Physical mapping of the G-protein coupled receptor 19 (GPR19) in the chromosome 12p12.3 region frequently rearranged in cancer cells

Rearrangements of the short arm of chromosome 12 are frequently observed in hematological malignancies and in certain solid neoplasias. Loss of heterozygosity studies identified a small genetic interval on chromosome 12p12.3 that is frequently deleted in childhood acute lymphoblastic leukemia (ALL). Two genes, ETV6 and CDKN1B, are located within this interval although evidence has accumulated that an as yet unidentified tumor suppressor gene is closely linked. Here we report the physical mapping of the G-protein coupled receptor 19 (GPR19) at approximately 40 kb from CDKN1B. The delineation of the gene order tel-ETV6-CDKN1B-GPR19-cen excluded GPR19 from the region commonly deleted in childhood ALL, but it could still be the target of genetic alterations found in other cancers. Electronic Publication     

Physical and radiation hybrid mapping of canine chromosome 12, in a region corresponding to human chromosome 6p12-q12

The positional cloning of the hypocretin receptor 2, the gene for autosomal recessive canine narcolepsy, has led to the development of a physical map spanning a large portion of canine chromosome 12 (CFA12), in a region corresponding to human chromosome 6p12-q13. More than 40 expressed sequence tags (ESTs) were used in homology search experiments, together with chromosome walking, to build both physical and radiation hybrid maps of the CFA12 13-21 region. The resulting map of bacterial artificial chromosome ends, ESTs, and microsatellite markers represents the longest continuous high-density map of the dog genome reported to date. These data further establish the dog as a system for studying disease genes of interest to human populations and highlight feasible approaches for positional cloning of disease genes in organisms where genomic resources are limited.     

Abnormal methylation of p16/CDKN2A AND p14/ARF genes GpG Islands in non-small cell lung cancer and in acute lymphoblastic leukemia

Multiplex methylation-sensitive PCR and methylation-specific PCR were employed in studying the methylation of CpG islands in the p16/CDKN2A and p14/ARF promoter and the first exon regions in non-small cell lung cancer (54 samples) and acute B-cell lymphoblastic leukemia (61 samples). Differences in methylation were detected between types of neoplasia as well as between CpG islands studied within the same types of tumors. High level of the p16/CDKN2A first exon CpC island methylation was revealed in non-small cell lung cancer (68%) and in acute B-cell lymphoblastic leukemia (55%) and the CpG island of p14/ARF first exon was nonmethylated in these types of tumors. The methylation of CpG-rich fragments of genes p16/CDKN2A and p14/ARF promoters was analysed. As was found out, CpG islands located in 5' areas of one and the same gene can differ in methylation frequencies. The comparison of sensitivity between methylation-specific PCR and methylation-sensitive PCR used in the methylations studies was carried out.     

Chromosomal localization of phospholipase A2 activating protein, an Ets2 target gene, to 9p21

A murine Ets2 target gene isolated by differential display cloning was identified as the phospholipase A2 activating protein (PLAA) gene. A 2.7-kb human cDNA demonstrating high homology to mouse and rat Plaa genes was then isolated and characterized. Human PLAA contains six WD-40 repeat motifs and three different protein kinase consensus domains. Fluorescence in situ hybridization (FISH) mapping placed PLAA on chromosome 9p21, a region frequently deleted in various cancers. A comprehensive mapping strategy was employed to define further the chromosomal localization of PLAA relative to CDKN2A within the 9p21 locus. Radiation hybrid mapping placed the gene 7.69 cR from WI-5735 (LOD >3.0), a marker in close proximity to CDKN2A and CDKN2B. Yeast artificial chromosome (YAC) mapping localized PLAA proximal to the CDKN2A/CDKN2B genes and to a region flanked by D9S171 and INFA commonly deleted in many neoplasms. Two YACs contained both PLAA and D9S259, a marker present in a second more proximal minimal deleted region observed in cutaneous melanoma and squamous cell lung carcinoma. Double-color fiber FISH mapping confirmed the location of PLAA centromeric to D9S171 and CDKN2A/CDKN2B. The mapping data suggest a possible tumor suppressor role for this gene.     

Characterization of the human laminin beta2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS).

The laminin beta2 chain is an important constituent of certain kidney and muscle basement membranes. We have generated a detailed physical map of a 110-kb genomic DNA segment surrounding the human laminin beta2 chain gene (LAMB2) on chromosome 3p21.3-->p21.2, a region paralogous with the chromosome 7q22-->q31 region that contains the laminin beta1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3' end of LAMB2 lies 16 kb from the 5' end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3' end of LAMB2L was similar to exons 8-12 of the laminin beta2 chain gene. The LAMB2L-LAMB2-QARS cluster lies telomeric to the gene encoding the laminin-binding protein dystroglycan (DAG1).     

A 2.5-Mb physical map within 3p21.1 spans the breakpoint associated with Greig cephalopolysyndactyly syndrome.

Numerous investigations suggest that one or more genes residing in the p14 to p21 region of human chromosome 3 are critical to the development of neoplastic diseases such as renal cell carcinoma and small-cell lung cancer (SCLC). This region is additionally involved in several interchromosomal translocations, one of which is associated with the developmental disorder Greig cephalopolysyndactyly syndrome. A series of five loci that map in close proximity to the Greig syndrome breakpoint [t(3;7)(p21.1;p13)] at 3p21.1 have been physically linked by pulsed-field gel analysis over a 2.5-Mb region. The probes include ACY1, cA84 (D3S92), cA199 (D3S93), pHF12-32 (D3S2), and MW-Not153 (D3S332). The Greig 3;7 translocation breakpoint was discovered between clones cA199 and MW-Not153, separated by 825 kb. Further analysis revealed comigration of a rearranged fragment detected by MW-Not153 and a chromosome 7 probe previously shown to be in close proximity to the breakpoint (CRI-R944). This latter probe also detects a rearrangement in a second Greig-associated translocation, (6;7)(q27;p13). The physical map resulting from this analysis orders the markers along the chromosome and identifies several locations for CpG islands, likely associated with genes. Although probe pEFD145.1 (D3S32) has been genetically linked to D3S2 (2 cM), physical linkage to the other five loci could not be demonstrated. One of the linked loci, D3S2, has been widely utilized in the analysis of chromosome 3p loss in several malignant diseases. Since expression of ACY1, a housekeeping gene, is specifically reduced in many cases of SCLC, knowledge of its precise chromosomal position and identification of neighboring putative gene loci should facilitate investigation into the mechanism of this reduction.     

Localization of the CDKN4/p27Kip1 gene to human chromosome 12p12.3

CDKN4/p27Kip1 is a cyclin-dependent kinase (Cdk) inhibitor implicated in G1 phase arrest, which negatively regulates G1 phase progression in response to TGF, and might represent a tumor suppressor gene. We report here the chromosomal assignment of the human CDKN4 gene to chromosome 12p12.3 in close proximity to highly polymorphic microsatellite markers.     

Juxta-centromeric region of human chromosome 21 is enriched for pseudogenes and gene fragments

A physical map including four pseudogenes and 10 gene fragments and spanning 500 kb in the juxta-centromeric region of the long arm of human chromosome 21 is presented. cDNA fragments isolated from a selected cDNA library were characterized and mapped to the 831B6 YAC and to two BAC contigs that cover 250 kb of the region. An 85 kb genomic sequence located in the proximal region of the map was analyzed for putative exons. Four pseudogenes were found, including psiIGSF3, psiEIF3, psiGCT-rel whose functional copies map to chromosome 1p13, chromosome 2 and chromosome 22q11, respectively. The TTLL1 pseudogene corresponds to a new gene whose functional copy maps to chromosome 22q13. Ten gene fragments represent novel sequences that have related sequences on different human chromosomes and show 97-100% nucleotide identity to chromosome 21. These may correspond to pseudogenes on chromosome 21 and to functional genes in other chromosomes. The 85 kb genomic sequence was analyzed also for GC content, CpG islands, and repetitive sequence distribution. A GC-poor L isochore spanning 40 kb from satellite 1 was observed in the most centromeric region, next to a GC-rich H isochore that is a candidate region for the presence of functional genes. The pericentric duplication of a 7.8 kb region that is derived from the 22q13 chromosome band is described. We showed that the juxta-centromeric region of human chromosome 21 is enriched for retrotransposed pseudogenes and gene fragments transferred by interchromosome duplications, but we do not rule out the possibility that the region harbors functional genes also.     

Alu-PCR Approach to Isolating NotI-Linking Clones from the 3p14-p21 Region Frequently Deleted in Renal Cell Carcinoma

In the mammalian genome CpG islands are associated with functional genes and cloning of these islands could be an alternative approach for cloning functional genes. Recently we have developed a new approach for cloning CpG islands and constructing NotI linking libraries. We have initiated the construction of a NotI restriction map for chromosome 3, especially focusing on the rearrangements in the 3p14-p21 region, which are associated with different malignancies. CpG islands from this region are useful for isolation of candidate tumor suppressor genes that map to this region and for isolating NotI-linking clones from 3p14-p21 for mapping purposes. Here we suggest a modification of Alu-PCR as an approach to isolating Not I sites (e.g., CpG islands) from defined regions of the chromosome. Instead of using whole chromosomal DNA for Alu-PCR, we have used representative NotI-linking libraries from hybrid cell lines containing either whole or deleted human chromosome 3 (MCH903.1 and MCH924.4, respectively). This decreases the complexity of the Alu-PCR products 10-100 times compared to the whole human genome. Using this modification, we can isolate NotI-linking clones, which are natural markers on the chromosome, rather than random genomic fragments. Among eight clones selected by this method, seven were from the region deleted in MCH924.4. The results clearly demonstrate the feasibility of Alu-PCR for isolating CpG islands from defined regions of the genome.     

Genomic sequence analysis of a potential QTL region for fat trait on pig chromosome 6

On pig chromosome 6, the SW71 microsatellite is located in the region corresponding to several quantitative trait loci (QTL), such as those for intramuscular fat content and for body weight at 4 weeks of age. The genomic sequence of approximately 909 kb was obtained from seven BAC clones encompassing the SW71 region corresponding to human 18q11.21-q11.22. By searching the NCBI GenBank using BLASTX and BLASTN, this 909-kb segment was found to contain eight genes, RAB31, TXNDC2, VAPA, APCDD1, NAPG, FAM38B, C18orf30, and C18orf58, and one putative gene (DN119777). The average G + C content in the sequence of this contig was 45.75% and 33 CpG islands were detected. CpG islands were scattered throughout the region in which most of the putative genes were located. Dense CpG islands of approximately 840 bp were observed, including within the 5' UTR and exon 1 of the orthologs of the RAB31, VAPA, APCDD1, and NAPG genes. Comparative analysis of conserved segments of six species showed that K(a)/K(s) ratios of the TXNDC2 gene in collinear and rearranged segments were significantly different at 4.1 and 1.3, respectively. In conclusion, we demonstrated the genomic organization of pig chromosome 6, including the gene order surrounding SW71, which provides important information for comparative mapping. Moreover, the genes revealed in this study may be positional candidate genes associated with QTL on chromosome 6 that affect fat deposition in pigs.     

The Fanconi anemia group E gene, FANCE, maps to chromosome 6p

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive disease with bone marrow failure and predisposition to cancer as major features, often accompanied by developmental anomalies. The cells of patients with FA are hypersensitive to DNA cross-linking agents in terms of cell survival and chromosomal breakage. Of the eight complementation groups (FA-A to FA-H) distinguished thus far by cell fusion studies, the genes for three-FANCA, FANCC, and FANCG-have been identified, and the FANCD gene has been localized to chromosome 3p22-26. We report here the use of homozygosity mapping and genetic linkage analysis to map a fifth distinct genetic locus for FA. DNA from three families was assigned to group FA-E by cell fusion and complementation analysis and was then used to localize the FANCE gene to chromosome 6p21-22 in an 18.2-cM region flanked by markers D6S422 and D6S1610. This study shows that data from even a small number of families can be successfully used to map a gene for a genetically heterogeneous disorder.     

Linkage and association mapping of the LRP5 locus on chromosome 11q13 in type 1 diabetes

Linkage of chromosome 11q13 to type 1 diabetes (T1D) was first reported from genome scans (Davies et al. 1994; Hashimoto et al. 1994) resulting in P <2.2 x 10(-5) (Luo et al. 1996) and designated IDDM4 ( insulin dependent diabetes mellitus 4). Association mapping under the linkage peak using 12 polymorphic microsatellite markers suggested some evidence of association with a two-marker haplotype, D11S1917*03-H0570POLYA*02, which was under-transmitted to affected siblings and over-transmitted to unaffected siblings ( P=1.5 x 10(-6)) (Nakagawa et al. 1998). Others have reported evidence for T1D association of the microsatellite marker D11S987, which is approximately 100 kb proximal to D11S1917 (Eckenrode et al. 2000). We have sequenced a 400-kb interval surrounding these loci and identified four genes, including the low-density lipoprotein receptor related protein (LRP5) gene, which has been considered as a functional candidate gene for T1D (Hey et al. 1998; Twells et al. 2001). Consequently, we have developed a comprehensive SNP map of the LRP5 gene region, and identified 95 SNPs encompassing 269 kb of genomic DNA, characterised the LD in the region and haplotypes (Twells et al. 2003). Here, we present our refined linkage curve of the IDDM4 region, comprising 32 microsatellite markers and 12 SNPs, providing a peak MLS=2.58, P=5 x 10(-4), at LRP5 g.17646G>T. The disease association data, largely focused in the LRP5 region with 1,106 T1D families, provided no further evidence for disease association at LRP5 or at D11S987. A second dataset, comprising 1,569 families from Finland, failed to replicate our previous findings at LRP5. The continued search for the variants of the putative IDDM4 locus will greatly benefit from the future development of a haplotype map of the genome.     

小鼠骨髓细胞中p16和Ralb基因启动区CpG岛的甲基化位点分析

抑癌基因p16和白血病致癌因子Ralb与白血病的发生密切相关,其启动子区CpG岛的甲基化对基因表达具有重要作用.本文旨在分析p16、Ralb基因启动子区CpG岛甲基化位点信息,并比较这两个基因在小鼠骨髓细胞和原代培养的骨髓细胞中甲基化状态的差异.运用"MethPrimer"软件预测p16、Ralb基因启动子区的CpG岛,设计甲基化特异性引物.利用重亚硫酸盐测序法(BSP)检测甲基化位点信息.结果显示,p16有1个CpG岛,岛上21个CpG位点全部未发生甲基化;Ralb有2个CpG岛,CpG岛1上的5个CpG位点全部呈甲基化状态,而CpG岛2上的17个CpG位点全部呈非甲基化状态,且小鼠骨髓细胞和体外原代培养的骨髓细胞中两基因的甲基化状态一致.表明p16、Ralb基因甲基化状态未受外界培养条件的影响而改变,提示在与两基因甲基化相关的研究中体外试验可替代体内试验.     

Genetic and physical map of the interferon region on chromosome 9p.

A region of chromosome 9, surrounding the interferon-beta (IFNB1) locus and the interferon-alpha (IFNA) gene cluster on 9p13-p22, has been shown to be frequently deleted or rearranged in a number of human cancers, including leukemia, glioma, non-small-cell lung carcinoma, and melanoma. To assist in better defining the precise region(s) of 9p implicated in each of these malignancies, a combined genetic and physical map of this region was generated using the available 9p markers IFNB1, IFNA, D9S3, and D9S19, along with a newly described locus, D9S126. The relative order and distances between these loci were determined by multipoint linkage analysis of CEPH (Centre d'Etude du Polymorphisme Humain) pedigree DNAs, pulsed-field gel electrophoresis, and fluorescence in situ hybridization. All three mapping approaches gave concordant results and, in the case of multipoint linkage analysis, the following gene order was supported for these and other closely linked chromosome 9 markers present in the CEPH database: pter-D9S33-IFNB1/IFNA-D9S126-D9S3-D9S19 -D9S9/D9S15-ASSP3-qter. This map serves to extend preexisting chromosome 9 maps (which focus primarily on 9q) and also reassigns D9S3 and D9S19 to more proximal locations on 9p.     

Congenital fibrosis of the extraocular muscles (autosomal dominant congenital external ophthalmoplegia): genetic homogeneity, linkage refinement, and physical mapping on chromosome 12.

Congenital fibrosis of the extraocular muscles (CFEOM) is an autosomal dominant syndrome of congenital external ophthalmoplegia and bilateral ptosis. We previously reported linkage of this disorder in two unrelated families to an 8-cM region near the centromere of human chromosome 12. We now present refinement of linkage in the original two families, linkage analysis of five additional families, and a physical map of the critical region for the CFEOM gene. In each of the seven families the disease gene is linked to the pericentromeric region of chromosome 12. D12S345, D12S59, D12S331, and D12S1048 do not recombine with the disease gene and have combined lod scores of 35.7, 35.6, 16.0, and 31.4, respectively. AFM136xf6 and AFMb320wd9 flank the CFEOM locus, defining a critical region of 3 cM spanning the centromere of chromosome 12. These data support the concept that this may be a genetically homogeneous disorder. We also describe the generation of a YAC contig encompassing the critical region of the CFEOM locus. This interval has been assigned cytogenetically to 12p11.2-q12 and spans the centromere of chromosome 12. These results provide the basis for further molecular analyses of the structure and organization of the CFEOM locus and will help in the identification of candidate genes.     

Long range restriction map of the von Hippel-Lindau gene region on human chromosome 3p

Von Hippel-Lindau disease is a heritable tumour syndrome caused by the loss of the function of a tumour suppressor gene on the short arm of human chromosome 3. The interval RAF1-D3S18 (3p25–3p26) has been identfied by genetic linkage studies to harbour the von Hippel-Lindau gene. We have constructed a long range restriction map of this region and have succeeded in demonstrating the physical linkage of loci D3S726 (DNA probe LIB31-38), D3S18 (c-LIB-1, L162E5), D3S601 (LIB1963) and D3S587 (LIB 12–48). Since multipoint analysis has located D3S601 proximal to D3S726, the physical map should be oriented with D3S726 towards the telomere. The order and distances of probes within the von Hippel-Lindau gene region is as follows: telomere — LIB3138 — (<280 kb) — c-LIB-1 — (overlapping) — L162E5 — (900–1600 kb) — (LIB 19-63, LIB 12–48) — centromere. In tissues that included blood, semen and Epstein-Barrvirus-transformed lymphocytes, we detected a putative CpG island flanking D3S18.     

The clustering of CpG islands may constitute an important determinant of the 3D organization of interphase chromosomes

We used the 4C-Seq technique to characterize the genome-wide patterns of spatial contacts of several CpG islands located on chromosome 14 in cultured chicken lymphoid and erythroid cells. We observed a clear tendency for the spatial clustering of CpG islands present on the same and different chromosomes, regardless of the presence or absence of promoters within these CpG islands. Accordingly, we observed preferential spatial contacts between Sp1 binding motifs and other GC-rich genomic elements, including the DNA sequence motifs capable of forming G-quadruplexes. However, an anchor placed in a gene/CpG island-poor area formed spatial contacts with other gene/CpG island-poor areas on chromosome 14 and other chromosomes. These results corroborate the two-compartment model of the spatial organization of interphase chromosomes and suggest that the clustering of CpG islands constitutes an important determinant of the 3D organization of the eukaryotic genome in the cell nucleus. Using the ChIP-Seq technique, we mapped the genome-wide CTCF deposition sites in the chicken lymphoid and erythroid cells that were used for the 4C analysis. We observed a good correlation between the density of CTCF deposition sites and the level of 4C signals for the anchors located in CpG islands but not for an anchor located in a gene desert. It is thus possible that CTCF contributes to the clustering of CpG islands observed in our experiments.     

Large-scale physical mapping within the region 22q12.3-13.1 in meningioma

The lack of physical mapping data strongly restricts the analysis of the meningioma chromosomal region that was assigned to the bands 22q12.3-qter. Recently, we reported a new marker D22S16 for chromosome 22 that was assigned to the region 22q13-qter by in situ hybridization. Utilizing somatic cell hybrids we now sublocalized the marker D22S16 within the band region 22q12-13.1, thus placing it in the vicinity of the gene for the platelet derived growth factor (PDGFB). A physical map was established for the regions surrounding the PDGFB gene and the D22S16 marker. By means of pulsed-field gel electrophoresis (PFGE) D22S16 and PDGFB were found to be physically linked within 900 kb. We also identified two CpG clusters bordering the PDGFB gene. For the enzyme NotI, a variation of the PDGFB restriction pattern was found between different individuals. PFGE analysis of the two loci (PDFGB and D22S16) failed to identify major rearrangements in meningioma.