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     

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.     

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.     

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.     

21- 三体综合征亲子鉴定一例暨文献复习

目的:对亲子鉴定中检出的三带型等位基因座进一步探讨其发生原因,与同行共享。方法:亲子鉴定中发现一个体两个等位基因座(D21S11和Penta D)检出三等位基因,进一步采集其静脉血进行血细胞培养作染色体分析。结果:该个体染色体核型为:47,XY,+21。结论:染色体为三体型的个体在基因检测时能检测到三等位基因。     

Non-invasive prenatal diagnosis by massively parallel sequencing of maternal plasma DNA

The presence of foetal DNA in the plasma of pregnant women has opened up new possibilities for non-invasive prenatal diagnosis. The use of circulating foetal DNA for the non-invasive prenatal detection of foetal chromosomal aneuploidies is challenging as foetal DNA represents a minor fraction of maternal plasma DNA. In 2007, it was shown that single molecule counting methods would allow the detection of the presence of a trisomic foetus, as long as enough molecules were counted. With the advent of massively parallel sequencing, millions or billions of DNA molecules can be readily counted. Using massively parallel sequencing, foetal trisomies 21, 13 and 18 have been detected from maternal plasma. Recently, large-scale clinical studies have validated the robustness of this approach for the prenatal detection of foetal chromosomal aneuploidies. A proof-of-concept study has also shown that a genome-wide genetic and mutational map of a foetus can be constructed from the maternal plasma DNA sequencing data. These developments suggest that the analysis of foetal DNA in maternal plasma would play an increasingly important role in future obstetrics practice. It is thus a priority that the ethical, social and legal issues regarding this technology be systematically studied.     

DSCR1 gene expression is dependent on NFATc1 during cardiac valve formation and colocalizes with anomalous organ development in trisomy 16 mice

The Down syndrome critical region 1 (DSCR1) gene is present in the region of human chromosome 21 and the syntenic region of mouse chromosome 16, trisomy of which is associated with congenital heart defects observed in Down syndrome. DSCR1 encodes a regulatory protein in the calcineurin/NFAT signal transduction pathway. During valvuloseptal development in the heart, DSCR1 is expressed in the endocardium of the developing atrioventricular and semilunar valves, the muscular interventricular septum, and the ventricular myocardium. Human DSCR1 contains an NFAT-rich calcineurin-responsive element adjacent to exon 4. Transgenic mice generated with a homologous regulatory region of the mouse DSCR1 gene linked to lacZ (DSCR1(e4)/lacZ) show gene activation in the endocardium of the developing valves and aorticopulmonary septum of the heart, recapitulating a specific subdomain of endogenous DSCR1 cardiac expression. DSCR1(e4)/lacZ expression in the developing valve endocardium colocalizes with NFATc1 and, endocardial DSCR1(e4)/lacZ, is notably reduced or absent in NFATc1(-/-) embryos. Furthermore, expression of the endogenous DSCR1(e4) isoform is decreased in the outflow tract of NFATc1(-/-) hearts, and the DSCR1(e4) intragenic element is trans-activated by NFATc1 in cell culture. In trisomy 16 (Ts16) mice, expression of endogenous DSCR1 and DSCR1(e4)/lacZ colocalizes with anomalous valvuloseptal development, and transgenic Ts16 hearts have increased beta-galactosidase activity. DSCR1 and DSCR1(e4)/lacZ also are expressed in other organ systems affected by trisomy 16 in mice or trisomy 21 in humans including the brain, eye, ear, face, and limbs. Together, these results show that DSCR1(e4) expression in the developing valve endocardium is dependent on NFATc1 and support a role for DSCR1 in normal cardiac valvuloseptal formation as well as the abnormal development of several organ systems affected in individuals with Down syndrome.