Anonymous Markers Research Articles

Genetic linkage mapping of 11 novel DNA markers and the ceruloplasmin (Cp) gene on rat chromosome 2.

We have mapped 11 novel, anonymous genetic markers to rat chromosome 2. The rat ceruloplasmin gene (Cp) had been previously mapped to chromosomes 2 and 7q11-->q13 by two different methods. To resolve the assignment and to localize the Cp gene on the rat genetic linkage map, we used linkage analysis to confirm that rat Cp lies on chromosome 2.

Location on the Human Genetic Linkage Map of 26 Genes Involved in Blood Coagulation

Several human genetic linkage maps have been constructed as part of the Human Genome Project. These maps show the positional order of closely linked, highly informative AC-repeat polymorphisms on each human chromosome, and are extremely useful in genetic linkage analysis of inheritable diseases. For a candidate gene approach the current linkage maps are less useful, since they consist mainly of anonymous markers rather than of specific genes. This situation also applies for inheritable disorders of blood coagulation. Numerous genes are involved in the blood coagulation cascade and its regulation, and can be considered as candidate genes for unexplained haemophilia and thrombophilia. We have selected 29 candidate genes that seem to be the ones most likely to be involved in thrombophilia. For 19 genes genotype data were already present in the CEPH database (version 7.0). We typed 7 additional genes in the CEPH reference families, i.e. the factor V, factor XII, protein C, protein S, prothrombin, thrombomodulin, and heparin cofactor II gene. The genotype data were used to integrate these 26 genes in the current genetic linkage map, and to identify closely linked AC-repeat polymorphisms. This information will benefit the investigation of inheritable disorders of blood coagulation, especially thrombophilia.

Translocations and Deletions of 5ql3.1 in Myelodysplasia and Acute Myelogenous Leukemia: Evidence for a Novel Critical Locus

Acquired partial and complete deletions of chromosome 5 (5q-, -5) are common cytogenetic anomalies associated with myelodysplasia (MDS) and acute myeloid leukemia (AML). A critical region of consistent loss at 5q31.1 (in > 90% of cases) has led us and others to postulate the presence of a key negative regulator(s) of leukemogenesis. Although the interstitial deletion limits vary among patients, del(5) (q13q33) and del(5)(q13q35) constitute major subsets. Furthermore, it is not rare to encounter deletions, translocations, or paracentric inversions involving 5q11 to 5q13, which indicates inactivation or disruption of important gene(s) at that locus. In this report, we have localized a novel locus at 5q13.1 to a 2.0-Mb interval between the anonymous markers D5S672 and GATA-P1804. This locus resided within the region of loss in 12 of 27 patients with anomalies of chromosome 5; one of these cases had apparent retention of both alleles of all the telomeric loci. Fluorescence in situ hybridization (FISH) studies demonstrate that the AML cell line ML3 is disrupted at 5q13.1 by a translocation involving chromosome 3, with apparent retention of the entire chromosome 5 sequence. Our results suggest that this novel proximal locus encodes a critical gene that may be deleted or disrupted in a subset of MDS/AML patients with chromosome 5 anomalies.

NHLBI Family Heart Study: objectives and design.

The NHLBI Family Heart Study is a multicenter, population-based study of genetic and nongenetic determinants of coronary heart disease (CHD), atherosclerosis, and cardiovascular risk factors. In phase I, 2,000 randomly selected participants and 2,000 with family histories of CHD were identified among 14,592 middle-aged participants in epidemiologic studies. Medical histories from these individuals, their parents, and their siblings were used to calculate family risk scores that compared the number of reported and validated CHD events with the number expected based on the size, sex, and age of family members. A total of 657 families with the highest risk scores and early-onset CHD and 588 randomly sampled families had clinic examinations that included electrocardiograms, carotid artery ultrasound scans, spirometry, measurements of body size, blood pressure, lipids, lipoproteins, hemostatic factors, insulin, glucose, and routine chemistries. Additional biochemical and genetic studies are being performed on selected participants. Serum, plasma, lymphocytes, red cells, and DNA are stored for future studies, including genotyping of candidate genes and anonymous markers. Contributions of genes, shared and individual environments, and behaviors to variations in risk factors, preclinical atherosclerosis, and CHD will be estimated. Linkage studies, including the quantitative trait loci approach, are planned.

TheSp4HDeletion May Contain a New Locus Essential for Postimplantation Development

Sp4His a semi-dominant mutation that maps to mouse chromosome 1. Heterozygous mice exhibit white spotting of the belly, whereas the fate of the homozygous embryos is unknown. We have previously shown that the entire coding region of thePax3gene is deleted in theSp4Hmutant. In this study, we have analyzed the fate of theSp4Hhomozygous embryos. NoSp4Hhomozygotes were detected by Southern blot or PCR analysis in 82 E9–E13-day embryos. We have also documented a significant increase in the number of resorption sites inSp4Hheterozygous matings compared to control litters. Sections of the resorption sites (moles) suggest that postimplantation development is arrested prior to gastrulation. We have mapped the extent of the deletion to a maximum of 1.53 ± 0.6 cM using markers flanking thePax3locus. Four anonymous markers,D1Mit215, D1Mit253, D1Mit332,andD1McG156,have been shown to be deleted in theSp4Hmutation. Further nondeleted markers have been used to extend the linkage map of this region. A total of 22 loci were analyzed in aSplotchintraspecific backcross. Using these data and deletion mapping data, we predict the following order of markers: (D1Mit46, Vil)–(D1Mit79)–(D1Mit132, D1McG153)–(D1Mit332)–(D1-McG156, D1Mit253, D1Mit215, Pax3)–(D1Mit134)–(D1Mit8, D1Mit9, D1Mit44, D1Ler3, D1Mit183)–(D1Mit53, D1Mit82, D1Mit182)–(Bcl2). As the deletion is large enough to include other genes, and it seems that deletion ofPax3is not likely to account for the early death of the embryos, we suggest that another developmentally important gene may be deleted in theSp4Hmouse mutant and that this may be responsible for the early death of the homozygous mutant embryos.

A High-Resolution Whole Genome Radiation Hybrid Map of Human Chromosome 17q22–q25.3 across the Genes forGHandTK

We have constructed a whole genome radiation hybrid (WG-RH) map across a region of human chromosome 17q, from growth hormone (GH) to thymidine kinase (TK). A panel of 128 WG-RH hybrid cell lines generated by X-irradiation and fusion has been tested for the retention of 39 sequence-tagged site (STS) markers by the polymerase chain reaction. This genome mapping technique has allowed the integration of existing VNTR and microsatellite markers with additional new markers and existing STS markers previously mapped to this region by other means. The WG-RH map includes eight expressed sequence tag (EST) and three anonymous markers developed for this study, together with 23 anonymous microsatellites and five existing ESTs. Analysis of these data resulted in a high-density comprehensive map across this region of the genome. A subset of these markers has been used to produce a framework map consisting of 20 loci ordered with odds greater than 1000:1. The markers are of sufficient density to build a YAC contig across this region based on marker content. We have developed sequence tags for both ends of a 2.1-Mb YAC and mapped these using the WG-RH panel, allowing a direct comparison of cRay6000to physical distance.

Framework for a Physical Map of the Human 22q13 Region Using Bacterial Artificial Chromosomes (BACs)

Detailed physical maps of entire chromosomes based on combined genetic, cytogenetic, and structural information are essential components for positional cloning and genomic sequencing. Despite the wealth of genetic information of the known diseases in the chromosome 22q13, the construction of a detailed physical map of the terminal region is difficult due to the sparsity of the genetic markers. We present here a map of bacterial artificial chromosome (BAC) contigs that cover a number of genetic loci in the 22q13 region. One hundred thirty-six BACs with an average insert size of 140 kb are assembled into 35 contigs defined by 64 markers in 22q13–qter. Twenty-three anonymous markers are now linked to the previously mapped genetic anchor points.

Molecular characterization of the 7q deletion in myeloid disorders.

Deletion of the long arm of chromosome 7 is a common karyotypic finding in myeloid disorders and in particular is found in association with secondary leukaemias. We have used restriction fragment length polymorphisms and gene dosage experiments to assess the loss or retention of sequences localized to chromosome 7q in five patients with clonal myeloid disorders and a 7q deletion. The deletion was interstitial in all cases with retention of the anonymous marker pS194 located at 7q36-qter. Three out of five cases also retained the more proximal gene T-cell receptor beta (TCR beta) located at 7q35. The proximal breakpoints of all five cases were localized to 7q22 by cytogenetic analysis. In two cases the proximal breakpoint lay between the genes for elastin (ELN) and collagen type 1 alpha (COL1A2) and in three cases distal to this region between the genes for erythropoietin (EPO) and acetylcholinesterase (ACHE). The genes of ACHE, plasminogen activator inhibitor 1 (PLANH1), CCAAT displacement protein (CUTL1) and Met proto-oncogene (MET) were deleted in all cases. Molecular analysis of the 7q deletion in myeloid leukaemias demonstrates heterogeneity of the breakpoints, supporting a recessive mechanism of tumourigenesis.

Fibulin-2: genetic mapping and exclusion as a candidate gene in Marfan syndrome type 2.

Fibulin-2 (FBLN2) is a new extracellular matrix protein that has been considered a candidate gene for Marfan syndrome type 2 (locus MFS2) based on chromosomal colocation at 3p24.2-p25 and disease phenotype. In the absence of polymorphic markers reported for FBLN2, direct sequencing of the gene was performed and two intragenic polymorphisms were identified. Linkage was excluded between FBLN2 and the MFS2 gene. Furthermore, two-point lod scores were generated between these markers and anonymous markers arrayed on the genetic map of 3p and closely linked to MFS2. These analyses placed FBLN2 at marker D3S1585.

A 3.1-Mb YAC contig within the Werner syndrome region, on the short arm of human chromosome 8

The locus for Werner syndrome (WRN) has been localized to human chromosome 8p21 --> p12, close to the anonymous marker D8S339. A 3.1-Mb contig of yeast artificial chromosomes (YACs) was assembled around D8S339. Results from analyses of somatic cell hybrids, FISH, and physical mapping suggest the following loci order: tel-NEFL-D8S131-D8S339-[D8S540/GSR]-D8S124 -D8S259-D8S87-FGFR1-cen. Close physical linkage between D8S540 and GSR was established within a DNA fragment of 200 kb. These two loci are not more than 400 kb from D8S339. In addition, D8S339, D8S540, D8S124, and GSR are within 1.1 Mb. These data establish a primary physical map of the Werner syndrome region and identify useful YAC clones for the isolation of new markers and of the corresponding gene.

A Precise Meiotic Map in the Class I Region of the Human Major Histocompatibility Complex

Human families in which recombinant meiotic event(s) are known to have occurred are powerful tools with which to analyze more precisely the structures of defined genomic regions, especially unstable areas. Such families allow the determination of the haplotypes of each member and, taking into account the recombinant event, it is possible to localize very precisely the point of crossover. Using families in which crossovers between the genes HLA-A and -B have occurred, we have constructed a meiotic map localizing the meiotic breakpoint events with respect to both anonymous markers and the principal genes of the region. Such mapping, which depends on the direct analysis of genomic DNA, is essential for fine structural analysis and is a powerful means of verification of the order and the localization of markers: physical mapping alone, using yeast artificial chromosomes, presents some uncertainties due to the numerous chimeras and inversions that can be produced. The establishment of this map will allow us to determine efficiently the precise location for new markers already localized to the map region. Three microsatellites (D6S265, D6S276, and D6S306), localized in the HLA region by linkage analysis, have been precisely located with respect to the points of recombination in the class I region. The sites of meiotic recombination in the MHC class I region seem to be not randomly distributed but in the majority of cases occurred between HLA-C and the microsatellite D6S265. This study also shows two cases of abnormal segregation of alleles. We discuss how these mutations correspond to a spontaneous mutation event at the somatic or germinal level.

Identification, characterization, and localization to chromosome 17q21-22 of the human TBX2 homolog, member of a conserved developmental gene family.

The T-box motif is present in a family of genes whose structural features and expression patterns support their involvement in developmental gene regulation. Previously, sequence comparisons among the T-box domains of ten vertebrate and invertebrate T-box (Tbx) genes established a phylogenetic tree with three major branches. The Tbx2-related branch includes mouse Mm-Tbx2 and Mm-Tbx3, Drosophila optomotor-blind (Dm-Omb), and Caenorhabditis elegans Ce-Tbx2 and Ce-Tbx2 and Ce-Tbx7 genes. From the localization of Mm-Tbx2 to Chromosome (Chr) 11, we focused our search for the human homolog, Hs-TBX2, within a region of synteny conservation on Chr 17q. We used Dm-Omb polymerase chain reaction (PCR) primers to amplify a 137-basepair(bp)_ product from human genomic, Chr 17 monochromosome hybrid, and fetal kidney cDNA templates. The human PCR product showed 89% DNA sequence identity and 100% peptide sequence identity to the corresponding T-box segment of Mm-Tbx2. The putative Hs-TBX2 locus was isolated within a YAC contig that included three anonymous markers, D17S792, and D17S948, located at Chr 17q21-22. Hybridization-and PCR-based screening of a 15-week fetal kidney cDNA library yielded several TBX2 clones. Sequence analysis of clone lambda omicron TBX2-1 confirmed homology to Mm-TBx2-90% DNA sequence identity over 283 nt, and 96% peptide sequence identity over 94 amino acids. Similar analysis of Hs-TBX2 cosmid 154F11 confirmed the cDNA coding sequence and also identified a 1.7-kb intron located at the same relative position as in Mm-Tbx2. Phylogenetic analyses of the T-box domain sequences found in several vertebrate and invertebrate species further suggested that the putative human TBX2 and mouse Tbx2 are true homologs. Northern blot analysis identified two major TBX2 expression fetal kidney and lung; and in adult kidney, lung, ovary, prostate, spleen, and testis. Reduced expression levels were seen in heart, white blood cells, small intestine, and thymus. These results suggest that Hs-TBX2 could play important roles in both developmental and postnatal gene regulation.

YAC analysis and minimal tiling path construction for chromosome 21q.

We have undertaken a detailed analysis of several hundred YACs from widely available YAC libraries which map to human chromosome 21 with the goal of improving the physical map of chromosome 21 and determining the feasibility of producing a minimal tiling path of well characterized, stable, non-chimeric YACs spanning the long arm of the chromosome (21q). We report information on over 500 YACs known to contain STS from 21q including information on size, stability, chimerism, marker content, and NotI restriction sites. YACs derive from the CEPH and St. Louis YAC libraries, and STSs include the set of 198 markers originally used do assemble a YAC contig of 21q, as well as additional anonymous probes and gene markers. This information has assisted in refinements of STS order, has defined a region of general instability in 2lq22.3, has identified an increased number of NotI restriction sites, and has defined cryptic gaps, particularly in 2lq2l, for which few or no markers are available. These results have allowed us to develop and assess a minimal tiling path of overlapping YACs consisting of 59 YACs (and two PI clones), largely non chimeric, stable, and of verified STS content. They total 30 mb of non-overlapping DNA, and contain all chromosome 21 specific STSs originally used to define the 810 YAC 21q YAC contig. When integrated with the analysis of a somatic cell hybrid mapping panel of chromosome 21 reported in the accompanying manuscript, a greatly enhanced understanding of the physical map of chromosome 21 is obtained.

Molecular Genetic Study on Schizophrenia

Schizophrenia is a disease of the brain that manifests with multiple signs and symptoms involving thought, perception, emotion, movement, and behavior. A number of studies have evidenced that schizophrenia is a familial disorder and that the familial aggregation is due largely to genetic factors. In order to find specific genes that confer susceptibility to schizophrenia, we employed two fundamentally distinct strategies: tests of association and linkage. The former was carried out using unrelated schizophrenic probands as subjects while the later is a family-based study. During the past two years, we have collected 130 muclear families each with no less than two individuals afflicted with schizophrenia, and 50 sporadic schizophrenic patients. Candidate genes with neuropsychopharmacological significance, such as genes of dopamine receptors and androgen receptors, were chosen as markers. Polymorphism of these genes were analyzed and their association with schizophrenia were tested. Genes with high polymorphic informative index such as DRD4 were used as marker loci in the sib-pair analysis. Advanced analysis using anonymous polymorphic markers and combining our samples with other races have been done. Collaborating with international schizophrenic gene hunters, the Taipei Group of Schizophrenic Genetic Study will probe further refining of schizophrenia phenotype and specific responsible genes. The present results reveal encouraging findings of linkage with anonymous markers at Chromosome 6p, 8p, 11, 19 and 22q. The candidate gene approach has not found sustaining evidence of association yet. We believe our approach, complemented by basic neuropsychopharmacological studies and refined phenotype definition, will offer substantial promise for finding at least some specific genes that underline the susceptibility to schizophrenia.

A 5.4-Mb Continuous Pulsed-Field Gel Electrophoresis Map of Human 9q34.1 between ABL and D9S114, Including the Tuberous Sclerosis (TSC1) Region

We constructed a long-range restriction map of the tuberous sclerosis (TSC1) region of human chromosome 9q34, extending from ABL (T39-2-2) to D9S114. The physical map includes five genes and seven anonymous markers. The maximum distance between ABL and D9S114 is 5.4 Mb. The TSC1 critical region, between D9S149 and D9S114, has a maximum distance of 2.7 Mb. The ratio of genetic and physical distance in the region is 1 cM:600 kb. We also used Southern blot analysis and a radiation hybrid cell line, E6B, to exclude 3 genes—PBX3A, RXR α, and TAN1—from the AK1 to D9S114 interval. This excludes them as disease genes for tuberous sclerosis.

Characterization of a t(10;11)(p13-14;q14-21) in the monoblastic cell line U937.

Previous analysis of the monoblastic cell line U937 has shown that several sublines contain a rearranged chromosome arm 11q. In order to determine the true nature of the rearrangement, fluorescence in situ hybridization (FISH) was carried out with various combinations of single copy anonymous markers, clones containing genes, a chromosome 10 paint, and an 11 centromere specific sequence. The rearrangement was deduced to be a reciprocal translocation between chromosomes 10 and 11 described as t(10;11)(p13-14;q14-21). The breakpoint on chromosome 11 is telomeric to the INT2 gene and the pHS11 probe at 11q13, and centromeric to the marker D11S36 localized to 11q14.3-q22.1 and the MLL gene at 11q23. Similar translocations have been reported in various acute leukemias, principally of the monocytic lineage, and also in T-cell precursor acute lymphocytic leukemias. Further characterization of the genetic rearrangements in U937 may lead to the isolation of genes important in leukemogenesis and provide an in vitro system for their study.

High-resolution linkage map in the vicinity of the Lp locus

Looptail ( Lp) is a mutation that profoundly affects neurulation in mouse and is characterized by craniorachischisis, an open neural tube extending from the midbrain to the tail in embryos homozygous for the mutation. Lp maps to the distal portion of mouse chromosome 1, and as part of a positional cloning approach, we have generated a high-resolution linkage map of the Lp chromosomal region. For this, we have carried out extensive segregation analysis in a total of 706 backcross mice informative for Lp and derived from two crosses, ( Lp/ + X SJL/J)F1 X SJL/J and ( Lp/ + X SWR/J)F1 X SWR/J. In addition, 269 mice from a ( Mus spretus X C57BL/6J)F1 X C57BL/6J interspecific backcross were also used to order marker loci and calculate intergene distances for this region. With these mice, a total of 28 DNA markers corresponding to either cloned genes or anonymous markers of the SSLP or SSCP-types were mapped within a 5-cM interval overlapping the Lp region, with the following locus order and interlocus distances (in cM): centromere- D1Mit110 / Atp1β1 / Cd3ζ / Cd3η / D1Mit145 — D1Hun14 / D1Mit15 — D1Mit111 / D1Mit112 — D1Mit114 — D1Mit148 / D1Mit205/ D1Mit36 / D1Mit146 / D1Mit147 / D1Mit270 / D1Hun13 — Fcgr2 — Mpp — Apoa2/Fcer1γ - Lp - D1Mit149 / Spna1/Fcer1α-Eph1-Hlx1/D1Mit62. These studies have allowed the delineation of a maximum genetic interval for Lp of 0.5 cM, a size amenable to physical mapping techniques.

Characterization of the human gene for microfibril-associated glycoprotein (MFAP2), assignment to chromosome 1p36.1–p35, and linkage to D1S170

Microfibril-associated glycoprotein, MAGP (gene symbol MFAP2), is a component of connective tissue microfibrils and a candidate for involvement in the etiology of inherited connective tissue diseases. We have cloned a human MAGP cDNA that is highly homologous to the previously characterized bovine and murine genes. Like the bovine and murine loci, the human gene has eight coding exons, but it contains two alternatively used 5′ untranslated exons, whereas only one untranslated exon was described in the bovine and murine Magp genes. By using rodent X human somatic cell hybrid panels and fluorescence chromosomal in situ hybridization, we have assigned the locus to human chromosome 1p36.1–p35. An insertion/ deletion polymorphism has been identified within intron 7. Linkage analysis between this polymorphism and markers on distal chromosome 1 revealed that MAGP is tightly linked to the anonymous marker D1S170. Physical mapping revealed a distance of <100 kb between the two markers. This information can be used to screen for linkage in families with microfibrillar abnormalities that are not linked to the fibrillin genes on chromosomes 15 or 5.

Cosmid contigs from the tuberous sclerosis candidate region on chromosome 9q34.

Tuberous sclerosis (TSC) is a heterogeneous multisystem disorder with loci on 9q34 (TSC1) and 16p13.3 (TSC2). The TSC2 gene has recently been isolated, while the TSC1 gene has been mapped to a 5-cM region between the markers D9S149 and D9S114. In our effort to localise and clone TSC1, we have obtained three adjacent cosmid contigs that cover the core of the candidate region. The three contigs comprise approximately 600 kb and include 80 cosmids, 2 P1 clones, 1 YAC, 5 anonymous markers and 4 sequence-tagged sites. The ABO blood group locus, the Surfeit gene cluster, the dopamine beta-hydroxylase gene (DBH) and VAV2, a homologue of the vav oncogene, have all been mapped within the contigs. Exon trapping and mutation screening experiments, aimed at identifying the TSC1 gene, are currently in progress.

Placement and refined mapping of established and new markers on human chromosome 11q using a small panel of somatic cell hybrids.

Constructing detailed chromosome maps relies on the ability to place new markers rapidly and accurately. Here we map two new anonymous DNA markers precisely on chromosome 11q and refine the location of established markers using a panel of just six fragmentation hybrids, with reference to four translocation chromosomes. As several of the hybrids retain multiple segments of chromosome 11, it is possible to quickly assign new markers to subintervals using this modest set of hybrid DNAs.