Overview of somatic cell hybrid mapping.

This unit provides an introduction to the various types of hybrid panels in use and reviews the status of commercially available panels.

A novel skeletal dysplasia with developmental delay and acanthosis nigricans is caused by a Lys650Met mutation in the fibroblast growth factor receptor 3 gene.

We have identified a novel fibroblast growth factor receptor 3 (FGFR3) missense mutation in four unrelated individuals with skeletal dysplasia that approaches the severity observed in thanatophoric dysplasia type I (TD1). However, three of the four individuals developed extensive areas of acanthosis nigricans beginning in early childhood, suffer from severe neurological impairments, and have survived past infancy without prolonged life-support measures. The FGFR3 mutation (A1949T: Lys650Met) occurs at the nucleotide adjacent to the TD type II (TD2) mutation (A1948G: Lys650Glu) and results in a different amino acid substitution at a highly conserved codon in the kinase domain activation loop. Transient transfection studies with FGFR3 mutant constructs show that the Lys650Met mutation causes a dramatic increase in constitutive receptor kinase activity, approximately three times greater than that observed with the Lys650Glu mutation. We refer to the phenotype caused by the Lys650Met mutation as "severe achondroplasia with developmental delay and acanthosis nigricans" (SADDAN) because it differs significantly from the phenotypes of other known FGFR3 mutations.

Molecular, radiologic, and histopathologic correlations in thanatophoric dysplasia.

Various mutations in the fibroblast growth factor receptor 3 (FGFR3) gene have recently been reported in thanatophoric dysplasia (TD). We examined the clinical, radiographic, and histologic findings in 91 cases from the International Skeletal Dysplasia Registry and correlated them with the specific FGFR3 mutation. Every case of TD examined had an identifiable FGFR3 mutation. Radiographically, all of the cases with the Lys650Glu substitution demonstrated straight femora with craniosynostosis, and frequently a cloverleaf skull (CS) was demonstrated. In all other cases, the femora were curved, and CS was infrequently present but was occasionally as severe as TD with the Lys650Glu substitution. Histopathologically, all of the cases shared similar abnormalities, but cases with the Lys650Glu substitution had better preservation of the growth plate. Cases with the Tyr373Cys substitution tended to have more severe radiographic manifestations than the Arg248Cys cases, but there was overlap in the phenotypic spectrum between them. One common classification of TD distinguishes affected infants based on the presence or absence of CS. In contrast, and as originally proposed by Langer et al. [1987: Am J Med Genet 3: 167-179], our data suggest that TD can be divided into at least two groups (TD1 and TD2) based on the presence of straight or curved femora. The variable presence of CS and severity of the radiologic and histologic findings in the other substitutions may be due to other genetic, environmental, or stochastic factors.

A radiation hybrid map of human chromosome 5 with integration of cytogenetic, genetic, and transcript maps.

One of the major goals of the human genome project is to establish a physical map of each human chromosome with a density of sequence-tagged site (STS) markers exceeding one every 100 kb. We report here the generation of a human chromosome 5-specific radiation hybrid (RH) map that includes 556 markers. Of these markers, 132 loci are ordered with a maximum likelihood ratio of >1000:1 compared with the next most likely order. An additional 113 loci were ordered relative to these backbone markers with a maximum likelihood ratio of >10:1 but <1000:1. Together, these 245 loci form an ordered framework map for the chromosome. Using this framework, >300 more markers were localized based on two-point analysis with the ordered set. On average, there are 50 markers in common with the RH map presented here and other chromosome 5 maps included in the current whole genome cytogenetic, genetic, and physical maps. The accuracy of all the maps is evident in that there are no more than two discrepancies between any one of them and these data. All of the maps encompassing chromosome 5 complement each other providing excellent STS coverage with >2200 loci combined. The chromosome 5-specific RH map contains 20% of these independent loci. In addition, our RH map contains STSs derived from clones suitable for fluorescent in situ hybridization, allowing alignment to the cytogenetic map. Together, these maps will assist in the assembly of sequence-ready contigs and will aid in the identification of disease loci on chromosome 5 by positional cloning and positional candidate approaches.

A high-resolution physical and transcript map of the Cri du chat region of human chromosome 5p.

A high-resolution physical and transcription map has been generated of a 3.5-Mb region of 5p15.2 that is associated with the Cri du chat (CDC) syndrome. Utilizing a variety of resources including a natural deletion panel, a chromosome specific radiation hybrid panel, and YAC, PAC, and BAC genomic clones we have ordered > 60 STSs within this region. Approximately 45% of these STSs were obtained from publicly available whole genome maps, thus allowing for integration of this map with currently available resources. Thirteen of these markers were ESTs. In addition, > 70 exon trapped products have been mapped on the natural deletion panel and bacterial clone resource. The combination of these resources has allowed for the identification of 17 transcripts within this region, all of which represent candidate genes for CDC. Further characterization of the genomic contig also revealed that this region of 5p15 contains a large number of repetitive elements.

Chimeras of the native form or achondroplasia mutant (G375C) of human fibroblast growth factor receptor 3 induce ligand-dependent differentiation of PC12 cells.

Mutations in the gene for human fibroblast growth factor receptor 3 (hFGFR3) cause a variety of skeletal dysplasias, including the most common genetic form of dwarfism, achondroplasia (ACH). Evidence indicates that these phenotypes are not due to simple haploinsufficiency of FGFR3 but are more likely related to a role in negatively regulating skeletal growth. The effects of one of these mutations on FGFR3 signaling were examined by constructing chimeric receptors composed of the extracellular domain of human platelet-derived growth factor receptor beta (hPDGFR beta) and the transmembrane and intracellular domains of hFGFR3 or of an ACH (G375C) mutant. Following stable transfection in PC12 cells, which lack platelet-derived growth factor (PDGF) receptors, all clonal cell lines, with either type of chimera, showed strong neurite outgrowth in the presence of PDGF but not in its absence. Antiphosphotyrosine immunoblots showed ligand-dependent autophosphorylation, and both receptor types stimulated strong phosphorylation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, an event associated with the differentiative response of these cells. In addition, ligand-dependent phosphorylation of phospholipase Cgamma and Shc was also observed. All of these responses were comparable to those observed from ligand activation, such as by nerve growth factor, of the native PC12 cells used to prepare the stable transfectants. The cells with the chimera bearing the ACH mutation were more rapidly responsive to ligand with less sustained MAPK activation, indicative of a preactivated or primed condition and consistent with the view that these mutations weaken ligand control of FGFR3 function. However, the full effect of the mutation likely depends in part on structural features of the extracellular domain. Although FGFR3 has been suggested to act as a negative regulator of long-bone growth in chrondrocytes, it produces differentiative signals similar to those of FGFR1, to which only positive effects have been ascribed, in PC12 cells. Therefore, its regulatory effects on bone growth likely result from cellular contexts and not the induction of a unique FGFR3 signaling pathway.

Reduced penetrance of the Huntington's disease mutation.

Controversy persists concerning the significance of Huntington disease (HD) alleles in the 36-39 repeat range. Although some clinically affected persons have been documented with repeats in this range, elderly unaffected individuals have also been reported. We examined 10 paternal transmissions of HD alleles of 37-39 repeats in collateral branches of families with de novo HD. All 10 descendants, including many who are elderly, are without symptoms of HD. Forty percent of the transmissions were unstable, although none varied by more than one repeat. The observation that individuals with alleles of 37-39 repeats may survive unaffected beyond common life expectancy supports the presence of reduced penetrance for HD among some persons with repeat sizes which overlap the clinical range. Non-penetrance may be increased in the collateral branches of de novo mutation families when compared to penetrance estimates from patient series. There was no CAA-->CAG mutation for the penultimate glutamine in either a de novo expanded 42 repeat allele or the corresponding non-penetrant 38 repeat allele in a family with fresh mutation to HD.

Treacher Collins syndrome may result from insertions, deletions or splicing mutations, which introduce a termination codon into the gene.

Treacher Collins syndrome is an autosomal dominant disorder of craniofacial development the features of which include conductive hearing loss and cleft palate. Recently, the Treacher Collins syndrome gene (TCOF1) has been positionally cloned and a series of five mutations within the coding sequence of the gene identified. In the current investigation, seven exons of TCOF1 have been identified which has permitted the identification of additional mutations in the gene. The mutations that have been identified are three distinct deletions and an insertion, which cause a frameshift, and a missense mutation which inactivates a donor splice site with extension of transcription into the intron. To date, all 10 of the mutations which have been reported result in a premature termination codon and are unique to a given family. As these mutations are spread throughout the gene, these observations provide further support for the hypothesis that Treacher Collins syndrome results from haploinsufficiency, although a dominant negative effect cannot, at this stage, be excluded.

The evolutionary distribution and structural organization of the homeobox-containing repeat D4Z4 indicates a functional role for the ancestral copy in the FSHD region.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disease that has been linked to deletions within a tandem array of 3.2 kb repeats adjacent to the telomere of 4q. These repeats are also present in other locations in the human genome, including the short arms of all the acrocentric chromosomes. Here, we examine two models for the role of this repeat in FSHD. First, because of the extensive similarity between the 3.2 kb repeats on 4q and those adjacent to rDNA on the acrocentric chromosomes, we investigated whether the FSHD region on 4q is involved in sub-nuclear localization, specifically to the nucleolus. The results likely exclude any involvement of nucleolar localization in the development of FSHD. Second, we investigated a model that suggests that a functional gene may be buried within the tandem array of 3.2 kb repeats. Toward this end, we evaluated the evolutionary conservation of the repeat and a double homeodomain sequence within the repeat in a variety of primate species. The genomic organization of the 3.2 kb repeat in humans, great apes and lower primates identified the FSHD-associated repeat on chromosome 4q as the likely ancestral copy. The sequence of the rhesus monkey double homeodomain reveals significant sequence identity with the human 4q sequence. These results strongly suggest a functional role for a component of the FSHD-associated repeat.

The Ras GTPase-activating-protein-related human protein IQGAP2 harbors a potential actin binding domain and interacts with calmodulin and Rho family GTPases.

We previously described IQGAP1 as a human protein related to a putative Ras GTPase-activating protein (RasGAP) from the fission yeast Schizosaccharomyces pombe. Here we report the identification of a liver-specific human protein that is 62% identical to IQGAP1. Like IQGAP1, the novel IQGAP2 protein harbors an N-terminal calponin homology motif which functions as an F-actin binding domain in members of the spectrin, filamin, and fimbrin families. Both IQGAPs also harbor several copies of a novel 50- to 55-amino-acid repeat, a single WW domain, and four IQ motifs and have 25% sequence identity with almost the entire S. pombe sar1 RasGAP homolog. As predicted by the presence of IQ motifs, IQGAP2 binds calmodulin. However, neither full-length nor truncated IQGAP2 stimulated the GTPase activity of Ras or its close relatives. Instead, IQGAP2 binds Cdc42 and Racl but not RhoA. This interaction involves the C-terminal half of IQGAP2 and appears to be independent of the nucleotide binding status of the GTPases. Although IQGAP2 shows no GAP activity towards Cdc42 and Rac1, the protein did inhibit both the intrinsic and RhoGAP-stimulated GTP hydrolysis rates of Cdc42 and Rac1, suggesting an alternative mechanism via which IQGAPs might modulate signaling by these GTPases. Since IQGAPs harbor a potential actin binding domain, they could play roles in the Cdc42 and Rac1 controlled generation of specific actin structures.

A contiguous high-resolution radiation hybrid map of 44 loci from the distal portion of the long arm of human chromosome 5.

A contiguous high-resolution map of 44 loci from a 35-Mb portion of the distal region of the long arm of human chromosome 5, q21-q35, was produced using radiation hybrid (RH) mapping in conjunction with a natural deletion mapping panel. The map includes 30 genes, four sequence-tagged site (STS) loci, and 10 DNA markers. Newly mapped markers fill two gap regions that were present in previous maps, between markers FER-IL4 and IL3-IL9. Identifying the position of genes on the physical map aids in positional cloning efforts and contributes to our understanding of the overall organization of the human genome.

Regional assignment and tissue expression of twenty-three expressed sequence tags (ESTs) from human chromosome 5.

Regional localization and expression patterns are reported for 19 expressed sequence tags (ESTs) from human chromosome 5, two of which were derived from the same transcript. Two of the ESTs correspond to genes not previously characterized in humans: a stress-activated protein kinase and nicotinamide nucleotide transhydrogenase. Expression was determined by three methods: Northern blots, PCR from tissue-specific cDNA libraries, and sequence sampling from EST sequencing projects. Six of the ESTs show no expression, and EST01986 appears to be expressed predominantly in the brain by all methods tested.

Genomic organization of the human heparan sulfate-N-deacetylase/N-sulfotransferase gene: exclusion from a causative role in the pathogenesis of Treacher Collins syndrome.

Heparan sulfate-N-deacetylase/N-sulfotransferase (HSST) catalyzes both the N-deacetylation and the N-sulfation of heparan sulfate. Previous studies have resulted in the isolation of the human HSST gene from within the Treacher Collins syndrome locus (TCOF1) critical region on 5q. In the present study, the genomic organization of the HSST gene has been elucidated, and the 14 exons identified have been tested for TCOF1-specific mutations. As a result of these studies, mutations within the coding sequence and adjacent splice junctions of HSST can be excluded from a causative role in the pathogenesis of Treacher Collins syndrome.

The generation and regional localization of 303 new chromosome 5 sequence-tagged sites.

With the ultimate goal of creating a sequence-ready physical map of all of chromosome 5, 303 new human chromosome 5-specific STS markers have been systematically generated and regionally ordered. Chromosome 5 DNA prepared from flow-sorted chromosomes was digested with restriction enzymes BamHI and HindIII and cloned in bacteriophage M13mp18. Random clones were sequenced, and appropriate PCR deoxyoligomers were synthesized. An acceptable sequence-tagged site (STS)-PCR assay yielded the appropriate size amplification product from both total human DNA and hybrid cell line DNA containing only human chromosome 5. Each STS has been regionally localized by breakpoint analysis using a set of hybrid cell panels consisting of natural deletions or translocations of human chromosome 5. This hybrid cell panel was able to localize the STSs to 1 of 51 bins on the short arm and 1 of 15 bins on the long arm. The STS markers appear to be randomly distributed along the length of this 194-Mb chromosome. The current overall density of these markers (approximately 1 STS/640 kb), combined with the numerous PCR-based physical and genetic markers generated by other groups, will provide sufficient "nucleation points" for YAC contig assembly and verification in any region of human chromosome 5.

Transcriptional map of the Treacher Collins candidate gene region.

Treacher Collins syndrome (TCOF1) is a dominant disorder of craniofacial development that has been linked previosuly to a region of chromosome 5q31.3-32. Identification of recombination events in affected individuals has reduced the candidate gene region to a 0.5-Mb area between the loci RPS14 (proximal) and ANX6 [distal]. A transcriptional map of this candidate gene region, generated by analysis of exon amplification clones, has identified the genomic location of four genes, heparan sulfate-N-sulfotransferase-N-deacetylase, glutathione peroxidase, as well as two novel, previously uncharacterized genes. Each of these genes, based on their location, must be considered candidates for TCOF1 locus.

The human and mouse receptors for hyaluronan-mediated motility, RHAMM, genes (HMMR) map to human chromosome 5q33.2-qter and mouse chromosome 11.

The gene for the receptor for hyaluronan-mediated motility, RHAMM (designated hyaluronan-mediated motility receptor, HMMR (human) and Hmmr (mouse), for mapping purposes), was localized to human chromosome 5q33.2-qter by somatic cell and radiation hybrid analyses. Investigation of two interspecific backcrosses localized the mouse RHAMM (Hmmr) locus 18 cM from the centromere of mouse chromosome 11 within a region of synteny homology with human chromosome 5q23-q35 genes. The map position of the human RHAMM gene places it in a region comparatively rich in disease-associated genes, including those for low-frequency hearing loss, dominant limb-girdle muscular dystrophy, diastrophic dysplasia, Treacher Collins syndrome, and myeloid disorders associated with the 5q- syndrome. The RHAMM gene location and its ability to transform cells when overexpressed implicate RHAMM as a possible candidate gene in the pathogenesis of the recently described t(5;14)(q33-q34;q11) acute lymphoblastic leukemias.