A high-resolution physical map of human chromosome 21p using yeast artificial chromosomes

The short arm of human chromosome 21 (21p) contains many different types of repetitive sequences and is highly homologous to the short arms of other acrocentric chromosomes. Owing to its repetitive nature and the lack of chromosome 21p-specific molecular markers, most physical maps of chromosome 21 exclude this region. We constructed a physical map of chromosome 21p using sequence tagged site (STS) content mapping of yeast artificial chromosomes (YACs). To this end, 39 STSs located on the short arm or near the centromere of chromosome 21 were constructed, including four polymorphic simple tandem repeats (STRs) and two expressed sequence tags (ESTs). Thirty YACs were selected from the St. Louis YAC library, the chromosome 21-enriched ICRF YAC library, and the CEPH YAC and megaYAC libraries. These were assembled in a YAC contig map ranging from the centromere to the rDNA gene cluster at 21p12. The total size of the region covered by YACs is estimated between 2.9 and 5 Mb. The integrity of the YAC contig was confirmed by restriction enzyme fingerprinting and fluorescence in situ hybridization (FISH). One gap with an estimated size of 400 kb remained near the telomeric end of the contig. This YAC contig map of the short arm of human chromosome 21 constitutes a basic framework for further structural and functional studies of chromosome 21p.     

Integrated YAC Contig Map of the Prader–Willi/Angelman Region on Chromosome 15q11–q13 with Average STS Spacing of 35 kb

Prader–Willi syndrome and Angelman syndrome are associated with parent-of-origin-specific abnormalities of chromosome 15q11–q13, most frequently a deletion of an ~4-Mb region. Because of genomic imprinting, paternal deficiency of this region leads to PWS and maternal deficiency to AS. Additionally, this region is frequently involved in other chromosomal rearrangements including duplications, triplications, or supernumerary marker formation. A detailed physical map of this region is important for elucidating the genes and mechanisms involved in genomic imprinting, as well as for understanding the mechanism of recurrent chromosomal rearrangments. An initial YAC contig extended from D15S18 to D15S12 and was comprised of 23 YACs and 21 STSs providing an average resolution of about one STS per 200 kb. To close two gaps in this contig, YAC screening was performed using two STSs that flank the gap between D15S18 and 254B5R and three STSs located distal to the GABRA5–149A9L gap. Additionally, we developed 11 new STSs, including seven polymorphic markers. Although several groups have developed whole-genome genetic and radiation hybrid maps, the depth of coverage for 15q11–q13 has been somewhat limited and discrepancies in marker order exist between the maps. To resolve the inconsistencies and to provide a more detailed map order of STSs in this region, we have constructed an integrated YAC STS-based physical map of chromosome 15q11–q13 containing 118 YACs and 118 STSs, including 38 STRs and 49 genes/ESTs. Using an estimate of 4 Mb for the size of this region, the map provides an average STS spacing of 35 kb. This map provides a valuable resource for identification of disease genes localized to this region as well as a framework for complete DNA sequencing.     

A yeast artificial chromosome contig from human chromosome 14q24 spanning the Alzheimer's disease locus AD3

Familial Alzheimer's disease has been previously linked to threegenetic loci on chromosomes 21, 19 and 14. The AD3 locus onchromosome 14 has not been cloned and the molecular defect inchromosome 14-linked AD3 families has yet to be identified.Genetic linkage analysis has placed the AD3 locus in band 14q24between the dinucleotide markers D14S61 and D14S289, a geneticdistance of approximately 6.4 cM. We have constructed a yeastartificial chromosome (YAC) contig that covers the entire minimalregion, encompassing all genetic markers that are non-recombinantfor the disease in AD3-linked families. This contig, constructedby using a combination of YAC end sequence walking and sequence-taggedsite (STS) mapping, consists of 63 YACs from three differentlibraries. The AD3 contig contains 12 polymorphic dinucleotiderepeat markers from D14S61 to D14S251, as well as an additional43 non-polymorphic STSs. This contiguous physical map of theregion will allow the physical distances between the markersto be determined, as well as providing a framework for the identificationof candidate genes.     

YAC contig mapping of six expressed sequences encoded by human chromosome 21

Six cDNA clones from human chromosome 21 have been mapped in a set of complete YAC contig spanning the entire chromosome 21q. The mapping positions between two STSs on the YAC contig and the NotI coordinates starting from the telomere of 21q were determined for the cDNA clones. The YAC contig mapping positions agree well with those using a comprehensive somatic cell hybrid mapping panel.     

Mapping of a chromosome 15 region involved in limb girdle muscular dystrophy

A gene responsible for an autosomal recessive form of limb girdlemuscular dystrophy (LGMD2, MIM number 253600 [OMIM] ) has been localizedon chromosome 15. After genotyping additional markers of thischromosome, two were found to flank the disease locus withinan interval that was assessed as 7 centiMorgans. The screeningof the CEPH YAC libraries with the corresponding probes allowedthe isolation of YACs which were used in fluorescence In situhybridization to define the LGMD2 cytogenetic interval as 15q15.1-15q21.1.Four different approaches were pursued for the establishmentof the physical map of this area which allowed the assemblyof an uninterrupted YAC contig spanning an estimated 10–12 megabases, with an average STS resolution of 140 kb or forthe 25 polymorphic microsatellites on this map, of 400 kb. Twelvegenes and 25 genetic markers were positioned in this contig,which is constituted of a minimum of 10 clones.     

Refinement of the RP17 locus for autosomal dominant retinitis pigmentosa, construction of a YAC contig and investigation of the candidate gene retinal fascin

The RP17 locus for autosomal dominant retinitis pigmentosa has previously been mapped to chromosome 17q by linkage analysis. Two unrelated South African families are linked to this locus and the identification of key recombination events assigned the RP17 locus to a 10 cM interval on 17q22. The work reported here refines the mapping of the locus from a 10 cM to a 1 cM interval between the microsatellite markers D17S1604 and D17S948. A physical map of this interval was constructed using information from the Whitehead/MIT YAC contig WC 17.8. Sequence-tagged site (STS) content mapping of seven overlapping YACs from this contig was employed in order to build the map. A BAC library was screened to cover a gap in the YAC contig and two positive BACs were identified. Intragenic polymorphisms in the retinal fascin gene provided evidence for the exclusion of this candidate as the RP17 disease gene.     

An integrated physical map for the short arm of human chromosome 5

The short arm of human chromosome 5 contains approximately 48 Mb of DNA and comprises 1.5% of the genome. We have constructed a mega-YAC/ STS map of this region that includes 436 YACs anchored by 216 STSs. By combining and integrating our map with the 5p maps of other groups using the same recombinant DNA library, a comprehensive map was constructed that includes 552 YACs and 504 markers. The YAC map covers >94% of 5p in four YAC contigs, bridges the centromere, and includes an additional 5 Mb of 5q DNA. The average marker density is 95 kb. This integrated 5p map will serve as a resource for the continuing localization of genes on the short arm of human chromosome 5 and as a framework for both generating and aligning the DNA sequence of this region.     

Genetic and physical characterization of the early-onset Alzheimer's disease AD3 locus on chromosome 14q24.3

Genetic linkage studies have provided significant evidence thata major gene defect, AD3, for familial early-onset Alzheimer'sdisease (EOAD) is located at chromosome 14q24.3, between theshort tandem repeat (STR) markers D14S52 and D14S53 defininga genetic size of 22.7 cM for the AD3 candidate region. We constructeda physical map of the AD3 region using yeast artificial chromosomes(YACs) selected from both the CEPH and megaCEPH YAC librariesusing the AD3 linked STR markers as well as new sequence-taggedsites (STSs) designed based on YAC terminal sequences. The YACmap is contiguous in the region between D14S258 and D14S53,a region of 8.2 cM, and has an estimated physical size of 4–8Mb. The YAC contig map was used as a framework to localize threeknown genes, a pseudogene and two brain expressed sequence tags(ESTs). Linkage analysis studies in two Belgian chromosome 14EOAD families AD/A and AD/B, identified obligate recombinantsin family AD/A with D14S289 and D14S61 reducing the geneticsize of the candidate AD3 region substantially. The minimalAD3 candidate region measured 6.4 cM on the genetic map andis contained within six overlapping megaCEPH YACs that covereda physical distance estimated between 2 and 6 Mb. These YACsas well as other YACs in the YAC contig map are valuable resourcesin gene cloning efforts or genomic sequencing experiments aimingat isolating the AD3 gene.     

Localisation of two candidate genes for mental retardation using a YAC physical map of the Xq21.1-21.2 subbands.

Genetic studies in families with X linked mental retardation have suggested the location of several MR genes in the human q21 region. Since the establishment of cloned resources is an essential step towards the cloning of genes involved in inherited diseases, we built a yeast artificial chromosome (YAC) contig and an STS map of this part of the X chromosome. The contig, which extends from PGK1 in Xq13.3 to DXS1002 in Xq21.2, consists of 30 YACs mapped with 21 markers and spans about 6 Mb. The YAC contig was used as a framework to localise several previously known genes and CEPH/Genethon polymorphic markers, as well as to construct a physical map of the region surrounding one of these genes. We recently localised a presumed MR locus to the region flanked by DXS233 (proximal) and CHM (distal). In the present work, the zinc finger gene, ZNF6, has been shown to lie within this region and to be highly expressed in brain, making it a good candidate MR gene. Similarly the VDAC1 gene has been mapped between DXS986 and DXS72 and its candidate gene status for the Allan-Herndon-Dudley syndrome is discussed.     

Cloning of the Huntington disease region in yeast artificial chromosomes.

The gene responsible for Huntington disease has been localized to a 2.5 million base pair (Mb) region between the loci D4S10 and D4S168 on the short arm of chromosome 4. As part of a strategy to clone the HD gene on the basis of its chromosomal location, we isolated genomic DNA from the HD region as a set of overlapping yeast artificial chromosome (YAC) clones. Twenty-eight YAC clones were identified by screening human YAC libraries with twelve PCR-based sequence-tagged sites (STSs) from the region. We assembled the YAC clones into overlapping sets by hybridizing them to a large number of DNA probes from the HD region, including the STSs. In addition, we isolated the ends of the human DNA inserts of most of the YAC clones to assist in the construction of the contig. Although almost half of the YACs appear to contain chimeric inserts and several contain internal deletions or other rearrangements, we were able to obtain over 2.2 Mb of the HD region in YACs, including one continuous segment of 2.0 Mb covering the region that most likely contains the HD gene. Ten of the twenty eight YAC clones comprise a minimal set spanning the 2.2 Mb. These clones provide reagents for the complete characterization of this region of the genome and for the eventual isolation of the HD gene.     

Integration of physical, breakpoint and genetic maps of chromosome 22. Localization of 587 yeast artificial chromosomes with 238 mapped markers

Detailed physical maps of the human genome are important resourcesfor the Identification and isolation of disease genes and forstudying the structure and function of the genome. We used datafrom STS content mapping of YACs and natural and induced chromosomalbreakpoints to anchor contigs of overlapping yeast artificialchromosome (YAC) clones spanning extensive regions of humanchromosome 22. The STSs were assigned to specific regions (bins)on the chromosome using cell lines from a somatic hybrid mappingpanel defining a maximum of 25 intervals. YAC libraries werescreened by PCR amplification of hierarchical pools of yeastDNA with 238 markers, and a total of 587 YAC clones were identified.These YACs were assembled into contigs based upon their sharedSTS content using a simulated annealing algorithm. Fifteen contigs,containing between 2 and 74 STSs were assembled, and orderedalong the chromosome based upon the cytogenetic breakpoint,meiotic and PFG maps. Additional singleton YACs were assignedto unique chromosomal bins. These ordered YAC contigs will beuseful for identifying disease genes and chromosomal breakpointsby positional cloning and will provide the foundation for higherresolution physical maps for large scale sequencing of the chromosome.     

A 12-Mb Complete Coverage BAC Contig Map in Human Chromosome 16p13.1–p11.2

We have constructed a complete coverage BAC contig map that spans a 12-Mb genomic segment in the human chromosome 16p13.1-p11.2 region. The map consists of 68 previously mapped STSs and 289 BAC clones, 51 of which-corresponding to a total of 7.721 Mb of genomic DNA-have been sequenced, and provides a high resolution physical map of the region. Contigs were initially built based mainly on the analysis of STS contents and restriction fingerprint patterns of the clones. To close the gaps, probes derived from BAC clone ends were used to screen deeper BAC libraries. Clone end sequence data obtained from chromosome 16-specific BACs, as well as from public databases, were used for the identification of BACs that overlap with fully sequenced BACs by means of sequence match. This approach allowed precise alignment of clone overlaps in addition to restriction fingerprint comparison. A freehand contig drawing software tool was developed and used to manage the map data graphically and generate a real scale physical map. The map we present here is approximately 3.5 x deep and provides a minimal tiling path that covers the region in an array of contigous, overlapping BACs.     

A contiguous 3-Mb sequence-ready map in the S3-MX region on 21q22.2 based on high- throughput nonisotopic library screenings

Progress in complete genomic sequencing of human chromosome 21 relies on the construction of high-quality bacterial clone maps spanning large chromosomal regions. To achieve this goal, we have applied a strategy based on nonradioactive hybridizations to contig building. A contiguous sequence-ready map was constructed in the Down syndrome congenital heart disease (DS-CHD) region in 21q22.2, as a framework for large-scale genomic sequencing and positional candidate gene approach. Contig assembly was performed essentially by high throughput nonisotopic screenings of genomic libraries, prior to clone validation by (1) restriction digest fingerprinting, (2) STS analysis, (3) Southern hybridizations, and (4) FISH analysis. The contig contains a total of 50 STSs, of which 13 were newly isolated. A minimum tiling path (MTP) was subsequently defined that consists of 20 PACs, 2 BACs, and 5 cosmids covering 3 Mb between D21S3 and MX1. Gene distribution in the region includes 9 known genes (c21-LRP, WRB, SH3BGR, HMG14, PCP4, DSCAM, MX2, MX1, and TMPRSS2) and 14 new additional gene signatures consisting of cDNA selection products and ESTs. Forthcoming genomic sequence information will unravel the structural organization of potential candidate genes involved in specific features of Down syndrome pathogenesis.     

Quality Assessment of Whole Genome Mapping Data in the Refined Familial Spastic Paraplegia Interval on Chromosome 14q

Autosomal dominant familial spastic paraplegia (AD-FSP) is a genetically heterogeneous neurodegenerative disorder characterized by progressive spasticity of the lower limbs. Three loci on chromosome 14q (SPG3), 2p (SPG4), and 15q (SPG6) were shown to be responsible for AD-FSP. Analysis of recombination events in three SPG3-linked families allowed us to narrow the critical interval from 9 to 5 cM. An ~5-Mb YAC contig comprising 32 clones and 90 STSs was built from D14S301 to D14S991, encompassing this region of 14q21. Fifty-six ESTs assigned previously to this region with radiation hybrid (RH) panels Genebridge 4 and G3 were precisely localized on the YAC contig. The 90 STSs positioned on the contig were tested on the TNG RH panel to compare our YAC-based map with an RH map at a high level of resolution. Comparison between our map and the whole genome mapping data on this interval of chromosome 14q is discussed.     

Three genes that escape X chromosome inactivation are clustered within a 6 Mb YAC contig and STS map in Xp11.21-p11.22

In order to study the distribution of genes that escape X chromosomeinactivation, a high density yeast artificial chromosome (YAC)contig and STS map spanning approximately 6 Mb has been constructedin Xp11.21-p11.22. The contig contains 113 YACs mapped with53 markers, including 10 genes. Four genes have been assayedfor their expression status on both the active and inactivehuman X chromosomes, and these data have been combined withprevious results on two other genes in the contig. Three ofthese genes escape X inactivation and have been localized toa single YAC clone of {small tilde}1075 kb. The other threegenes are subject to inactivation, with two of them lying amongthe genes that escape inactivation. These results suggest thatthere are both regional control signals as well as gene-specificelements that determine the X inactivation status of genes onthe proximal short arm of the human X chromosome.     

A yeast artificial chromosome contig spanning the Charcot-Marie-Tooth disease type 1A duplication region.

A contiguous set of 43 overlapping yeast artificial chromosome (YAC) clones has been developed for the Charcot-Marie-Tooth disease type 1A (CMT1A) duplication region of chromosome 17p11.2. The contig spans approximately 2.0 Mb and can be represented in a minimum of five overlapping YACs. The YAC clones were isolated from two total human genomic YAC libraries and from YAC libraries made from rodent-human hybrid cell lines. YAC clones were isolated from the libraries by polymerase chain reaction (PCR) technique. Localization to chromosome 17p11.2 was confirmed by fluorescence in situ hybridization. Overlap between the YAC clones was detected by inter-Alu PCR amplification of the YACs and by cross hybridization of the YACs with YAC insert ends obtained by Vectorette PCR. This YAC contig is a useful resource for analyzing and mapping all the genes contained within the CMT1A duplication.     

Physical localisation of the breakpoints of a constitutional translocation t(5;6)(q21;q21) in a child with bilateral Wilms'' tumour.

A 6 month old boy presented with bilateral Wilms' tumour. Cytogenetic analysis of the lymphocytes from the patient showed a de novo balanced translocation t(5;6)(q21;q21), which was also present in the tumour material as the sole cytogenetic abnormality. To facilitate the identification of the translocation breakpoints, we have established a lymphoblastoid cell line (MA214L) from the patient which maintains the translocation in culture. We have used Genethon microsatellite markers as sequence tagged sites (STSs) to isolate yeast artificial chromosome (YAC) clones to 5q and 6q from human genomic libraries. Using fluorescence in situ hybridisation (FISH) on metaphase preparations of MA214L, we have physically defined the translocation breakpoints between YAC clones on each chromosome arm. The genetic distance separating the flanking YACs on 6q21 is 3 cM, while that on 5q21 is 4 cM. To date this is the first report of these chromosomal regions being implicated in Wilms' tumourigenesis.     

Paired STSs amplified from radiation hybrids, and from associated YACs, identify highly polymorphic loci flanking the ataxia telangiectasia locus on chromosome 11q22-23

The high resolution mapping of the ataxia telangiectasia (A-T)locus on chromosome 11q22–23 requires the generation ofnew polymorphic markers specifically within the segment of 11q22–23to which the locus has been assigned. We have made use of alibrary of Alu-PCR clones, amplified from a radiation reducedsomatic cell hybrid containing the relevant chromosome 11 segment,to generate sequence tagged sites (STS) within the 11q22–23region and have used YAC clones to extend the loci identifiedby these STSs. The identification of paired polymorphisms (fromAlu-PCR and the associated YAC derived clone), which are physicallylinked, but which show minimal linkage disequilibrium, providesa highly informative haplotype for use in genetic linkage analysisin A-T famllies. We describe the characterisation of 2 suchpolymorphic locl, D11S535 and D11S611, which map between existingflanking markers, and which provide additional information onthe location of the major A-T locus.