Relationship between Charcot - Marie-Tooth 1A and Smith - Magenis regions. snU3 may be a candidate gene for the Smith - Magenis syndrome

The juxtacentromeric region of the human chromosome 17 shortarm (17p11.2-p12) contains genes Involved in the Charcot - Marie- Tooth type 1A disease (CMT1A) and the Smith-Magenis syndrome(SMS). CMT1A Is associated with a duplication of a short segmentwhereas SMS is linked to microdeletions, extending toward thecentromere. We describe the construction and analysis of a 5Mb YAC contig spanning the CMT1A duplicated segment and thedistal part of four SMS microdeletions. We concluded that theYAC contig contains about 1Mb of genomic DNA which is deletedin the four SMS patients analysed. Moreover two YACs containboth STS deleted in SMS (U3) and STS duplicated in CMT1A (5H5),but the proximal breakpoint associated with the CMT1A duplicationis not the same as the distal SMS breakpoint we studied. Finallywe located five new STS In SMS deletion. Two of them, a microsatellite(D17S805(23)) and the gene coding for small nuclear RNA U3,have been localized In the contig we described. We may alsonote that snU3 Is the first expressed sequence localized Inan SMS deletion so far. The possible participation of this genein the SMS phenotype is discussed.     

Delimitation of duplicated segments and identification of their parental origin in two partial chromosome 3p duplications

Two chromosome 3 short arm duplications identified through G-banding were further investigated using fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) of microsatellite markers, aiming at mapping breakpoints and disclosing mechanisms of origin of these chromosome aberrations. Patient 1 was found to be a mosaic: a 3p12 --> 3p21 duplication was observed in most of his cells, and a normal cell line occurred with a frequency of about 3% in blood. In situ hybridization of chromosome 3 short- and long-arm libraries confirmed the short-arm duplication. Using FISH of short-arm sequences, the YAC 961_h_3 was shown to contain the proximal breakpoint (3p12.1 or 3p12.2), and the distal breakpoint was located between the YACs 729_c_3 and 806_h_2, which are adjacent in the WC 3.10 contig (3p21.1). In Patient 2, G-banding indicated a 3p21 --> 3p24 duplication, without mosaicism. In situ hybridization of chromosome 3 short- and long-arm libraries confirmed the duplication of short-arm sequences. FISH of chromosome 3 sequences showed that the YAC 749_a_7 spanned the proximal breakpoint (3p21.33). The distal breakpoint mapped to the interval between YACs 932_b_6 (3p24.3) and 909_b_6 (3p25). In both cases, microsatellite genotyping pointed to a rearrangement between paternal sister chromatids.     

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.     

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.     

Mapping of the breakpoints on the short arm of chromosome 17 in neoplasms with an i(17q).

Isochromosomes are monocentric or dicentric chromosomes with homologous arms that are attached in a reverse configuration as mirror images. With an incidence of 3-4%, the i(17q) represents the most frequent isochromosome in human cancer. It is found in a variety of tumors, particularly in blast crisis of chronic myeloid leukemia (CML-BC), acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), and medulloblastoma (MB), and indicates a poor prognosis. To determine the breakpoints on the molecular genetic level, we analyzed 18 neoplasms (six CML, four AML, one NHL, and seven MB) with an i(17q) and two MB with a pure del(17p) applying fluorescence in situ hybridization (FISH) with yeast artificial chromosome (YAC) clones, P1-artificial chromosome (PAC) clones, and cosmids from a well-characterized contig covering more than 6 Mb of genomic DNA. We identified four different breakpoint cluster regions. One is located close to or within the centromere of chromosome 17 and a second in the Charcot-Marie-Tooth (CMT1A) region at 17(p11.2). A third breakpoint was found telomeric to the CMT1A region. The fourth, most common breakpoint was detected in MB, AML, and in CML-BC specimens and was bordered by two adjacent cosmid clones (clones D14149 and M0140) within the Smith-Magenis syndrome (SMS) region. These results indicate that the low copy number repeat gene clusters which are present in the CMT and SMS regions may be one of the factors for the increased instability that may trigger the formation of an i(17q).     

Characterization of a 1.0 Mb YAC contig spanning two chromosome breakpoints related to Menkes disease.

Menkes disease, an X-linked recessive disorder of copper metabolism, has recently been mapped to Xq13.3 by two Menkes patients carrying chromosome rearrangements within this region. The breakpoints have been investigated by nonisotopic in situ suppression hybridization using YACs isolated from this region with the flanking markers DXS56 and PGK1. Three YACs were extending over the breakpoints at Xq13.3 and were shown to be overlapping by partial digest restriction maps, IRS-PCR fingerprinting and by the presence of common cosmid clones. These cosmids were subcloned and one of the single copy probes detected both breakpoints using rare-cutting restriction enzyme digests of the patients. All the results together localize the breakpoints to about 100 kb within the overlapping region of the YACs. Mapping of both breakpoints in a 1 Mb YAC contig implies that these YACs contain at least partially, the gene responsible for Menkes disease.     

Inheritance of CMT1A duplication from a mosaic father.

We describe a case with molecular duplication of chromosome 17 (p11.2-p12) whose duplicated chromosome was inherited from a mosaic father. The patient has clinical manifestations consistent with Charcot-Marie-Tooth disease type 1A (CMT1A), while the mosaic father has minimal findings of CMT1A. The father was found to be homozygous with DNA markers VAW409R3A (D17S122) and p132G8RI (PMP-22) which are duplicated in CMT1A cases. Fluorescence in situ hybridisation (FISH) analysis with YAC clone 49H7 confirmed the duplication in the affected patient and diagnosed the mosaicism in his father. These findings based on clinical diagnosis and FISH analysis suggest that the mosaicism may have occurred early in embryogenesis leading to the disease in the father. This is the only reported case of CMT1A with transmission from a mildly affected mosaic father.     

Vector–Hexamer PCR Isolation of All Insert Ends from a YAC Contig of the Mouse Igh Locus

We have developed a simple PCR strategy, termed vector–hexamer PCR, that is unique in its ability to easily recover every insert end from large insert clones in YAC and BAC vectors. We used this method to amplify and isolate all insert ends from a YAC contig covering the mouse Igh locus. Seventy-seven ends were amplified and sequenced from 36 YAC clones from four libraries in the pYAC4 vector. Unexpectedly, 40% of the insert ends of these YACs were LINE1 repeats. Nonrepetitive ends were suitable for use as probes on Southern blots of digested YACs to identify overlaps and construct a contig. The same strategy was used successfully to amplify insert ends from YACs in the pRML vector from the Whitehead Institute/MIT-820 mouse YAC library and from BACs in pBeloBAC11. The simplicity of this technique and its ability to isolate every end from large insert clones are of great utility in genomic investigation.     

The isolation of a yeast artificial chromosome (YAC) contig extending for 2 megabases in the vicinity of the Von Hippel Lindau disease gene

Von Hippel LIndau disease (VHL) is a rare autosomal dominantdisease associated with tumors and cysts in multiple organ systems.The VHL disease gene is tightly linked to the polymorphic DNAmarker 233E2 (D3S720) and flanked by 479H4 (D3S719) on its telomericand RAF1 on its centromeric side. Two additional markers, D3S1038and D3S601, have also been identified, and these markers, likeD3S720, are very tightly linked to VHL. Previously 93 cosmidclones were mapped to the larger region, 3p24.2 - pter, surroundingthe VHL disease gene (1). Using a Southern-based screening strategyon pools of YAC clones we have Isolated a contig of overlappingYAC clones that extends about 0.7 megabase centromeric, andabout 1.3 megabases telomeric of D3S720 and contains all threetightly linked VHL markers. Individual YACs In this contig werehybridized to grids containing cosmids localized between 3p24.2-pterand to several cosmids localized by fluorescent in situ hybridization(FISH) to 3p25. A total of 28 cosmids were positioned on thiscontig of overlapping YAC clones. We have also identified homologousYAC clones to many additional cosmid clones localized between3p24.2–p25, although these have not yet been preciselylocalized relative to the contig of YAC clones. This contigof YAC clones probably contains the VHL disease gene and shouldfacilitate the isolation and characterization of this 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.     

Assignment of microsatellite sequences to the region duplicated in CMT1A (17p12): a useful tool for diagnosis.

Charcot-Marie-Tooth disease type 1A (CMT1A), the most prevalent form of the peripheral hereditary neuropathies, has been associated with a duplication of a genomic segment of 1.5 Mb, located in 17p11.2. Recently, the same segment has been found to be deleted in patients with another peripheral neuropathy, hereditary neuropathy with liability to pressure palsies (HNPP). Highly polymorphic markers are rare in this area, rendering the diagnosis highly dependent either on invasive examinations (like nerve biopsy) or not totally reliable (like gene dosage). Thus, we used a contig of YACs, including the whole region duplicated in CMT1A, to map highly polymorphic microsatellite loci, designed in Genethon. We showed that four of these loci are located in the duplicated region, allowing us to propose them as diagnostic markers for CMT1A and HNPP.     

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.     

YAC cloning Mus musculustelomeric DNA: physical, genetic, in situand STS markers for the distal telomere of chromosome 10

Three Mus musculusDBA/2 YAC libraries were constructed usinga half-YAC telomere cloning vector. This functional complementationapproach yields libraries which include terminal restrictionfragments of the mouse genome. Screening all three librariesled to the isolation of 32 independent clones which carry linearYACs containing the mouse terminal repeat sequence, (TTAGGG)_n.These YACs provide a resource to isolate regions of the mousegenome close to chromosome termini and excluded from existingconventional YAC libraries. To demonstrate their utility, ahybridization probe was isolated from Mtel-1, the first (TTAGGG)_n-containingYAC isolated. This probe detects a 70 kb Kpnl fragment in themouse genome which is sensitive to pretreatment with BAL31 exonuclease.A PCR-based genetic marker generated from the sequence of thisprobe maps 4.4 cM from the most distal anchor locus on chromosome10 in the EUCIB interspecific backcross. STS primers for thislocus, D10Hgu1, were used to isolate YAC 110F4 from a commerciallyavailable mouse YAC library. Fluorescence in situ hybridizationdemonstrates that YAC 110F4 hybridizes to the distal telomereof chromosome 10. Clones in this collection of telomere YACstherefore partially overlap clones in conventional YAC libraries,and thus the previously unavailable terminal regions of themouse genome can now be linked with the developing mouse STSYAC contig. Genetic markers such as D10Hgu1 allow the ends ofthe mouse genetic map to be defined, thus closing the map.     

A YAC-based binning strategy facilitating the rapid assembly of cosmid contigs: 1.6 Mb of overlapping cosmids in Xp22

We have applied a yeast artificial chromosome (YAC)-based cosmidisolation and binning strategy to convert a YAC contig in Xp22into 1.6 Mb of overlapping cosmids. This strategy is based onthe screening of a high-density arrayed X chromosome-specificcosmid library with large YAC-derived restriction fragmentsand entire YAC probes. Cosmids selected in this way were griddedon dot blots and further mapped into bins defined by the overlapintervals of the YACs and YAC fragments. This rapid binningof cosmids simplified the subsequent assembly of cosmid contigsby restriction fingerprint hybridization. In total, we identified139 cosmids spanning the entire 1.6 Mb region with a minimaloverlap set of 53 clones. These cosmids were assigned to 17bins and 9 contigs. One of the contigs is 665 kb in length andis one of the largest uninterrupted cosmid contigs in humansreported to date. The gaps between the contigs are minor and,together, they represent less than 7% of the region covered.Two previously identified genes are contained in these cosmids,the gene for amelogenin (AMG) and the recently isolated putativechloride channel gene CICN4. In addition, two disease loci havebeen mapped to this region: X-linked ocular albinism type 1(OA1) and the microphthalmia with linear skin defects (MLS)syndrome. The assembly of the cosmid maps allowed us to determinethe size of the deletion intervals for these two loci, whichwere estimated to be 110 kb for OA1 and 570 kb for MLS. Thesecosmid contigs will greatly facilitate the positional cloningof the OA1 and MLS disease genes. Together with the Huntingtondisease gene region on chromosome 4, this region in Xp22 representsone of the best characterized large regions in the human genome.     

Analysis of 5S rDNA arrays in Arabidopsis thaliana: physical mapping and chromosome-specific polymorphisms

A physical map of a pericentromeric region of chromosome 5 containing a 5S rDNA locus and spanning approximately 1000 kb was established using the CIC YAC clones. Three 5S rDNA arrays were resolved in this YAC contig by PFGE analysis and we have mapped different types of sequences between these three blocks. 5S rDNA units from each of these three arrays of chromosome 5, and from chromosomes 3 and 4, were isolated by PCR. A total of 38 new DNA sequences were obtained. Two types of 5S rDNA repeated units exist: the major variant with 0.5-kb repeats and one with short repeats (251 bp) only detected on YAC 11A3 from chromosome 3. Although the 38 sequences displayed noticeable heterogeneity, we were able to group them according to their 5S array origin. The presence of 5S array-specific variants was confirmed with the restriction polymorphism study of all the YACs carrying 5S units.     

Molecular dissection of the Prader-Willi/Angelman syndrome region (15q11-13) by YAC cloning and FISH analysis.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct mental retardation disorders associated with deletions of proximal 15q (q11-q13) of different parental origin. Yeast artificial chromosome (YAC) clones were isolated for 9 previously mapped DNA probes from this region, and for one newly derived marker, LS6-1 (D15S113). A YAC contig of 1-1.5 Mb encompassing four markers (ML34, IR4-3R, PW71, and TD189-1) was constructed. Multi-color fluorescence in situ hybridization (FISH) analysis of interphase nuclei was combined with YAC contig information to provide the following order of markers: cen-IR39-ML34-IR4-3R-PW71-TD189-1-LS6++ +-1-TD3-21-GABRB3-IR10-1-CMW1-tel. FISH analysis was performed on 8 cases of PWS and 3 cases of AS, including 5 patients with normal karyotypes. All eleven patients were deleted for YACs in the interval from IR4-3R to GABRB3. On the proximal side of the deletion interval, 10/10 breakpoints fell within a single ML34 YAC of 370 kb. On the distal side, 8/9 breakpoints fell within a single IR10-1 YAC of 200 kb. These results indicate a striking consistency in the location of the proximal and distal breakpoints in PWS and AS patients. FISH analysis on a previously reported case of familial AS confirmed a submicroscopic deletion including YACs corresponding to LS6-1, TD3-21 and GABRB3 and supports the separation of the PWS and AS critical regions. Since these three YACs do not overlap each other, the minimum size of the AS critical region is > or = 650 kb.     

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.