Comparative mapping on the mouse X Chromosome defines a myotubular myopathy equivalent region

The gene for X-linked myotubular myopathy (MTM1) has been localized to a 300-kb critical region in human Xq28 between IDS and GABRA3. As part of an effort to clone this gene, we developed a YAC contig on the mouse X Chromosome (Chr) which includes loci homologous to those within the human MTM1 critical region. The murine contig consists of 18 YACs and spans 2.5–3.0 Mb. We have aligned the human and murine physical maps by isolating conserved mouse genomic fragments, including CpG islands and trapped exons. We believe that the simultaneous isolation of genes from both mouse and human and continued comparative mapping will prove helpful in the eventual identification of MTM1 and other genes in the region. Received: 9 February 1996 / Accepted: 30 March 1996     

Clinical and genetic heterogeneity in Meckel syndrome

Meckel syndrome (MKS) is a lethal malformation syndrome characterised by posterior meningoencephalocele, polycystic kidneys, fibrotic changes of the liver, and polydactyly. We have previously shown a linkage to chromosome 17q in 17 Finnish Meckel families. In this study we have analysed one Italian, one Austrian (of Turkish origin) and three British MKS families (Caucasian, Pakistani, and Bangladeshi families) for linkage to the MKS locus on chromosome 17q22–q24. We did not observe co-segregation of the disease and marker haplotypes in the Austrian family or in the three British families, of which two represented classical MKS and one a slightly atypical MKS phenotype with longer survival of the patient. In the Italian family the affected and non-affected children did not share the same maternal chromosome and thus this family could represent the same allelic disease as the Finnish MKS families. These results suggest locus heterogeneity in Meckel syndrome – a feature previously suspected based on the highly variable clinical phenotype. Received: 2 June 1997 / Accepted: 12 June 1997     

Genomic structure, mapping, and expression analysis of the mammalian Lunatic, Manic, and Radical fringe genes

The three members of the mammalian fringe gene family, Manic fringe (Mfng), Radical fringe (Rfng), and Lunatic fringe (Lfng), were identified on the basis of their similarity to Drosophila fringe (fng) and their participation in the evolutionarily conserved Notch receptor signaling pathway. Fringe genes encode pioneer secretory proteins with weak similarity to glycosyltransferases. Both expression patterns and functional studies support an important role for Fringe genes in patterning during embryonic development and an association with cellular transformation. We have now further characterized the expression and determined the chromosomal localization and genomic structure of the mouse Mfng, Rfng, and Lfng genes; the genomic structure and conceptual open reading frame of the human RFNG gene; and the refined chromosomal localization of the three human fringe genes. The mouse Fringe genes are expressed in the embryo and in adult tissues. The mouse and human Fringe family members map to three different chromosomes in regions of conserved synteny: Mfng maps to mouse Chr 15, and MFNG maps to human Chr 22q13.1 in the region of two cancer-associated loci; Lfng maps to mouse Chr 5, and LFNG maps to human Chr 7p22; Rfng maps to mouse Chr 11, and RFNG maps to human Chr 17q25 in the minimal region for a familial psoriasis susceptibility locus. Characterization of the genomic loci of the Fringe gene family members reveals a conserved genomic organization of 8 exons. Comparative analysis of mammalian Fringe genomic organization suggests that the first exon is evolutionarily labile and that the Fringe genes have a genomic structure distinct from those of previously characterized glycosyltransferases. Received: 19 February 1999 / Accepted: 22 February 1999     

Comparative fine maps of bovine toll-like receptor 4 and toll-like receptor 2 regions

Abstract Toll-like receptors are cell-surface receptors that activate innate and adaptive immune responses. We have used a 5000-rad, whole-genome radiation hybrid panel to map Toll-like receptor 4 (TLR4) to the distal end of bovine Chromosome (Chr) 8, and Toll-like receptor 2 (TLR2) to the proximal end of bovine Chr 17. To facilitate comparative mapping and contig construction, we have also used 5000- and 12,000-rad, whole-genome radiation hybrid panels to produce fine maps of the regions surrounding these genes in cattle. These fine maps triple the number of available markers in the TLR4 region and more than double the number of available markers in the TLR2 region. Comparative analyses show gene order conservation between the bovine Chr 8 region and human Chr 9, and between the bovine Chr 17 region and human Chr 4. In addition, the bovine Chr 8 region refines an evolutionary chromosomal breakpoint from a 10-megabase region to a 2.5-megabase region, and the bovine Chr 17 map suggests a new evolutionary chromosomal breakpoint.     

Identification of CC2D2A as a Meckel syndrome gene adds an important piece to the ciliopathy puzzle

Meckel syndrome (MKS) is a lethal malformation disorder characterized classically by encephalocele, polycystic kidneys, and polydactyly. MKS is also one of the major contributors to syndromic neural tube defects (NTDs). Recent findings have shown primary cilia dysfunction in the molecular background of MKS, indicating that cilia are critical for early human development. However, even though four genes behind MKS have been identified to date, they elucidate only a minor proportion of the MKS cases. In this study, instead of traditional linkage analysis, we selected 10 nonrelated affected fetuses and looked for the homozygous regions shared by them. Based on this strategy, we identified the sixth locus and the fifth gene, CC2D2A (MKS6), behind MKS. The biological function of CC2D2A is uncharacterized, but the corresponding polypeptide is predicted to be involved in ciliary functions and it has a calcium binding domain (C2). Immunofluorescence staining of patient's fibroblast cells demonstrates that the cells lack cilia, providing evidence for the critical role of CC2D2A in cilia formation. Our finding is very significant not only to understand the molecular background of MKS, but also to obtain additional information about the function of the cilia, which can help to understand their significance in normal development and also in other ciliopathies, which are an increasing group of disorders with overlapping phenotypes.     

Chromosomal Localization of the Human and Mouse Hyaluronan Synthase Genes

We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designatedHAS1, HAS2,andHAS3in humans andHas1, Has2,andHas3in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to theStreptococcus pyogenesHA synthase, HasA. Furthermore, expression of any oneHASgene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the threeHASgenes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes.HAS1was localized to the human chromosome 19q13.3–q13.4 boundary andHas1to mouse Chr 17.HAS2was localized to human chromosome 8q24.12 andHas2to mouse Chr 15.HAS3was localized to human chromosome 16q22.1 andHas3to mouse Chr 8. The map position forHAS1reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17.HAS2mapped outside the predicted critical region delineated for the Langer–Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome.     

Ts65Dn -- localization of the translocation breakpoint and trisomic gene content in a mouse model for Down syndrome

Fluorescent in situ hybridization (FISH) -- using mouse chromosome paints, probes for the mouse major centromeric satellite DNA, and probes for genes on chromosomes (Chr) 16 and 17 -- was employed to locate the breakpoint in a translocation used to produce a mouse model for Down syndrome. The Ts65Dn trisomy is derived from the reciprocal translocation T(16;17)65Dn. The Ts65Dn mouse carries a marker chromosome containing the distal segment of Chr 16, a region that shows linkage conservation with human Chr 21, and the proximal end of Chr 17. This chromosome confers trisomy for most of the genes in the Chr 16 segment and Ts65Dn mice show many of the phenotypic features characteristic of Down syndrome. We used FISH on metaphase chromosomes from translocation T65Dn/+ heterozygotes and Ts65Dn mice to show that the Chr 17 breakpoint is distal to the heterochromatin of Chr 17, that the Ts65Dn marker chromosome contains a small portion of Chr 17 euchromatin, that the Chr 16 breakpoint lies between the Ncam2 and Gabpa/App genes, and that the Ts65Dn chromosome contains >80% of the human Chr 21 homologs. The significance of this finding is discussed in terms of the utility of this mouse model.     

Conserved Genetic Interactions between Ciliopathy Complexes Cooperatively Support Ciliogenesis and Ciliary Signaling

Mutations in genes encoding cilia proteins cause human ciliopathies, diverse disorders affecting many tissues. Individual genes can be linked to ciliopathies with dramatically different phenotypes, suggesting that genetic modifiers may participate in their pathogenesis. The ciliary transition zone contains two protein complexes affected in the ciliopathies Meckel syndrome (MKS) and nephronophthisis (NPHP). The BBSome is a third protein complex, affected in the ciliopathy Bardet-Biedl syndrome (BBS). We tested whether mutations in MKS, NPHP and BBS complex genes modify the phenotypic consequences of one another in both C. elegans and mice. To this end, we identified TCTN-1, the C. elegans ortholog of vertebrate MKS complex components called Tectonics, as an evolutionarily conserved transition zone protein. Neither disruption of TCTN-1 alone or together with MKS complex components abrogated ciliary structure in C. elegans. In contrast, disruption of TCTN-1 together with either of two NPHP complex components, NPHP-1 or NPHP-4, compromised ciliary structure. Similarly, disruption of an NPHP complex component and the BBS complex component BBS-5 individually did not compromise ciliary structure, but together did. As in nematodes, disrupting two components of the mouse MKS complex did not cause additive phenotypes compared to single mutants. However, disrupting both Tctn1 and either Nphp1 or Nphp4 exacerbated defects in ciliogenesis and cilia-associated developmental signaling, as did disrupting both Tctn1 and the BBSome component Bbs1. Thus, we demonstrate that ciliary complexes act in parallel to support ciliary function and suggest that human ciliopathy phenotypes are altered by genetic interactions between different ciliary biochemical complexes.     

Sequence-ready physical map of the mouse Chromosome 16 region with conserved synteny to the human Velocardiofacial syndrome region on 22q11.2

Proximal mouse Chromosome (Chr) 16 shows conserved synteny with human Chrs 16, 8, 22, and 3. The mouse Chr 16/human Chr 22 conserved synteny region includes the DiGeorge/Velocardiofacial syndrome region of human Chr 22q11.2. A physical map of the entire mouse Chr 16/human Chr 22 region of conserved synteny has been constructed to provide a substrate for gene discovery, genomic sequencing, and animal model development. A YAC contig was constructed that extends ca. 5.4 Mb from a region of conserved synteny with human Chr 8 at Prkdc through the region conserved with human Chr 3 at DVL3. Sixty-one markers including 37 genes are mapped with average marker spacing of 90 kb. Physical distance was determined across the 2.6-Mb region from D16Mit74 to Hira with YAC fragmentation. The central region from D16Jhu28 to Igl-C1 was converted into BAC and PAC clones, further refining the physical map and providing sequence-ready template. The gene content and borders of three blocks of conserved linkage between human Chr 22q11.2 mouse Chr 16 are refined. Received: 4 November 1998 / Accepted: 21 December 1998     

MKS5 and CEP290 Dependent Assembly Pathway of the Ciliary Transition Zone

Cilia have a unique diffusion barrier (“gate”) within their proximal region, termed transition zone (TZ), that compartmentalises signalling proteins within the organelle. The TZ is known to harbour two functional modules/complexes (Meckel syndrome [MKS] and Nephronophthisis [NPHP]) defined by genetic interaction, interdependent protein localisation (hierarchy), and proteomic studies. However, the composition and molecular organisation of these modules and their links to human ciliary disease are not completely understood. Here, we reveal Caenorhabditis elegans CEP-290 (mammalian Cep290/Mks4/Nphp6 orthologue) as a central assembly factor that is specific for established MKS module components and depends on the coiled coil region of MKS-5 (Rpgrip1L/Rpgrip1) for TZ localisation. Consistent with a critical role in ciliary gate function, CEP-290 prevents inappropriate entry of membrane-associated proteins into cilia and keeps ARL-13 (Arl13b) from leaking out of cilia via the TZ. We identify a novel MKS module component, TMEM-218 (Tmem218), that requires CEP-290 and other MKS module components for TZ localisation and functions together with the NPHP module to facilitate ciliogenesis. We show that TZ localisation of TMEM-138 (Tmem138) and CDKL-1 (Cdkl1/Cdkl2/Cdkl3/Cdlk4 related), not previously linked to a specific TZ module, similarly depends on CEP-290; surprisingly, neither TMEM-138 or CDKL-1 exhibit interdependent localisation or genetic interactions with core MKS or NPHP module components, suggesting they are part of a distinct, CEP-290-associated module. Lastly, we show that families presenting with Oral-Facial-Digital syndrome type 6 (OFD6) have likely pathogenic mutations in CEP-290-dependent TZ proteins, namely Tmem17, Tmem138, and Tmem231. Notably, patient fibroblasts harbouring mutated Tmem17, a protein not yet ciliopathy-associated, display ciliogenesis defects. Together, our findings expand the repertoire of MKS module-associated proteins—including the previously uncharacterised mammalian Tmem80—and suggest an MKS-5 and CEP-290-dependent assembly pathway for building a functional TZ.     

A physical map of the mouse shaker-2 region contains many of the genes commonly deleted in Smith-Magenis syndrome (del17p11.2p11.2)

We report the construction of a physical map of the region of mouse chromosome 11 that encompasses shaker-2 (sh2), a model for the human nonsyndromic deafness DFNB3. DFNB3 maps within the common deletion region of Smith-Magenis syndrome (SMS), del(17)(p11.2p11.2). Eleven of the genes mapping within the SMS common deletion region have murine homologs on the sh2 physical map. The gene order in this region is not perfectly conserved between mouse and human, a finding to be considered as we engineer a mouse model of Smith-Magenis syndrome.     

TMEM231, mutated in orofaciodigital and Meckel syndromes,organizes the ciliary transition zone

The Meckel syndrome (MKS) complex functions at the transition zone, located between the basal body and axoneme, to regulate the localization of ciliary membrane proteins. We investigated the role of Tmem231, a two-pass transmembrane protein, in MKS complex formation and function. Consistent with a role in transition zone function, mutation of mouse Tmem231 disrupts the localization of proteins including Arl13b and Inpp5e to cilia, resulting in phenotypes characteristic of MKS such as polydactyly and kidney cysts. Tmem231 and B9d1 are essential for each other and other complex components such as Mks1 to localize to the transition zone. As in mouse, the Caenorhabditis elegans orthologue of Tmem231 localizes to and controls transition zone formation and function, suggesting an evolutionarily conserved role for Tmem231. We identified TMEM231 mutations in orofaciodigital syndrome type 3 (OFD3) and MKS patients that compromise transition zone function. Thus, Tmem231 is critical for organizing the MKS complex and controlling ciliary composition, defects in which cause OFD3 and MKS.     

Comparative mapping of the DiGeorge syndrome region in mouse shows inconsistent gene order and differential degree of gene conservation

We have constructed a comparative map in mouse of the critical region of human 22q11 deleted in DiGeorge (DGS) and Velocardiofacial (VCFS) syndromes. The map includes 11 genes potentially haploinsufficient in these deletion syndromes. We have localized all the conserved genes to mouse Chromosome (Chr) 16, bands B1-B3. The determination of gene order shows the presence of two regions (distal and proximal), containing two groups of conserved genes. The gene order in the two regions is not completely conserved; only in the proximal group is the gene order identical to human. In the distal group the gene order is inverted. These two regions are separated by a DNA segment containing at least one gene which, in the human DGS region, is the most proximal of the known deleted genes. In addition, the gene order within the distal group of genes is inverted relative to the human gene order. Furthermore, a clathrin heavy chain-like gene was not found in the mouse genome by DNA hybridization, indicating that there is an inconsistent level of gene conservation in the region. These and other independent data obtained in our laboratory clearly show a complex evolutionary history of the DGS-VCFS region. Our data provide a framework for the development of a mouse model for the 22q11 deletion with chromosome engineering technologies. Received: 8 July 1997 / Accepted 11 August 1997     

High-resolution mapping of mouse Chromosome 8 identifies an evolutionary chromosomal breakpoint

The central region of mouse Chromosome (Chr) 8, containing the myodystrophy (myd) locus, is syntenic with human Chr 4q28-qter. The human neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD) maps to Chr 4q35, and myd has been proposed as a mouse homolog of FSHD. We have employed a comparative mapping approach to investigate this relationship further by extending the mouse genetic map of this region. We have ordered 12 genes in a single cross, 8 of which have human homologs on 4q28-qter. The results confirm a general relationship between the most distal genes on human 4q and the most proximal genes in the mouse 8 syntenic region. Despite chromosomal rearrangements of syntenic groups in this region, conservation of gene order is maintained between the group of genes in the human telomeric region of 4q35 and MMU8. Furthermore, this conserved telomeric HSA4q35 syntenic group maps proximal to the myd mutation and is flanked by genes with homologs on HSA8p22. At the proximal boundary of the MMU8 linkage group we have identified a single 300-kb YAC containing the genes Frgl and Pcml, which have human homologs on 4q35 and 8p22, respectively. Thus, this YAC spans an evolutionary chromosomal breakpoint. As well as providing clues about chromosomal evolution, this map of the FSHD syntenic mouse region should prove invaluable in the isolation of candidate genes for this disease. Received: 20 January 1998 / Accepted: 10 April 1998     

High-resolution mapping of the human 4q21 and the mouse 5E3 SCYB chemokine cluster by fiber-fluorescence in situ hybridization

The CXC chemokine or small inducible cytokine B (SCYB) subfamily includes the T-cell chemoattractants MIG (CXCL9, SCYB9), IP-10 (CXCL10, SCYB10), and I-TAC (CXCL11, SCYB11). These three highly homologous chemokines lack the glutamic acid-leucine-arginine (ELR) motif and signal via the CXCR3 receptor. Previous work showed that the genes encoding these chemokines are localized in an individual mini-cluster on human Chromosome (Chr) 4 at position 4q21.2. Recently, we identified mouse Scyb11 and mapped this gene by fluorescence in situ hybridization (FISH) to mouse Chr 5E3, the orthologous locus to human 4q21 where the other two homologous mouse genes, Scyb9 and Scyb10, have also been localized. Since SCYB10 and SCYB11 are not represented in the recently published draft sequence of the human genome, we wanted to clarify exactly the order and distances of the three chemokine genes using two-color FISH on stretched DNA fiber preparations. Here, we report the simultaneous localization of all three genes and provide high-resolution visual maps of this chemokine cluster from both mouse and human. The three chemokine genes were found within a range of 32 kb on mouse and 29 kb on human DNA fiber targets. The precise physical distances were defined, and an almost identical arrangement of the human and mouse homologues was identified, indicating that this CXC chemokine mini-cluster has been completely conserved evolutionarily since the divergence of mouse and human. Our results refine previous maps of the three genes, support the hypothesis that they resulted from gene duplication that took place in a common ancestor of mouse and human, and provide complementary information on a region of the draft sequence of human Chr 4 that is not yet covered.     

Human homologs of two testes-expressed loci on mouse chromosome 17 map to opposite arms of chromosome 6

Our laboratory has recently cloned and characterized two testes-expressed loci--the Tcp-10 gene family cluster and the D17Si11 gene--that map to the proximal portion of mouse chromosome 17. Human homologs of both loci have been identified and cloned. Somatic cell hybrid lines have been used to map the human homolog of D17Si11 to the short arm of chromosome 6 (p11-p21.1) along with homologs of other genes from the (Pim-1)-(Pgk-2) region of the mouse chromosome. The human TCP 10 locus maps to the long arm of chromosome 6 (q21-qter) along with homologs of other genes from the mouse chromosome 17 region between the centromere and Pim-1. The mapping of large portions of the mouse t haplotype to unlinked regions on human chromosome 6 rules out the possibility that a t-haplotype-like chromosome could exist in humans.     

Localization to chicken Chromosome 5 of a novel locus determining salmonellosis resistance

Clear genetic differences in the susceptibility of chickens to visceral infection by Salmonella have been observed and it has been possible to identify resistant and susceptible lines of inbred chickens. We report here the results of experiments to map directly the gene(s) controlling this trait in chickens by examining crosses between highly susceptible and highly resistant lines. In the mapping panel, a region on chicken Chromosome (Chr) 5 was found to have a large effect on resistance, and this effect was observed in three separate resource populations. Mapping of additional marker loci in the region of the resistance gene further localized it to a region of approximately 2 cM, close to the genes for creatine kinase (CKB) and dynein (DNCH1). This region shows conserved synteny with telomeric regions of human Chr 14 and mouse Chr 12. On the basis of this conserved synteny, this resistance gene seems unlikely to correspond to the previously identified salmonellosis resistance genes Lps (located on mouse Chr 4) or Nos(2) (located on mouse Chr 11). There was no association between Nramp1 and resistance in these crosses, although this gene was shown to contribute to resistance in other crosses. The homologous human and mouse regions at present contain no likely candidate genes for this trait. Thus this appears to be a novel resistance gene, which we designate SAL1.