Down syndrome (DS), or trisomy 21, is a common disorder associated with several complex clinical phenotypes. Although several hypotheses have been put forward, it is unclear as to whether particular gene loci on chromosome 21 (HSA21) are sufficient to cause DS and its associated features. Here we present a high-resolution genetic map of DS phenotypes based on an analysis of 30 subjects carrying rare segmental trisomies of various regions of HSA21. By using state-of-the-art genomics technologies we mapped segmental trisomies at exon-level resolution and identified discrete regions of 1.8-16.3 Mb likely to be involved in the development of 8 DS phenotypes, 4 of which are congenital malformations, including acute megakaryocytic leukemia, transient myeloproliferative disorder, Hirschsprung disease, duodenal stenosis, imperforate anus, severe mental retardation, DS-Alzheimer Disease, and DS-specific congenital heart disease (DSCHD). Our DS-phenotypic maps located DSCHD to a <2-Mb interval. Furthermore, the map enabled us to present evidence against the necessary involvement of other loci as well as specific hypotheses that have been put forward in relation to the etiology of DS-i.e., the presence of a single DS consensus region and the sufficiency of DSCR1 and DYRK1A, or APP, in causing several severe DS phenotypes. Our study demonstrates the value of combining advanced genomics with cohorts of rare patients for studying DS, a prototype for the role of copy-number variation in complex disease.

Genetic contributions to human cognition and behavior are clear but difficult to define. Williams syndrome (WS) provides a unique model for relating single genes to visual-spatial cognition and social behavior. We defined a approximately 1.5 Mb region of approximately 25 genes deleted in >98% of typical WS and then rare small deletions, showing that visual-spatial construction (VSC) in WS was associated with the genes GTF2IRD1 and GTF2I. To distinguish the roles of GTF2IRD1 and GTF2I in VSC and social behavior, we utilized multiple genomic methods (custom high resolution oligonucleotide microarray, multicolor FISH and somatic cell hybrids analyzed by PCR) to identify individuals deleted for either gene but not both. We analyzed genetic, cognitive and social behavior in a unique individual with WS features (heart defects, small size, facies), but with an atypical deletion of a set of genes that includes GTF2IRD1, but not GTF2I. The centromeric breakpoint localized to the region 72.32-72.38 Mb and the telomeric breakpoint to 72.66 Mb, 10 kb downstream of GTF2IRD1. Cognitive testing (WPPSI-R, K-BIT, and PLS-3) demonstrated striking deficits in VSC (Block Design, Object Assembly) but overall performance 1.5-3 SD above WS means. We have now integrated the genetic, clinical and cognitive data with previous reports of social behavior in this subject. These results combine with previous data from small deletions to suggest the gene GTF2IRD1 is associated with WS facies and VSC, and that GTF2I may contribute to WS social behaviors including increased gaze and attention to strangers.

William's syndrome (WS) features a spectrum of neurocognitive and behavioral abnormalities due to a rare 1.5 MB deletion that includes about 24–28 genes on chromosome band 7q11.23. Study of the expression of these genes from the single normal copy provides an opportunity to elucidate the genetic and epigenetic controls on these genes as well as their roles in both WS and normal brain development and function. We used quantitative RT-PCR to determine the transcriptional level of 14 WS gene markers in a cohort of 77 persons with WS and 48 normal controls. Results reported here: (1) show that the expression of the genes deleted in WS is decreased in some but not all cases, (2) demonstrate that the parental origin of the deletion contributes to the level of expression of GTF2I independently of age and gender and (3) indicate that the correlation of expression between GTF2I and some other genes in the WS region differs in WS subjects and normal controls, which in turn points toward a regulatory role for this gene. Interspecies comparisons suggest GTF2I may play a key role in normal brain development.

Purpose: Humans with small deletions of chromosome 21 provide important models for understanding the role of dosage-sensitive genes in brain morphogenesis. To identify chromosome 21 genes responsible for defects of the central nervous system, we determined the deleted regions and brain malformations in three unrelated individuals with overlapping partial deletions of chromosome 21.Methods: Fluorescent in situ hybridization and magnetic resonance imaging were used to define the chromosomal structure and structural brain abnormalities present in these three individuals.Results: The regions of chromosome 21 found to be deleted in these individuals were as follows: case 1: KCNJ6 to the telomere; case 2: ITSN1 to the telomere; and case 3: ITSN1 to PCNT2. The abnormalities of brain structure shared by all included microcephaly, pachygyria, polymicrogyria, colpocephaly, hypoplastic corpus callosum and white matter, hypoplastic cerebellum, and enlarged ventricular system. The clinical features in common included mental retardation, microcephaly, facial dysmorphism, and epilepsy (severe in one patient).Conclusion: From analyses of the molecular, cytogenetic, and neuroimaging data from these three individuals, combined with those from previously reported cases, we infer that deletion of an 8.4-Mb region in chromosome band 21q22.2-22.3 (KCNJ6-COL6A2) is associated with cortical dysplasia. We propose that one or more dosage-sensitive genes in this region contributes to cortical development and that deletion of 21q22.2-22.3 should be considered in the diagnosis of mentally retarded patients with facial dysmorphism and cerebral dysplasia.

]The ultimate goal of the Human Genome Project is to establish the DNA sequence of human and model organism genomes as the critical first step in understanding disease, development and evolution. To accomplish this goal and a broad spectrum of applications requires integration of genome sequence information to genetic markers (expressed sequence tag/cDNA/gene transcripts content) and to reagents that can be seen through a microscope and linked to cytogenetic landmarks. Such linkage/integration should be dense large fragments and for reagents well characterized with respect to low-copy repeats that are present at multiple other points in the genome. Therefore, ideally, the same templates should be used as an integrating framework of entry points for sequencing and then applied to gene isolation and mapping, studies of genome organization and evolution, and a myriad of clinical applications (1).

Objective To explore the differences between radiation-induced translocation and dicentric chromosome aberrations detected by multicolor fluorescence in situ hybridization (M-FISH) method. Methods The translocation and dicentric chromosome aberrations in lymphocytes of umbilical cord blood induced by different doses of 60 Co gamma rays were analyzed by M-FISH.The specific telomeres and centromeres BAC clones of chromosomes 1,2,3,7,8,9,14 and 15 were used for M-FISH probes. Results (1)The dose-response curves of translocation and dicentric aberrations with absorbed dose levels accorded to linear-quadratic dose-response model.The translocation frequency was not equal to the dicentric frequency at most dose levels;(2)The proportion of cells with complete translocation but without unstable chromosome aberration decreased with increasing absorbed dose;(3)The observed values and the predicted values of chromosome aberrations,induced by 3 Gy gamma radiation,were not significantly different in most of the labeled chromosomes,but chromosome 9 had observed values significantly higher than predicted values ( P 0.05 or P 0.01),and chromosome 15 vice versa ( P 0.01). Conclusion The frequency of chromosome translocation induced by ionizing radiation is not equal to that of dicentric aberration.The frequencies of translocation and dicentric chromosome aberrations induced by ionizing radiation are at random for most chromosomes. ;

Background Chromosome band 10q24 is a gene-rich domain and host to a number of cancer, developmental, and neurological genes. Recurring translocations, deletions and mutations involving this chromosome band have been observed in different human cancers and other disease conditions, but the precise identification of breakpoint sites, and detailed characterization of the genetic basis and mechanisms which underlie many of these rearrangements has yet to be resolved. Towards this end it is vital to establish a definitive genetic map of this region, which to date has shown considerable volatility through time in published works of scientific journals, within different builds of the same international genomic database, and across the differently constructed databases. Results Using a combination of chromosome and interphase fluorescent in situ hybridization (FISH), BAC end-sequencing and genomic database analysis we present a physical map showing that the order and chromosomal orientation of selected genes within 10q24 is CEN- CYP2C9 - PAX2-HOX11-NFKB2- TEL. Our analysis has resolved the orientation of an otherwise dynamically evolving assembly of larger contigs upstream of this region, and in so doing verifies the order and orientation of a further 9 cancer-related genes and GOT1 . This study further shows that the previously reported human papillomavirus type 6a DNA integration site HPV6AI1 does not map to 10q24, but that it maps at the interface of chromosome bands 14q13.3-q21.1. Conclusions This revised map will allow more precise localization of chromosome rearrangements involving chromosome band 10q24, and will serve as a useful baseline to better understand the molecular aetiology of chromosomal instability in this region. In particular, the relocation of HPV6AI1 is important to report because this HPV6a integration site, originally isolated from a tonsillar carcinoma, was shown to be rearranged in other HPV6a-related malignancies, including 2 of 25 genital condylomas, and 2 of 7 head and neck tumors tested. Our finding shifts the focus of this genomic interest from 10q24 to the chromosome 14 site.

Objective To establish an multicolor FISH (M\|FISH) method and to investigate its applicability in retrospective dose reconstruction. Methods Bacterial artificial chromosome (BAC) clones specific for telomeres and centromeres of 8 pairs of chromosomes were selected,and an M\|FISH method was to be established.Chromosome aberrations in lymphocytes of two new\|borns umbilical cord blood having been irradiated with 60 Co γ\|rays in vitro were analyzed with the M\|FISH method.Then the dose\|response curves of chromosome aberrations were established.The cumulative doses for two individuals having been exposed to ionizing radiation were estimated with the M\|FISH method according to the corresponding dose response curve. Results An M\|FISH method,using BAC clones specific for telomeres and centromeres of chromosomes 1,2,3,7,8,9,14 and 15,labeled with biotin\|14\|dATP and/or digoxigenin\|11\|dUTP,was established.The 8 pairs of chromosomes could be easily distinguished.The dose\|response curve between chromosome aberrations rate and the absorbed dose levels for lymphocytes in umbilical cord blood were of linear\|quadratic dose\|response model,except for fragments without centromere.The cumulative dose estimated for the two individuals were similar with reciprocal translocation rate in all analyzed cells or in stable cells. Conclusion Dose reconstruction of previous radiation exposure victims can be carried out with the M\|FISH method established in this study.\;

Regions close to human centromeres contain DNA fragments spanning hundreds of kilobases that exhibit a high degree of sequence identity (>95%). Here we report the genomic structure and evolution of a family of four paralogous regions related to a 220-kb genomic fragment present on the long arm of human chromosome 21 (21q22.1). Phylogenetic classification of the paralogous sequences obtained from the draft of the Human Genome Project are in agreement with results from comparative fluorescence in situ hybridization on metaphase chromosomes from human and great apes. The original copy present in 21q22.1 in human was duplicated in great apes after the divergence of the orang-utan and inserted in a pericentromeric region, most likely the ancestor of HSA2q, then disseminated by transposition of a larger fragment to other pericentromeric locations: HSA18p11, HSA13q11 and HSA21q11.1. The degree of dissemination varies among species.

Purpose: To identify the relationship between specific genes and phenotypic features of Williams syndrome.Methods: Subjects were selected based on their deletion status determined by fluorescence in situ hybridization using a panel of 24 BACs and cosmids spanning the region commonly deleted and single gene analysis using Southern blotting. From the cohort of subjects, three had atypical deletions. Physical examinations and cognitive tests were administered to the three subjects and the results were compared to those from a cohort of typical WS subjects.Results: The molecular results indicate smaller deletions for each subject. In all three cases, typical Williams facies were absent and visual spatial abilities were above that of full deletion WS subjects, particularly in the qualitative aspects of visual spatial processing.Conclusions: Combining the molecular analysis with the cognitive results suggest that the genes GTF2IRD1 and GTF2I contribute to deficits on visual spatial functioning.

Bacterial artificial chromosomes (BACs) are ideal materials to use for the purpose of integrating DNA sequence with cytogenetic markers. They have been the major vectors used in genome sequencing. BACs are also well suited for fluorescent in situ hybridization (FISH) in that they represent a stable and easily manipulated form of cloned DNA that produces bright, well-defined signals on metaphase and interphase chromosome preparations (). To link chromosomal position with DNA sequence throughout the human genome, we have developed an integrated BAC resource () by using FISH, PCR, and sequencing. The Resource contains a total of 6000 randomly mapped BAC clones, out of which, 1021 are BAC-sequence-tagged sites (STS) pairs representing 957 BACs (Fig. 1). This tool can be now used to rapidly identify genes affected by chromosomal rearrangements seen in genetic disorders and cancers. After this initial development, an international effort has assembled a collection of BAC clones that are both sequence-tagged and mapped relative to cytogenetic bands using FISH, resulting in a collection of 7600 clones (; see Note 1). Open image in new window Open image in new window Fig. 1. An Integrated BAC Resource was developed using FISH and PCR to link chromosomal position with DNA sequence in the human genome (). This map contains a total of 1021 BAC-STS pairs representing 957 BACs, each mapping to the position indicated by the vertical lines at the right of each chromosome ideogram. The length of the line indicates the resolution of the assignment, i.e, the outer boundaries of the band(s) within which the BAC signal is located as described in the methods. Although higher resolution may be obtained from our archived images, within a single band region, BACs are also ordered by the genetic and RH maps (,).

Deletion 3p syndrome is associated with characteristic facial features, growth failure, and mental retardation. Typically, individuals with deletion 3p syndrome have terminal deletions that result in loss of material from 3p25 to 3pter. We present a child with a clinical phenotype consistent with deletion 3p syndrome (ptosis, microcephaly, growth retardation, and developmental delay) and a subtle interstitial deletion in the distal portion of the short arm of chromosome 3, del(3)(p25.3p26.2). Fluorescence in situ hybridization (FISH) studies using 3p subtelomeric probes confirmed the terminal region of chromosome 3 was present. Sequence tagged sites (STS)-linked BAC clones mapping to chromosomal region 3p25-p26 were used to characterize the interstitial deletion by FISH. The results indicate the deletion is within a region of approximately 4.5 Mb between STS markers D3S3630 and D3S1304. This interstitial deletion lies within all previously reported terminal deletions in deletion 3p syndrome individuals, and represents the smallest reported deletion associated with deletion 3p syndrome. Characterization of the deletion may help identify genes important to growth and development that contribute to the deletion 3p syndrome phenotype when present in a hemizygous state.

Central nervous system (CNS) development involves neural patterning, neuronal and axonal migrations, and synapse formation. DSCAM, a chromosome 21 axon guidance molecule, is expressed by CNS neurons during development and throughout adult life. We now report that DSCAM and its chromosome 11 paralog DSCAML1 exhibit inverse ventral–dorsal expression patterns in the developing spinal cord and distinct, partly inverse, expression patterns in the developing cortex, beginning in the Cajal–Retzius cells. In the adult cortex, DSCAM predominates in layer 3/5 pyramidal cells and DSCAML1 predominates in layer 2 granule cells. In the cerebellum, DSCAM is stronger in the Purkinje cells and DSCAML1 in the granule cells. Finally, we find that the predicted DSCAML1 protein contains 60 additional N-terminal amino acids which may contribute to its distinct expression pattern and putative function. We propose that the DSCAMs comprise novel elements of the pathways mediating dorsal–ventral patterning and cell-fate specification in the developing CNS.