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Chromosome 1 Workshop 97 Report

Abstracts

Genetic Mapping of Human Chromosome 1.

Matise TC 1 and White PS 2Lab of Statistical Genetics, The Rockefeller University, New York, NY 10021 1

 

Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, PA 19104 2

Many new polymorphic markers have been developed and deposited in public databases since the previous Chromosome 1 workshop. Using a set of 689 markers that represents 810 loci, we have constructed two genetic mapsof chromosome 1. Of these 810 loci, 767 are anonymous DNA segments (692are PCR-based and 75 are hybridization-based) and 43 are genes. Both ofthe genetic maps are termed 'comprehensive' because an attempt was made toidentify and map as many markers as possible.

The first map is a 'comprehensive positional map.' We first identified a well-supported (odds > 1000:1) framework map, based on the Généthon maps published in 1996. This framework consists of 126 markerswith an average inter-marker distance of 2.3 cM and a total length of 289cM. Next, using MultiMap and CRIMAP to compute multipoint lod scores, theremaining 684 markers were positioned or binned relative to thisframework.

The second map is a 'comprehensive expanded map.' This map used the 126-marker framework map as a starting point for additional map building. Using the results from the positional map, markers whose 1000:1odds map position encompassed a single interval on the framework map wereplaced on the map in their respective intervals. Several rounds of addingmarkers to the map and re-analyzing the remaining binned markers led to afinal map of 154 markers, with a total length of 289 cM and an average resolution of 1.9 cM.

The fact that only 28 of 684 non-framework markers could be added to the framework indicates that the 126-marker framework has nearly reached the limits of resolution available from the small-subset of CEPH reference pedigrees that are currently being genotyped. The combined effort by us and Généthon that led to the development of this framework map has resulted in a map whose order is quite well supported. There is no reason to believe that the additional 28 markers added to the framework have been mapped with any less precision. Therefore, these maps are well suited to use for map integration, combining mapping information from genetic, radiation hybrid, and physical mapping resources.

Both maps are viewable on the Chromosome 1 web site. This site contains detailed information on the positional map and a graphical display of both maps. Disseminating maps of this size is challenging, and we have decided to use GDB to display these maps more effectively. MultiMap has been edited so that its maps can be output in a format that is readable by GDB's Mapview.


High Density Physical Map of a Refined Neuroblastoma Consensus Deletion at Human Chromosome 1p36.3

Onyango P 1; Gardellin P 1; Lubyova B 1; Lummerstorfer JA 1; Paiha K 1; Ambros P 2; Biegel
JA 3; and Weith A 1

 

Division of Human Genetics and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA, U.S.A 1

Research Institute of Molecular Pathology (I.M.P.), Vienna, Austria 2

 

Children‚s Cancer Research Institute, St. Anna Children‚s Hospital, Vienna, Austria 3

The subtelomeric region of the human 1p arm displays significant allelic deletions in human neuroblastomas indicating the location of one or more tumor suppressor genes in this genomic interval. In order to precisely determine the location of these genes and to generate tools for gene identification strategies, we used region-specific microclones to isolate corresponding long insert clones from YAC, PAC, P1 and cosmid libraries. The long insert clones were employed for FISH mapping on karyotypes with chromosomal aberrations in 1p36, the breakpoints serving as interval landmarks. Three neuroblastoma-associated cell lines with allelic deletions, SK-N-AS, Kelly and CH91-074, were used to delimit a consensus deletion in 1p36. Long range restriction mapping assigned 3.5 Mbp of DNA to this consensus deletion. Distal and proximal borders were assigned to the flanking markers D1S1224 and NPPA, respectively. In further support of the importance of this interval, we have mapped the balanced translocation breakpoints of two neuroectodermal tumor cell lines, NGP and SK-N-MC, to the consensus deletion. This region has been cloned to saturation using 24 ICI library YAC clones, 4 CEPH megaYACs, 9 PAC, 24 P1 and 218 Cosmid clones. These clones were ordered into three different intervals relative to the deletion/translocation breakpoints. Furthermore, several contigs of Cosmid and P1 clones could be established. This marker map is expected to be of substantial value for subsequent gene isolation experiments.


Molecular Cloning and Expression Analysis of Five Novel Genes in Chromosome 1p36

Onyango P 1; Lubyova B 1; Gardellin P 1; Kurzbauer R 1; and Weith A 2

 

Division of Human Genetics and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA, U.S.A 1

Research Institute of Molecular Pathology,Vienna, Austria 2

 

The human chromosome 1p36 region displays frequent non random chromosomal deletions and translocations in a number of human malignancies; these are thought to inactivate tumour suppressor genes. In order to identify these putative tumour suppressors we employed exon trapping, cDNA selection and zoo blot analysis to clone five new genes located in 1p36. Two of these represent novel genes and were designated PO42 and human xylosidase. Two further genes represented new members of known gene families: HTPZP2 was a tyrosine phosphatase and FRAP2 represented a FKBP12-rapamycin associated protein. The fifth gene identified, ENO1L1, was significantly homologous to c-myc promoter binding protein, MBP-1 and to enolase 1 (ENO1). It co-localised with alpha enolase (ENO1) on a single P1 clone. ENO1L1 differed from both ENO1 and MBP-1 in the organisation at the 5´ untranslated sequences. Secondly, MBP-1 contained two single base insertions not present in both ENO1 and ENO1L1 sequences which led to a shift in the MBP-1 reading frame. Expression analysis revealed two brain specific transcripts of 7.9 and 6.5 kb for HTPZP2. In contrast, PO42, FRAP2, ENO1L1 and human xylosidase appeared to be ubiquitously expressed in the tissues tested with transcript sizes of 4.5 kb, 8.7 kb, 1.75 kb and 4.5 kb, respectively. Using fluorescence in situ hybridisation (FISH) we mapped the 5 novel genes relative to chromosome 1p36 located breakpoints present in 3 established tumour and one non tumour cell lines. The karyotypic abnormatilties in these cell lines were exploited as chromosomal landmarks, we could thus show that the telomere to centromere gene order was: HTPZP2-(MBP-1/ENO1/ENO1L1)-(PO42/human xylosidase)-FRAP2. The location of these genes to a chromosomal region that is prone to deletions in human cancers makes them potential candidate tumour suppressors.


A review of physical mapping on the long arm of chromosome 1.

Schutte BC

 

Department of Pediatrics, University of Iowa, Iowa City, IA (USA)

A search of GDB, OMIM, and Medline databases was performed to identify new (since the previous Chromosome 1 Workshop - 10/95) physical mapping data on the long arm of chromosome 1. The results of the searches were compiled into three categories - FISH mapping, contig mapping and gene structures. A total of 51 new loci were localized to 1q by FISH analysis. A study by Bray-Ward et al. (1996), integrated genetic and cytogenetic maps by performing FISH analysis on 17 YAC clones that each contained a genetic marker. Numerous groups contributed to the 31 single gene mapping reports. Several multi-gene reports were found including FISH mapping of LERK1, LERK3, and LERK4 to 1q21-q22 (Cerretti et al., 1996), SPTA1 - H4F2 - H3F2 - IFI-12 - CRP - CRPP1 - APCS - FCER1A to 1q23 (Walsh et al., 1996), and HF1/F13B - PTPRC - REN - C4BP/MCP to 1q31-q32 (Pardo-Manuel de Villena et al., 1996).

Physical mapping on 1q also included the construction of 10 YAC contigs, analysis of the S100 gene cluster by 3 independent groups and the identification of a microsatellite marker from a telomere YAC from 1qtel. Of the 10 YAC contigs, 4 spanned the critical regions of disease loci - GLC1A at 1q23-q25 (2 groups), VWS at 1q32-q41, USH2A at 1q41 and CHS at 1q42-q43. The genes for GLC1A and CHS were also recently reported. Finally, the complete intron/exon structure for 5 genes were reported - S100A12 at 1q21, NTRK1 at 1q21-q22, PPOX at 1q22-q23 and SCM-1a and SCM-1b at 1q23


Integrated transcript maps of chromosome 1.

White PS 1; Matise TC 2; Sulman EP 2; Jensen SJ 1; Maris JM 1; and Brodeur GM 1

 

Division of Oncology, Children's Hospital of Philadelphia, Philadelphia PA, 19104 1

 

Lab of Statistical Genetics, The Rockefeller University, New York, NY, 10021 2

Comprehensive maps achieving an integration of multiple marker types and reflecting physical distance are essential to genetic disease locus searches, gene identification, and sequencing efforts. Although several high resolution maps of chromosome 1 have recently been constructed, these efforts have often relied upon non-overlapping sets of markers, making direct comparison difficult. We have experimented with radiation-reduced hybrid (RH) panels to determine if high resolution integrated maps can be constructed from RH typing data. To test this hypothesis, we constructed a radiation-reduced hybrid (RH) panel specific for 1p35-36. To anchor the integrated map, a framework genetic map was constructed with 24 genetic markers and a marker order of >1000:1 odds, yielding an average resolution of 2.8 centimorgans. An additional 106 genetic markers were localized relative to the framework genetic map. Individual DNA fragments of the RH panel were identified and ordered by PCR with the framework genetic map. A total of 250 markers, including 142 genes and ESTs, were mapped by PCR against the RH panel. The resulting map had an observed average resolution of 800 kilobases, and the results closely matched and more precisely defined previous mapping information for most markers. The actual identification and mapping of individual RH fragments, rather than the statistical ordering normally used to construct RH maps, allowed a much higher map resolution to be achieved. An analysis of RH typing data from the radiation hybrid database (RHdb) is being performed upon chromosome-specific sets of markers to determine if the RH fragment approach can be applied to whole chromosomes. Markers typed on the GeneBridge 4 RH panel are selected for each chromosome (>2700 for chromosome 1), and duplicate markers and retention patterns are removed. The resulting data set is used to create an RH map using MultiMap. An initial set of accurately-typed markers from the Wellcome Trust Center for Human Genetics is used as a fixed skeletal map to which additional markers are placed in succession onto a framework with >1000:1 odds. Marker addition to the framework proceeds until no additional markers can be placed with sufficient likelihood support. The 1000:1 odds-defined locations of the remaining markers, relative to the framework, are then calculated. These are screened against the Unigene database to identify markers grouped in identical EST clusters. Markers mapping to identical clusters that are also closely mapped are removed from the set. Marker orders are then regionally refined using contig assembly algorithms that identify and order RH fragments. Preliminary analysis of marker subsets for 1p35-p36 and chromosome 21 suggest that markers can be ordered with 10-fold greater precision than the current Science RH map. The resulting maps can serve as frameworks for integrating genetic, transcript, and cytogenetic markers, as well as physical clones.


 

Mapping of the CMT2A region of 1p36.

Vance JM, Richards C, Blomeier H, Tao Y, Stajich JM, and Pericak-Vance, MA

 

Division of Neurology, Department of Medicine, Duke University Medical Center, Durham, NC 27710 (USA)

We have constructed a YAC contig across the region of D1S450 to D1S228. Using this contig as a framework we have begun a PAC/BAC contig spanning the region. This has been used to order and map markers and ESTs in the region. This demonstrates that a large deletion lies in the YACs in this region, but is covered well by the PAC library. Preliminary evidence suggests a hot spot of recombination in the region, which overlaps with neuroblastoma and several eye disorders, on the order of 5:1 genetic to physical distance.


Mapping and Sequence Analysis of Human Chromosome 1

Wooster R, Vaudin M, and Gregory S

 

Chromosome 1 Mapping and Sequencing Teams, The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, UK CB10 1SA

Our aim is to construct physical clone maps covering chromosome 1 and use these to determine the DNA sequence of the whole chromosome. The strategy we are following involves establishing a high density framework map (in the order of 15 markers per Megabase sequence) using radiation hybrid (RH) mapping. The framework markers are then used to identify bacterial genomic clones covering the chromosome.

More than 3500 established markers were imported by anonymous ftp from the Genome Database (GDB), Généthon, the Whitehead Institute (WI) and the Cooperative Human Linkage Centre (CHLC), in order to facilitate the integration of mapping data. Incoming primer pairs were tested and then used to screen the Généthon/Cambridge (GB4) panel of 85 radiation hybrids. Two point analysis of the data was used to define the intervals across the chromosome. Totally linked (i.e. inseparable) markers were subsequently masked from further analysis and the RH map was constructed using maximum likelihood and stepwise locus ordering algorithms through the RHMAP program. We have increased the power of the RH map by generating a further 2500 STS markers in-house, following partial sequencing of clones from libraries of HindIII digests of flow-sorted chromosome 1 DNA ligated into Bluescript plasmid.

In the initial stage of bacterial clone map construction, markers from from 1p35 to the p telomere, 1q22 and the centromere to 1p13 were used to screen primary pools from a whole genome P1-derived artificial chromosome (PAC) library of approximately 7 genome equivalents, by a combination of PCR and hybridisation of radiolabelled pooled STS PCR products. Colony PCR and radiolabelled hybridisation to high density gridded arrays of PACs have been performed to determine the STS content of each PAC clone. Clones identified from this screening were picked into microtitre plates for analysis by HindIII/Sau3AI restriction enzyme fingerprinting. Overlapping clones are assembled in to contigs in FPC. The contigs are extended by walking, and a minimally overlapping subset is selected for sequencing. Clone localisation is verified by FISH.

For sequence production, our main approach is shotgun sequencing of 1.2-1.8 Kb fragments in M13, followed by directed finishing. Primary analysis of human sequence is semi automated and involves the masking of several repeat families. After the searching of public databases and exon/splice site (gene) prediction the results are examined in an ACEDB database prior to the final submission to EMBL.

The current RH map and sequence-ready contigs established below the RH map as well as sequencing strategies and progress of the sequence from 1q22 will be presented. All this infor-mation is available from our anonymous ftp and WWW sites at http://ftp.sanger.ac.uk and http://www.sanger.ac.uk respectively.