The evolution of human chromosome 21: evidence from in situ hybridization in marsupials and a monotreme.

We have mapped five human chromosome 21 (HSA 21) markers in marsupials and a monotreme, two major groups of mammals that diverged from eutherians 130-150 and 150-170 million years before present (MYrBP), respectively. We have found that these genes map to two distinct autosomal sites, one containing SOD1/CBR/BCEI and the other containing ETS2/INFAR, in the marsupials Macropus eugenii and Sminthopsis macroura (which belong to orders that diverged 40-80 MYrBP), as well as in the monotreme Ornithorhynchus anatinus (the platypus). Since marsupials and monotremes diverged independently from eutherians, these data suggest that HSA 21 genes were originally located in two separate autosomal blocks. In another Sminthopsis species, SOD1 is linked to TRF (a marker on HSA 3q), suggesting that the ancestral SOD1/CBR/BCEI region also included HSA 3 markers. We suggest that these blocks became fused early in the eutherian evolution to form a HSA 3/21 chromosome, which has remained intact in artiodactyls, but has been independently disrupted in both the primate and rodent lineages.     

Rearrangement of chromosome 21 in Alzheimer's disease

We have analyzed the DNA from four patients suffering from a dementia of the Alzheimer type and of four controls of identical age. Estimates of the copy numbers of two genes located on chromosome 21, SOD1 and ETS2, gave the following results: in all the cases there was duplication of ETS2 whereas SOD1 was normal. These preliminary results indicate that the segment of chromosome 21(q21----q22.1) is rearranged in all the four patients who were investigated.     

The ETS genes on chromosome 21 are distal to the breakpoint of the acute myelogenous leukemia translocation (8;21)

The definition of the genetic linkage map of human chromosomes may be helpful in the analysis of cancer-specific chromosome abnormalities. In the translocation (8;21)(q22;q22), a nonrandom cytogenetic abnormality of acute myelogenous leukemia (AML), we previously observed the transposition of the ETS2 gene located at the 21q22 region from chromosome 21 to chromosome 8. However, no ETS2 rearrangements were detected in the DNA of t(8;21)-positive AML cells. Genetic linkage analysis has allowed us to locate the ETS2 gene relative to other loci and to establish that the breakpoint is at an approximate genetic distance of 17 cM from ETS2. When the information from the linkage map is combined with that from molecular studies, it is apparent that (a) the t(8;21) breakpoint does not affect the ETS2 gene structure or the structure of the other four loci proximal to ETS2: D21S55, D21S57, D21S17, and ERG, and ETS-related gene; and (b) the actual DNA sequence involved in the t(8;21) must reside in a 3-cM genetic region between the D21S58 and the D21S55/D21S57 loci, and remains to be identified.     

Characterisation of three microsatellite polymorphisms (D21S1262, D21S1419 and D21S1421) from band 21q22.1

We have isolated three clones, containing highly polymorphic CA-repeat sequences, from a human chromosome 21 phage library (LA21NS01). These clones have been localised to band q22.1 by using a chromosome 21 somatic cell hybrid panel. D21S1262 is located between breakpoints 6918-8a1 and 32S, and D21S1419 and D21S1421 are localised between breakpoints JC6-A and MRC2G. Their observed heterozygosities range between 0.75 and 0.85 as shown by unrelated reference parents from the Centre d'Etude du Polymorphisme Humain. These highly polymorphic markers should be useful for improving the analysis of this region of chromosome 21, which contains important genes such as SOD1, GART and IFNAR.     

A genetic linkage map of human chromosome 21: analysis of recombination as a function of sex and age.

A genetic linkage map of human chromosome 21 has been constructed using 22 anonymous DNA markers and five complementary DNAs (cDNAs) encoding the amyloid beta protein precursor (APP), superoxide dismutase 1 (SOD1), the ets-2 proto-oncogene (ETS2), the estrogen inducible breast cancer locus (BCEI), and the leukocyte antigen, CD18 (CD18). Segregation of RFLPs detected by these DNA markers was traced in the Venezuelan Reference Pedigree (VRP). A comprehensive genetic linkage map consisting of the 27 DNA markers spans 102 cM on the long arm of chromosome 21. We have confirmed our initial findings of a dramatically increased rate of recombination at the telomere in both females and males and of significantly higher recombination in females in the pericentromeric region. By comparing patterns of recombination in specific regions of chromosome 21 with regard to both parental sex and age, we have now identified a statistically significant downward trend in the frequency of crossovers in the most telomeric portion of chromosome 21 with increasing maternal age. A less significant decrease in recombination with increasing maternal age was observed in the pericentromeric region of the chromosome. These results may help in ultimately understanding the physical relationship between recombination and nondisjunction in the occurrence of trisomy 21.     

Structure,chromosomal location,and analysis of the canine Cu/Zn superoxide dismutase (SOD1) gene

Mutations in Cu/Zn superoxide dismutase (SOD1), a major cytosolic antioxidant enzyme in eukaryotic cells, have been reported in approximately 20% of familial amyotrophic lateral sclerosis (FALS) patients. Hereditary canine spinal muscular atrophy (HCSMA), a fatal inherited motor neuron disease in Brittany spaniels, shares many clinical and pathological features with human motor neuron disease, including FALS. The SOD1 coding region has been sequenced and cloned from several animal species, but not from the dog. We have mapped the chromosomal location, sequenced, and characterized the canine SOD1 gene. Extending this analysis, we have evaluated SOD1 as a candidate for HCSMA. The 462 bp SOD1 coding region in the dog encodes 153 amino acid residues and exhibits more than 83% and 79% sequence identity to other mammalian homologues at both the nucleotide and amino acid levels, respectively. The canine SOD1 gene maps to CFA31 close to syntenic group 13 on the radiation hybrid (RH) map in the vicinity of sodium myo/inositol transporter (SMIT) gene. The human orthologous SOD1 and SMIT genes have been localized on HSA 21q22.1 and HSA 21q21, respectively, confirming the conservation of synteny between dog syntenic group 13 and HSA 21. Direct sequencing of SOD1 cDNA from six dogs with HCSMA revealed no mutations. Northern analysis indicated no differences in steady-state levels of SOD1 mRNA.     

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.     

Fine mapping of the long arm of human chromosome 11 by in situ hybridization using different translocations, including the t(11;22) of Ewing sarcoma

The progesterone receptor gene (PGR), the gene coding for porphobilinogen deaminase (PBGD), the gene coding for a neural cell adhesion molecule (NCAM), the oncogene ETS1, and the anonymous genomic sequence D11S29 have been previously located on the long arm of chromosome 11. However, gene localizations obtained with different gene-mapping procedures have led to occasional discrepancies. To localize these genes more precisely, we hybridized five human DNA sequences with different chromosomal rearrangements, including four balanced and one unbalanced translocations. We show here that the order of these five sequences is cen-PGR-PBGD-DIIS29/NCAM/ETS1-tel.     

Linkage mapping of highly informative DNA polymorphisms within the human interferon-alpha receptor gene on chromosome 21.

Two polymorphic loci within the interferon-alpha receptor (IFNAR) gene on human chromosome 21 have been identified and mapped by linkage analysis in 40 CEPH families. These markers are (1) a multiallelic RFLP with an observed heterozygosity of 0.72 and (2) a variable (AT3)n short sequence repeat at the poly(A) tail of an Alu sequence (AluVpA) with an observed heterozygosity of 0.83. This locus is close to D21S58 (theta = 0.02, zeta = 36.76) and D21S17 (theta = 0.02, Zeta = 21.76) with chromosomal band 21q22.1. Multipoint linkage analysis suggests the most likely locus order to be 21cen-D21S58-IFNAR-D21S17-21qter. Given its high heterozygosity, the IFNAR gene can be used as an index marker on human chromosome 21.     

Bone morphogenetic protein: chromosomal localization of human genes for BMP1, BMP2A, and BMP3

Bone morphogenetic protein (BMP) induces endochondral bone formation in vivo. The human genes have been cloned for a group of proteins containing BMP activity (BMP1, BMP2A, and BMP3). Two of the proteins are members of the transforming growth factor-beta supergene family (BMP2A and BMP3), while BMP1 is a novel regulatory protein. Using somatic cell hybrid lines, cDNA probes were used to map BMP1 to chromosome 8, BMP2A to chromosome 20, and BMP3 to the p14-q21 region of chromosome 4. This analysis reveals that the BMP2A and BMP3 genes map to conserved regions between mouse and human, while the BMP1 gene does not. The locations of the BMP genes were found to overlap with the loci for several disorders of cartilage and bone formation.     

Mapping of aminoacylase-1 and beta-galactosidase-A to homologous regions of human chromosome 3 and mouse chromosome 9 suggests location of additional genes.

Conserved linkage groups have been found on the X and autosomal chromosomes in several mammalian species. The identification of conserved chromosomal regions has potential for predicting gene location in mammals, particularly in humans. The genes for human aminoacylase-1 (ACY1, N-acylamino acid aminohydrolase, E.C.3.5.1.14), an enzyme in amino acid metabolism, and beta-galactosidase-A (GLB1, E.C.3.2.1.23), deficient in GM1-gangliosidosis, have been assigned to human chromosome 3. Using human-mouse somatic cell hybrids segregating translocations of human chromosome 3, expression of both ACY1 and GLB1 correlated with the presence of the p21 leads to q21 region of chromosome 3. In a previous study, assignment of these genes to mouse chromosome 9 used mouse-Chinese hamster somatic cell hybrids, eliminating mouse chromosomes. To approximate the size of the conserved region in the mouse, experiments were performed with recombinant inbred mouse strains. An electrophoretic variant of ACY-1 in mouse strains was used to map the Acy-1 gene 10.7 map U from the beta-galactosidase locus. These data suggest that there is a region of homology within the p21 leads to q21 region of human chromosome 3 and a segment of mouse chromosome 9. Since the mouse transferrin gene (Trf) is closely linked to the aminoacylase and beta-galactosidase loci, we predict that the human transferrin (TF) gene is on chromosome 3.     

Regional assignment of human genes TPI1, GAPDH, LDHB, SHMT, and PEPB on chromosome 12.

Karyological analysis was performed on a series of human-Chinese hamster cell hybrids containing deletions of human chromosome 12. Chromosome breakage was produced by treatment of the cells with either X-rays or 5-bromodeoxyuridine and near-visible light. The hybrid clones were analyzed for the presence or absence of the following five human gene markers known to be located on chromosome 12: triosephosphate isomerase-1 (TPI1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase-B (LDHB), serine hydroxymethyltransferase (SHMT), and peptidase-B (PEPB). Based on the correlation between the isozyme markers and karyological analysis of these clones, a regional map of the five human genes on chromosome 12 was established. The linear order for these genes is: pter-TPI1-GAPDH-LDHB-centromere-SHMT-PEPB-qter. The locations of these genes are: TPI1, GAPDH, LDHB: pter leads to p12; SHMT: q12 leads to q14; PEPB: q14 leads to qter. Statistical analysis similar to that of Goss and Harris (1975, 1977a, b) has been performed on the segregation data in the hybrid clones. The statistical map, in general, agrees with the cytogenetic map and further localizes PEPB to 12q21.     

Linkage analysis of the human HMG14 gene on chromosome 21 using a GT dinucleotide repeat as polymorphic marker

A (GT)n repeat in intron 4 of the functional human HMG14 gene on chromosome 21 was used as polymorphic marker to map this gene relative to the genetic linkage map of human chromosome 21. Variation in the length of the (GT)n repeat was detected by electrophoresis on polyacrylamide gels of DNA amplified by the polymerase chain reaction using primers flanking the repeat. The observed heterozygosity of this polymorphism in 40 CEPH families was 58% with six different alleles. Linkage analysis localized the HMG14 gene close to the ETS2 gene and locus D21S3 in chromosomal band 21q22.3.     

Chromosomal localization of the human apoprotein CI gene and of a polymorphic apoprotein AII gene

Human apoprotein(apo) CI and apo AII cDNA probes have been used to analyze the segregation of the human genes in panels of human-mouse hybrids. The apo CI (APOCI) gene segregates with chromosome 19 and the apo AII (APOA2) gene with chromosome 1. Somatic cell hybrids containing chromosome translocations were used to map the apo AII gene to the 1p21-1qter region. Human APOA2 is polymorphic for the restriction endonuclease Msp I. Comparison of human and mouse chromosome 1 reveals a conserved group including apo AII, renin and peptidase genes and suggests that APOA2 will be found distal to this group on human chromosome 1. The mouse apo AII gene is closely linked with genes that regulate HDL structure. Similar HDL regulatory genes will probably be found near human APOA2.     

Genetic linkage map of human chromosome 21

Two of the most common disorders affecting the human nervous system, Down syndrome and Alzheimer's disease, involve genes residing on human chromosome 21. A genetic linkage map of human chromosome 21 has been constructed using 13 anonymous DNA markers and cDNAs encoding the genes for superoxide dismutase 1 (SOD1) and the precursor of Alzheimer's amyloid beta peptide (APP). Segregation of restriction fragment length polymorphisms (RFLPs) for these genes and DNA markers was traced in a large Venezuelan kindred established as a "reference" pedigree for human linkage analysis. The 15 loci form a single linkage group spanning 81 cM on the long arm of chromosome 21, with a markedly increased frequency of recombination occurring toward the telomere. Consequently, 40% of the genetic length of the long arm corresponds to less than 10% of its cytogenetic length, represented by the terminal half of 21q22.3. Females displayed greater recombination than males throughout the linkage group, with the difference being most striking for markers just below the centromere. Definition of the linkage relationships for these chromosome 21 markers will help refine the map position of the familial Alzheimer's disease gene and facilitate investigation of the role of recombination in nondisjunction associated with Down syndrome.     

Structure of the human CRFB4 gene: Comparison with its IFNAR neighbor

The cytokine receptor family consists of a growing number of structurally and evolutionarily related transmembrane receptors. CRFB4 and IFNAR are two of the most similar members of this family. They are encoded by two neighboring genes on both human chromosome 21 and murine chromosome 16. The sequence of the human CRFB4 gene was determined from the first exon to the last intron. The nature of the repetitive sequences present in the introns was analyzed and compared with those present in the human IFNAR gene. This analysis leads to considerations of the antiquity of the duplication that gave rise to both genes from a common ancestor. A pseudogene for USF has been identified in the IFNAR gene and a new definition for the repetitive sequence MER37 is proposed. The polymorphism associated with two CA repeats present in the CRFB4 gene is described.The nucleotide sequence reported in this paper has been deposited to GenBank with accession numbers U08988 and U12021 Correspondence to: G. Lutfalla