Cosmid walking and chromosome jumping in the region of PKD1 reveal a locus duplication and three CpG islands.

The locus responsible for the most common form of autosomal dominant polycystic kidney disease (PKD1) is located on chromosome 16p13.3. Genetic mapping studies indicate that PKD1 is flanked on the proximal side by the DNA marker 26.6 (D16S125). Here we show that 26.6 has undergone a locus duplication and that the two loci are less than 150kb apart. One of the two loci contains a polymorphic TaqI site that has been used in genetic studies and represents the proximal boundary for the PKD1 locus. We demonstrate that the polymorphic locus is the more proximal of the two 26.6-hybridizing loci. Therefore, four cosmids isolated from the distal 26.6-hybridizing locus contain candidate sequences for the PKD1 gene. These cosmids were found to contain two CpG islands that are likely markers for transcribed regions. A third CpG island was detected and cloned by directional chromosome jumping.     

Determination and Regional Assignment of Grouped Sets of Microclones in Chromosome 1pter–p35

In an approach to mapping physically the most distal 30 Mb of human chromosome 1p, region-specific clone libraries were generated by microdissection and microcloning. PFGE blot hybridization of single or low-copy microclones against rare-cutter digests of genomic DNA revealed physical linkage for groups of markers. Supplementary PFGE analysis of 31 1p36–p35-specific probes for genetically mapped loci established a total of 15 grouped sets, consisting of altogether 69 markers. Twelve of the grouped sets were located in 1pter–p36.12, as revealed by microcell hybrid mapping; the remaining three were localized proximal to 1p36.12. Regional assignment and ordering of most grouped sets was achieved either by evaluating the included genetic markers or by fluorescencein situhybridization of representative probes. The genomic extent of individual grouped sets encompassed between 1100 and 2100 kb, covering a total of approximately 22 Mb of the distal chromosome 1p region. One particular grouped set was shown to contain seven polymorphic marker loci that were previously suggested to be distributed across the entire 1pter–p35 region. The increase in the number of hybridization marker probes in 1p36 and their physical mapping is expected to facilitate positional cloning experiments in this region; in particular, the construction of clone contigs may be greatly facilitated.     

Fine genetic localization of the gene for autosomal dominant polycystic kidney disease (PKD1) with respect to physically mapped markers.

PKD1, the gene for the chromosome 16-linked form of autosomal dominant polycystic kidney disease, has previously been genetically mapped to an interval bounded by the polymorphic loci Fr3-42/EKMDA2 distally and O327hb/O90a proximally. More recently, 26.6PROX was identified as the closest proximal flanking locus. We set out to refine the localization of PKD1 by identifying a series of single recombinant events between the flanking markers Fr3-42/EKMDA2 and O327hb/O90a and analyzing them with a new set of polymorphic loci that have been physically mapped within the PKD1 interval. We identified 11 such crossovers in eight families; 6 of these fell into the interval between GGG1 and 26.6PROX, a distance of less than 750 kb. Three of these crossovers placed PKD1 proximal to GGG1 and two crossovers placed PKD1 distal to 26.6PROX. Both of the latter also placed PKD1 telomeric to a locus 92.6SH1.0, which lies 200-250 kb distal to 26.6PROX. The sixth recombinant, however, placed the disease mutation proximal to the locus 92.6SH1.0. Several possible explanations for these observations are discussed. An intensive study to locate deletions, insertions, and other chromosomal rearrangements associated with PKD1 mutations failed to detect any such abnormalities. Thus we have defined, in genetic and physical terms, the segment of 16p13.3 where PKD1 resides and conclude that a gene-by-gene analysis of the region will be necessary to identify the mutation(s).     

Identification of a locus which shows no genetic recombination with the autosomal dominant polycystic kidney disease gene on chromosome 16.

The major site for mutations leading to autosomal dominant polycystic kidney disease (ADPKD) is at the PKD1 locus, previously mapped to 16p13. Three additional probes have now been mapped within an existing array of genetic markers flanking this locus. One of these, CMM65b (D16S84), shows no recombination with PKD1 in 201 informative meioses. The others, Fr3-42 (D16S21) and EKMDA2 (D16S83), are shown to be the closest telomeric flanking markers. Somatic cell hybrids containing derivative chromosome 16s were used to construct a physical map of the region. Cosmid overlap cloning of the D16S84 region allowed a t(16;1) translocation breakpoint to be mapped at the molecular level, orientating the extended D16S84 locus with respect to the chromosome. The new markers and physical map described here provide an improved framework for attempts to clone the PKD1 region and to identify polycystic kidney disease mutations.     

Physical analysis of murine albino deletions that disrupt liver-specific gene regulation or mesoderm development.

Complementation analyses of radiation-induced deletion mutations involving the albino (c) locus in Chromosome (Chr) 7 of the mouse have identified several loci, in addition to c, that have important roles in development. The "mesoderm-deficient" (msd) and "hepatocyte-specific developmental regulation-1" (hsdr-1) loci, which are proximal and tightly linked to c, are important in the formation of mesoderm and in the regulation of liver- and kidney-specific induction of various enzymes and proteins, respectively. Cloning deletion-breakpoint-fusion fragments caused by lethal albino deletions that genetically define the extents of the msd and hsdr-1 loci is one way of generating molecular probes for studying the gene(s) involved in these phenotypes. The distal breakpoints of five such deletions were positioned on a long-range (PFGE) map of approximately 1.7 Mb of wild-type DNA surrounding the c, D7Was12, and Emv-23 loci. In addition, the distal breakpoints of two viable albino deletions, which remove part of the tyrosinase gene and extend distally, were localized in the vicinity of the lethal deletion breakpoints. Therefore, the viable deletions can be exploited to generate additional DNA probes that should facilitate the isolation of breakpoint clones from chromosomes carrying lethal deletions defining hsdr-1 and msd.     

Stable length polymorphism of up to 260 kb at the tip of the short arm of human chromosome 16

We have completed a long-range restriction map of the terminal region of the short arm of human chromosome 16 (16p13.3) by physically linking a distal genetic locus (alpha-globin) with two recently isolated probes to telomere-associated repeats (TelBam3.4 and TelBam-11). Comparison of 47 chromosomes has revealed major polymorphic length variation in this region: we have identified three alleles in which the alpha-globin genes lie 170 kb, 350 kb, or 430 kb from the telemere. The two most common alleles contain different terminal segments, starting 145 kb distal to the alpha-globin genes. Beyond this boundary these alleles are nonhomologous, yet each contains sequences related to other (different) chromosome termini. This chromosome size polymorphism has probably arisen by occasional exchanges between the subtelomeric regions of nonhomologous chromosomes; analogous length variation is likely to be present at other human telomeres.     

Organization of the human monoamine oxidase genes and long-range physical mapping around them.

A 265-kb yeast artificial chromosome containing sequences for human monoamine oxidase A and B (MAO-A and MAO-B) genes has been characterized. These two genes are localized within a region of about 240 kb and are arranged in a tail-to-tail configuration, with the 3' coding sequences separated by about 50 kb. A region about 2.5 Mb around the MAO loci was mapped by pulsed-field gel electrophoresis (PFGE). Comparisons between the restriction maps derived from the YAC and the long-range map derived from genomic digestions were in general agreement. The important features identified include a CpG island at the 5' end of the MAO-A and MAO-B genes, respectively. The combined information supports the order of markers within this region to be DXS77-DXS7-MAOA-MAOB.     

Long-range genomic map of the Duchenne muscular dystrophy (DMD) gene: Isolation and use of J66 (DXS268), a distal intragenic marker

By cloning the endpoints of a DMD-associated deletion, we have "jumped" 1100 kb from pERT87-1 (DSX164) to a new locus designated J66 (DXS268), mapping distally within the Duchenne muscular dystrophy (DMD) gene. Both J66 and JBir are mapped by field-inversion gel electrophoresis and detect abnormal SfiI fragments in DMD patients and distal DMD-associated X; autosome translocations. Our long-range map extends the physical map of the DMD gene from 800 to 2000 kb (2 Mb) and increases the mapped portion of Xp21 to approximately 8 Mb. The position of the glycerol kinase gene and the adrenal hypoplasia locus are further confined to the region between J66 and the nearest distal probe L1-4. This region spans at least 1.5 Mb. The multiallelic J66 polymorphism has immediate application in the diagnosis of DMD and generally appears to be distal to DMD mutations.     

The long-range restriction map surrounding the mouse agouti locus reveals a disparity between physical and genetic distances

Isolation of a gene based on its location, which depends on aligning physical landmarks with the genetic map, can yield basic information about genome structure and organization. As a first step toward isolating the mouse agouti (A) locus, we have begun to define the physical position of this gene relative to genetically linked DNA probes from the Psp, Emv-15, and Src loci. Using a combination of pulsed-field gel techniques that include partial digestion with rare-cutting restriction enzymes and analysis of polymorphic sites present in certain inbred strains, we have constructed long-range restriction maps for each of the probes that span a total of more than 3000 kb. The Src and Emv-15 probes are less than 600 kb apart, but are separated from the Psp probe by at least 1500 kb. By determining the position of a 75-kb deletion that inactivates agouti function, we have localized the A locus to within 500 kb of the Psp probe, but more than 600 kb away from the Emv-15 probe. These physical distances contrast with the known recombination frequencies, 3 +/- 3 cM for A-Psp and less than 0.3 cM for A-Emv-15, and suggest that recombination between A and Emv-15 may be suppressed.     

The gene for autosomal dominant polycystic kidney disease lies in a 750-kb CpG-rich region.

PKD1, the locus most commonly affected by mutations that produce autosomal dominant polycystic kidney disease (ADPKD), has previously been localized to chromosome 16p13.3. Since no cytogenetic abnormalities have been found in association with ADPKD, flanking genetic markers have been required to define an interval--the PKD1 region--that contains the PKD1 gene. In this report we demonstrate, through the construction of a long-range restriction map that links the flanking genetic markers GGG1 (D16S84) and 26.6PROX (D16S125), that the PKD1 gene lies within an extremely CpG-rich 750-kb segment of chromosome 16p13.3. Approximately 90% of this region has been cloned in three extensive cosmid/bacteriophage contigs. The cloned DNA is a valuable resource for identifying new closer flanking genetic markers and for isolating candidate genes from the region.     

Regional localization of the autosomal dominant polycystic kidney disease locus

The localization of the autosomal dominant polycystic kidney disease locus (PKD1) within an array of anonymous polymorphic DNA sequences on chromosome 16 band p13 was determined by multipoint mapping. Nine polymorphic DNA markers, including two hypervariable sequences, were used to study 19 PKD1 and 21 reference families. PKD1 was found to lie proximal to the 3' and 5' hypervariable regions of alpha-globin and distal to the anonymous sequence CRI-0327. Somatic cell hybrid mapping places PKD1 within the region 16p13.11-16pter. The availability of an array of linked markers which bracket the PKD1 locus provides a framework for further attempts to identify the PKD1 gene and offers an improved method of presymptomatic diagnosis of the disease.     

Isolation and field-inversion gel electrophoresis analysis of DNA markers located close to the Huntington disease gene

A radiation-induced hybrid cell line containing 10-20 million base pairs of DNA derived from the terminal part of human 4p16 in a background of hamster chromosomes has been used to construct a genomic library highly enriched for human sequences located close to the Huntington disease (HD) gene. Recombinant phage containing human inserts were isolated from this library and used as hybridization probes against two other radiation hybrids containing human fragments with chromosomal breaks in 4p16 and against a human-hamster somatic cell hybrid that retains only the 4p15-4pter part of chromosome 4. Of 121 human phage tested, 6 were mapped distal to the HD-linked D4S10 locus. Since the HD gene is located between D4S10 and the 4p telomere, all of these sequences are likely to be closer to HD than D4S10, and any one of them may be a distal flanking marker for the disease locus. Long-range restriction map analysis performed with a field-inversion gel system shows that the six new loci are distributed in different places within 4p16. Although it is not possible to establish an order for the six sequences with the FIGE data, the results demonstrate that the region detected by these probes must span at least 2000 kb of DNA.     

A physical map of the human Y-chromosome short arm

A physical map of the Y-chromosome short arm was constructed using DNA probes p19B, Y-286/la5, pZFY, Y-280, and Y-227. These probes hybridize with four NotI fragments of 400 kb (p19B and Y-286/la5), 350 kb, 1.9 Mb, and 3.0 Mb, respectively. The restriction fragments were shown to be adjacent to each other by analysis of NotI partial digests, overlapping restriction fragments, and/or the detection of rearranged restriction fragments in a 46,XX male. The present map covers approximately 5.6 Mb of contiguous DNA of Yp. Previously, the size of the pseudoautosomal region was estimated to be 2.3 Mb, and a 5.3-Mb NotI fragment containing Y-specific repeated DNA was assigned to proximal Yp. These and the present data account for approximately 13 Mb and thus for most of the DNA content of the Y short arm.     

High-resolution physical and transcript map of the locus for venous malformations with glomus cells (VMGLOM) on chromosome 1p21-p22

Vascular anomalies are congenital lesions that usually occur sporadically, but can be inherited. Previously, we have described that venous malformations, localized bluish-purple skin lesions, are caused by an activating mutation in the TIE2/TEK receptor. Moreover, we mapped another locus to chromosome 1p21-p22, for venous malformations with glomus cells (VM-GLOM). Here we report a physical map, based on 18 overlapping YAC clones, spanning this 5-Mb VMGLOM locus, from marker GATA63C06 to D1S2664. In addition, we report a sequence-ready PAC map of 46 clones covering 1.48 Mb within the YAC contig, a region to which we have restricted VMGLOM. We describe 21 new STSs and nine novel CA repeats, seven of which are polymorphic. These data will enable positional cloning of genes for diseases mapped to this locus, including the VMGLOM gene, likely a currently unknown regulator of vasculogenesis and/or angiogenesis.     

Physical mapping in the region of human Xq12-21.1 using pulsed field gel electrophoresis

A number of human disease genes have been localised to Xq12-21.1. A genetic map of this region has previously been constructed using family linkage studies and has been complemented by physical mapping studies using hybrid and deletion cell lines. We have constructed a preliminary long-range physical map of the region, which incorporates thirteen polymorphic and non-polymorphic probes, using pulsed field gel electrophoresis. The order of loci that can be inferred from all the genetic and physical mapping data is: cen-DXS133-[DXS153, DXS159]-DXS132-DXS135-[DXS131, DXS162]-[DXS325, DXS-347, DXS441]-PGKl-DXS447-DXS72-tel. The detection of several large non-overlapping MluI fragments by these probes implies that the minimum extent of the genomic DNA containing these loci is 16Mb. This information should be useful in the eventual identification and isolation of the genes responsible for diseases that map to this region.     

Genetic linkage studies of autosomal dominant polycystic kidney disease: search for the second gene in a large Sicilian family

Summary Polycystic kidney disease (PKD) is a common autosomal dominant genetic disorder caused by mutation in at least two different gene loci. The PKD1 gene has been localized on the short arm of chromosome 16. The location of a second genetic locus in the human genome is not yet known. A large PKD kindred, unlinked to chromosome 16, with over 250 members was studied using both DNA and classical markers. In total, 29 informative marker loci on 11 autosomes have been analyzed for linkage with PKD. The data significantly exclude the linkage with disease locus from 17 marker loci and show no evidence of close linkage with the other loci.     

Toward a physical map of the Xq28 region in man: Linking color vision, G6PD, and coagulation factor VIII genes to an X-Y homology region

We are using pulsed-field gel electrophoresis (PFGE) to establish a physical map of the human Xq28 region. We have identified a new probe 35.239 (DXYS64), localized in Xq28 by somatic hybrid mapping and belonging to a region of greater than 99% homology between the X and the Y chromosomes. PFGE data show that probes 35.239 and the polymorphic locus DXS115 (probe 767) map within a common 300-kb BssHII fragment. Both probes, in addition, hybridize to 575-kb BssHII and 590-kb ClaI fragments that contain the gene coding for coagulation factor VIII (F8C). The order F8C-DXS115-DXYS64 could be determined. Our results also provide evidence for linkage between the red/green color vision locus (RCP,GCP) and probes MD13 and T1.7 (GdX, DXS254) within a 750-kb ClaI fragment. Although the latter two probes are located within 50 kb of the 3' end of the G6PD gene, a G6PD cDNA probe did not hybridize to this fragment. G6PD, on the other hand, could be linked to F8C on a 290-kb BssHII fragment. All these data allow us to propose the order (RCP,GCP)-MD13-GdX-G6PD-F8C-DXS115-DXYS 64. We also linked probes St14 (DXS52), MN12 (DXS33), and DX13 (DXS15) to a member of a small family of X-linked dispersed sequences (DNF22S3) within a 575-kb BssHII fragment. The preliminary physical map presented here should be useful for further fine mapping of disease genes in the Xq28 region and should be helpful in orientating efforts toward the cloning of sequences close to the fragile X syndrome.