyl directed mismatch repair system increased repeat stability only when (CTG)n comprised the leading template strand. Replication errors made with the opposite repeat orientation were apparently not recognized. For the (CAG)n leading strand orientation, mutation rates were reduced as much as 3000-fold in a recA- strain. In a second assay, out-of-frame mutation inserts underwent complete deletion at rates ranging from about 5 x 10(-9) to 1 x 10(-7) per cell per generation. These assays allow careful quantitation of triplet repeat instability in E. coli and provide a way to examine the effects of mutations in replication, repair, and recombination on repeat instability.     

Distribution of extracellular matrix components in nuchal skin from fetuses carrying trisomy 18 and trisomy 21

We have investigated histologically the elevations of the skin in dorsal and lateral neck (nuchal) regions of human fetuses carrying karyotypes of trisomy 18 (Edwards' syndrome) and trisomy 21 (Down's syndrome). Cavities filled with interstitial fluid were found in the dermis, epidermal basement membrane and occasionally in the epidermis of trisomy-18 fetuses, but were not delineated by an epithelium or basement membrane as judged by the absence of immunostaining for laminin, collagen IV and collagen VII. Dilated vessels were also found at the interface between dermis and subcutis. Neither normal fetal skin nor that of trisomy-21 fetuses contained cavities or dilated vessels. In order to detect possible alterations of the extracellular matrix in trisomy-18 and trisomy-21 skin, the distribution of glycoproteins, glycosaminoglycans and proteoglycans was studied immunohistochemically. In trisomy-21 and control skin, the dermis stained intensely for fibronectin, whereas the subcutis reacted only weakly. In trisomy-18 skin, the stronger staining for fibronectin appeared in the subcutis, and the prevailing collagen type was collagen III, collagen type I being absent. In the skin of trisomy-21 fetuses, collagen VI was more irregularly arranged and densely packed, whereas collagen I was more widely spaced than in normal fetuses. More hyaluronan was present in the dermis and subcutis of trisomy-21 fetuses than in that of trisomy-18 and control fetuses. A correlation seems to exist between undelimited cavities and collagen III in trisomy-18 skin, and between hyaluronan and the specific arrangement of collagen in trisomy-21 skin.Abbreviations bm Basement membrane - ep epidermis - d dermis - sc subcutis - hf hair follicle - c capillary This article is dedicated to Professor Dr. Konrad Märkel on the occasion of his 70^th birthday     

Contribution of ultrasonographic examination to the prenatal detection of trisomy 21: experience from 19 European registers

The objective of this study was to evaluate the contribution of ultrasound scanning to the prenatal detection of trisomy 21 in a large unselected European population. Data from 19 congenital malformation registers in 11 European countries were included. The prenatal ultrasound screening programs in the countries ranged from no routine screening to three ultrasound investigations per patient. Routine serum screening was offered in four of the 11 countries and routine screening on the basis of maternal age amniocentesis in all. The results show that overall 53% of cases of trisomy 21 were detected prenatally with a range from 3% in Lithuania to 88% in Paris. Ninety-eight percent of women whose babies were diagnosed before 24 weeks gestation chose to terminate the pregnancy. Centres/countries that offer serum screening do not have a significantly higher detection rate of trisomy 21 when compared to those that offer maternal age amniocentesis and anomaly scanning only. Fifty percent of trisomy 21 cases were born to women aged 35 years or more. In conclusions, second trimester ultrasound plays an important role in the prenatal diagnosis of trisomy 21. Of those cases prenatally diagnosed, 64% of cases in women <35 years and 36% of those in women >or=35 years were detected because of an ultrasound finding. Ultrasound soft markers accounted for 84% of the scan diagnoses. There is evidence of increasing maternal age across Europe with 50% of cases of trisomy 21 born to women aged 35 years or more.     

Predictors of trisomy 21 in the offspring of older and younger women

BACKGROUND: Advanced maternal age is the only well‐established risk factor for trisomy 21, yet the majority of affected individuals are born to younger women. To identify factors associated with the risk of trisomy 21 in the offspring of younger and older women, we analyzed data for cases with trisomy 21 from the Texas Birth Defects Registry for 1999 to 2007. METHODS: Data were analyzed separately for younger (i.e., <35 years of age at delivery; n = 2306) and older (i.e., ≥35 years of age at delivery; n = 1811) women using Poisson regression. RESULTS: After adjustment for maternal age and several other covariates, the prevalence of trisomy 21 in the offspring of women in both maternal age groups was higher in male than in female infants and in offspring of women who were Hispanic (compared with non‐Hispanic white women) or who had at least one previous liveborn child compared to those with none. In the offspring of older women only, the prevalence of trisomy 21 was also significantly higher when the father was 20to 24 years old (compared with 25 to 29 years old; adjusted prevalence ratio [aPR], 2.27; 95% confidence interval [CI], 1.47–3.49) and Hispanic (compared with non‐Hispanic white; aPR, 1.34; 95% CI, 1.13–1.58) and among women with less than a high school education (compared with greater than high school). CONCLUSIONS: This study identified several factors, in addition to maternal age, that were associated with trisomy 21 risk. In general, these factors were similar for both maternal age groups, although paternal characteristics were significantly associated with risk of trisomy 21 only in offspring of older women. Birth Defects Research (Part A), 2012. © 2011 Wiley Periodicals, Inc.     

The impact of trisomy 21 on early human hematopoiesis

Mitogen-activated protein kinases (MAPKs) are components of signaling cascades regulated by environmental stimuli. In addition to participating in the stress response, the MAPKs c-Jun N-terminal Kinases JNK1 and JNK2 regulate the proliferation of normal and neoplastic cells. JNKs contribute to these processes largely by phosphorylating c-Jun and thus contributing to the activation of the AP-1 complex. We here report that JNKs control entry into mitosis. We have observed that JNK activity and phosphorylation of c-Jun become elevated during the G2/M transition of the cell cycle in immortalized fibroblasts and ovarian granulosa cells. Pharmacological inhibition of JNK causes a profound cell cycle arrest at the G2/M transition in both cell types. This effect is specific as it occurs with two distinct small molecule compounds. Inactivation of JNK prior to mitosis prevents expression of Aurora B and phosphorylation of Histone-H3 at Ser 10. Silencing of JNK1 and 2 causes a similar effect, whereas overexpression of JNK1 and 2 causes the opposite effect. Inhibition of JNK delays activation of cdc-2 and prevents downregulation of Cyclin B1. We conclude that JNK signaling promotes entry into mitosis by promoting expression of Aurora B and thereby phosphorylation of Histone-H3.     

A possible cause of non-disjunction of additional chromosome 21 in Down syndrome

Summary A possible cause of non-disjunction of chromosome 21 in Down Syndromes has been cytogenetically evaluated by examining the parents by Ag-staining technique. In all the cases studied so far, the contributing parents have active ribosomal cistrons on both chromosomes 21 i.e. both chromosomes are stained positively by silver staining. These results show that the active NORs might play an essential role in meiotic non-disjunction. Furthermore, the preliminary results demonstrate that the acrocentric associations of homologous and non-homologous nature involving chromosome 21 are the most frequent in the contributing parent which may further indicate the role of multiple cellular factors affecting the associations in promoting the non-disjunction in addition to active NORs. The possible mechanisms regarding the non-disjunction of chromosome 21 have been described.Presented at the 34th Annual Meeting of the American Society of Human Genetics, Norfolk, VA, USA     

Chromosome studies of in vitro senescent lymphocytes: nonrandom trisomy 2

Chromosome studies were carried out in long-term (142 and 184 d) human lymphocyte in vitro cultures in order to investigate the cytogenetic status of aging lymphocytes. The female donors were subdivided into three subgroups according to their age: 20-40 year-old (three individuals), 70-90 year-old (five persons), and centenarians (three persons). Besides some aneuploidy and structural abnormalities, telomere fusions were detected in all donor cells, and associations of acrocentric chromosomes were found in six persons in the three age-groups. Clonal trisomy 2 was present in three individuals (two from the 70-90 year-group and one centenarian with a clone +2, +8). While telomeric fusions and acrocentric associations seem to be more related to in vitro aging, trisomy 2 also appears dependent on the age of the cell donors.     

Severe psychomotor retardation in a boy with a supernumerary derivative chromosome resulting in partial trisomy 21 and partial trisomy 7p

We report on a 12-year-old boy with a supernumerary chromosome der(21)t(7; 21)(p21; q21.3)mat, resulting in a partial trisomy 21 and a partial trisomy 7p. The patient has a severe psychomotor retardation. Although he has most of chromosome 21 in three copies, he does not have a phenotype of Down syndrome (DS). In addition to cytogenetic analysis, molecular analysis confirmed that the "DS critical region" on chromosome 21 (21q22) is not present in three copies, since the breakpoint of the partial trisomy 21 was found to be located distal to the marker locus D21S145 but proximal to D21S226. The patient's severe mental retardation is probably due to the small telomeric 7p trisomy, having the breakpoint between markers D7S507 and D7S488. In comparison with previously published cases of partial trisomy 7p, the phenotype of this patient indicates that there is a region around the distal part of band 7p21 that in three copies might contribute to many of the facial features common to patients with partial trisomy 7p.     

Generation of a novel isogenic trisomy panel in human embryonic stem cells via microcell-mediated chromosome transfer

Aneuploidy is the gain or loss of a chromosome. Down syndrome or trisomy (Ts) 21 is the most frequent live-born aneuploidy syndrome in humans and extensively studied using model mice. However, there is no available model mouse for other congenital Ts syndromes, possibly because of the lethality of Ts in vivo, resulting in the lack of studies to identify the responsible gene(s) for aneuploid syndromes. Although induced pluripotent stem cells derived from patients are useful to analyse aneuploidy syndromes, there are concerns about differences in the genetic background for comparative studies and clonal variations. Therefore, a model cell line panel with the same genetic background has been strongly desired for sophisticated comparative analyses. In this study, we established isogenic human embryonic stem (hES) cells of Ts8, Ts13, and Ts18 in addition to previously established Ts21 by transferring each single chromosome into parental hES cells via microcell-mediated chromosome transfer. Genes on each trisomic chromosome were globally overexpressed in each established cell line, and all Ts cell lines differentiated into all three embryonic germ layers. This cell line panel is expected to be a useful resource to elucidate molecular and epigenetic mechanisms of genetic imbalance and determine how aneuploidy is involved in various abnormal phenotypes including tumourigenesis and impaired neurogenesis.     

Detection of gonadal mosaicism in parents of offspring with down syndrome

A review of data of a standard clinical karyological analysis and study of QFQ chromosomal polymorphism in 151 families of children with Down syndrome is performed. A total of eight families with proved and predicted trisomy 21 gonadal mosaicism were identified; in all the cases the mothers were younger than 35 years of age. The prevalence of carriers of mosaicism in young families amounted to 6.5% (8/123). In a standard analysis, if the capabilities of the QFQ method had not been employed, this prevalence would have amounted to only 1%. A comparison of the results of a retrospective analysis of the data obtained by the QFQ method with the expected results of molecular testing of the same families leads to the conclusion that both methods should be used.     

Quantitative proteomic analysis of amniocytes reveals potentially dysregulated molecular networks in Down syndrome

Background

Down syndrome (DS), caused by an extra copy of chromosome 21, affects 1 in 750 live births and is characterized by cognitive impairment and a constellation of congenital defects. Currently, little is known about the molecular pathogenesis and no direct genotype-phenotype relationship has yet been confirmed. Since DS amniocytes are expected to have a distinct biological behaviour compared to normal amniocytes, we hypothesize that relative quantification of proteins produced from trisomy and euploid (chromosomally normal) amniocytes will reveal dysregulated molecular pathways.

Results

Chromosomally normal- and Trisomy 21-amniocytes were quantitatively analyzed by using Stable Isotope Labeling of Amino acids in Cell culture and tandem mass spectrometry. A total of 4919 unique proteins were identified from the supernatant and cell lysate proteome. More specifically, 4548 unique proteins were identified from the lysate, and 91% of these proteins were quantified based on MS/MS spectra ratios of peptides containing isotope-labeled amino acids. A total of 904 proteins showed significant differential expression and were involved in 25 molecular pathways, each containing a minimum of 16 proteins. Sixty of these proteins consistently showed aberrant expression from trisomy 21 affected amniocytes, indicating their potential role in DS pathogenesis. Nine proteins were analyzed with a multiplex selected reaction monitoring assay in an independent set of Trisomy 21-amniocyte samples and two of them (SOD1 and NES) showed a consistent differential expression.

Conclusions

The most extensive proteome of amniocytes and amniotic fluid has been generated and differentially expressed proteins from amniocytes with Trisomy 21 revealed molecular pathways that seem to be most significantly affected by the presence of an extra copy of chromosome 21.     

Mosaicism for trisomy 21 and ring (21) in a male born to normal parents: A case report

We present a case of a ring (21) in a mentally challenged patient with mosaicism for trisomy 21 showing karyotype 47, XY,+21/47,XY,+21(r)/46,XY, born to normal parents. The parents and female sibling were phenotypically normal. This is a unique case report from Central India, on occurrence of trisomy 21 and r (21) in the same individual born to normal parents. Also being documented for the first time is the immuno-FISH analysis revealing differential expression of hTERT and a linked over expression of TRF2 in proband, probably corresponding to a high percentage of acrocentric associations.     

Alterations in erythrocyte membrane fluidity in children with trisomy 21: a fluorescence study.

Membrane fluidity of erythrocytes obtained from 15 children with trisomy 21 and 20 healthy controls were studied by measuring steady-state fluorescence anisotropy and fluorescence lifetime of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated in hemoglobin-free erythrocyte membranes. Our results demonstrate a significant decrease in DPH fluorescence anisotropy and a significant increase in TMA-DPH fluorescence anistropy in erythrocytes from subjects with trisomy 21. No significant differences between the two groups were observed in the fluorescence lifetime of DPH and TMA-DPH. These data suggest an increase in membrane fluidity in the interior part of the membrane and a decrease in fluidity at the lipid-water interface region. This could be in part attributed to an increased oxidative damage in trisomy 21.     

A Novel Gene Isolated from Human Placenta Located in Down Syndrome Critical Region on Chromosome 21

Down syndrome is the most common birth defect, which is causedby trisomy 21. We identified a novel gene in the so-called Downsyndrome critical region by EST mapping to genomic DNA and followingcDNA cloning. The gene, designated DCRB (Down syndrome CriticalRegion gene B), consisted ofthree exons of1095 bp in total andencoded a large open reading frame of118 amino acid residues.The amino acids sequence ofDCRB showed no significant homologyto any known protein. Northern blot analysis showed that DCRBis mainly expressed in the placenta, in which a major 1.1-kbband and a minor 2.0-kb band were detected. Minor bands of 1.4kb and 2.2 kb were also detected in adult heart and skeletalmuscle.     

Simian crease incidence and the correlation with thenar and hypothenar pattern types in Swedish patients with trisomy 21 (Down's syndrome)

In the present study the frequency of the simian crease among 57 Down patients is compared to the corresponding figures of related and unrelated individuals. A study of the correlation with the dermatoglyphic patterns of the thenar and hypothenar areas is presented, the palm print classification being carried out according to a Swedish method.     

An investigation of the molecular mechanisms engaged before and after the development of Alzheimer disease neuropathology in Down syndrome: a proteomics approach

Down syndrome (DS) is one of the most common causes of intellectual disability, owing to trisomy of all or part of chromosome 21. DS is also associated with the development of Alzheimer disease (AD) neuropathology after the age of 40 years. To better clarify the cellular and metabolic pathways that could contribute to the differences in DS brain, in particular those involved in the onset of neurodegeneration, we analyzed the frontal cortex of DS subjects with or without significant AD pathology in comparison with age-matched controls, using a proteomics approach. Proteomics represents an advantageous tool to investigate the molecular mechanisms underlying the disease. From these analyses, we investigated the effects that age, DS, and AD neuropathology could have on protein expression levels. Our results show overlapping and independent molecular pathways (including energy metabolism, oxidative damage, protein synthesis, and autophagy) contributing to DS, to aging, and to the presence of AD pathology in DS. Investigation of pathomechanisms involved in DS with AD may provide putative targets for therapeutic approaches to slow the development of AD.     

Stem and progenitor cell dysfunction in human trisomies

Trisomy 21, the commonest constitutional aneuploidy in humans, causes profound perturbation of stem and progenitor cell growth, which is both cell context dependent and developmental stage specific and mediated by complex genetic mechanisms beyond increased Hsa21 gene dosage. While proliferation of fetal hematopoietic and testicular stem/progenitors is increased and may underlie increased susceptibility to childhood leukemia and testicular cancer, fetal stem/progenitor proliferation in other tissues is markedly impaired leading to the characteristic craniofacial, neurocognitive and cardiac features in individuals with Down syndrome. After birth, trisomy 21‐mediated premature aging of stem/progenitor cells may contribute to the progressive multi‐system deterioration, including development of Alzheimer's disease.     

Double trisomy (48,XXY,+21) in monozygotic twins: case report and review of the literature

The occurrence of double aneuploidy in the same individual is a relatively rare phenomenon. We describe twin newborns with typical clinical features of Down's syndrome, of which one revealed 48,XXY,+21 GTG-band karyotype. The second newborn died 2 days after its birth, and was clinically diagnosed having Down syndrome. Due to the same clinical features of the twins, the common placenta and amniotic sac, we speculate that they were monozygotics and as a result the second newborn should also be a Klinefelter. The purpose of this report is to present a rare case of possible coincidence of double aneuploidy in newborn twins. A review of the literature showed that double trisomy (48,XXY,+21) in a twin newborn infant has never occurred.