Localization of the mouse kidney disease (kd) gene to a YAC/BAC contig on Chromosome 10

Mice that are homozygous for the kidney disease (kd) gene on Chromosome (Chr) 10 spontaneously develop a progressive and fatal interstitial nephritis. The disease phenotype is similar to that of the human disease, juvenile nephronophthisis. Using a backcross and intercross breeding strategy and analysis of over 900 resultant progeny, this genetic locus has now been mapped to a minimal co-segregating region of approximately two megabases between D10Mit 193 and D10Mit 38. The location assigned to kd by this study is over 3 cM from the current Mouse Genome Database location. The entire interval has been cloned in yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) clones. Recombinant analysis has permitted assignment of 13 Mit microsatellite markers to positions near or within the region. Two new markers have been identified by using single-strand conformation polymorphism (SSCP) analysis of sequenced BAC ends. Several BAC end sequences align with human BAC clones from Chr 6q21 that contain NR2E1, Snx3, and Ros1. Three murine genes, CD24a, fyn, and ColX reported to map in or near the kd region as defined by this study have been evaluated. Though not definitely excluded, they appear to be unlikely candidates. Received: 23 July 1999 / Accepted 23 June 2000     

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.     

Fine mapping of the circling (cir) gene on the distal portion of mouse chromosome 9

Circling mice manifest profound deafness, head-tossing, and bi-directional circling behavior, which they inherit in autosomal recessive manner. Histologic examination of the inner ear reveals abnormalities of the region around the organ of Corti, spiral ganglion neurons, and outer hair cells. A genetic linkage map was constructed for an intraspecific backcross between cir and C57BL/6J mice. The cir gene was mapped to a region between D9Mit116/D9Mit15 and D9Mit38 on mouse chromosome (Chr) 9. Estimated distances between cir and D9Mit116, and between cir and D9Mit38 were 0.70 +/- 0.40 and 0.23 +/- 0.23 cM, respectively. Order of the markers was defined as follows: centromere - D9Mit182 - D9Mit51/D9Mit79/D9Mit310 - D9Mit212/D184 - D9Mit116/D9Mit15 - cir - D9Mit38 - D9Mit20 - D9Mit243 - D9Mit16 - D9Mit55/D9Mit125 - D9Mit281. On the basis of genetic mapping, we constructed a yeast artificial chromosome (YAC) contig across the cir region. The cir gene is located between the lactotransferrin (ltf) and microtubule-associated protein (map4) genes. The distal portion of mouse Chr 9 encompassing the cir region is homologous with human chromosome 3p21, which contains the Deafness, form B: Autosomal Recessive Deafness (DFNB6) locus. Therefore, the circling mouse is a potential animal model for DFNB6 deafness in humans.     

Allelic loss mapping and physical delineation of a region harboring a thymic lymphoma suppressor gene on mouse chromosome 16

Our previous mapping of allelic loss in gamma-ray induced thymic lymphomas in F(1) hybrid and backcross mice between BALB/c and MSM strains identified three regions with high frequencies of allelic loss which probably harbor a tumor suppressor gene. One region, Tlsr7, exists near the D16 Mit122 locus on chromosome 16. This study has further localized Tlsr7 by constructing a physical map and scanning a total of 587 thymic lymphomas. The map consists of 13 overlapping BAC clones and isolation of BAC-derived polymorphic probes leads to fine mapping of allelic losses. Eleven lymphomas show informative breakpoints of allelic loss regions relative to the flanking markers on the map. Pulsed-field gel electrophoresis of NotI digests of the clones shows that the commonly lost region is localized within an approximately 300 kb interval near D16Mit192. This map is invaluable to facilitate the identification of genes in the Tlsr7 region.     

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.     

A sequence-ready BAC/PAC contig and partial transcript map of approximately 1.5 Mb in human chromosome 17q25 comprising multiple disease genes

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant recurrent neuropathy mapped to a 4-cM interval on chromosome 17q25 between the short tandem repeat (STR) markers D17S1603 and D17S802. Chromosome 17q25 in general and the 4-cM HNA region in particular are also implicated in the pathogenesis of a number of tumors (tylosis with esophageal cancer, sporadic breast and ovarian tumors) and harbor a psoriasis susceptibility locus. Initial attempts to construct a yeast artificial chromosome contig failed. Therefore, we have now constructed a complete P1 artificial chromosome (PAC) and bacterial artificial chromosome (BAC) contig of the region flanked by the STR markers D17S1603 and D17S802. The contig contains 22 PAC and 64 BAC clones and covers a physical distance of approximately 1. 5 Mb. A total of 83 sequence-tagged site (STS) markers (10 known STSs and STRs, 56 STSs generated from clone end-fragments, 12 expressed sequence tags, and 5 known genes) were mapped on the contig, resulting in an extremely dense physical map with approximately 1 STS per 20 kb. This sequence-ready PAC and BAC contig will be pivotal for the positional cloning of the HNA gene as well as other disease genes mapping to this region.     

A 3-Mb Sequence-Ready Contig Map Encompassing the Multiple Disease Gene Cluster on Chromosome 11q13.1-q13.3

Despite the presence of several human disease genes on chromosome11q13, few of them have been molecularly cloned. Here, we reportthe construction of a contig map encompassing 11q13.1–q13.3using bacteriophage P1 (P1), bacterial artificial chromosome(BAC), and P1-derived artificial chromosome (PAC). The contigmap comprises 32 P1 clones, 27 BAC clones, 6 PAC clones, and1 YAC clone and spans a 3-Mb region from D11S480 to D11S913.The map encompasses all the candidate loci of Bardet-Biedlesyndrome type I (BBS1) and spinocerebellar ataxia type 5 (SCA5),one-third of the distal region for hereditary paraganglioma2 (PGL2), and one-third of the central region for insulin-dependentdiabetes mellitus 4 (IDDM4). In the process of map construction,61 new sequence-tagged site (STS) markers were developed fromthe Not I linking clones and the termini of clone inserts. Wehave also mapped 30 ESTs on this map. This contig map will facilitatethe isolation of polymorphic markers for a more re.ned analysisof the disease gene region and identi.cation of candidate genesby direct cDNA selection, as well as prediction of gene functionfrom sequence information of these bacterial clones.     

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.     

Autistic disorder and chromosome 15q11-q13: construction and analysis of a BAC/PAC contig

Autistic disorder (AD) is a neurodevelopmental disorder that affects approximately 2-10/10,000 individuals. Chromosome 15q11-q13 has been implicated in the genetic etiology of AD based on (1) cytogenetic abnormalities; (2) increased recombination frequency in this region in AD versus non-AD families; (3) suggested linkage with markers D15S156, D15S219, and D15S217; and (4) evidence for significant association with polymorphisms in the gamma-aminobutyric acid receptor subunit B3 gene (GABRB3). To isolate the putative 15q11-q13 candidate AD gene, a genomic contig and physical map of the approximately 1.2-Mb region from the GABA receptor gene cluster to the OCA2 locus was generated. Twenty-one bacterial artificial chromosome (BAC) clones, 32 P1-derived artificial chromosome (PAC) clones, and 2 P1 clones have been isolated using the markers D15S540, GABRB3, GABRA5, GABRG3, D15S822, and D15S217, as well as 34 novel markers developed from the end sequences of BAC/PAC clones. In contrast to previous findings, the markers D15S822 and D15S975 have been localized within the GABRG3 gene, which we have shown to be approximately 250 kb in size. NotI and numerous EagI restriction enzyme cut sites were identified in this region. The BAC/PAC genomic contig can be utilized for the study of genomic structure and the identification and characterization of genes and their methylation status in this autism candidate gene region on human chromosome 15q11-q13.     

Refining the DFNB17 interval in consanguineous Indian families

We previously mapped the DFNB17 locus to a 3-4 cM interval on human chromosome 7q31 in a large consanguineous Indian family with congenital profound sensorineural hearing loss. To further refine this interval, 30 new highly polymorphic markers and 8 SNPs were analyzed against the pedigree. Re-analysis in the original DFNB 17 family and additional data from a second unrelated consanguineous family with congenital deafness found to map to the interval, limited the area of shared homozygosity-by-descent (HBD) to approximately 4 megabase (Mb) between markers D7S2453 and D7S525. Nineteen known genes and over 20 other cDNAs have been identified in the refined DFNB 17 interval, including the SLC26A4 gene. We have analyzed 4 other cochlear-expressed genes that map to the DFNB17 interval as candidate genes. Analysis of coding and splice site regions of these cochlear expressed genes did not reveal any disease causing mutations. Further study of other candidate genes is currently underway.     

Structure and localization of mouse Pmscl1 and Pmscl2 genes

Sera from some patients with polymyositis-scleoderma overlap syndrome (PM-SCL) recognize two antigenically unrelated proteins, PMSCL1 and PMSCL2. Complete mouse Pmscl1 and Pmscl2 cDNA sequences, chromosomal localizations, exon/intron structure, and promoter region sequences of the mouse Pmscl2 gene are presented. The PMSCL1 gene was found to overlap significantly with cyclin A2 in both human and mouse. As such, it may be deduced that PMSCL1 sequences map to human chromosome 4q27 and the proximal portion of mouse chromosome (Chr) 3 where human and mouse cyclin A2 genes reside. Analysis of human and mouse PMSCL1 cDNA sequences provides evidence that the PMSCL1 protein is 68 amino acids longer than previously thought. A BAC containing mouse Pmscl2 was localized to distal mouse Chr 4 by FISH. This BAC contains the microsatellite D4Mit310. D4Mit310 colocalizes with a number of genes that map to human 1p36. In fact, a STS (G25404) located 54.6 cR from the top of human chromosome 1 was found to contain PMSCL2 sequence upon BLAST search.     

A Radiation Hybrid Map of Mouse Chromosome 13

A mouse radiation hybrid (RH) panel was used to make a framework map for the entire length of mouse chromosome (Chr) 13. Forty-one loci were typed, and while most used primers flanking simple sequence repeats, some genes were included. The most proximal and distal loci are D13Mit132 and D13Mit35. The estimate of map length for Chr 13 is 1328 cR. The map is compared with the same set of loci from the consensus map for Chr 13, which is 70 cM in length, and also with a recombinational map derived from an intraspecies cross typed for many of the same loci. The mouse RH panel gave good resolution for Chr 13 and at the distal end allowed separation of previously nonrecombinant markers that are present on a single 620-kb YAC clone. Data analysis was performed using the RH option for Map Manager QT. This framework RH map of Chr 13 is the second of a series of RH maps for mouse chromosomes.     

The positions of 12 simple sequence repeat markers relative to reference loci on mouse Chromosome 16

The genetic map positions of 12 simple sequence repeat (SSR) markers spanning mouse Chromosome (Chr) 16 were determined relative to reference markers on that chromosome. Interval mapping data were obtained with a panel of DNAs from two intersubspecific backcrosses. All but one of the markers were typed by use of nonradioactive polymerase chain reaction (PCR) products analyzed on agarose gels. The marker order was determined to be Prm-1, D16Mit9, Igl-1, D16Mit29, D16Mit1/D16Mit2, Smst, D16Mit4, D16Mit11, Gap43, D16Mit14, D16Mit30, D16Mit5, Pit-1, D16Mit27, D16H21S16 (formerly D21S16h), D16Mit19, App, D16Mit7, Sod-1. Two of these markers mapped to the known human Chr 21 (HSA21)/Chr 16 conserved linkage group. Nine additional SSR markers could not be typed because they were not polymorophic (four markers), did not amplify MOLD/Rk DNA (three markers), or failed to give PCR products under a range of conditions (two markers). A subset of the most robust SSRs provide a useful marker set for the analysis of previously unmapped crosses.