A mouse-human hybrid cell panel for mapping human chromosome 16

A mouse-human hybrid cell panel for human chromosome 16 was constructed from human cell lines with breakpoints on chromosome 16 at p13.11, q13, q22 and q24. Fusions with the human fibroblast line GM3884, t(X;16)(q26;q24) allowed the isolation of clones with either the derivative X or the derivative 16 as the only human chromosome. This was a consequence of both the genes APRT and HPRT being involved in the translocation. The breakpoints of the line GM3884 were confirmed by aphidicolin induction of the common fragile site at 16q23. The results of the fusions with this line suggest a localisation of the APRT gene at 16q24 and confirm the localisation of HPRT to Xq26 to Xq27.3. These hybrid cell lines enable the localisation of genes and DNA fragments to six clearly defined regions. Further localisation within three of these regions is possible by use of the three fragile sites on chromosome 16. In situ hybridisation with the probe pBLUR confirmed that of three lines tested all contained a single human chromosome.     

A detailed analysis of chromosomal changes in heritable and non-heritable retinoblastoma

Summary Full cytogenetic analysis of 27 different retinoblastoma tumors is presented. Gross aneuploidy of chromosome arms 6p and 1q were very common, being observed in 15/27 and 21/27 tumors, respectively. However, we found that chromosome 13 was rarely missing: only 3/27 had a detectable monosomy affecting 13q14. Monosomy of chromosome 13 by small deletion or rearrangement was also not observed in any of 12 retinoblastoma tumor lines analyzed detail at the 300–400 chromosome band level. A novel observation in retinoblastoma was the discovery of non-random translocations at three specific breakpoints, 14q32 (4/12), 17p12 (5/12), and 10q25 (3/12). Genomic rearrangements similar to those described involving C-myc in Burkitt lymphoma 14q+ cells could not be demonstrated in the four 14q+ retinoblastoma lines using molecular techniques, and a probe mapping to the site implicated to have an activating role in lymphoma. These data suggest that there is a target for rearrangement at 14q32 but it is not the same sequence used in some Burkitt lymphomas. Two other breakpoints (2p24 and 8q24) coincided with the mapped position of cellular oncogenes, but also failed to show a molecular rearrangement with the oncogene probes. The breakpoints, 10q25 and 17p12, are constitutional fragile sites which may predispose these regions to act as acceptors of translocations in malignant cells. One line had double minute chromosomes, and was the only one of 16 (6%) tested with the N-myc probe which had an amplification. Different tumors from single patients with multifocal heritable retinoblastoma showed independent karyotype evolution. Unilateral non-heritable tumors exhibited a high level of karyotype stability throughout both in vivo and in vitro growth. The various common patterns of aneuploidy and translocations probably confer an early selective advantage to malignant cells, rather than induce malignant transformation.     

Physical mapping studies on the human X chromosome in the region Xq27-Xqter

We have characterized three terminal deletions of the long arm of the X chromosome. Southern analysis using Xq27/q28 probes suggests that two of the deletions have breakpoints near the fragile site at Xq27.3. Flow karyotype analysis provides an estimate of 12 X 10(6) bp for the size of the deleted region. We have not detected the deletion breakpoints by pulsed-field gel electrophoresis (PFGE) using the closet DNA probes, proximal to the fragile site. The physical distance between the breakpoints and the probes may therefore be several hundred kilobases. The use of the deletion patients has allowed a preliminary physical map of Xq27/28 to be constructed. Our data suggest that the closest probes to the fragile site on the proximal side are 4D-8 (DXS98), cX55.7 (DXS105), and cX33.2 (DXS152). PFGE studies provide evidence for the physical linkage of 4D-8, cX55.7, and cX33.2. We have also found evidence for the physical linkage of F8C, G6PD, and 767 (DXS115), distal to the fragile site.     

Mapping of the versican proteoglycan gene (CSPG2) to the long arm of human chromosome 5 (5q12-5q14).

Versican is a major chondroitin sulfate proteoglycan of vascularized connective tissues whose eponym reflects its functional versatility in macromolecular affinity and interactions. In this report we have localized the versican gene (CSPG2) to the long arm of human chromosome 5 by utilizing a combination of somatic cell hybrids, Southern blotting, polymerase chain reaction, and chromosomal in situ hybridization. The proteoglycan gene segregated concordantly with hybrid cell lines containing the long arm of chromosome 5, comprising the 5q12-q14 band regions. To refine this locus further, we screened a chromosome 5-specific library and isolated several genomic clones encoding a portion of the 5' end of versican. One of these genomic clones was used as a probe for in situ hybridization of human chromosome metaphases. The results corroborated the data obtained using somatic cell hybrids and further refined the assignment of the versican gene to the narrow band region of 5q12-5q14, with the primary site likely to be 5q13.2. The availability of novel genomic clones and the mapping data presented here will make possible the identification of any defect genetically linked to this proteoglycan gene.     

Localization of the rhabdomyosarcoma t(2;13) breakpoint on a physical map of chromosome 13.

Previous investigations of the pediatric soft tissue tumor alveolar rhabdomyosarcoma have identified a characteristic translocation t(2;13)(q35;q14). We have employed a physical mapping strategy to localize the site of this translocation breakpoint on chromosome 13. Using a panel of somatic cell hybrid and lymphoblast cell lines with deletions and unbalanced translocations involving chromosome 13, we have mapped numerous probes from the 13q12-q14 region and demonstrate that this region is divisible into five physical intervals. These probes were then mapped with respect to the t(2;13) rhabdomyosarcoma breakpoint by quantitative Southern blot analysis of an alveolar rhabdomyosarcoma cell line with two copies of the derivative chromosome 13 and one copy of the derivative chromosome 2. Our findings demonstrate that the t(2;13) breakpoint is localized within a map interval delimited by the proximal deletion breakpoints in lymphoblast lines GM01484 and GM07312. Furthermore, the breakpoint is most closely flanked by loci D13S29 and TUBBP2 within this map interval. These findings will facilitate chromosomal walking strategies for cloning the regions disrupted by the alveolar rhabdomyosarcoma translocation. In addition, this physical map will permit rapid determination of the proximity of new cloned sequences to the translocation breakpoint.     

Physical mapping of new DNA probes near the fragile X mutation (FRAXA) by using a panel of cell lines

The fragile X syndrome is a very common disorder, but there has been little progress toward isolating the fragile X mutation (FRAXA). We describe a panel of 14 somatic cell hybrid lines, lymphoblastoid cell lines, and peripheral lymphocytes with X-chromosome translocation or deletion breakpoints near FRAXA. The locations of the breakpoints were defined with 16 established probes between pX45d (DXS100) and St14-1 (DXS52). Seven of the cell lines had breakpoints between the probes RN1 (DXS369) and U6.2 (DXS304), which flank FRAXA at distances of 3-5 centimorgans. The panel of cell lines was used to localize 16 new DNA probes in this region. Six of the probes-VK16, VK18, VK23, VK24, VK37, and VK47--detected loci near FRAXA, and it was possible to order both the X-chromosome breakpoints and the probes in relation to FRAXA. The order of probes and loci near FRAXA is cen-RN1,VK24-VK47-VK23-VK16,FRAXA-++ +VK21A-VK18-IDS-VK37-U6.2-qter. The breakpoints near FRAXA are sufficiently close together that probes localized with this panel can be linked on a large-scale restriction map by pulsed-field gel electrophoresis. This panel of cell lines will be valuable in rapidly localizing other probes near FRAXA.     

Fine mapping of gene probes and anonymous DNA fragments to the long arm of chromosome 16

The fragile site, FRA16B, at 16q22.100 and four different translocations with breakpoints at 16q22.102, 16q22.105, 16q22.108, and 16q22.3 were used to locate and order DNA probes. This was achieved by Southern analysis of a somatic cell hybrid panel containing portions of chromosome 16 and by in situ hybridization. The anonymous DNA fragments D16S6, D16S10, and D16S11 were proximal to FRA16B and located at 16q13----q22.100. D16S4 and LCAT were located at 16q22.100----q22.102. TAT and HP were located at 16q22.105----q22.108. CTRB was located distal to 16q22.105 and therefore is in the distal half of 16q22. The order of markers in this region was determined as centromere-D16S6, D16S11, D16S10, MT-FRA16B-D16S4, LCAT-HP,TAT,CTRB-APRT- telomere. Linkage studies to determine map distances between the closest markers flanking the fragile site are now in progress.     

A new unbalanced chromosomal abnormality in 1q31.1 to 1q32 without phenotypic consequences

A familial duplication in the long arm of one chromosome 1 was detected due to recurrent abortions in a couple. The duplication was present in the male partner of the couple and in his mother, both clinically healthy. By reverse FISH, the duplication was determined to be located in 1q31. Multicolor banding (MCB) and application of locus-specific probes narrowed down the breakpoints to 1q31.1 and 1q32. The duplication spans a region of 13.9 Mb. None of the two breakpoints was colocalized with a known fragile site in 1q31.2, which, however, was duplicated. To the best of our knowledge, this is the first report of an unbalanced chromosome abnormality in this region that is not correlated with any clinical consequences.     

An expanded mouse-human hybrid cell panel for mapping human chromosome 16

A mouse/human hybrid cell panel of human chromosome 16 has been extended to a total of 31 hybrids. These hybrids were derived from constitutional translocations and deletions ascertained during clinical cytogenetic studies. This panel of hybrids, together with four fragile sites, have the potential to divide chromosome 16 into 38 regions. Rapid detailed physical mapping of gene probes or anonymous DNA probes is possible using this hybrid panel. This hybrid cell panel also allows the physical mapping of other chromosomes with three breakpoints on chromosomes 1, 4, 11 and 13 and two on chromosomes 3, 10 and 18.     

Regional localization of a trophoblast antigen-related sequence and 16 other sequences to human chromosomes 6q using somatic cell hybrids.

Using a panel of 13 hybrid cell lines, we have regionally localized 22 markers to the long arm of chromosome 6. Revised or new locations are provided for 17 of the markers, and preliminary assignments to chromosome 6 of 11 loci are confirmed. The location of NT5, previously determined by antigen expression in hybrids, has been confirmed at 6q14-q15 by using a cDNA probe. Other DNA probes include one new anonymous sequence, designated D6S130, that maps to 6q12 and 4 VNTR probes that map to the proterminal band, 6q27. Probe CRI-L1065 also maps to 6q21, CRI-994 maps to 6q21-qter, and CRI-L322 maps to 6q14-15, information that may assist the merging of physical and genetic maps.     

Regional mapping panel for human chromosome 17: Application to neurofibromatosis type 1

A somatic cell hybrid mapping panel was constructed to localize cloned DNA sequences to any of 15 potentially different regions of human chromosome 17. Relatively high-resolution mapping is possible for 50% of the chromosome length in which 12 breakpoints are distributed over approximately 45 megabases, with an average spacing estimated at 1 breakpoint every 2-7 megabases. This high-resolution capability includes the pericentromeric region of 17 to which von Recklinghausen neurofibromatosis (NF1) has recently been mapped. Using 20 cloned genes and anonymous probes, we have tested the expected order and location of panel breakpoints and confirmed, refined, or corrected the regional assignment of several cloned genes and anonymous probes. Four markers with varying degrees of linkage to NF1 have been physically localized and ordered by the panel: the loosely linked markers myosin heavy chain 2 (25 cM) to p12----13.105 and nerve growth factor receptor (14 cM) to q21.1----q23; the more closely linked pABL10-41 (D17S71, 5 cM) to p11.2; and the tightly linked pHHH202 (D17S33) to q11.2-q12. Thus, physical mapping of linked markers confirms a pericentromeric location of NF1 and, along with other data, suggests the most likely localization is proximal 17q.     

Fragile site long arm chromosome 16

Summary A fragile site at the long arms (q21) of chromosome 16 was found in two persons, each of whom became the parent of a child with a de novo structural chromosome abnormality—a balanced autosomal translocation and an autosomal deletion. The question of an increased risk of structural chromosome abnormalities in the offspring of persons with fragile site long arm 16 is discussed.     

Analysis of human chromosome 21: correlation of physical and cytogenetic maps; gene and CpG island distributions.

Human chromosome 21 has been analyzed by pulsed-field gel electrophoresis using somatic cell hybrids containing limited regions of the chromosome and greater than 60 unique sequence probes. Thirty-three independent NotI fragments have been identified, totalling 43 million bp. This must account for essentially the entire long arm, and therefore gaps remaining in the map must be small. The extent of the pulsed-field map has allowed the direct correlation of the physical map with the cytogenetic map: translocation breakpoints can be unambiguously positioned along the long arm and the distances between them measured in base pairs. Three breakpoints have been identified, providing physical confirmation of cytogenetic landmarks. Information on sequence organization has been obtained: (i) 60% of the unique sequence probes are located within 11 physical linkage groups which can be contained in only 20% of the long arm; (ii) 9/21 genes are clustered within 4%; (iii) translocation breakpoints appear to occur within CpG island regions, making their identification difficult by pulsed-field techniques. This analysis contributes to the human genome mapping effort, and provides information to guide the rapid investigation of the biology of chromosome 21.     

Molecular analysis of the 18q- syndrome--and correlation with phenotype.

Seven individuals with deletions of the distal long arm of chromosome 18 were evaluated at the clinical, cytogenetic, and molecular levels. The patients had varying degrees of typical clinical findings associated with the 18q- syndrome. Cytogenetic analysis revealed deletions from 18q21.3 or 18q22.2 to qter. Somatic cell hybrids derived from the patients were molecularly characterized using ordered groups of probes isolated from a chromosome 18-specific library. In general, the size of the deletion could be correlated with the severity of the phenotype. Based on the clinical pictures of these seven patients, a preliminary phenotypic map for the clinical features associated with deletions of the distal portion of the long arm has been generated. Furthermore, genes previously localized to 18q21 were mapped relative to the chromosome breakpoints present in these patients.     

Unbalanced 1q whole-arm translocation resulting in der(14)t(1;14)(q11-12;p11) in myelodysplastic syndrome

Unbalanced whole-arm translocations (WATs) of the long arm of chromosome 1, resulting in complete trisomy 1q, are chromosomal abnormalities detectable in both solid tumors and hematologic neoplasms. Among the WATs of 1q to acrocentric chromosomes, a few patients with der(1;15) described as a dicentric chromosome have been reported so far, whereas cases of der(1;14) are much rarer. We report on a case of der(1;14) detected as single anomaly in a patient with myelodysplastic syndrome. The aim of our work was to investigate the breakpoints of the (1;14) translocation leading to the der(1;14). Fluorescence in situ hybridization (FISH) experiments have been performed on chromosome preparations from bone marrow aspirate, using specific centromeric probes of both chromosomes, as well as a probe mapping to 1q11 band. FISH results showed that in our patient the derivative chromosome was monocentric with a unique centromere derived from chromosome 14. The breakpoints of the translocation were located in the short arm of chromosome 14 and in the long arm of chromosome 1, between the alphoid D1Z5 and the satellite II domains. The 1q breakpoint was within the pericentromeric region of chromosome 1, which is notoriously an unstable chromosomal region, involved in different chromosomal rearrangements.     

Localization of HeLa cell tumor-suppressor gene to the long arm of chromosome II.

Cytogenetic and molecular genetic analyses of human intraspecific HeLa x fibroblast hybrids have provided evidence for the presence of a tumor-suppressor gene(s) on chromosome 11 of normal cells. In the present study, we have carried out extensive RFLP analysis of various nontumorigenic and tumorigenic hybrids with at least 50 different chromosome 11-specific probes to determine the precise location of this tumor-suppressor gene(s). Two different hybrid systems, (1) microcell hybrids derived by the transfer of a normal chromosome 11 into a tumorigenic HeLa-derived hybrid cell and (2) somatic cell hybrids derived by the fusion of the HeLa (D98OR) cells to a retinoblastoma (Y79) cell line, were particularly informative. The analysis showed that all but one of the nontumorigenic hybrid cell lines contained a complete copy of the normal chromosome 11. This variant hybrid contained a segment of the long arm but had lost the entire short arm of the chromosome. The tumorigenic microcell and somatic cell hybrids had retained the short arm of the chromosome but had lost at least the q13-23 region of the chromosome. Thus, these results showed a perfect correlation between the presence of the long arm of chromosome 11 and the suppression of the tumorigenic phenotype. We conclude therefore that the gene(s) involved in the suppression of the HeLa cell tumors is localized to the long arm (q arm) of chromosome 11.     

A somatic cell hybrid panel and DNA probes for physical mapping of human chromosome 7p.

To identify by reverse genetics genes on the short arm of human chromosome 7 expected to be involved in the regulation of human craniofacial and limb development, we have set up a human mouse somatic cell hybrid panel that divides 7p into 9 fragments. The breakpoints are defined by deletions or translocations involving one chromosome 7 in the cells of the human cell fusion partners. Particularly densely covered with these cytogenetic anchor points is the proximal area of 7p within and around 7p13. The number of cytogenetic mapping points within proximal 7p could be increased by four, using two diploid human cell lines with small interstitial deletions in this region for dosage studies. We used Southern blots of this panel to assign to 7q or subregions of 7p more than 300 arbitrary DNA probes or genes that provide reference points for physical mapping of 7p. Three reciprocal translocations with one of the breakpoints in 7p13 mark the location of a gene involved in Greig cephalopolysyndactyly syndrome. To define an area in which we could identify candidates for this developmental gene, we established a macrorestriction map using probes flanking the putative gene region. The Greig translocations were found to be located within a 630-kb NotI restriction fragment.     

Characterization of three de novo derivative chromosomes 16 by “Reverse chromosome painting” and molecular analysis

We have analyzed three de novo chromosome 16 rearrangements—two with a 16p+ chromosome and one a 16q+—none of which could be fully characterized by conventional cytogenetics. In each case, flow karyotypes have been produced, and the aberrant chromosome has been isolated by flow sorting. The origin of the additional material has been ascertained by amplifying and labeling the DNA of the abnormal chromosome by degenerate-oligonucleotide-primer–PCR and hybridizing it in situ to normal metaphase spreads (reverse chromosome painting). Both 16p+ chromosomes contain more than 30 Mb of DNA from the short arm of chromosome 9 (9p21.2-pter), while the 16q+ contains approximately 9 Mb of DNA from 2q37. The breakpoints on chromosome 16 have been localized in each case; the two breakpoints on the short arm are at different points within the terminal band, 16p13.3. The breakpoint on the long arm of chromosome 16 is very close to (within 230 kb of) the 16q telomere. Determination of the regions of monosomy and trisomy allowed the observed phenotypes to be compared with other reported cases involving aneuploidy for these regions.     

Fine structure DNA mapping studies of the chromosomal region harboring the genetic defect in neurofibromatosis type 1

To better map the location of the von Recklinghausen neurofibromatosis (NF1) gene, we have characterized a somatic cell hybrid designated 7AE-11. This microcell-mediated, chromosome-transfer construct harbors a centromeric segment and a neo-marked segment from the distal long arm of human chromosome 17. We have identified 269 cosmid clones with human sequences from a 7AE-11 library and, using a panel of somatic cell hybrids with a total of six chromosome 17q breakpoints, have mapped 240 of these clones on chromosome 17q. The panel included a hybrid (NF13) carrying a der(22) chromosome that was isolated from an NF1 patient with a balanced translocation, t(17;22) (q11.2;q11.2). Fifty-three of the cosmids map into a region spanning the NF13 breakpoint, as defined by the two closest flanking breakpoints (17q11.2 and 17q11.2-q12). RFLP clones from a subset of these cosmids have been mapped by linkage analysis in normal reference families, to localize the NF1 gene more precisely and to enhance the potential for genetic diagnosis of this disorder. The cosmids in the NF1 region will be an important resource for testing DNA blots of large-fragment restriction-enzyme digests from NF1 patient cell lines, to detect rearrangements in patients' DNA and to identify the 17;22 NF1 translocation breakpoint.     

Different breakage-prone regions on chromosome 1 detected in t(11;14)-positive mantle cell lymphoma cell lines and multiple myeloma cell lines are associated with different tumor progression-related mechanisms

To better define secondary aberrations that occur in addition to translocation t(11;14)(q13;q32) in mantle cell lymphomas (MCL) and in multiple myelomas (MM), seven t(11;14)-positive MCL cell lines and four t(11;14)-positive MM cell lines were analysed by fluorescence R-banding and spectral karyotyping (SKY). Compared with published data obtained by G-banding, most chromosome aberrations were redefined or further specified. Furthermore, several additional chromosome aberrations were identified. Thus, these cytogenetically well defined t(11;14)-positive MCL and MM cell lines may be useful tools for the identification and characterization of genes that might be involved in the pathogenesis of MCL and MM, respectively. Since MCL and MM were found to have different alterations of chromosome 1, these were investigated in more detail by fluorescence in situ hybridization (FISH) and multicolor banding (MCB) analyses. The most frequently altered and deletion-prone loci in MCL cell lines were regions 1p31 and 1p21. In contrast, breakpoints in MM cell lines most often involved the heterochromatic regions 1p12-->p11, and the subcentromeric regions 1q12 and 1q21. These data are in accordance with previously published data of primary lymphomas. Our findings may indicate that different pathways of clonal evolution are involved in these morphologically distinct lymphomas harboring an identical primary chromosome aberration, t(11;14).