Prenatal exclusion of segmental trisomy in familial chromosome 21 pericentric inversion by fluorescence in situ hybridization

We report the prenatal exclusion of partial trisomy in a family with maternal pericentric inversion of chromosome 21 by fluorescence in situ hybridization (FISH). After determining the structural rearrangement in the mother and her affected son with 46,XY,rec(21)dup(21q)inv(21)(p11q22) resulting in Down syndrome (DS), a chorionic villus sample from the current pregnancy was analysed for the copy number of the DS critical region with a cosmid contig. The signal distribution was normal and the cytogenetic analysis revealed that the fetus had inherited the inverted chromosome 21 in a balanced form. FISH probes specific for the DS region are of great value in supporting cytogenetic results, regardless of the structural status of chromosome 21.     

Allelic variability in D21S11, but not in APP or APOE, is associated with cognitive decline in Down syndrome

Genetic variation in the APOE gene and variation in chromosome 21 genotypes, including the APP locus, may influence age-associated cognitive decline in adults with Down syndrome. Molecular genetic and longitudinal neuropsychological analysis was performed for 41 unrelated Caucasian individuals (mean age 48.1 +/- 1.1 years (s.c.m.)) with free trisomy 21. Allele frequencies and genotype distributions were compared among subgroups with or without evidence of cognitive decline. Genetic variability at APOE and APP was not significantly associated with evidence of cognitive decline. However, aged individuals with Down syndrome, without evidence of cognitive decline, demonstrated unusual allelic variability at D21S11. These findings are discussed in the context of current hypotheses of Alzheimer-type dementia in Down syndrome and in the general population.     

Nicotine replacement: a new approach to reducing tobacco-related harm

A case of transient abnormal myelopoiesis in a normal newborn without features of Down syndrome is described. The majority of bone marrow cells analysed belonged to a chromosomally abnormal clone with trisomy for chromosomes 18 and 21. Complex intrachromosomal rearrangements of one chromosome 21, demonstrated by fluorescence in situ hybridization using locus-specific probes, were found in a minor population of the clonal cells. These rearrangements involved loci previously shown to be rearranged in the leukaemic cells from patients with Down syndrome and leukaemia. However, the child's myeloproliferation resolved rapidly, with disappearance of the abnormal clone, and 3.5 years later she remains well.     

Poly-FISH: a technique of repeated hybridizations that improves cytogenetic analysis of fetal cells in maternal blood

Prenatal diagnosis of fetal chromosomal abnormalities using interphase fetal nucleated erythrocytes (FNRBCs) separated from maternal peripheral blood can be technically challenging due to the limited number of FNRBCs available for analysis, the limited number of probes that can be used simultaneously, and low FISH efficiency on the formaldehyde-fixed and immunohistochemically stained interphase FNRBCs. We developed a technique of sequential FISH analysis that involves removal of the previous hybridized probe under denaturing conditions, and rehybridization with different probes to improve FISH efficiency. This technique facilitates the analysis of multiple chromosome-specific probes on the same nuclei. Results from our experiments show that FISH can be performed at least nine times on the same interphase nucleus and at least three different probes can be used simultaneously. Thus, theoretically, at least 24 different chromosomes can be analysed on a single interphase fetal cell isolated from maternal blood. We have termed this technique 'Poly-FISH', and have successfully diagnosed trisomy 21, triploidy, and other chromosome abnormalities in FNRBCs using this technique.     

Deletions of chromosome 21 restricted to the leukemic cells of children with Down syndrome and leukemia

Down syndrome (DS) is associated with an increased risk of developing hematological malignancies, but the basis for this predisposition is so far unknown. Using fluorescence in situ hybridization with a panel of locus-specific probes on normal and leukemic metaphases, we have found long-arm interstitial deletions of one of the chromosome 21s in the leukemic cells from five patients with DS and leukemia. This finding provides strong evidence for a gene or genes present on chromosome 21 having an important function in the development of leukemia in individuals with Down syndrome.     

Fighting the cold war: how information technology can help

OBJECTIVE: To detect the parental origin of extra chromosome 21 in Down syndrome, using two (GT)n polymorphic markers-D21S215 and D21S120. METHODS: The alleles of D21S215 and D21S120 were amplified by polymerase chain reaction and identified with denaturing polyacrylamide gel electrophoresis followed with Ag-staining. The parental origin of extra chromosome 21 was determined by comparing genotypes of probands with their parents. RESULTS: The parental origin of extra chromosome 21 was determined in 17 Down syndrome out of 24, with 12 and 5 inherited from mother and father respectively. CONCLUSION: The parental origin of extra chromosome 21 in Down syndrome can be determined by analyzing the polymorphisms of D21S215 and D21S120. Etiological study of trisomy 21 should be focused on maternal meiosis.     

Maternal serum alpha-fetoprotein and dimeric inhibin A detect aneuploidies other than Down syndrome

OBJECTIVE: Our purpose was to determine whether the combination of maternal serum alpha-fetoprotein, free human chorionic gonadotropin-beta, dimeric inhibin A, and maternal age detects aneuploidies other than Down syndrome. STUDY DESIGN: We retrieved stored serum from pregnancies complicated by aneuploidies other than Down syndrome from 1988 to 1997 (n = 55, mean maternal age 35.2 +/- 5.6 years). Alpha-fetoprotein levels were obtained from our database, and free human chorionic gonadotropin-beta and dimeric inhibin A levels were measured in the thawed serum with use of commercial assays. Analyte values were used in both 3-analyte and 2-analyte multiple-marker screening tests; detection rates were determined at several different Down syndrome risk-positive cutoff values. RESULTS: In the 3-analyte test 58% (32/55) of all aneuploidies were detected with use of both the Down syndrome protocol at a screen-positive risk cutoff value of 1:300 (false-positive rate 17%) and a novel trisomy 18 screening algorithm. However, 67% (37/55) detection was obtained with use of the 2-analyte combination of alpha-fetoprotein and dimeric inhibin A, with both the Down syndrome protocol (screen positive cutoff value 1:300) and the trisomy 18 algorithm: 12 of 13 trisomy 18 (92%), 9 of 17 Turner's syndrome (53%), 10 of 17 other sex chromosome aneuploidies (59%), 1 of 1 trisomy 22 (100%), and 5 of 7 trisomy 13 (71%). CONCLUSIONS: The combination of maternal serum alpha-fetoprotein, dimeric inhibin A, and maternal age detects autosomal trisomies other than Down syndrome at a rate superior to that of the traditional analyte combination.     

Ventricular tachycardia originating at the right ventricular outflow tract--surgical treatment of extrasystole

A boy with Down syndrome who developed acute nonlymphocytic leukemia (ANLL/M2) at the age of 40 months is presented. Chromosomal analysis of cultured peripheral blood cells without mitogen revealed a constitutional abnormality, trisomy 21, associated with the acquired chromosome change t(8;21)(q22;q22).     

Isolation and refined regional mapping of expressed sequences from human chromosome 21

To increase candidate genes from human chromosome 21 for the analysis of Down syndrome and other genetic diseases localized on this chromosome, we have isolated and studied 9 cDNA clones encoded by chromosome 21. For isolating cDNAs, single-copy microclones from a chromosome 21 microdissection library were used in direct screening of various cDNA libraries. Seven of the cDNA clones have been regionally mapped on chromosome 21 using a comprehensive hybrid mapping panel comprising 24 cell hybrids that divide the chromosome into 33 subregions. These cDNA clones with refined mapping positions should be useful for identification and cloning of genes responsible for the specific component phenotypes of Down syndrome and other diseases on chromosome 21, including progressive myoclonus epilepsy in 21q22.3.     

Rapid prenatal diagnosis of Down's syndrome in the first trimester of pregnancy by fluorescence in situ hybridization

OBJECTIVE: To assess whether fluorescence in situ hybridization (FISH) with chromosome 21, specific DNA probe is applicable as a prenatal diagnostic tool for Down's syndrome. METHOD: We used FISH with chromosome 21 specific probe on 30 uncultured chorionic villi cell samples to detect the Down's fetus, and we also performed the conventional chromosome analysis of chorion cells from parallel samples. RESULTS: In samples with disomic karyotype, an average of 1 percent (0-5 percent) of the nuclei had three hybridization signals. By contrast, in the samples of trisomy 21 fetus, an average of 86 percent (78-91 percent) of the nuclei displayed three signals. CONCLUSION: FISH can provide a rapid and accurate method for the first trimester prenatal diagnosis of Down's syndrome.     

Trisomy 21 in childhood acute lymphoblastic leukemia: a Pediatric Oncology Group study (8602)

Of 1,036 children with newly diagnosed non-T, non-B acute lymphoblastic leukemia (ALL) and a demonstrated cytogenetic abnormality treated on the frontline Pediatric Oncology Group (POG) therapeutic trial 8602, there were 33 patients with trisomy 21 as the sole abnormality. Of these 33, 14 had Down syndrome (DS). Although the non-DS (NDS) trisomy 21 cases tended to be older than the DS cases, there were no other significant differences in clinicobiologic features nor in treatment outcomes between the DS and NDS groups, nor between the entire trisomy 21 group and the other chromosome abnormality group. Among NDS patients with +21 and one additional abnormality, +X, +16, -20, and structural abnormalities involving 6q or 12p were common findings. Kaplan-Meier event-free survival (EFS) curves showed a 4-year EFS of 80% (SE, 12%) in NDS trisomy 21 cases, 71% (SE, 22%) in DS cases with trisomy 21 as the sole abnormality, and 69% (SE, 2%) in cases with other chromosome abnormalities. Trisomy 21 as a sole acquired abnormality in NDS patients suggests a good prognosis.     

Hearing the voices of women in war through continuous quality improvement

Double color in situ hybridization was used to determine the specificity of cloned alpha-satellite cosmid DNA sequences belonging to satellite DNA sequences shared by chromosomes 13 and 21. It was determined that cosmid clone cos56D8 is more specific to chromosome 13, cosmid clone cos37E is more specific to chromosome 21 and cosmid clones cosA5130 is specific to both chromosomes 13 and 21. These new cosmid probes could bw useful in the molecular-cytogenetic analysis of trisomic cells at Patau or Down syndromes as well as in analysis of marker chromosomes originated from the chromosomes 13 and 21.     

Several cytogenetic subclones may be identified within plasma cells from patients with monoclonal gammopathy of undetermined significance, both at diagnosis and during the indolent course of this condition

Monoclonal gammopathy of undetermined significance (MGUS) is a frequent condition in patients over 50 years old, that ultimately leads to multiple myeloma (MM) in 20% of patients after 20 to 35 years of follow-up. Little is known about cytogenetic changes associated with this condition. We studied 19 MGUS patients both at diagnosis and after 12 to 35 months of follow-up (mean = 26), using DNA content measurement of bone marrow plasma cells (BMPC), and a new interphase fluorescence in situ hybridization technique (FISH) allowing the simultaneous identification of monotypic BMPC (fluorescent anti light-chain antibodies) and the determination of the number of copies for two different chromosomes within the same PC nucleus (one biotin-labeled probe coupled next to texas red avidin and one FITC-labeled probe). At diagnosis of the MGUS, single interphase FISH showed at least one numeric chromosome change in 13 of 19 patients, after the use of centromeric probes directed against chromosomes no. 3, no. 7, no. 9, and no. 11. At follow-up, abnormalities found at diagnosis in 13 patients were still shown. Moreover, abnormalities occurred in three of the last six patients (trisomy for one to three different chromosomes), although no patient evolved into MM. Dual interphase FISH showed that some BMPC bore numeric changes with both probes tested whereas other BMPC bore abnormality with only one of the probes tested. In patients who showed trisomy for at least three different chromosomes, distribution of numeric changes within BMPC defined significant numbers of up to seven different BMPC clones. All these various clones were shown both at diagnosis and at follow-up. In every patient, these various clones differed only for the number of abnormalities they exhibited, and could be related to each other in a model of gradual acquisition of chromosome changes. Eventually, data reported here show that MGUS patients acquire slowly, gradually, but ineluctably chromosome changes, distributed within several related subclones. However, these changes are not related to transformation into MM: among the various clones coexisting within the same patient, a peculiar change, still to demonstrate, might develop and lead to overt MM.     

2q21-qter trisomy in hepatoblastoma

Cytogenetic findings and fluorescens in situ hybridization results of a hepatoblastoma of a boy of two and half years of age are presented. Cytogenetic analysis and fluorescens in situ hybridization technique were performed using tumor tissue obtained by biopsy. The direct culture was harvested after 16 hour colcemid treatment. The results of G-banding were as follows: 47,XY,add(4) (q26),-9,+20. There were considerable variation in the degree of condensation and hence in the number of visible G-bands both between metaphases and between homologous chromosomes in the same metaphases. Fluorescens in situ hybridisation were carried out by whole chromosome painting probes: 2,3,4, and 20. The karyotype of the malignant cells was adjusted accordingly: 47,XY,der(4)(q35),dir ins(9;2)(p22;q?21q?25),+20. The results confirm the most common primary chromosome abnormalities in hepatoblastoma are the following: trisomy 2, trisomy 20 and 4q structural rearrangement. Fluorescens in situ hybridization confirms the importance of trisomy 2q21-qter in hepatoblastoma. Authors recommend the use of fluorescens in situ hybridization to correct any tumor karyotype with difficult or ambiguous chromosome morphology.     

Bacterial contig map of the 21q11 region associated with Alzheimer's disease and abnormal myelopoiesis in Down syndrome

We present a high-resolution bacterial contig map of 3.4 Mb of genomic DNA in human chromosome 21q11-q21, encompassing the region of elevated disomic homozygosity in Down Syndrome-associated abnormal myelopoiesis and leukemia, as well as the markers, which has shown a strong association with Alzheimer's Disease that has never been explained. The map contains 89 overlapping PACs, BACs, or cosmids in three contigs (850, 850, and 1500 kb) with two gaps (one of 140-210 kb and the second <5 kb). To date, eight transcribed sequences derived by cDNA selection, exon trapping, and/or global EST sequencing have been positioned onto the map, and the only two genes so far mapped to this cytogenetic region, STCH and RIP140 have been precisely localized. This work converts a further 10% of chromosome 21q into a high-resolution bacterial contig map, which will be the physical basis for the long-range sequencing of this region. The map will also enable positional derivation of new transcribed sequences, as well as new polymorphic probes, that will help in elucidation of the role the genes in this region may play in abnormal myelopoiesis and leukemia associated with trisomy 21 and Alzheimer's Disease.     

Chromosome spatial order in human cells: evidence for early origin and faithful propagation

We have investigated the origin and nature of chromosome spatial order in human cells by analyzing and comparing chromosome distribution patterns of normal cells with cells showing specific chromosome numerical anomalies known to arise early in development. Results show that all chromosomes in normal diploid cells, triploid cells and in cells exhibiting nondisjunction trisomy 21 are incorporated into a single, radial array (rosette) throughout mitosis. Analysis of cells using fluorescence in situ hybridization, digital imaging and computer-assisted image analysis suggests that chromosomes within rosettes are segregated into tandemly linked "haploid sets" containing 23 chromosomes each. In cells exhibiting nondisjunction trisomy 21, the distribution of chromosome 21 homologs in rosettes was such that two of the three homologs were closely juxtaposed, a pattern consistent with our current understanding of the mechanism of chromosomal nondisjunction. Rosettes of cells derived from triploid individuals contained chromosomes segregated into three, tandemly linked haploid sets in which chromosome spatial order was preserved, but with chromosome positional order in one haploid set inverted with respect to the other two sets. The spatial separation of homologs in triploid cells was chromosome specific, providing evidence that chromosomes occupy preferred positions within the haploid sets. Since both triploidy and nondisjunction trisomy 21 are chromosome numerical anomalies that arise extremely early in development (e.g., during meiosis or during the first few mitoses), our results support the idea that normal and abnormal chromosome distribution patterns in mitotic human cells are established early in development, and are propagated faithfully by mitosis throughout development and into adult life. Furthermore, our observations suggest that segregation of chromosome homologs into two haploid sets in normal diploid cells is a remnant of fertilization and, in normal diploid cells, reflects segregation of maternal and paternal chromosomes.     

NG-nitro-L-arginine methyl ester inhibits bone metastasis after modified intracardiac injection of human breast cancer cells in a nude mouse model

A retrospective evaluation of alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), and unconjugated oestriol (uE3) levels in maternal blood in the second trimester was conducted for cases of aneuploid pregnancies identified from a series of women who underwent amniocentesis. Blood samples were collected from 1078 women just before genetic amniocentesis was performed, mainly for individuals of advanced maternal age (greater than 35 years). Twenty-five maternal serum samples from pregnant women with an aneuploid fetus, including 14 with Down's syndrome, were available for analysis of all three parameters. An algorithm to detect Down's syndrome was used for this analysis with a risk of > or = 1:299 classified as screen-positive, this being found for 20.4 per cent of the cases (220/1078). The actual Down's syndrome detection rate was 85.7 per cent (12/14), whereas the detection rate for all aneuploidies was 72.0 per cent (18/25). Those that were not detected were two cases of trisomy 21, one trisomy 18, two trisomy 13, three sex chromosome abnormalities, and one case of an additional marker chromosome. The data indicate that this tri-analyte test should be provided after thorough genetic counselling and informed decision-making regarding maternal serum screening for women who wish for a prenatal diagnosis.     

Gene identification and DNA sequence analysis in the GC-poor 20 megabase region of human chromosome 21

In contrast to the distal half of the long arm of chromosome 21, the proximal half of approximately 20 megabases of DNA, including 21q11-21 bands, is low in GC content, CpG islands, and identified genes. Despite intensive searches, very few genes and cDNAs have been found in this region. Since the 21q11-21 region is associated with certain Down syndrome pathologies like mental retardation, the identification of relevant genes in this region is important. We used a different approach by constructing microdissection libraries specifically for this region and isolating unique sequence microclones for detailed molecular analysis. We found that this region is enriched with middle and low-copy repetitive sequences, and is also heavily methylated. By sequencing and homology analysis, we identified a significant number of genes/cDNAs, most of which appear to belong to gene families. In addition, we used unique sequence microclones in direct screening of cDNA libraries and isolated 12 cDNAs for this region. Thus, although the 21q11-21 region is gene poor, it is not completely devoid of genes/cDNAs. The presence of high proportions of middle and low-copy repetitive sequences in this region may have evolutionary significance in the genome organization and function of this region. Since 21q11-21 is heavily methylated, the expression of genes in this region may be regulated by a delicate balance of methylation and demethylation, and the presence of an additional copy of chromosome 21 may seriously disturb this balance and cause specific Down syndrome anomalies including mental retardation.