Proc. Nati. Acad. Sci. USA Vol. 86, pp. 1914-1918, March 1989 Genetics Molecular mechanism in the formation of a human ring chromosome 21 CORINNE WONG*, HAIG H. KAZAZIAN, JR.*, GAIL STETTENt, WILLIAM C. EARNSHAWt, MARGARET L. VAN KEUREN§¶, AND STYLIANOS E. ANTONARAKIS*II *Program in Human Genetics, Genetics Unit, Department of Pediatrics, tDepartment of Gynecology and Obstetrics, and tDepartment of Anatomy and Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; and §Eleanor Roosevelt Institute for Cancer Research, 4200 East Ninth Avenue, Denver, CO 80206 Communicated by Victor A. McKusick, October 5, 1988 ABSTRACT We have characterized the structural re- D21K9 of locus D21S13, were used (18). The superoxide arrangements of a chromosome 21 that led to the de novo dismutase 1 probe (SOD-1) (20) and 0-amyloid probe (APP) formation of a human ring chromosome 21 [r(21)]. Molecular (21) were also employed. The DNA fragment CW21A is a cloning and chromosomal localization of the DNA regions 650-nucleotide (nt) Pvu II-Sph I fragment, cloned in flanking the ring junction provide evidence for a long arm to M13mp9, that spans the break point. The DNA fragment long arm fusion in formation of the r(21). In addition, the CW21B of locus D21S111 is a 530-nt HindIII-Sph I fragment centromere and proximal long arm region of a maternal located on the 5' side of the break point, cloned in M13mp9 chromosome 21 are duplicated in the r(21). Therefore, the and pGEM-4. mechanism in formation ofthe r(21) was complex involving two Somatic Cell Hybrids. A somatic cell hybrid (R2-10) con- sequential chromosomal rearrangements. (i) Duplication ofthe taining r(21) as the only detectable human chromosome was centromere and long arm of one maternal chromosome 21 made (19). The R2-10 line was also back selected for loss of occurred forming a rearranged intermediate. (ii) Chromo- the r(21) (cell line R2-1OW-S5) by growth in nonselective somal breaks in both the proximal and telomeric long arm (purine supplemented) medium, followed by selective me- regions on opposite arms of this rearranged chromosome dium with the addition of bromodeoxyuridine and light (22). occurred with subsequent reunion producing the r(21). For mapping, a panel of somatic cell hybrids, 72532X-6, 153E7bx, and 2FuR1 (23), with characterized rearrangements Ring chromosomes in humans represent a class of aberrant of human chromosome 21 was used (19). chromosomes observed frequently in congenital anomalies Cloning the Chromosomal Break Points. DNA from the and occasionally with normal phenotypes (1). Most ring patient's blood lymphocytes was digested with EcoRI and a chromosomes arise de novo, yet occasional familial trans- size-selected library (7-9 kb) was made in Agtwes B. About mission has been reported (2-5). The frequency of ring 300,000 recombinant phage were screened (24) with chromosomes is 1 in 25,000 recognized conceptions (6), and pPW231C and a single clone, A21BP (Fig. 1B), was purified. all human chromosomes have been observed as rings. Almost DNA from the region on the 3' side of the break point was 50% of ring autosomes are derived from the acrocentric cloned (A2lpq) by using probe CW21A from a flow-sorted, chromosomes (7). The proposed mechanism of ring forma- HindIII-digested chromosome 21 library in Charon 21A (ID tion, breakage of both short and long arms of a chromosome code, LL21NS02; American Type Culture Collection num- with subsequent end to end fusion (8), remains unproven. In ber, 57713). several reports of acrocentric ring chromosomes, a Robert- Immunofluorescence Studies. For quantification of DNA, sonian translocation produced by short arm fusion has been photographic negatives of 4',6-diamidino-2-phenylindole observed in the patient, a parent, or a sibling (9-16). In each (DAPI)-stained (25) images were scanned using a Loats case, one or both of the chromosomes involved in the (Westminster, MD) video densitometer equipped with a two- translocation were present in the ring, suggesting that ring dimensional gel-scanning software package. Indirect immu- formation was related to or derived from the translocation. In nofluorescence was performed on mitotic spreads (26). Chro- this study, we characterize a ring chromosome 21 [r(21)] and mosomes were first stained with DAPI and then with a provide molecular evidence** for the formation of a rear- monoclonal anti-centromere antibody prepared against the ranged chromosome 21 with a duplicated centromere and cloned 80-kDa human autoantigen CENP-B (27). Bound long arm that preceded ring formation. antibody was detected with a second antibody coupled to streptavidin linked to Texas red dye. MATERIALS AND METHODS Sources of DNA. DNA was isolated from cultured amnio- RESULTS cytes and fibroblasts and peripheral blood from the proband Cytogenetic Characterization and Origin of r(21). r(21) was with the r(21) and his parents (17). Fibroblast and lympho- observed in 90% of the cultured amniocytes, fibroblasts, and blastoid cell lines were established from the proband (GM lymphocytes (28, 29); the remaining 10% of the cells were 6137 and GM 6779) and a lymphoblastoid line was established monosomic for chromosome 21. Break points were located at from his mother (GM 8779) and stored in the Coriell Institute band 21q22.3 and in the pericentromeric region. r(21)s were for Medical Research (Camden, NJ). variable in size, the majority (84%) being slightly larger than DNA fragment pPW231C of locus D21S3 is a 2.1-kilobase (kb) EcoRI fragment cloned in pBR328 (18, 19). Additional Abbreviations: r(21), ring chromosome 21; nt, nucleotide(s); DAPI, chromosome 21-specific probes, pPW228C of locus D21S1, 4',6-diamidine-2-phenylindole; MAR, matrix-association region; pPW236B of locus D21S11, pPW245D of locus D21S8, and LINE, long interspersed repetitive element. VPresent address: Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI 48109. The publication costs of this article were defrayed in part by page charge I1To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" **The sequence reported in this paper is being deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. EMBL/GenBank data base (accession no. J04504). 1914 Downloaded by guest on September 25, 2021 Genetics: Wong et al. Proc. Natl. Acad. Sci. USA 86 (1989) 1915 of the child and his parents was analyzed. Probe pPW231C 17 nt reat A Brealokit detected new DNA fragments in the child's DNA that were t MAR homolg - UNE pat absent in the DNA of the parents with several restriction I TOPO II homology enzymes. Analysis with EcoRI, for example, showed a normal 2.1-kb fragment in all family members and a new 7.5-kb fragment in DNA isolated from the child's blood and fibroblasts, indicating that a chromosomal break occurred X21lpq i ,szc (D2lSll1) within the 2.1-kb EcoRI fragment pPW231C (29). 1m l ,I1 A. 5' 1.6 . P 3' The 7.5-kb EcoRI junction fragment was cloned (A21BP) proximal long arm from blood lymphocytes of the child and the corresponding CW21 B normal region of DNA brought into juxtaposition to pPW231C was also cloned as a 3.0-kb HindIII fragment from II a chromosome 21 library. This latterfragment is A2lpq ofFig. 72 1A and has been designated locus D21S111. The restriction X21BP I maps of the cloned 1 indicated that 5'. 3 fragments (Fig. A-C) 5. g,,,,"n,,,,,,,JJ>,,,z,,,,,,,,,,,,XW,,,%XiR,2222i~yMM0X~s 3 breakage occurred in A21pq and pPW231C and that fusion tol Z, f junction fragment was within A21BP. DNA fragments on both sides of the i CW21 A junction were mapped to chromosome 21 by using somatic cell hybrids containing chromosome 21 (72532X-6) as the only human chromosome, thereby excluding involvement of A , Tr CT chromosomes other than 21 in ring formation. D21S3 Nucleotide of C. 5' I Sequence Comparisons pPW231C, A21BP, Ito distal long arm and A21pq. DNA sequences of the 2.1-kb pPW231C frag- pPW23I C ment, the 2.7-kb from the 5' end of A21BP, and the 1.5-kb from the 5' half of A21pq were determined (Fig. 3). The FIG. 1 . Identification and cloning the ringjunction fragment and sequences of A21BP and pPW231C 5' to nt 623 are identical surrounding normal DNA. The junction fragment A21BP (B) as well except for a nt substitution at position 574, a probable neutral as 3.5 kb from the surrounding region A21pq (A) were cloned. The sequence polymorphism. No differences were noted between DNA fragment pPW231C is also shown (C). Restriction maps ofthe A21BP and A21pq in the 1.5-kb region sequenced 3' to the three cloned regions are aligned by the vertical dotted line repre- senting the reunion site. The 5' and 3' orientations ofthe clones were junction point. arbitrarily designated. Regions of identity are indicated by the At the ring junction, 9 nt, CATTCACCA, of unknown hatched or open bars. Probes CW21B and CW21A are underlined in origin were inserted (Fig. 3). Both break points occurred A and B, respectively. Symbols identify DNA sequence determinants within single-copy sequences; pPW231C is single-copy and shown in Fig. 3. the first 1 kb at the 5' end of A21pq is also free of repetitive sequences. Two single-copy probes flanking the ringjunction the corresponding homologue. Dicentric rings approximately (CW21B, just 5' to the break point, and CW21A, on the 3' twice the size of the normal chromosome 21 were 12% ofthe side) represent contiguous fragments in normal DNA, yet r(21), and the remaining 4% of the rings appeared equivalent they were derived from two separate clones, A21pq and in size to the corresponding homologue.