Rapid communication: the human FEM1B gene maps to chromosome 15q22 and is excluded as the gene for Bardet-Biedl syndrome, type 4

We have identified a novel human gene, FEM1B, that encodes a protein virtually identical to that encoded by the mouse gene Fem1b. These mammalian proteins are homologs of the FEM-1 protein of Caenorhabditis elegans, which acts as a signal-transduction component within the nematode sex-determination pathway. We report here the mapping of FEM1B to chromosome 15q22, a region that is homologous to the region of mouse chromosome 9, where Fem1b resides. The BBS4 locus, one of the loci causing the autosomal recessive Bardet-Biedl syndrome, maps to this region of chromosome 15. Therefore, we sought to determine whether the FEM1B gene might be involved in this disorder. Radiation hybrid mapping demonstrates that FEM1B does not reside within the interval of chromosome 15 containing the BBS4 locus.     

Conserved synteny in rat and mouse for a blood pressure QTL on human chromosome 17

Evidence for blood pressure quantitative trait loci (QTLs) on rat chromosome 10 has been found in multiple independent studies. Analysis of the homologous region on human chromosome 17 revealed significant linkage to blood pressure. The critical segment on human chromosome 17 spans a large interval containing the genes Itga2b, Gfap, and Itgb3. Therefore, findings in the rat may help to refine the position of blood pressure-regulating loci, assuming a common molecular cause across species. However, it has recently been suggested that the gene order in human, rat, and mouse is not conserved in this region, leaving uncertainty about the overlap of the blood pressure- regulating region between human chromosome 17 and rat chromosome 10. We have performed a detailed comparative analysis among human, mouse, and rat, defining the segment in question, by obtaining gene structure information in silico and by radiation hybrid mapping. It is of interest that this region also contains Wnk4, a gene previously identified to cause pseudohypoaldosteronism type II and human hypertension. Our results definitively show that the conserved synteny extends among human chromosome 17, rat chromosome 10, and mouse chromosome 11, demonstrating an overlap between previously localized blood pressure QTLs in humans and rats.     

Chromosomal localization of glutamate receptor genes: relationship to familial amyotrophic lateral sclerosis and other neurological disorders of mice and humans.

Receptors for the major excitatory neurotransmitter glutamate may play key roles in neurodegeneration. The mouse Glur-5 gene maps to chromosome 16 between App and Sod-1. The homologous human GLUR5 gene maps to the corresponding region of human chromosome 21, which contains the locus for familial amyotrophic lateral sclerosis. This location, and other features, render GLUR5 a possible candidate gene for familial amyotrophic lateral sclerosis. In addition, dosage imbalance of GLUR5 may have a role in the trisomy 21 (Down syndrome). Further characterization of the murine glutamate receptor family includes mapping of Glur-1 to the same region as neurological mutants spasmodic, shaker-2, tipsy, and vibrator on chromosome 11; Glur-2 near spastic on chromosome 3; Glur-6 near waltzer and Jackson circler on chromosome 10; and Glur-7 near clasper on chromosome 4.     

Comparative mapping of DNA markers from the familial Alzheimer disease and Down syndrome regions of human chromosome 21 to mouse chromosomes 16 and 17.

Mouse trisomy 16 has been proposed as an animal model of Down syndrome (DS), since this chromosome contains homologues of several loci from the q22 band of human chromosome 21. The recent mapping of the defect causing familial Alzheimer disease (FAD) and the locus encoding the Alzheimer amyloid beta precursor protein (APP) to human chromosome 21 has prompted a more detailed examination of the extent of conservation of this linkage group between the two species. Using anonymous DNA probes and cloned genes from human chromosome 21 in a combination of recombinant inbred and interspecific mouse backcross analyses, we have established that the linkage group shared by mouse chromosome 16 includes not only the critical DS region of human chromosome 21 but also the APP gene and FAD-linked markers. Extending from the anonymous DNA locus D21S52 to ETS2, the linkage map of six loci spans 39% recombination in man but only 6.4% recombination in the mouse. A break in synteny occurs distal to ETS2, with the homologue of the human marker D21S56 mapping to mouse chromosome 17. Conservation of the linkage relationships of markers in the FAD region suggests that the murine homologue of the FAD locus probably maps to chromosome 16 and that detailed comparison of the corresponding region in both species could facilitate identification of the primary defect in this disorder. The break in synteny between the terminal portion of human chromosome 21 and mouse chromosome 16 indicates, however, that mouse trisomy 16 may not represent a complete model of DS.     

Chromosomal location of three spectrin genes: relationship to the inherited hemolytic anemias of mouse and man.

Three genetic loci in the mouse affect the synthesis and assembly of the erythrocyte membrane skeleton. The spherocytosis and jaundiced loci affect the membrane skeletal protein known as spectrin. The normoblastosis locus affects the spectrin binding protein called ankyrin. We have obtained genetic data that define the linkage relationships among three spectrin genes and the spherocytosis and jaundiced loci. The erythroid alpha-spectrin gene is tightly linked to the spherocytosis locus on chromosome 1 and the jaundiced locus is on chromosome 12, tightly linked to the erythroid beta-spectrin gene. The brain alpha-spectrin (alpha-fodrin) gene is located on the centromeric end of chromosome 2 and is not closely linked to any previously mapped erythroid or neurological mutation. These results are consistent with the hypothesis that defects in the alpha- and beta-spectrin genes cause the spherocytosis and jaundiced hemolytic anemias in mice. All five loci studied are located within chromosomal segments that are conserved between mouse and man. Analysis of the data from the chromosome 12 study defines a new order for the genes on that chromosome and delineates the largest mouse/human conserved chromosomal segment yet known.     

Structural analysis of chromosomal rearrangements associated with the developmental mutations Ph, W19H, and Rw on mouse chromosome 5.

We are studying the chromosomal structure of three developmental mutations, dominant spotting (W), patch (Ph), and rump white (Rw) on mouse chromosome 5. These mutations are clustered in a region containing three genes encoding tyrosine kinase receptors (Kit, Pdgfra, and Flk1). Using probes for these genes and for a closely linked locus, D5Mn125, we established a high-resolution physical map covering approximately 2.8 Mb. The entire chromosomal segment mapped in this study is deleted in the W19H mutation. The map indicates the position of the Ph deletion, which encompasses not more than 400 kb around and including the Pdgfra gene. The map also places the distal breakpoint of the Rw inversion to a limited chromosomal segment between Kit and Pdgfra. In light of the structure of the Ph-W-Rw region, we interpret the previously published complementation analyses as indicating that the pigmentation defect in Rw/+ heterozygotes could be due to the disruption of Kit and/or Pdgfra regulatory sequences, whereas the gene(s) responsible for the recessive lethality of Rw/Rw embryos is not closely linked to the Ph and W loci and maps proximally to the W19H deletion. The structural analysis of chromosomal rearrangements associated with W19H, Ph, and Rw combined with the high-resolution physical mapping points the way toward the definition of these mutations in molecular terms and isolation of homologous genes on human chromosome 4.     

Cognate homeo-box loci mapped on homologous human and mouse chromosomes.

The homeotic genes of Drosophila, which regulate pattern formation during larval development, contain a 180-base-pair DNA sequence termed the "homeo-box." Nucleotide sequence comparisons indicate that the homeo-box motif is highly conserved in a variety of motazoan species. As in Drosophila, homeo-box sequences of mammalian species are expressed in a temporal and tissue-specific pattern during embryogenesis. These observations suggest functional homologies between dipteran and mammalian homeo-box gene products. To identify possible relationships between homeo-box genes of mice and humans, we have compared the chromosomal location of homeo-box genes in these species. Using in situ hybridization and somatic cell genetic techniques, we have mapped the chromosome 6-specific murine Hox-1 homolog to the region p14-p21 on human chromosome 7. We have also regionally mapped the murine Hox-3 locus to 15F1-3 and its human cognate to 12q11-q21. These comparative mapping data indicate that a syntenic relationship in mice and humans is maintained for all homeo-box loci examined to date. We suggest these regions represent evolutionarily conserved genomic domains encoding homologous protein products that function in regulating patterns of mammalian development.     

Polymorphisms in the human haptoglobin gene cluster: chromosomes with multiple haptoglobin-related (Hpr) genes.

We have found polymorphisms for the number of tandemly arranged haptoglobin-related (Hpr) genes in the haptoglobin gene cluster of Blacks. Genomic mapping and nucleotide sequence analysis indicate that two copies of the Hpr gene first resulted from unequal but homologous crossing-over in a region 3' to the haptoglobin (Hp) and the haptoglobin-related genes. Subsequent increases in the number of Hpr loci have occurred in some chromosomes. Among 25 American Blacks studied (15 were unrelated), 2 related individuals have one extra copy of the Hpr gene and 5 unrelated individuals have more than two extra Hpr genes. None of 26 Whites and one Oriental studied have extra copies. In one of the Blacks, six tandemly arranged Hpr genes were demonstrated in one chromosome by pulsed field gradient electrophoresis. His other chromosome had one Hpr gene. The tandem Hpr genes were found in individuals with the haptoglobin genotypes Hp2/Hp2 (3 of 3 tested) and Hp2/Hp1 (4 of 11 tested), but none were found in the Hp1/Hp1 individuals (11 tested). Fibroblast cell cultures from two Hp2/Hp1 heterozygotes were fused to mouse cells to obtain cell lines retaining a human chromosome 16 on which the haptoglobin gene cluster is located. DNA analysis of the hybrid cells showed that in both individuals the tandemly arranged Hpr genes are linked to the Hp2 allele. These results suggest that the multiple copies are associated with the Hp2 gene.     

A 1.5-megabase yeast artificial chromosome contig from human chromosome 10q11.2 connecting three genetic loci (RET, D10S94, and D10S102) closely linked to the MEN2A locus.

The genetic loci RET, D10S94, and D10S102 from human chromosome 10q11.2 are very closely linked to a locus responsible for the multiple endocrine neoplasia type 2 (MEN2A and MEN2B) and medullary thyroid carcinoma (MTC1) familial cancer syndromes. We have constructed a 1.5-megabase contig consisting of six genomic yeast artificial chromosome clones which include these loci and define their physical order. A critical crossover event has been identified within the map interval; this event places the MEN2A locus centromeric to D10S102 and defines the orientation of the physical map on the chromosome. The orientation of the contig and order of the markers are centromere-RET-D10S94-D10S102-telomere. In addition, a microsatellite repeat polymorphism with a heterozygosity of 71% at the RET locus and a restriction fragment length polymorphism with a heterozygosity of 42% detected by a lambda clone from the D10S94 locus have been developed for high-resolution genetic linkage mapping and predictive diagnostic testing. These data place three important markers on a contiguous physical map, narrow the MEN2 disease locus interval, and provide a framework for further candidate gene identification efforts. Placement of these genetic loci along a clone-based map and continued expansion of the contig will also facilitate efforts to determine the relationship of physical to genetic distance near the centromeres of human chromosomes.     

Chromosomal location of the gene encoding the neural cell adhesion molecule (N-CAM) in the mouse.

The gene encoding the neural cell adhesion molecule, N-CAM, has been localized on mouse chromosome 9. A BALB/cJ mouse genomic library prepared in lambda bacteriophage EMBL4 was screened by using a cDNA probe, pEC204, that corresponds to the coding region of the chicken N-CAM gene. Four weakly reactive and one strongly reactive recombinant phage were isolated. A region of the latter that was strongly homologous to pEC204 was subcloned to yield a new probe, pEC501. RNA transfer blots and nucleotide sequencing indicated that pEC501 encoded part of the mouse N-CAM gene. This probe defined a unique genetic locus, Ncam, associated with a restriction fragment length polymorphism that allowed the definition of two alleles. The locus could be provisionally assigned either to chromosome 9 or to chromosome 10 by correlating the presence or absence of mouse-specific DNA fragments reactive with the probe in a panel of somatic hybrid cell lines with the presence or absence of the various mouse chromosomes. Analysis of the inheritance of the Ncam-associated DNA polymorphism in recombinant inbred strains of mice revealed close linkage between Ncam and the Lap-1, Sep-1, and Thy-1 loci on chromosome 9. This result suggests an additional linkage between Ncam and the locus for the cerebellar mutation staggerer (sg). The Ncam locus provides an important reference point for mapping the genes for additional cell adhesion molecules as well as genes for other molecules involved in neural development and function.     

Ankyrin and the hemolytic anemia mutation, nb, map to mouse chromosome 8: presence of the nb allele is associated with a truncated erythrocyte ankyrin.

Mice with normoblastosis, nb/nb, have a severe hemolytic anemia. The extreme fragility and shortened lifespan of the mutant erythrocytes result from a defective membrane skeleton. Previous studies in our laboratory indicated a 50% deficiency of spectrin and an absence of normal ankyrin in erythrocyte membranes of nb/nb mice. We now report genetic mapping data that localize both the nb and erythroid ankyrin (Ank-1) loci to the centromeric end of mouse chromosome 8. Using immunological and biochemical methods, we have further characterized the nature of the ankyrin defect in mutant erythrocytes. We do not detect normal sized (210 kDa) erythroid ankyrin by immunoblot analysis in nb/nb reticulocytes. However, nb/nb reticulocytes do contain a 150-kDa ankyrin immunoreactive protein. The 150-kDa protein is present with normal-sized ankyrin in nb/+ reticulocytes but is not found in +/+ reticulocytes. Our genetic and biochemical data indicate that the nb mutation results from a defect in the erythroid ankyrin gene. A human hereditary spherocytosis putatively resulting from an ankyrin defect maps to a segment of human chromosome 8 that is homologous to the nb-ankyrin region of mouse chromosome 8. The linkage data suggest that the mouse and human diseases result from mutations in homologous loci.     

Direct detection and isolation of restriction landmark genomic scanning (RLGS) spot DNA markers tightly linked to a specific trait by using the RLGS spot-bombing method.

We have developed a technique for isolating DNA markers tightly linked to a target region that is based on RLGS, named RLGS spot-bombing (RLGS-SB). RLGS-SB allows us to scan the genome of higher organisms quickly and efficiently to identify loci that are linked to either a target region or gene of interest. The method was initially tested by analyzing a C57BL/6-GusS mouse congenic strain. We identified 33 variant markers out of 10,565 total loci in a 4.2-centimorgan (cM) interval surrounding the Gus locus in 4 days of laboratory work. The validity of RLGS-SB to find DNA markers linked to a target locus was also tested on pooled DNA from segregating backcross progeny by analyzing the spot intensity of already mapped RLGS loci. Finally, we used RLGS-SB to identify DNA markers closely linked to the mouse reeler (rl) locus on chromosome 5 by phenotypic pooling. A total of 31 RLGS loci were identified and mapped to the target region after screening 8856 loci. These 31 loci were mapped within 11.7 cM surrounding rl. The average density of RLGS loci located in the rl region was 0.38 cM. Three loci were closely linked to rl showing a recombination frequency of 0/340, which is < 1 cM from rl. Thus, RLGS-SB provides an efficient and rapid method for the detection and isolation of polymorphic DNA markers linked to a trait or gene of interest.     

Molecular structure and chromosomal mapping of the human homolog of the agouti gene.

The agouti (a) locus in mouse chromosome 2 normally regulates coat color pigmentation. The mouse agouti gene was recently cloned and shown to encode a distinctive 131-amino acid protein with a consensus signal peptide. Here we describe the cloning of the human homolog of the mouse agouti gene using an interspecies DNA-hybridization approach. Sequence analysis revealed that the coding region of the human agouti gene is 85% identical to the mouse gene and has the potential to encode a protein of 132 amino acids with a consensus signal peptide. Chromosomal assignment using somatic-cell-hybrid mapping panels and fluorescence in situ hybridization demonstrated that the human agouti gene maps to chromosome band 20q11.2. This result revealed that the human agouti gene is closely linked to several traits, including a locus called MODY (for maturity onset diabetes of the young) and another region that is associated with the development of myeloid leukemia. Initial expression studies with RNA from several adult human tissues showed that the human agouti gene is expressed in adipose tissue and testis.     

Linkage map of the short arm of human chromosome 11: location of the genes for catalase, calcitonin, and insulin-like growth factor II.

The following order of genes on the short arm of human chromosome 11 (11p) was determined previously: parathyroid hormone (PTH)-the beta-globin gene cluster (HBBC)-HRAS1/insulin. Although it is generally agreed that HRAS1 (formerly termed c-Ha-ras-1) and the insulin gene are close to each other [1-4 centimorgans (cM)], their order on chromosome 11p is still in question. We have now added three other genes, those for catalase, calcitonin, and insulin-like growth factor II (IGF-II), to this map of chromosome 11p by use of restriction site polymorphisms adjacent to these genes in classical linkage analysis. Most importantly, we find no evidence of linkage between the catalase and HBBC loci. In addition, our data indicate that the calcitonin gene is located between the catalase gene and the PTH gene. Our best estimate of the distance between the catalase and calcitonin gene is approximately 16 cM, while that between the calcitonin and PTH genes is approximately equal to 8 cM. In agreement, very loose linkage was found between the catalase and PTH loci (approximately 26 cM). Since the catalase locus has been mapped to 11p13, these data support the view that the PTH, HBBC, HRAS1, and insulin loci are located on the distal short arm of chromosome 11. The IGF-II gene is tightly linked to both the HRAS1 oncogene and the insulin gene since no recombinants were observed between the IGF-II and the HRAS1/insulin loci. Thus, based on our linkage analysis we propose that the most likely gene order for the short arm of chromosome 11 is centromere-catalase-calcitonin-PTH-HBBC-HRAS1/insulin-tel ome re and that the IGF-II gene is very close to both the HRAS1 and the insulin genes.     

Loss of heterozygosity in sporadic parathyroid tumours: involvement of chromosome 1 and the MEN1 gene locus in 11q13

OBJECTIVE: Hyperparathyroidism (HPT) is a common endocrine disorder. Several loci of genetic interest have been identified in parathyroid tumours, including the MEN1 gene locus at 11q13; the HPT-JT region at 1q21-q32; and a putative tumour suppressor gene on 1p. We analysed these intervals, which harbour known genes or putative loci associated with familial hyperparathyroidism, in order to clarify the involvement of the respective regions in parathyroid tumourigenesis. DESIGN: We performed loss of heterozygosity (LOH) studies on 33 sporadic parathyroid tumours using a PCR based technique. A total of 22 microsatellite markers were used to analyse loci at 11q13, 1q21-q32 and 1p. Ten markers located distal on 1p, eight markers encompassed the HPT-JT region at 1q21-q32 and four markers surrounded the MEN1 gene locus at 11q13. MEN1 mutations were screened for using Single Strand Conformation Polymorphism analysis (SSCP) and automated sequencing of SSCP variants. PATIENTS: Thirty-three parathyroid glands and the corresponding blood samples were obtained from 33 patients (26 females and seven males) who underwent parathyroidectomy for primary hyperparathyroidism. RESULTS: Loss of heterozygosity was detected in 13 of 33 (39%) cases at 11q13, 6 of 33 (18%) cases at 1p, and in three of 33 (9%) cases at 1q (in conjunction with 1p loss). Only one of the 18 tumours in which LOH was detected, showed LOH at both chromosome 1 and chromosome 11. Additionally, those tumours found to exhibit LOH at 11q13 were screened for MEN1 mutations using single strand conformation polymorphism analysis (SSCP) and automated sequencing. Nine novel somatic mutations were found on the remaining allele in 13 (69%) tumours. CONCLUSIONS: This study consolidates the role of multiple loci in the pathogenesis of sporadic parathyroid tumours. The results indicate that there are at least two genetic loci involved in sporadic parathyroid tumourigenesis on chromosome 1, one of which has been linked to the distinct familial parathyroid condition, hyperparathyroidism-jaw tumour (HPT-JT) syndrome. The high frequency of loss of heterozygosity at 1p suggests the presence of a tumour suppressor at this locus.     

Chromosomal localization of the proteasome Z subunit gene reveals an ancient chromosomal duplication involving the major histocompatibility complex.

Proteasomes are the multi-subunit protease thought to play a key role in the generation of peptides presented by major histocompatibility complex (MHC) class I molecules. When cells are stimulated with interferon gamma, two MHC-encoded subunits, low molecular mass polypeptide (LMP) 2 and LMP7, and the MECL1 subunit encoded outside the MHC are incorporated into the proteasomal complex, presumably by displacing the housekeeping subunits designated Y, X, and Z, respectively. These changes in the subunit composition appear to facilitate class I-mediated antigen presentation, presumably by altering the cleavage specificities of the proteasome. Here we show that the mouse gene encoding the Z subunit (Psmb7) maps to the paracentromeric region of chromosome 2. Inspection of the mouse loci adjacent to the Psmb7 locus provides evidence that the paracentromeric region of chromosome 2 and the MHC region on chromosome 17 most likely arose as a result of a duplication that took place at an early stage of vertebrate evolution. The traces of this duplication are also evident in the homologous human chromosome regions (6p21.3 and 9q33-q34). These observations have implications in understanding the genomic organization of the present-day MHC and offer insights into the origin of the MHC.     

Actively transcribed genes in the raf oncogene group, located on the X chromosome in mouse and human.

Murine and human cDNAs, related to but distinct from c-raf-1, have been isolated and designated mA-raf and hA-raf, respectively. The mA-raf and hA-raf cDNAs detect the same murine and human fragments in Southern blots of restriction enzyme-cleaved murine and human cellular DNA. The murine restriction enzyme fragments homologous to mA-raf cDNA cosegregate with mouse chromosome X in a panel of Chinese hamster-mouse hybrid cells, thus localizing the mA-raf locus to mouse chromosome X. Two independently segregating loci, detected by the hA-raf cDNA (or mA-raf cDNA), hA-raf-1 and hA-raf-2, are located on human chromosomes X and 7, respectively. The mA-raf locus and the hA-raf-1 locus are actively transcribed in several mouse and human cell lines.