Xp21 contiguous gene syndromes: deletion quantitation with bivariate flow karyotyping allows mapping of patient breakpoints.

Bivariate flow karyotyping was used to estimate the deletion sizes for a series of patients with Xp21 contiguous gene syndromes. The deletion estimates were used to develop an approximate scale for the genomic map in Xp21. The bivariate flow karyotype results were compared with clinical and molecular genetic information on the extent of the patients' deletions, and these various types of data were consistent. The resulting map spans > 15 Mb, from the telomeric interval between DXS41 (99-6) and DXS68 (L1-4) to a position centromeric to the ornithine transcarbamylase locus. The deletion sizing was considered to be accurate to +/- 1 Mb. The map provides information on the relative localization of genes and markers within this region. For example, the map suggests that the adrenal hypoplasia congenita and glycerol kinase genes are physically close to each other, are within 1-2 Mb of the telomeric end of the Duchenne muscular dystrophy (DMD) gene, and are nearer to the DMD locus than to the more distal marker DXS28 (C7). Information of this type is useful in developing genomic strategies for positional cloning in Xp21. These investigations demonstrate that the DNA from patients with Xp21 contiguous gene syndromes can be valuable reagents, not only for ordering loci and markers but also for providing an approximate scale to the map of the Xp21 region surrounding DMD.     

A deletion hot spot in the Duchenne muscular dystrophy gene

We have made a detailed study of a deletion hot spot in the distal half of the Duchenne muscular dystrophy (DMD) gene, using intragenic probe P20 (DXS269), isolated by a hybrid cell-mediated cloning procedure. P20 detects 16% deletions in patients suffering from either DMD or Becker muscular dystrophy (BMD), in sharp contrast to the adjacent intragenic markers JBir (7%) and J66 (less than 1%), mapping respectively 200-320 kb proximal and 380-500 kb distal to P20. Of the P20 deletions, 30% start within a region of 25-40 kb, the majority extending distally. P20 was confirmed to map internal to a distal intron of the DMD gene. This region was recently shown by both cDNA analysis (M. Koenig et al., 1987; Cell 50: 509-517), and field inversion electrophoresis studies (J.T. Den Dunnen et al., 1987, Nature (London) 329: 640-642) to be specifically prone to deletions. In addition, P20 detects MspI and EcoRV RFLPs, informative in 48% of the carrier females. Together, these properties make P20 useful for carrier detection, prenatal diagnosis, and the study of deletion induction in both DMD and BMD.     

A physical map of 4 million bp around the Duchenne muscular dystrophy gene on the human X-chromosome

Employing pulsed field gradient electrophoresis, we constructed a 4.5 million bp (Mb) Sfil restriction map of the human X-chromosomal region p21, harboring genes for Duchenne (DMD) and Becker Muscular Dystrophy. In a DMD patient with additional chronic granulomatosis and retinitis pigmentosa, the proximal 3.5 Mb is deleted. Another DMD patient, with additional glycerol kinase deficiency and adrenal hypoplasia, lacks at least 3.3 Mb in the middle region, including marker C7 but not B24, placing C7 closer to DMD. Another DMD patient has a partial pERT-87 deletion of minimally 140 kb. Truncated Sfil fragments in a female X:21 translocation patient place the junction probe XJ1.1 115 kb from the distal end of the normal fragment. Probe pERT-84 maps to the same fragment, within 750 kb of XJ1.1.     

A long-range restriction map between the alpha-globin complex and a marker closely linked to the polycystic kidney disease 1 (PKD1) locus

Two polymorphic loci and two additional probes that map close to CMM65, which is tightly linked to the polycystic kidney disease 1 (PKD1) locus in chromosome band 16p13.3, are described. These new probes were isolated from a library that was enriched by preparative pulsed-field gel electrophoresis (PFGE) for sequences from a 320-kb NotI fragment that includes CMM65. Through the use of a panel of somatic cell hybrids and PFGE, the new polymorphic loci, PNL56S and NKISP1, were localized within 60 kb and approximately 250 kb distal to CMM65, respectively. A long-range restriction map linking these new probes and the distal markers EKMDA2, CMM103, and alpha-globin was constructed. These latter probes have been localized to regions approximately 900 kb, 1.2 Mb, and 1.9 Mb distal to CMM65, respectively. The entire region was found to be unusually rich in CpG dinucleotides. The new polymorphic probes and the long-range map will aid both the search for the PKD1 locus and the detailed characterization of this distal region of 16p.     

Preliminary study on the molecular structure of 3'''' region of Duchenne muscular dystrophy (DMD) gene in Chinese

Number and order of HindⅢ exon-containing fragments (Hd) at 3' region of DMD gene were studied systematically using 16 partly-overlapping cDNA subprobes which were produced from dystrophin cDNA 9- 14 with each of 9 restriction endonudeases. There are 25 Hd fragments corresponding to cDNA 9 -14 in DMD gene. Since then, the exact length and the new order of Hd fragments are established. A new 2.1 kb fragment (Hd 55) is revealed, a 5.2 kb fragment (formely designated as Hd 59) is excluded and the existence of a controversial 3.2 kb fragment (Hd 64) is confirmed. Besides, three new exons were revealed by comparing the PvuⅡ and the XbaⅠ hybridization patterns with the Hindlll hybridization patterns for these cDNA subprobes. It is concluded that there are at least 66 Hd fragments, or 79 exons in DMD gene basing on the discovery of three additional exons. The corresponding relationship between the 66 Hd fragments and the SfiⅠ large scale physical map has been studied, and at least 17 Hd fragments or 19 exo     

Localisation of the endpoints of deletions in the 5'' region of the Duchenne gene using a sequence isolated by chromosome jumping.

We have used chromosome jumping technology to move from within a large intron sequence in the Duchenne muscular dystrophy (DMD) gene to a region adjacent to exons of the gene. The single copy jump clone, HH1, was used to characterise deletions in patients previously shown to be deleted for DNA markers in the 5' end of the gene. 12 out of 15 such patients have breakpoints which lie between HH1 and the genomic locus J-47. Thus the vast majority of the deletions in these patients have proximal breakpoints in a similar region distal to the 5' end of the gene. HH1 was mapped with respect to the X;1 translocation in a DMD female and was shown to lie at least 80 kb from the starting point of the chromosome jump, HIP25.     

A 2-Mb YAC/BAC-based physical map of the ovum mutant (Om) locus region on mouse chromosome 11

The embryonic lethal phenotype observed when DDK females are crossed with males from other strains results from a deleterious interaction between the egg cytoplasm and the paternal pronucleus soon after fertilization. We have previously mapped the Om locus responsible for this phenotype, called the DDK syndrome, to an approximately 2-cM region of chromosome 11. Here, we report the generation of a physical map of 28 yeast and bacterial artificial chromosome clones encompassing the entire genetic interval containing the Om locus. This contig, spanning approximately 2 Mb, was used to map precisely genes and genetic markers of the region. We determined the maximum physical interval for Om to be 1400 kb. In addition, 11 members of the Scya gene family were found to be organized into two clusters at the borders of the Om region. Two other genes (Rad51l3 and Schlafen 2) and one EST (D11Wsu78e) were also mapped in the Om region. This integrated map provides support for the identification of additional candidate genes for the DDK syndrome.     

Physical mapping distal to the DMD locus

We report a new locus, designated JC-1, which maps between the gene responsible for adrenal hypoplasia (AHC) and the gene that encodes glycerol kinase (GK) in Xp21.2-21.3. The probe identifying this locus was obtained by cloning the distal sequence of a junction fragment from a Duchenne muscular dystrophy (DMD) patient with a large deletion. Pulsed-field gel electrophoresis analysis shows that a region of at least 4 Mb separates the 3' end of the dystrophin gene and the closest distal marker to AHC, DXS28. This region of the human genome contains few genes whose deletion results in a clinical phenotype. JC-1 is a useful probe from which to initiate strategies directed at cloning the AHC and GK loci.     

Positional cloning of "Lisch-Like", a candidate modifier of susceptibility to type 2 diabetes in mice

In 404 Lep(ob/ob) F2 progeny of a C57BL/6J (B6) x DBA/2J (DBA) intercross, we mapped a DBA-related quantitative trait locus (QTL) to distal Chr1 at 169.6 Mb, centered about D1Mit110, for diabetes-related phenotypes that included blood glucose, HbA1c, and pancreatic islet histology. The interval was refined to 1.8 Mb in a series of B6.DBA congenic/subcongenic lines also segregating for Lep(ob). The phenotypes of B6.DBA congenic mice include reduced beta-cell replication rates accompanied by reduced beta-cell mass, reduced insulin/glucose ratio in blood, reduced glucose tolerance, and persistent mild hypoinsulinemic hyperglycemia. Nucleotide sequence and expression analysis of 14 genes in this interval identified a predicted gene that we have designated "Lisch-like" (Ll) as the most likely candidate. The gene spans 62.7 kb on Chr1qH2.3, encoding a 10-exon, 646-amino acid polypeptide, homologous to Lsr on Chr7qB1 and to Ildr1 on Chr16qB3. The largest isoform of Ll is predicted to be a transmembrane molecule with an immunoglobulin-like extracellular domain and a serine/threonine-rich intracellular domain that contains a 14-3-3 binding domain. Morpholino knockdown of the zebrafish paralog of Ll resulted in a generalized delay in endodermal development in the gut region and dispersion of insulin-positive cells. Mice segregating for an ENU-induced null allele of Ll have phenotypes comparable to the B.D congenic lines. The human ortholog, C1orf32, is in the middle of a 30-Mb region of Chr1q23-25 that has been repeatedly associated with type 2 diabetes.     

Genetic and physical maps of jerker (Espn(je)) on mouse chromosome 4

The jerker mutation causes degeneration of cochlea and vestibular sensory hair cells in mice. A frame-shift mutation in the actin bundling gene Espin (Espn) leads to hair bundle defects by disrupting the actin filament assembly in stereocilia. Previously, jerker was mapped to distal mouse chromosome 4. Here, analyzing 2536 informative meioses derived from two intersubspecific intercrosses, we localize jerker to a 0.51+/-0.14cM interval on chromosome 4. The following order and distances of genes and markers were determined: D4Mit180-0.44+/-0.13cM-Hes2, Espn(je)-0.08+/-0.06cM-D4Mit356-0.28+/-0.1cM-D4Mit208. A 300kb physical bacterial artificial chromosome (BAC) contig was generated containing the Espn(je) locus. The human homologous region maps to 1p36.31. We present a detailed high-resolution genetic and physical map of markers located at distal chromosome 4 and demonstrate concordance of Espn with jerker.     

Physical and transcriptional map of a 3-Mb region of mouse chromosome 1 containing the gene for the neural tube defect mutant loop-tail (Lp)

The Lp mouse mutant provides a model for the severe human neural tube defect (NTD), cranio-rachischisis. To identify the Lp gene, a positional cloning approach has been adopted. Previously, linkage analysis in a large intraspecific backcross was used to map the Lp locus to distal mouse chromosome 1. Here we report a detailed physical map of this region. The interval surrounding Lp has been cloned in a yeast artificial chromosome (YAC) contig consisting of 63 clones spanning approximately 3.2 Mb. Fifty sequence tagged sites (STSs) have been used to construct the contig and establish marker order across the interval. Based on the high level of conserved synteny between distal mouse chromosome 1 and human 1q21-q24, many of these STSs were designed from expressed sequences identified by cross-screening human and mouse databases of expressed sequence tags. Added to other known genes in the region, a total of 29 genes were located and ordered within the contig. Seven novel polymorphisms were identified within the region, allowing refinement of the genetic map and a reduction in the size of the physical interval containing the Lp gene. The Lp interval, between D1Mit113 and Tagln2, can be spanned by two nonchimeric overlapping YACs that define a physical distance of approximately 1 Mb. Within this region, 10 potential candidate genes have been mapped. The materials and genes described here will provide a resource for the identification and further study of the mutated Lp gene that causes this severe neural tube defect and will provide candidates for other defects known to map to the homologous region on human chromosome 1q.     

人Xp21．1－p21．3上3．5MbYAC重叠群构建及物理图谱分析

用Ａｌｕ－ＰＣＲ指纹图谱法分析了人Ｘｐ２１．１－ｐ２１．３上一系列的酵母人工染色体（ｙｅａｓｔａｒｔｉｆｉｃｉａｌｃｈｒｏｍｏｓｏｍｅ,ＹＡＣ）克隆,发现其中的两个ＹＡＣ克隆构成包含ＤＸＳ１６６位点的重叠群,而且这一重叠群与以前构建的包含ＤＭＤ基因全序列的ＹＡＣ重叠群相连接,ＹＡＣ克隆末端探针交叉杂交证实了这一重叠,使这一ＹＡＣ重叠群至少延伸至ＤＸＳ１６６位点,形成一个跨度为３．５Ｍｂ的ＹＡＣ重叠群。基于这些重叠的ＹＡＣ克隆绘制了这一区域的大尺度限制酶切图谱,并在这一图谱上定位了ＤＸＳ１６６位点,从而确定了ＤＸＳ１６６位点与ＤＭＤ基因的物理关系。这一工作为ＤＭＤ基因的５＇远端调控作用研究及该区域未知基因的克隆奠定了基础。     

An integrated map of human 6q22.3-q24 including a 3-Mb high-resolution BAC/PAC contig encompassing a QTL for fetal hemoglobin

Genetic studies have previously assigned a quantitative trait locus (QTL) for hemoglobin F and F cells to a region of approximately 4 Mb between the markers D6S408 and D6S292 on chromosome 6q23. An initial yeast artificial chromosome contig of 13 clones spanning this region was generated. Further linkage analysis of an extended kindred refined the candidate interval to 1-2 cM, and key recombination events now place the QTL within a region of <800 kb. We describe a high-resolution bacterial clone contig spanning 3 Mb covering this critical region. The map consists of 223 bacterial artificial chromosome (BAC) and 100 P1 artificial chromosome (PAC) clones ordered by sequence-tagged site (STS) content and restriction fragment fingerprinting with a minimum tiling path of 22 BACs and 1 PAC. A total of 194 STSs map to this interval of 3 Mb, giving an average marker resolution of approximately one per 15 kb. About half of the markers were novel and were isolated in the present study, including three CA repeats and 13 single nucleotide polymorphisms. Altogether 24 expressed sequence tags, 6 of which are unique genes, have been mapped to the contig.     

A BAC contig map over the proximal approximately 3.3 Mb region of horse chromosome 21

A total of 207 BAC clones containing 155 loci were isolated and arranged into a map of linearly ordered overlapping clones over the proximal part of horse chromosome 21 (ECA21), which corresponds to the proximal half of the short arm of human chromosome 19 (HSA19p) and part of HSA5. The clones form two contigs - each corresponding to the respective human chromosomes - that are estimated to be separated by a gap of approximately 200 kb. Of the 155 markers present in the two contigs, 141 (33 genes and 108 STS) were generated and mapped in this study. The BACs provide a 4-5x coverage of the region and span an estimated length of approximately 3.3 Mb. The region presently contains one mapped marker per 22 kb on average, which represents a major improvement over the previous resolution of one marker per 380 kb obtained through the generation of a dense RH map for this segment. Dual color fluorescence in situ hybridization on metaphase and interphase chromosomes verified the relative order of some of the BACs and helped to orient them accurately in the contigs. Despite having similar gene order and content, the equine region covered by the contigs appears to be distinctly smaller than the corresponding region in human (3.3 Mb vs. 5.5-6 Mb) because the latter harbors a host of repetitive elements and gene families unique to humans/primates. Considering limited representation of the region in the latest version of the horse whole genome sequence EquCab2, the dense map developed in this study will prove useful for the assembly and annotation of the sequence data on ECA21 and will be instrumental in rapid search and isolation of candidate genes for traits mapped to this region.     

Mapping of 12 translocation breakpoints in the Xp21 region with respect to the locus for Duchenne muscular dystrophy

Over 20 females have been reported to carry reciprocal X; autosome translocations with breakpoints in Xp21 and to suffer from Duchenne muscular dystrophy (DMD). We have positioned nine of these breakpoints with respect to the Duchenne gene by mapping probes from the DMD region against a panel of somatic cell hybrids, each containing one of the translocation chromosomes from a different female patient; further information has also been obtained by in situ hybridization, including the breakpoint location in a tenth DMD patient. We have also characterized two translocation breakpoints that lie in the same chromosomal region but which are not associated with the expression of DMD. All the DMD-associated translocation breakpoints examined lie at several sites within the DMD locus and between the two non-DMD breakpoints.     

Human chromosome 17 NotI linking clones and their use in long-range restriction mapping of the Miller-Dieker chromosome region (MDCR) in 17p13.3

A NotI linking library constructed from flow-sorted human chromosome 17 material was screened to aid in construction of a long-range restriction map of the Miller-Dieker chromosome region (MDCR) in 17p13.3. A total of 66 clones were mapped to one of eight regions of chromosome 17 using a somatic cell hybrid panel, and 44/66 (67%) of these clones cross-hybridized to rodent DNA on Southern blots. Of these, 24 clones were tested and all mapped to mouse chromosome 11, the homolog of human chromosome 17. Four linking clones mapped to 17p13.3 and were used for pulsed-field gel electrophoresis studies along with six other anonymous probes previously mapped to this region. Clone L132 was found to be deleted in all Miller-Dieker patients tested (n = 15) and therefore lies within the critical region for this disorder. It detects two NotI fragments (180 and 320 kb), one of which (320 kb) was shared by YNZ22 and YNH37, two probes previously shown to be co-deleted in all patients with the Miller-Dieker syndrome (MDS). These results indicate that all MDS patients share a minimum deletion region of greater than 370 kb. Two other NotI clones, L53 and L125, mapped telomeric to the MDS critical region and share a 600-kb MluI fragment with each other and with YNZ22/YNH37. This provides a 930-kb MluI map that encompasses the distal boundary of the MDS critical region but does not include the proximal boundary. A total of over 2 Mbp is represented in the MluI fragments by probes in subband p13.3, a cytogenetic region estimated to be 3-4 Mbp.     

Superhelicity induces hypersensitivity of a human polypyrimidine . polypurine DNA sequence in the human alpha 2-alpha 1 globin intergenic region to S1 nuclease digestion--high resolution mapping of the clustered cleavage sites.

Supercoiled recombinant DNAs containing the human adult alpha-globin gene region have been probed with nuclease S1 in vitro. While agarose gel electrophoresis showed only one predominant, double-stranded cleavage generated by S1 within 6 kb of human DNA and 4 kb of pBR322 sequence, a high resolution gel analysis reveals that the unique S1-hypersensitive locus in the human adult alpha-globin gene region actually contains more than 15 authentic S1 cleavage sites closely spaced together. The mapping approach used here locates the specific S1 cleavage sites on both DNA strands at the nucleotide sequence level. Interestingly, most of these sites are mapped within a 90 bp stretch of GC-rich (66%) polypyrimidine . polypurine DNA that is located 1060 to 1150 bp upstream from alpha 1-globin gene. These results provide the first high resolution map of double-stranded S1-cleavage sites induced within a specific DNA sequence under supercoil strain. The distribution and relative cutting frequencies of these sites mapped are consistent with a slippage mechanism in which the simple repeating sequences are organized into base-mismatched duplex on supercoiled DNA.     

Physical mapping at a potential X-linked retinitis pigmentosa locus (RP3) by pulsed-field gel electrophoresis.

A genetic locus (RP3) for X-linked retinitis pigmentosa (XLRP) has been assigned to Xp21 by genetic linkage studies and has been supported by two Xp21 male deletion patients with XLRP. RP3 appears to be the most centromeric of several positioned loci, including chronic granulomatous disease (CGD), McLeod phenotype (XK), and Duchenne muscular dystrophy (DMD). In one patient, BB, the X-chromosome deletion includes RP3 and extends to within the DMD locus. Using a DMD cDNA, the centromeric endpoint of this patient was cloned and used as a starting point for chromosome walking along a normal X chromosome. A single-copy probe, XH1.4, positioned near the centromeric junction but deleted in BB, was used along with a CGD cDNA probe to establish a refined long-range physical map. Both probes recognized a common SfiI fragment of 205 kb. As the CGD gene covers approximately 30-60 kb, the RP3 locus has been restricted to approximately 150-170 kb. A CpG island, potentially marking a new gene, was identified within the SfiI fragment at a position approximately 35 kb from the deletion endpoint in BB.