Isolation, characterization, and regional mapping of microclones from a human chromosome 21 microdissection library.

Thirty-four unique-sequence microclones were isolated from a previously described microdissection library of human chromosome 21 and were regionally mapped using a cell hybrid mapping panel which consists of six cell hybrids and divides chromosome 21 into eight regions. The mapping results showed that the microclones were unevenly distributed along chromosome 21, with the majority of microclones located in the distal half portion of the long arm, between 21q21.3 and 21qter. The number of unique-sequence clones began to decrease significantly from 21q21.2 to centromere and extending to the short arm. This finding is consistent with those reported in other chromosome 21 libraries. Thus, it may be inferred that the proximal portion of the long arm of chromosome 21 contains higher proportions of repetitive sequences, rather than unique sequences or genes. The microclones were also characterized for insert size and were used to identify the corresponding genomic fragments generated by HindIII. In addition, we demonstrated that the microclones with short inserts can be efficiently used to identify YAC (yeast artificial chromosome) clones with large inserts, for increased genomic coverage for high-resolution physical mapping. We also used 200 unique-sequence microclones to screen a human liver cDNA library and identified two cDNA clones which were regionally assigned to the 21q21.3-q22.1 region. Thus, generation of unique-sequence microclones from chromosome 21 appears to be useful to isolate and regionally map many cDNA clones, among which will be candidate genes for important diseases on chromosome 21, including Down syndrome, Alzheimer disease, amyotrophic lateral sclerosis, and one form of epilepsy.     

Analysis of chromosome 21 yeast artificial chromosome (YAC) clones.

Chromosome 21 contains genes relevant to several important diseases. Yeast artificial chromosome (YAC) clones, because they span > 100 kbp, will provide attractive material for initiating searches for such genes. Twenty-two YAC clones, each of which maps to a region of potential relevance either to aspects of the Down syndrome phenotype or to one of the other chromosome 21-associated genetic diseases, have been analyzed in detail. Clones total approximately 6,000 kb and derive from all parts of the long arm. Rare restriction-site maps have been constructed for each clone and have been used to determine regional variations in clonability, methylation frequency, CpG island density, and CpG island frequency versus gene density. This information will be useful for the isolation and mapping of new genes to chromosome 21 and for walking in YAC libraries.     

Human chromosome 21-encoded cDNA clones

We have employed two strategies to isolate random cDNA clones encoded by chromosome 21. In the first approach, a cDNA library representing expressed genes of WA17, a mouse-human somatic cell hybrid carrying chromosome 21 as its sole human chromosome, was screened with total human DNA to identify human chromosome 21-specific cDNAs. The second approach utilized previously characterized single-copy genomic fragments from chromosome 21 as probes to retrieve homologous coding sequences from a human fetal brain cDNA library. Six cDNA clones on chromosome 21 were obtained in this manner. Two were localized to the proximal long arm of chromosome 21, two to the distal portion of the long arm, and one to the region of 21q22 implicated in the pathology of Down syndrome.     

Isolation and regional mapping of NotI and EagI clones from human chromosome 21

NotI and EagI boundary libraries were constructed for human chromosome 21. One hundred forty-seven clones were isolated from the somatic cell hybrid 72532X-6 and localized using a hybrid mapping panel. After identification of those clones, which were isolated more than once, as well as those probes derived from a previously unrecognized integrated non-chromosome-21 fragment, 58 individual boundary clones (plus 2 additional NotI-EcoRI clones isolated from a flow-sorted library) were localized to 11 separate regions. The distribution of these probes is highly nonrandom, with 50% of the clones located in the distal band 21q22.3. Two probes, Not50 and Eag101, map to regions in the very proximal long arm which may contain the gene responsible for familial Alzheimer's disease (AD1), and Not50 would appear to be more proximal than D21S16 (E9). Twenty-eight probes map to the region between superoxide dismutase (SOD1) and the ETS2 oncogene, which appears to contain genes responsible for many of the phenotypic features of Down syndrome. Twenty clones contain (GT)n repeats, as determined by hybridization to a CA polymer, and should provide additional highly polymorphic probes. Closure of gaps in the physical linkage map of chromosome 21 should be facilitated by the isolation of these probes, as they identify many of the unmethylated CpG-rich islands that have hindered pulsed-field gel analysis. They will also be useful in identifying a set of genes in proximity to NotI and EagI restriction sites, as well as conserved DNA sequences for comparative mapping studies.     

Identification of a Novel Human Gene Containing the Tetratricopeptide Repeat Domain from the Down Syndrome Region of Chromosome 21

The Down syndrome (DS) region on chromosome 21, which is responsiblefor the DS main features, has been defined by analysis of DSpatients with partial trisomy 21. Within the DS region, we constructeda 1.6-Mb P1 contig map previously. To isolate gene fragmentsfrom the 1.6-Mb region, we performed direct cDNA library screeningand exon trapping using the P1 clones and a human fetal braincDNA library, and obtained 67 cDNA fragments and 52 possibleexons. Among them, 23 cDNA fragments and 4 exons were interpretedto be derived from a single gene by localization on P1 clonesand by Northern analysis. To obtain the full-length cDNA sequence,longer cDNA clones were further screened from another humancDNA library which was enriched with longer cDNA species. Theseclones were sequenced and assembled to a sequence of 9045 bp.This transcribed sequence encodes a novel 2025 amino-acid proteincontaining tetratricopeptide repeat (TPR) motifs and thereforethe gene was designated as TPRD (a gene containing the TPR motifson the Down syndrome region). The TPR domain has been foundin a certain protein phosphatase and in other proteins involvedin the regulation of RNA synthesis or mitosis. The TPRD gene,the novel gene which was proved to be in the 1.6-Mb region andto have the interesting features described above, is a candidatefor genes responsible for the DS phenotypes.     

The gene encoding DRAP (BACE2), a glycosylated transmembrane protein of the aspartic protease family, maps to the down critical region

We applied cDNA selection methods to a genomic clone (YAC 761B5) from chromosome 21 located in the so-called 'Down critical region' in 21q22.3. Starting from human fetal heart and brain mRNAs we obtained and sequenced several cDNA clones. One of these clones (Down region aspartic protease (DRAP), named also BACE2 according to the gene nomenclature) revealed a striking nucleotide and amino acid sequence identity with several motifs present in members of the aspartic protease family. In particular the amino acid sequences comprising the two catalytic sites found in all mammalian aspartic proteases are perfectly conserved. Interestingly, the predicted protein shows a typical membrane spanning region; this is at variance with most other known aspartic proteases, which are soluble molecules. We present preliminary evidence, on the basis of in vitro translation studies and cell transfection, that this gene encodes a glycosylated protein which localizes mainly intracellularly but to some extent also to the plasma membrane. Furthermore DRAP/BACE2 shares a high homology with a newly described beta-secretase enzyme (BACE-1) which is a transmembrane aspartic protease. The implications of this finding for Down syndrome are discussed.     

Highly polymorphic sequence at D21S1448 mapping close to D21S55, within the Down syndrome critical region

We have isolated a highly polymorphic sequence from the Down syndrome critical region on human chromosome 21. This is a particularly useful marker because it lies adjacent to the locus D21S55, which is most closely associated with the major defects on Down syndrome. Other than this marker, few other variable sequences are known in this region (including other restriction fragment length polymorphisms or CA repeats) and therefore D21S1448 will be extremely helpful not only for people studying the inheritance of portions of chromosome 21 with respect to Down syndrome, but also for those carrying out linkage analysis of the chromosome.     

Physical Mapping of the Holoprosencephaly Critical Region in 21q22.3, Exclusion of SIM2 as a Candidate Gene for Holoprosencephaly, and Mapping of SIM2 to a Region of Chromosome 21 Important for Down Syndrome

We set out to define the holoprosencephaly (HPE) critical region on chromosome 21 and also to determine whether there were human homologues of the Drosophila single-minded (sim) gene that might be involved in HPE. Analysis of somatic cell hybrid clones that contained rearranged chromosomes 21 from HPE patients defined the HPE minimal critical region in 21q22.3 as D21S113 to qter. We used established somatic cell hybrid mapping panels to map SIM2 to chromosome 21 within subbands q22.2-q22.3. Analysis of the HPE patient–derived somatic cell hybrids showed that SIM2 is not deleted in two of three patients and thus is not a likely candidate for HPE1, the HPE gene on chromosome 21. However, SIM2 does map within the Down syndrome critical region and thus is a candidate gene that might contribute to the Down syndrome phenotype.     

Evolutionary breakpoints on human chromosome 21.

Segments of the long arm of human chromosome 21 are conserved, centromere to telomere, in mouse chromosomes 16, 17, and 10. There have been 28 genes identified in human chromosome 21 between TMPRSS2, whose orthologue is the most distal gene mapped to mouse chromosome 16, and PDXK, whose orthologue is the most proximal gene mapped to mouse chromosome 10. Only 6 of these 28 genes have been mapped in mouse, and all are located on chromosome 17. To better define the chromosome 17 segment and the 16 to 17 transition, we used a combination of mouse radiation hybrid panel mapping and physical mapping by mouse: human genomic sequence comparison. We have determined the mouse chromosomal location of an additional 12 genes, predicted the location of 7 more,and defined the endpoints of the mouse chromosome 17 region. The mouse chromosome 16/chromosome 17 evolutionary breakpoint is between human genes ZNF295 and UMODL1, showing there are seven genes in the chromosome 16 segment distal to Tmprss2. The chromosome 17/chromosome 10 breakpoint seems to have involved a duplication of the gene PDXK, which on chromosome 21 lies immediately distal to the KIAA0179 gene. These data suggest that there may be as few as 21 functional genes in the mouse chromosome 17 segment. This information is important for defining existing and constructing more complete mouse models of Down syndrome.     

The mouse homolog of the human amyloid beta protein (AD-AP) gene is located on the distal end of mouse chromosome 16: further extension of the homology between human chromosome 21 and mouse chromosome 16

The human amyloid beta protein is the major constituent of the brain amyloid plaques found in Alzheimer disease. The gene that encodes this protein is located on chromosome 21, and individuals with Down syndrome (trisomy 21) also exhibit an early onset form of Alzheimer disease. We have used the cloned human amyloid beta protein gene and a panel of somatic cell hybrids to map the location of the mouse homolog of this gene. We report here that the mouse gene is located on chromosome 16 within the region 16C3----ter, in common with three other genes which map within the Down syndrome region of human chromosome 21.     

Down syndrome and the genes of human chromosome 21: current knowledge and future potentials. Report on the Expert workshop on the biology of chromosome 21 genes: towards gene-phenotype correlations in Down syndrome. Washington D.C., September 28-October 1, 2007

Down syndrome (DS), trisomy of human chromosome 21, is the most common genetic cause of intellectual disability. With an incidence in some countries as high as one in approximately 700 live births, and a complex, extensive and variably severe phenotype, Down syndrome is a significant medical and social challenge. In recent years, there has been a rapid increase in information on the functions of the genes of human chromosome 21, as well as in techniques and resources for their analysis. A recent workshop brought together experts on the molecular biology of Down syndrome and chromosome 21 with interested researchers in other fields to discuss advances and potentials for generating gene-phenotype correlations. An additional goal of the workshop was to work towards identification of targets for therapeutics that will correct features of DS. A knowledge-based approach to therapeutics also requires the correlation of chromosome 21 gene function with phenotypic features.     

Gene-dosage effects in Down syndrome and trisomic mouse models

The abnormalities found in human Down syndrome (trisomy 21) have been thought to result from increased expression of genes on chromosome 21 because of their higher gene dosage. Now, several groups have shown this to be generally the case, but some inter-individual variability and other exceptions were found.     

The alpha-A-crystallin and cystathionine beta-synthase genes are physically very closely linked in proximal mouse chromosome 17

Murine genes homologous to those contributing to the Down syndrome (DS) phenotype in man are currently of interest because of their potential for providing animal models for the study of specific DS symptoms. Most of the genes mapping to human chromosome 21q22, where the DS genes are concentrated, are related to sequences located on mouse chromosome 16. Others, however, are known to map to mouse chromosome 10, and two genes, cystathionine beta-synthase (Cbs) and alpha-A-crystallin (Crya-1), have been localized to the proximal portion of mouse chromosome 17. In this paper, we show that the two genes mapping to human chromosome 21q22 and mouse chromosome 17 are very tightly linked in mouse, being separated by at least 70 kb, but not more than 130 kb. The very close physical linkage of mouse Cbs and Crya-1, combined with data that localize homologs of the closely flanking markers H2k and Pim-1 to human chromosome 6, suggests that the human 21q22/mouse chromosome 17 conserved segment is of a very limited total physical size and is likely to contain a relatively small number of genes.