Partial physical map of human chromosome 21 from fibroblast and lymphocyte DNA.

A partial physical map of the human chromosome 21 including 26 genes and anonymous sequences was established by pulsed-field gel electrophoresis analysis of restriction fragments obtained from lymphocyte and fibroblast DNAs. The sizes of the restriction fragments obtained by total digestion with eight different enzymes were compared in these two tissues. Differences resulting from the variations in the methylation state of the restriction sites were frequently observed. These differences and partial digestions were used to estimate the order and the distances between genes and sequences. Six linkage groups were defined: D21S13-D21S16, D21S1-D21S11, D21S65-D21S17, (D21S55,ERG)-ETS2, BCEI-D21S19-D21S42-D21S113-CBS-CRYA1, and COL6A2-S100B. For six intergenic distances the resolution of previous maps was significantly increased.

Mapping the Down syndrome chromosome region. Establishment of a YAC contig spanning 1.2 megabases.

The triplication of a region of chromosome 21 around D21S55 in 21q22.2-22.3 has been involved in the main features of Down syndrome including mental retardation (Down syndrome chromosome region: DCR). To improve the physical map of this region, we screened yeast artificial chromosome (YAC) libraries with ETS2 and ERG sequences. Five selected clones were analyzed by AluPCR, pulsed-field gel electrophoresis, and in situ hybridization. A 1.2-Mg contig, encompassing the protooncogenes ETS2 and ERG, was identified, its restriction map established and compared to the genomic map. ERG is distal to D21S55 and proximal to ETS2. ERG and ETS2 genes are 400 kb apart and in opposite orientations. The contig contains the distal boundary and part of the DCR. Three putative HTF islands were identified.

New chromosome 21 DNA markers isolated by pulsed field gel electrophoresis from an ETS2-containing Down syndrome chromosomal region.

To generate new chromosome 21 markers in a region that is critical for the pathogenesis of Down syndrome (D21S55-MX1), we used pulsed field gel electrophoresis (PFGE) to isolate a 600-kb NruI DNA fragment from the WA17 hybrid cell line, which has retained chromosome 21 as the only human material. This fragment, which contains the oncogene ETS2, was used to construct a partial genomic library. Among the 14 unique sequences that were isolated, 3 were polymorphic markers and contained sequences that are conserved in mammals. Five of these markers mapped on the ETS2-containing NruI fragment and allowed us to define an 800-kb high-resolution PFGE map.

Mapping around the Xq13.1 breakpoints of two X/A translocations in hypohidrotic ectodermal dysplasia (EDA) female patients.

Cellular hybrids were obtained from a t(X;12) identified in a female patient with hypohidrotic ectodermal dysplasia (EDA). This rearrangement had the same Xq13.1 cytogenetic breakpoint as a t(X;9) found in a previously observed EDA patient. A comparative analysis of these two rearrangements with nine probes was performed at the molecular level. These probes could define three subregions: three are proximal, two are distal, and four are between the two breakpoints. These last probes should prove useful for cloning the gene.

Molecular analysis of 12 patients with the t(8;21) translocation and M2 acute myelogenous leukemia.

The t(8;21)(q22;q22) is a nonrandom cytogenetic abnormality associated with acute myelogenous leukemia of the M2 subtype (FAB classification). The 8q- and 21q+ derivative chromosomes have previously been isolated in somatic cell hybrids and used to map the anonymous sequences D21S65 and D21S17, which were proximal and distal, respectively, to the breakpoint on chromosome 21. DNA from a series of 12 t(8;21) patients and 7 controls was analyzed by pulsed field gel electrophoresis. Physical linkage of probes D21S65 and D21S17 on a 2100 kb NruI fragment was established by partial digestion experiments. In all the patients, the translocation generated a rearranged D21S65 NruI fragment of 650 to 750 kb, suggesting heterogeneity in the breakpoints. This heterogeneity was confirmed by using BssHII, SacII, and EagI enzymes. Our results are consistent with the presence of a 100 Kb breakpoint cluster region on chromosome 21 encompassing the AML1 gene. Interestingly, in half of the patients, demethylation of an NruI site located 7 kb proximal to the last exon of the AML1 gene occurred on the nontranslocated chromosome 21.

Accurate evaluation of the sizes of DNA fragments (from 30 to 4700 kb) in pulse field gel electrophoresis.

Construction of long-range genomic maps by pulse field gel electrophoresis requires optimum resolution of large DNA fragments. Using the transverse-alternating field electrophoresis system, we describe a method to accurately evaluate the sizes of fragments generated by rare-cutter digestions within the 30-4700-kb range. A protocol generating large (greater than 1000 kb) molecules by partial digestion is also reported.

Cytogenetic and molecular analysis of a de novo tandem duplication of chromosome 21.

We have characterised by cytogenetic and molecular analysis a de novo tandem duplication of chromosome 21. High resolution chromosome examination of lymphocytes revealed the following karyotype in 90% of the cells: 46,XY,dir dup (21)(pter----q22.300::q11.205----qter). Of these cells, 10% showed a normal karyotype. Gene dosage of chromosome 21 sequences by a slot blot method indicated that the duplication extends from D21S16 to D21S55. In situ hybridization with probes close to the borders of the duplicated segment confirmed the gene dosage data and gave results consistent with a true tandem duplication of chromosome 21. Pulsed field gel electrophoresis of the patient's DNA showed an abnormal restriction band common to D21S55 and D21S16, confirming that the junction point between the two homologous parts of the tandem chromosome brings these two sequences into proximity. Restriction fragment length polymorphism analysis indicated that the abnormal chromosome was maternal in origin and that the rearrangement of chromosome 21 could not have occurred at a post-zygotic stage of development but resulted from a recombination event during maternal gametogenesis. The possible mechanisms of formation of the abnormal chromosome are discussed, as is the presence of cells with normal chromosomes 21, in the patient.

Increased SOD1 enzymatic activity and gene modifications in orangutans: evolutionary implications.

Superoxide dismutase CuZn (SOD1) enzymatic activity was measured in five orangutans (Pongo pygmaeus, PPY) and compared to that of man, chimpanzee, and gorilla. It was found to be increased by a factor of two in one orangutan (Ralfina) and by a factor of 1.5 in the four others. In situ hybridization of the SOD1 cDNA human probe showed a heterozygous intra-chromosomal rearrangement of pair PPY XXI, possibly an insertion, in Ralfina. Southern blotting showed that the SOD1 gene is modified in the three orangutans that were investigated and that a further modification of the 5'-end of the gene had occurred in Ralfina. The evolutionary implications of these observations are discussed.

Physical map around the retinoblastoma gene: possible genomic imprinting suggested by NruI digestion.

A long-range restriction map constructed around the retinoblastoma (RB) gene by means of PFGE analysis allowed further definition of chromosomal rearrangements with a breakpoint within the gene, as well as of submicroscopic deletions. A serendipitous observation was that the NruI restriction pattern differs according to the parental origin of the rearrangement.

Molecular detection of constitutional deletions in patients with retinoblastoma.

The recent cloning of the retinoblastoma (RB) gene as well as the identification of intragenic polymorphisms afford the necessary tools for the analysis of rearrangements using molecular hybridization. We searched for constitutional deletions by Southern blotting in 67 independent patients with normal karyotype comprising 15 familial and 52 sporadic cases. Among the latter, 33 were bilaterally and 19 unilaterally affected. We detected 6 deletions using cDNA probes covering almost all of the RB gene, as well as a genomic probe of the 5' part of the gene. With this approach, the incidence of detectable deletions was around 10%. No hot spots for deletion breakpoints were found. Asymptomatic carriers were detected in 2 families. The effectiveness of genetic counselling was largely improved by this approach.

Down syndrome critical region around D21S55 on proximal 21q22.3.

We have analysed the DNA of 2 patients with many manifestations of Down syndrome and partial duplication of distinct regions of chromosome 21, respectively, q11.205----q22.300 and q22.300----qter (Rahmani et al.: Proceedings of the National Academy of Sciences of the United States of America 86:5958-5962, 1989). Assessment of the copy number of five chromosome 21 sequences (SOD1, D21S17, D21S55, ETS2, and D21S15) has shown that D21S55 was duplicated in both cases. The size of the common duplicated region can be estimated between 400 and 3,000 Kb, after the results of pulsed-field gel analysis and from the knowledge of regional mapping of the probes D21S17, D21S55, and ETS2. This region, located on the proximal part of 21q22.3, is postulated to contain genes the overexpression of which plays a major role in the pathogenesis of Down syndrome.

Critical role of the D21S55 region on chromosome 21 in the pathogenesis of Down syndrome.

The duplication of a specific region of chromosome 21 could be responsible for the main features of Down syndrome. To define and localize this region, we analyzed at the molecular level the DNA of two patients with partial duplication of chromosome 21. These patients belong to two groups of Down syndrome patients characterized by different partial trisomies 21: (i) duplication of the long arm, proximal to 21q22.2, and (ii) duplication of the end of the chromosome, distal to 21q22.2 We assessed the copy number of five chromosome 21 sequences (SOD1, D21S17, D21S55, ETS2, and D21S15) and found that D21S55 was duplicated in both cases. By means of pulsed-field gel analysis and with the knowledge of regional mapping of the probes D21S17, D21S55 and ETS2, we estimated the size of the common duplicated region to be between 400 and 3000 kilobases. This region, localized on the proximal part of 21q22.3, is suspected to contain genes the overexpression of which is crucial in the pathogenesis of Down syndrome.

Twofold CuZnSOD activity suggesting homozygous submicroscopic duplication of chromosome 21 in the orangutan.

A twofold CuZnSOD activity was observed in an orangutan as compared to man and a chimpanzee. This suggests that a submicroscopic duplication of chromosome 21 (similar to those demonstrated in patients with the trisomy 21 phenotype but with a normal karyotype) has occurred in the homozygous state during evolution of the orangutan phylum. Such duplications could be important evolutionary mechanisms, together with visible chromosome rearrangements and classical gene mutations.

Studies on the human chromosome 3 centromere with a newly cloned alphoid DNA probe.

Starting from a chromosome-specific DNA library, we have isolated a human chromosome-specific satellite DNA sequence. This sequence of 635 base pairs (bp) consists of 3.7 alpha DNA monomers of 170-171 bp. Under high stringency it hybridizes to the centromere of chromosome 3 in a region composed of 2,750 bp tandem repeats characterized by the regular spacing of Hind III and TaqI restriction enzyme recognition sites. It has diverged and undergone amplification after the human speciation. The amplification allows an easy monitoring of the chromosome 3 centromere by in situ hybridization with a nonradioactive probe.

Maternal origin of a de novo balanced t(21q21q) identified by ets-2 polymorphism.

Molecular investigations were done in a woman with a de novo balanced t(21q21q) discovered because of the birth of a trisomic 21 baby. Polymorphisms detected with probe ets-2 after Msp I digestion showed that both chromosomes 21 involved in the rearrangement were of maternal origin. The most likely hypothesis is that of a disomic 21 oocyte fertilized by a nullisomic 21 sperm.

Submicroscopic duplication of chromosome 21 and trisomy 21 phenotype (Down syndrome).

A patient with the phenotype of trisomy 21 (Down syndrome) was found to have a normal karyotype in blood lymphocytes and fibroblasts. Assessment of the chromosome 21 markers SOD1, CBS, ETS2, D21S11, and BCEI showed partial trisomy by duplication of a chromosome segment carrying the SOD1, CBS, and ETS2 loci and flanked by the BCEI and D21S11 loci, which are not duplicated. This submicroscopic duplication at the interface of 21q21 and 21q22.1 reduces to about 2000-3000 kb the critical segment the trisomy of which is responsible for the phenotype of trisomy 21.

De novo t(2;13)(p24.3;q14.2) and retinoblastoma. Mapping of two 13q14 probes by in situ hybridization.

The two probes H3-8 and H2-42, known to be located in 13q14, were mapped by in situ hybridization to either side of the 13 breakpoint of an apparently balanced de novo t(2;13)(p24.3;q14.2) detected in a patient with retinoblastoma as the only phenotypic manifestation.