A radiation hybrid map of 48 loci including the clouston hidrotic ectodermal dysplasia locus in the pericentromeric region of chromosome 13q

To facilitate the identification of the gene responsible for Clouston hidrotic ectodermal dysplasia (HED), we used a chromosome 13-specific radiation hybrid panel to map 54 loci in the HED candidate region. The marker retention data were analyzed using RHMAP version 3. The 54 markers have an average retention frequency of 31.6% with decreasing retention as a function of distance from the centromere. Two-point analysis identified three linkage groups with a threshold lod score of 4.00; one linkage group consisted of 49 loci including the centromeric marker D13Z1 and the telomeric flanking marker for the HED candidate region D13S143. Assuming a centromeric retention model, multipoint maximum likelihood analysis of these 49 loci except D13Z1 provided a 1000:1 framework map ordering 29 loci with 21 unique map positions and approximately 2000 times more likely than the next order. Loci that could not be ordered with this level of support were positioned within a range of adjacent intervals. This map spans 347 cR9000, has an average resolution of 17.3 cR9000, and includes 3 genes (TUBA2, GJbeta2, and FGF-9), 18 ESTs, 19 polymorphic loci, and 8 single-copy DNA segments. Comparison of our RH map to a YAC contig showed an inconsistency in order involving a reversed interval of 6 loci. Fiber-FISH and FISH on interphase nuclei analyses with PACs isolated from this region supported our order. We also describe the isolation of 8 new chromosome 13q polymorphic (CA)n markers that have an average PIC value of 0.67. These data and mapping reagents will facilitate the isolation of disease genes from this region.     

Molecular mapping of the shrunken endosperm genes seg8 and sex1 in barley (Hordeum vulgare L.).

A number of mutations affecting seed development in barley (Hordeum vulgare L.) have been known for many years; however, to date, no research has been reported that elucidates the molecular structure of the causal genes. As a first step, we initiated the linkage mapping of the two shrunken endosperm genes seg8 and sex1 using microsatellite markers. The recessive gene seg8 was mapped in the centromeric region of chromosome 7H to a 4.6 cM interval flanked by markers GBM1516 and Bmag341. The recessive sex1 gene showed xenia effects and was located in the centromeric region of barley chromosome 6H, which is in accordance to the previously reported chromosomal location in the classical linkage map. It was flanked by markers GBM5012 and GBM1063 in a 4.2 cM interval. EST-derived microsatellite markers were used to establish the syntenic relationships to the genomic rice sequences. Two orthologous sites on rice chromosome 2 flanking a 4.1 Mb sequence had homology to the respective barley markers in the sex1 region. For the markers in the seg8 region orthologous sites on rice chromosome 6 were detected.     

A refined comparative map between porcine chromosome 13 and human chromosome 3

We report here the localisation of BAIAP1 (13q24), HTR1F (13q45), PTPRG (13q23) and UBE1C (13q24) by fluorescence in situ hybridisation (FISH), and BAIAP1 (Swr2114; 21 cR; LOD = 11.03), GATA2 (Sw2448; 37 cR; LOD = 8.26), IL5RA (Swr2114; 64 cR; LOD = 3.85), LMCD1 (Sw2450; 61 cR; LOD = 4.73), MME (CP; 50 cR; LOD = 7.75), RYK (Swc22; 12 cR; LOD = 18.62) and SGU003 (Sw1876; 6 cR; LOD = 16.99) by radiation hybrid (RH) mapping to porcine chromosome 13 (SSC13). The mapping of these 10 different loci (all mapped to human chromosome 3; HSA3) not only confirms the extended conservation of synteny between HSA3 and SSC13, but also defines more precisely the regions with conserved linkage. The syntenic region of the centromeric part of SSC13 was determined by isolating porcine bacterial artificial chromosome (BAC) clones (842D4 and 1031H1) using primers amplifying porcine microsatellite markers S0219 and S0076 (mapped to this region). Sequence comparison of the BAC end sequences with the human genome sequence showed that the centromeric part of SSC13 is homologous with HSA3p24.     

Radiation-associated loss of heterozygosity at the Znfn1a1 (Ikaros) locus on chromosome 11 in murine thymic lymphomas

Although information on the molecular pathways in radiation carcinogenesis is accumulating, the data are still relatively scanty. To find the tumor suppressor locus associated with radiation carcinogenesis, we determined the frequency and distribution of loss of heterozygosity (LOH) of X-ray-induced thymic lymphomas of B6C3F(1) mice using 58 microsatellite markers and compared the results with those for spontaneous lymphomas and N-ethylnitrosourea (ENU)-induced lymphomas. Based on the results, we describe a unique locus with frequent LOH in the centromeric region of chromosome 11 of X-ray-induced lymphomas. This locus has never been observed to be altered similarly in either ENU-induced or spontaneous lymphomas, suggesting radiation-specific molecular alteration. The LOH patterns of individual thymic lymphomas indicated that the common region of LOH was located within 1.6 cM between D11Mit62 and D11Mit204, a region syntenic to human chromosome 7p13. Linkage analysis revealed that the markers of the common LOH region were genetically linked to Ikaros (now known as Znfn1a1), a master gene of lymphopoiesis. Although the presence of radiation-associated LOH in other loci cannot be ruled out, these results suggest a novel molecular pathway in induction of thymic lymphomas by ionizing radiation.     

Fine mapping of a region of rat chromosome 12 close to the aspermia (as) locus and comparison with the human orthologous regions.

The aspermia mutation of the rat exhibits male sterility caused by arrest of spermatogenesis, which is controlled by an autosomal single recessive gene (as). The as locus has been mapped on rat chromosome 12. We recently identified a causative mutation for the aspermia phenotype of the as homozygous rats in the gene encoding Fkbp6, a member of the immunophilins FK506 binding proteins. In this paper, we report the fine mapping of the as locus by linkage analysis combined with comparative mapping using rat, mouse, and human genomic sequences and expression analysis of genes located in the as region. We constructed a fine linkage map of the region of rat chromosome 12 close to the as locus by using 13 microsatellite markers and localized the as locus to a 1.0-cM interval. Comparison of the linkage map with physical maps of rat, mouse, and human refined the as critical region in a 2.2-Mb segment of the rat physical map between the D12Nas3 and D12Nas8 genes, which includes the Fkbp6 gene. A centromeric part of this segment corresponds to the region commonly deleted in Williams syndrome, a human complex developmental disorder, on human chromosome 7q11.23. The expression analysis of 23 genes located on the 2.2-Mb segments in various mouse tissues identified genes exclusively or strongly expressed in the testis.     

Construction of a GT polymorphism map of human 9q.

To construct a framework map of human chromosome 9 consisting of highly informative markers, we identified 36 cosmid clones from chromosome 9 that contained long GT repeat sequences. The cosmids were found to cluster on the long arm of the chromosome, particularly in the q32-34 region. Thirteen highly informative polymorphisms from 9q were identified, with median observed heterozygosity 0.75 and median calculated heterozygosity based upon allele frequencies of 0.75. These new GT repeat polymorphisms (D9S56, D9S58-67), as well as anchor GT polymorphisms for D9S15 (MCT112, 9q13), and ABL and ASS (both 9q34.1) were utilized to construct a linkage map of human 9q by the typing of the Venezuelan Reference Pedigree. Care was taken to avoid errors, including analysis of the data with CHROMLOOK and verification of all double crossover events detected within a 30 cM interval by repetition of the marker analysis. The map was generated using the MAPMAKER program. All positions in the resulting map are favored by odds of greater than 10(4):1. The map has a sex-averaged length of 90 cM (Kosambi function) with a single maximum intermarker recombination fraction of 26%. All other intermarker recombination fractions are less than 15%. As D9S15 is known to be closely linked to markers on proximal 9p, and ASS/ABL are in band 34.1, this set of GT polymorphisms spans the length of 9q and provides a useful panel for linkage analysis of disease genes to this region. The marker order was confirmed by in situ hybridization of the cosmid clones to metaphase spreads of normal human chromosomes, which indicated an excess of recombination in the telomeric region in comparison to centromeric 9q, in agreement with previous chiasmata distribution observations. Two spontaneous new mutations for these GT repeat markers were identified, giving an overall observed spontaneous mutation rate of 0.00045 per locus per gamete. Direct observation of new mutations has not been previously reported for dinucleotide polymorphisms, but the observed rate is consistent with frequencies observed for other VNTR polymorphisms.     

An 800-kb region of deletion at 13q14 in human prostate and other carcinomas

Deletions of regions at 13q14 have been detected by various genetic approaches in human cancers including prostate cancer. Several studies have defined one region of loss of heterozygosity (LOH) at 13q14 that seems to reside in a DNA segment of 7.1 cM between genetic markers D13S263 and D13S153. To define the smallest region of overlap (SRO) for deletion at 13q14, we first applied tissue microdissection and multiplex PCR to detect homozygous deletion and/or hemizygous deletion at 13q14 in 134 prostate cancer specimens from 114 patients. We detected deletions at markers D13S1227, D13S1272, and A005O48 in 13 (10%) of these tumor specimens. Of the 13 tumors with deletions, 12 were either poorly differentiated primary tumors or metastases of prostate cancer. To fine-map the deletion region, we then constructed a high-resolution YAC/BAC/STS/EST physical map based on experimental and database analyses. Several markers encompassing the deletion region were analyzed for homozygous deletion and/or hemizygous deletion in 61 cell lines/xenografts derived from human cancers of the prostate, breast, ovary, endometrium, cervix, and bladder, and a region of deletion was defined by duplex PCR assay between markers A005X38 and WI-7773. We also analyzed LOH at 13q14 in the 61 cell lines/xenografts using the homozygosity mapping of deletion approach and 26 microsatellite markers. We found 24 (39%) of the cell lines/xenografts to show LOH at 13q14 and defined a region of LOH by markers M1 and M5. Combination of homozygous or hemizygous deletion and LOH results defined the SRO for deletion to be an 800-kb DNA interval between A005X38 and M5. There are six known genes located in or close to the SRO for deletion. This region of deletion is at least 2 Mb centromeric to the RB1 tumor-suppressor gene and the leukemia-associated genes 1 and 2, each of which is located at 13q14. These data suggest that the 800-kb DNA segment with deletion contains a gene whose deletion may be important for the development of prostate and other cancers. This study also provides a framework for the fine-mapping, cloning, and identification of a novel tumor-suppressor gene at 13q14.     

Loss of NOTCH2 positively predicts survival in subgroups of human glial brain tumors

The structural complexity of chromosome 1p centromeric region has been an obstacle for fine mapping of tumor suppressor genes in this area. Loss of heterozygosity (LOH) on chromosome 1p is associated with the longer survival of oligodendroglioma (OD) patients. To test the clinical relevance of 1p loss in glioblastomas (GBM) patients and identifiy the underlying tumor suppressor locus, we constructed a somatic deletion map on chromosome 1p in 26 OG and 118 GBM. Deletion hotspots at 4 microsatellite markers located at 1p36.3, 1p36.1, 1p22 and 1p11 defined 10 distinct haplotypes that were related to patient survival. We found that loss of 1p centromeric marker D1S2696 within NOTCH2 intron 12 was associated with favorable prognosis in OD (P = 0.0007) as well as in GBM (P = 0.0175), while 19q loss, concomitant with 1p LOH in OD, had no influence on GBM survival (P = 0.918). Assessment of the intra-chromosomal ratio between NOTCH2 and its 1q21 pericentric duplication N2N (N2/N2N-test) allowed delineation of a consistent centromeric breakpoint in OD that also contained a minimally lost area in GBM. OD and GBM showed distinct deletion patterns that converged to the NOTCH2 gene in both glioma subtypes. Moreover, the N2/N2N-test disclosed homozygous deletions of NOTCH2 in primary OD. The N2/N2N test distinguished OD from GBM with a specificity of 100% and a sensitivity of 97%. Combined assessment of NOTCH2 genetic markers D1S2696 and N2/N2N predicted 24-month survival with an accuracy (0.925) that is equivalent to histological classification combined with the D1S2696 status (0.954) and higher than current genetic evaluation by 1p/19q LOH (0.762). Our data propose NOTCH2 as a powerful new molecular test to detect prognostically favorable gliomas.     

Rat Chromosome 2: assignment of the genes encoding cyclin B1, interleukin 6 signal transducer, and proprotein convertase 1 to the Mcs1-containing region and identification of new microsatellite markers

The rat Chromosome (Chr) 2 harbors several genes controlling tumor growth or development, blood pressure, and non-insulin-dependent diabetes mellitus. We report that the region (2q1) containing the mammary susceptibility cancer gene Mcs1 also harbors the genes encoding cyclin B1, interleukin 6 signal transducer (gp130), and proprotein convertase 1. We also generated 13 new anonymous microsatellite markers from Chr 2-sorted DNA. These markers, as well as a microsatellite marker in the cyclin B1 gene, were genetically mapped in combination with known markers. A cyclin B1-related gene was also cytogenetically assigned to rat Chr 11q22-q23. Received: 21 July 1998 / Accepted: 28 August 1998     

Identification of three new microsatellite markers in the spinocerebellar ataxia type 2 (SCA2) region and 1.2 Mb physical map

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease recently mapped to chromosome 12q close to the locus D12S84 by genetic linkage analysis. To generate additional genetic markers in the SCA2 region, we constructed a physical map of the region using yeast artificial chomosome (YAC), P1 artificial chromosome (PAC) and cosmid clones. The physical map was found to agree well with the genetic map. Three novel microsatellite markers were isolated and physically mapped. A novel approach to isolate CAG repeats directly from YAC DNAs is described. Received: 25 January 1995 / Revised: 26 September 1995     

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.     

Genetic analysis of meiotic recombination in humans by use of sperm typing: reduced recombination within a heterozygous paracentric inversion of chromosome 9q32-q34.3.

To investigate patterns of genetic recombination within a heterozygous paracentric inversion of chromosome 9 (46XY inv[9] [q32q34.3]), we performed sperm typing using a series of polymorphic microsatellite markers spanning the inversion region. For comparison, two donors with cytogenetically normal chromosomes 9, one of whom was heterozygous for a pericentric chromosome 2 inversion (46XY inv[2] [p11q13]), were also tested. Linkage analysis was performed by use of the multilocus linkage-analysis program SPERM, and also CRI-MAP, which was adapted for sperm-typing data. Analysis of the controls generated a marker order in agreement with previously published data and revealed no significant interchromosomal effects of the inv(2) on recombination on chromosome 9. FISH employing cosmids containing appropriate chromosome 9 markers was used to localize the inversion breakpoint of inv(9). Analysis of inv(9) sperm was performed by use of a set of microsatellite markers that mapped centromeric to, telomeric to, and within the inversion breakpoints. Three distinct patterns of recombination across the region were observed. Proximal to the centromeric breakpoint, recombination was similar to normal levels. Distal to the telomeric breakpoint, there was an increase in recombination found in the inversion patient. Finally, within the inversion, recombination was dramatically reduced, but several apparent double recombinants were found. A putative model explaining these data is proposed.     

A high-density microsatellite map of the ataxia-telangiectasia locus

The locus of the autosomal recessive disorder ataxia-telangiectasia (A-T) has been assigned by linkage analysis with biallelic markers to a 4-Mb interval on chromosome 11q22-23, between GRIA4 and D11S1897. We have undertaken to saturate the A-T region with highly polymorphic microsatellite markers. To this end, we have identified seven new polymorphic CA-repeats in this region, and have mapped to it five new markers generated by Genethon and the Cooperative Human Linkage Center. These markers are in addition to 12 others that we have previously mapped or generated at the A-T locus. All 24 markers have been integrated into a high-density microsatellite map spanning some 6 Mb DNA. This map, which contains the A-T locus and flanking sequences, allows the construction of extensive, highly informative haplotypes.     

Association of the chromosomal region 2q35 with type 1 diabetes mellitus in the Russian patients from Moscow

To map human chromosome 2 region associated with type 1 diabetes mellitus, 89 families with concordant and discordant sib pairs were analyzed. Linkage and association with type 1 diabetes were examined using polymorphic microsatellite markers spanning the region of about 4 Mb. The linkage plot was constructed, and association of the five microsatellite markers within the chromosomal region 2q35 was examined. Polymorphic marker D2S137 (Z' = 3.225, p(c) = 0.0048) demonstrated maximum linkage and association with type 1 diabetes.     

Genomic mapping of chromosomal region 2p15-p21 (D2S378-D2S391): integration of Genemap'98 within a framework of yeast and bacterial artificial chromosomes

The region of chromosome 2 encompassed by the polymorphic markers D2S378 (centromeric) and D2S391 (telomeric) spans an approximately 10-cM distance in cytogenetic bands 2p15-p21. This area is frequently involved in cytogenetic alterations in human cancers. It also harbors the genes for several genetic disorders, including Type I hereditary nonpolyposis colorectal cancer (HNPCC), familial male precocious puberty (FMPP), Carney complex (CNC), Doyne's honeycomb retinal dystrophy (DHRD), and one form of familial dyslexia (DYX-3). Only a handful of known genes have been mapped to 2p16. These include MSH2, which is responsible for HNPCC, FSHR, the gene responsible for FMPP, EFEMP-1, the gene mutated in DHRD, GTBP, a DNA repair gene, and SPTBN1, nonerythryocytic beta-spectrin. The genes for CNC and DYX-3 remain unknown, due to lack of a contig of this region and its underrepresentation in the existing maps. This report presents a yeast- and bacterial-artificial chromosome (YAC and BAC, respectively) resource for the construction of a sequence-ready map of 2p15-p21 between the markers D2S378 and D2S391 at the centromeric and telomeric ends, respectively. The recently published Genemap'98 lists 146 expressed sequence tags (ESTs) in this region; we have used our YAC-BAC map to place each of these ESTs within a framework of 40 known and 3 newly cloned polymorphic markers and 37 new sequence-tagged sites. This map provides an integration of genetic, radiation hybrid, and physical mapping information for the region corresponding to cytogenetic bands 2p15-p21 and is expected to facilitate the identification of disease genes from the area.     

Genetic and radiation hybrid mapping of the hyperekplexia region on chromosome 5q.

Hyperekplexia, or startle disease (STHE), is an autosomal dominant neurologic disorder characterized by muscular rigidity of central nervous system origin, particularly in the neonatal period, and by an exaggerated startle response to sudden, unexpected acoustic or tactile stimuli. STHE responds dramatically to the benzodiazepine drug clonazepam, which acts at gamma-aminobutyric acid type A (GABA-A) receptors. The STHE locus (STHE) was recently assigned to chromosome 5q, on the basis of tight linkage to the colony-stimulating factor 1-receptor (CSF1-R) locus in a single large family. We performed linkage analysis in the original and three additional STHE pedigrees with eight chromosome 5q microsatellite markers and placed several of the most closely linked markers on an existing radiation hybrid (RH) map of the region. The results provide strong evidence for genetic locus homogeneity and assign STHE to a 5.9-cM interval defined by CSF1-R and D5S379, which are separated by an RH map distance of 74 centirays (roughly 2.2-3.7 Mb). Two polymorphic markers (D5S119 and D5S209) lie within this region, but they could not be ordered with respect to STHE. RH mapping eliminated the candidate genes GABRA1 and GABRG2, which encode GABA-A receptor components, by showing that they are telomeric to the target region.