High-Resolution Genetic and Physical Mapping of Modifier-of-deafwaddler (mdfw) and Waltzer (Cdh23)

Modifier-of-deafwaddler (mdfw) and waltzer (Cdh23^v) are loci on mouse chromosome 10 encoding factors that are essential for the function of auditory hair cells. The BALB/cByJ-specific mdfw allele encodes a necessary and sufficient modifier that induces progressive early onset hearing loss in CBy-dfw^2J heterozygotes. Recessive mutations in the waltzer locus result in circling behavior and congenital deafness. In this report we present a high-resolution integrated genetic and physical map of mdfw and Cdh23^v. Our genetic analyses localize mdfw between markers D10Mit60 and 148M13T7 within a 1.01-cM region. The Cdh23^v critical interval is fully contained within the mdfw region and localizes between markers 146O23T7 and 148M13T7 within a 0.35-cM interval that is represented in an ≈500-kb BAC contig. Our data suggest that mdfw and Cdh23^v are allelic.     

Mutations in Cdh23 cause nonsyndromic hearing loss in waltzer mice

Mutations at the waltzer (v) locus result in deafness and vestibular dysfunction due to degeneration of the neuroepithelium within the inner ear. Here, we use a positional cloning approach to show that waltzer encodes a novel cadherin (Cdh23), which is most closely related to the Drosophila Fat protein. A single nucleotide deletion in the v(J) allele and a single nucleotide insertion in the v allele are predicted to truncate each protein near the N-terminus and produce a functional null allele. In situ hybridization analysis showed that Cdh23 is expressed in the sensory hair cells of the inner ear, where it has been suggested to be a molecule critical for crosslinking of the stereocilia. In addition, Cdh23 is expressed in the urticulo-saccular foramen,the ductus reuniens, and Reissner's membrane, suggesting that Cdh23 may also be involved in maintaining the ionic composition of the endolymph. Finally, mutations in human CDH23 have recently been described for two loci, DFNB12 and USH1D, which cause nonsyndromic deafness, identifying waltzer as a mouse model for human hearing loss.     

Spatiotemporal pattern and isoforms of cadherin 23 in wild type and waltzer mice during inner ear hair cell development

Mutant alleles of the gene encoding cadherin 23 are associated with Usher syndrome type 1 (USH1D), isolated deafness (DFNB12) in humans, and deafness and circling behavior in waltzer (v) mice. Stereocilia of waltzer mice are disorganized and the kinocilia misplaced, indicating the importance of cadherin 23 for hair bundle development. Cadherin 23 was localized to developing stereocilia and proposed as a component of the tip link. We show that, during development of the inner ear, cadherin 23 is initially detected in centrosomes at E14.5, then along the length of emerging stereocilia, and later becomes concentrated at and subsequently disappears from the tops of stereocilia. In mature vestibular hair bundles, cadherin 23 is present along the kinocilium and in the region of stereocilia-kinocilium bonds, a pattern conserved in mammals, chicks, and frogs. Cadherin 23 is also present in Reissner's membrane (RM) throughout development. In homozygous v(6J) mice, a reported null allele, cadherin 23 was absent from stereocilia, but present in kinocilia, RM, and centrosomes. We reconciled these results by identifying two novel isoforms of Cdh23 unaffected in sequence and expression by the v(6J) allele. Our results suggest that Cdh23 participation in stereocilia links may be restricted to developing hair bundles.     

A YAC contig encompassing the recessive Stargardt disease gene (STGD) on chromosome 1p.

Stargardt disease (STGD) and fundus flavimaculatus are infrequent autosomal recessive conditions characterized by a juvenile macular dystrophy and variable degrees of peripheral retinal changes. Linkage analysis performed in 47 STGD/fundus flavimaculatus families demonstrated significant linkage to 13 polymorphic DNA markers on chromosome 1p. The maximum combined two-point lod score was 32.7 (maximum recombination fraction [phi max] = .006) with the polymorphic marker D1S188. Our data demonstrate that STGD and fundus flavimaculatus are the same disorder clinically and genetically and provide further evidence for genetic homogeneity of this phenotype. Analysis of recombination events on disease chromosomes placed the STGD gene within a 4-cM interval between markers D1S435 and D1S236. A physical map was constructed of a YAC contig flanking STGD, from markers D1S500 to D1S495, and includes the critical interval delineated by historical recombinants. This contig spans approximately 31 cM, with one gap (3-5 cM) that is outside the 4-cM critical region. Localization of STGD to a single YAC contig will facilitate its positional cloning.     

High-resolution meiotic and physical mapping of the best vitelliform macular dystrophy (VMD2) locus to pericentromeric chromosome 11.

Best vitelliform macular dystrophy (VMD2) has previously been linked to several microsatellite markers from chromosome 11. Subsequently, additional genetic studies have refined the Best disease region to a 3.7-cM interval flanked by markers at D11S903 and PYGM. To further narrow the interval containing the Best disease gene and to obtain an estimate of the physical size of the minimal candidate region, we used a combination of high-resolution PCR hybrid mapping and analysis of recombinant Best disease chromosomes. We identified six markers from within the D11S903-PYGM interval that show no recombination with the defective gene in three multigeneration Best disease pedigrees. Our hybrid panel localizes these markers on either side of the centromere on chromosome 11. The closest markers flanking the disease gene are at D11S986 in band p12-11.22 on the short arm and at D11S480 in band q13.2-13.3 on the proximal long arm. This study demonstrates that the physical size of the Best disease region is exceedingly larger than previously estimated from the genetic data, because of the proximity of the defective gene to the centromere of chromosome 11.     

Refined mapping of the Escherichia coli F4ab/F4ac receptor gene(s) on pig chromosome 13

Enterotoxigenic Escherichia coli expressing F4 fimbriae is the major cause of diarrhoea in neonatal and post-weaning piglets. Previous studies have revealed that the loci controlling the F4ab/F4ac receptors are located on SSC13q41, between markers SW207 and S0283. In this study, we refined their positions in a two generation population containing 366 piglets of three breeds (Large White, Landrace, and Songliao Black). Nine microsatellite markers within this region were selected from the MARC (U.S. Meat Animal Research Center) porcine linkage map, and the pedigree disequilibrium test was employed for fine-mapping. The F4abR gene was located in the interval between S0283 and SW1833, a 4.8-cM region, and the F4acR gene was located in the interval between S0283 and SW1876, a 1.6-cM region. Our results also suggest that the F4ab/F4ac receptors might be controlled by two different but closely linked loci. The results of microsatellite-based haplotype analysis in the corresponding region show that some specific haplotypes were overwhelmingly present in the adhesive or non-adhesive animals, indicating that there are mutations within the identified regions that are strongly associated with the F4ab/ac phenotypes.     

Genetic fine localization of the β-glucocerebrosidase (GBA) and prosaposin (PSAP) genes: implications for Gaucher disease

Mutations in the glucocerebrosidase (GBA) and prosaposin (PSAP) genes are responsible for Gaucher disease, the most prevalent sphingolipidosis. Somatic cell hybrid analysis and in situ hybridization experiments have localized the GBA gene to 1q21 and the PSAP gene to 10q21-q22. We performed pairwise and multi-point linkage analyses between the two genes and several highly polymorphic markers from the Généthon human linkage map. Our results show that six markers cosegregate with the GBA gene (Z_max = 8.73 at θ = 0.00 for marker D1S2714) and define a 3.2-cM interval between D1S305 and D1S2624 as the most probable location for the gene. Three of these markers (D1S2777, D1S303, and D1S2140), as well as the gene encoding pyruvate kinase (PKLR), are contained in a single YAC clone together with the GBA gene. A new polymorphism was identified within the PSAP gene (C16045T) and used for linkage studies. The multi-point analysis places the gene in a 9.8-cM interval between D10S1688 and D10S607. The fine localization of these genes provides a useful tool for cosegregation analysis, indirect molecular diagnosis, and population genetic studies. Received: 22 October 1996 / Accepted: 4 February 1997     

Founder effect at PGL1 in hereditary head and neck paraganglioma families from the Netherlands.

PGL1, a gene responsible for hereditary paragangliomas of the head and neck, recently was mapped to a 2-cM interval on chromosome 11q22-q23, by linkage and haplotype-sharing analysis of a large multibranch Dutch family. We determined the disease-linked haplotype, as defined by 13 markers encompassing a large interval on 11q21-q23, in 10 additional families ascertained from the same geographical locale. Alleles were identical for six contiguous markers, spanning a genetic distance of 6 cM and containing PGL1. Despite this strong indication of a common ancestor, no kinships between the families could be demonstrated through genealogical surveys going back to 1800 a.d. We conclude that a single ancestral mutation is responsible for most, if not all, hereditary paragangliomas, in this region of The Netherlands, and that strong founder effects may exist at the PGL1 locus.     

Primary autosomal recessive microcephaly (MCPH1) maps to chromosome 8p22-pter.

Primary (or "true") microcephaly is inherited as an autosomal recessive trait and is thought to be genetically heterogeneous. Using autozygosity mapping, we have identified a genetic locus (MCPH1) for primary microcephaly, at chromosome 8p22-pter, in two consanguineous families of Pakistani origin. Our results indicate that the gene lies within a 13-cM region between the markers D8S1824 and D8S1825 (maximum multipoint LOD score of 8.1 at D8S277). In addition, we have demonstrated the genetic heterogeneity of this condition by analyzing a total of nine consanguineous families with primary microcephaly.     

Identification and molecular characterization of de novo translocation t(8;14)(q22.3;q13) associated with a vascular and tissue overgrowth syndrome

Klippel-Trenaunay syndrome (KTS) is a disorder primarily characterized by capillary-venous vascular malformations associated with altered limb bulk and/or length. We report the identification of a balanced translocation involving chromosomes 8q22.3 and 14q13 in a patient with a vascular and tissue overgrowth syndrome consistent with KTS. We demonstrated that translocation t(8;14)(q22.3;q13) arose de novo. These data suggest that a pathogenic gene for a vascular and tissue overgrowth syndrome (KTS) may be located at chromosome 8q22.3 or 14q13. Fluorescence in situ hybridization (FISH) analysis was used to define the breakpoint on chromosome 8q22.3 to a <5-cM interval flanked by markers AFMA082TG9 and GATA25E10, and the 14q13 breakpoint within a 1-cM region between STSs WI-6583 and D14S989. This study provides a framework for the fine-mapping and ultimate cloning of a novel vascular gene at 8q22.3 or 14q13.     

A major locus for myoclonus-dystonia maps to chromosome 7q in eight families

Myoclonus-dystonia (M-D) is an autosomal dominant disorder characterized by myoclonic and dystonic muscle contractions that are often responsive to alcohol. The dopamine D2 receptor gene (DRD2) on chromosome 11q has been implicated in one family with this syndrome, and linkage to a 28-cM region on 7q has been reported in another. We performed genetic studies, using eight additional families with M-D, to assess these two loci. No evidence for linkage was found for 11q markers. However, all eight of these families showed linkage to chromosome 7 markers, with a combined multipoint LOD score of 11.71. Recombination events in the families define the disease gene within a 14-cM interval flanked by D7S2212 and D7S821. These data provide evidence for a major locus for M-D on chromosome 7q21.     

Two novel microsatellite markers for prenatal prediction of spinal muscular atrophy (SMA)

Autosomal recessive spinal muscular atrophy (SMA) has been mapped to a 6-cM interval on chromosome 5q12–13.3, flanked proximally by locus D5S6 and distally by locus D5S112. In this study we describe the isolation of two new microsatellite markers (EF1/2a and EF13/14) near locus D5S125, which lies 2 cM distal to D5S6. We show by linkage analysis and the study of the recombinants in 55 SMA pedigrees that the disease lies in the 4-cM interval between EF1/2a and D5S112. Fluorescence in situ analysis of cosmids from D5S6, EF1/2a and D5S112 confirms the genetic order and relative distance of markers. The microsatellites EF1/2a and EF13/14 are the first highly polymorphic PCR based proximal markers in SMA to be described, and will be of value in prental prediction of the disorder.     

A pericentric inversion of chromosome six in a patient with Peutz-Jeghers’ syndrome and the use of FISH to localise the breakpoints on a genetic map

Karyotypic analysis in a patient with Peutz-Jeghers’ syndrome demonstrated a pericentric inversion on chromosome 6. Further investigation was undertaken using fluorescence in situ hybridisation (FISH) with yeast artificial chromosome clones selected to contain genetic markers from chromosome 6, and a probe for the centromeric alphoid repeat array. This analysis located one inversion breakpoint within the alphoid array, in a 1-cM interval between D6S257 and D6S402, and the other in a 4-cM interval between D6S403 and D6S311. The oestrogen receptor gene locus (ESR) is excluded from the latter interval. Received: 23 January 1996 / Revised: 26 February 1996     

Genomic targeting and high-resolution mapping of the domestication gene Q in wheat.

The Q locus is largely responsible for the domestication of bread wheat. Q confers the free-threshing character of the spike and influences other important agronomic traits. Using chromosome deletion lines, Q was placed on the physical map within a submicroscopic segment of the long arm of chromosome 5A. We targeted markers to the segment by comparative mapping of anonymous RFLP clones, AFLP, and mRNA differential display analysis of deletion lines 5AL-7 and -23, which have deletion breakpoints that flank the Q locus. Differentially expressed sequences detected fragments at various loci on group 5 chromosomes suggesting that Q may be a regulatory gene. We identified 18 markers within the Q gene deletion interval and used them to construct a genetic linkage map of the region in F2 populations derived from chromosome 5A disomic substitution lines. The genetic map corresponding to the deletion segment was 20-cM long, and we identified markers as close as 0.7 cM to the Q gene. An estimate of base pairs per centimorgan within the region is 250 kb/cM, an 18-fold increase in recombination compared with the genomic average. Genomic targeting and high-density mapping provide a basis for the map-based cloning of the Q gene.     

Homozygosity and linkage-disequilibrium mapping of the urofacial (Ochoa) syndrome gene to a 1-cM interval on chromosome 10q23-q24.

The urofacial (Ochoa) syndrome (UFS) is a rare autosomal recessive disease characterized by congenital obstructive uropathy and abnormal facial expression. The patients present with enuresis, urinary-tract infection, hydronephrosis, and voiding dysfunctions as a result of neurogenic bladders. To map the UFS gene, a genome screen using a combination of homozygosity-mapping and DNA-pooling strategies was performed in 20 selected patients, one patient pool, and three control pools (unaffected relatives). After analyses of 36 randomly chosen markers, D10S677 was identified as being linked to and associated with UFS, as suggested by a significant excess of homozygosity in patients compared with that in unaffected relatives (P < 10(-6)), as well as by the allelic-frequency differences between the patient pool and control pools. Ten additional markers flanking D10S677 and covering a 22-cM region then were analyzed to fine-map the UFS gene by use of haplotype (linkage disequilibrium) analysis. All 31 patients were found to be homozygous for two closely linked markers (D10S1726 and D10S198) located approximately 5 cM telomeric to D10S677, whereas only 12% of the unaffected relatives were homozygous for both markers (P < 10(-19)). Several patients are heterozygous at two markers immediately flanking D10S1726/D10S198, one on the centromeric side (D10S1433) and the other on the telomeric side (D10S603). These recombinational events place the UFS gene near D10S1726/D10S198 and within a 1-cM interval defined by D10S1433 and D10S603 on chromosome 10q23-q24.     

Confirmation of linkage of Duane's syndrome and refinement of the disease locus to an 8.8-cM interval on chromosome 2q31

Duane's syndrome is a congenital abnormality of eye movement, which may be inherited as an autosomal dominant trait but usually occurs sporadically. Genetic mapping in a Mexican family has recently identified a locus for Duane's syndrome within a 17.8-cM region of chromosome 2q31. The region was flanked by the microsatellite markers D2S2330 and D2S364. We performed linkage and haplotype analysis in a four-generation UK family with autosomal dominant transmission of Duane's syndrome. Linkage to 2q31 was confirmed with a maximum logarithm of differences (lod) score of 3.3 at theta = 0. The genetic interval was reduced to an 8.8-cM region between markers D2S326 and D2S364 that includes the candidate homeobox D gene cluster.     

A 1.5-Mb physical map of the hidrotic ectodermal dysplasia (Clouston syndrome) gene region on human chromosome 13q11

The HED (hidrotic ectodermal dysplasia) or Clouston syndrome gene (named ED2) has been mapped to the pericentromeric region of chromosome 13 (13q11) to a 2.4-cM interval flanked by markers D13S1828 and D13S1830. We have developed a BAC/PAC-based contig map of this region. This contig, comprising 23 clones and spanning 1.5 Mb, was established by mapping of 27 BAC/PAC end-derived STSs, 11 known polymorphic markers, 2 previously mapped genes, and 14 ESTs. The genomic clone overlaps were confirmed by restriction fragment fingerprint analysis. This contig provides the basis for genomic sequencing and gene identification in the ED2 critical region. Of the 14 ESTs mapped to the contig, 6 show homology to human genes and 8 appear to be novel. Expression patterns of the genes/ESTs were tested by Northern blot and RT-PCR. Full characterization of some of these genes, as well as the novel ESTs, will be useful in assessing their involvement in the HED/Clouston syndrome.     

Genetic and Physical Mapping of Sex-Linked AFLP Markers in Nile Tilapia (Oreochromis niloticus)

Identification of the sex-determining genes of the Nile tilapia (Oreochromis niloticus) has important implications for commercial aquaculture. We previously identified an XX/XY sex-determining locus in this species within a 10-cM interval between markers GM201 and UNH995 on linkage group one (LG1). In order to refine this region, we developed new AFLP markers using bulked segregant analysis of the mapping families. We identified three AFLP markers that showed a sex-specific pattern of segregation. All three mapped near, but just outside, the previously identified sex-determining region on LG1. Hybridization of BAC clones containing these markers to chromosome spreads confirmed that the XX/XY sex-determining locus is on one of the small chromosomes in O. niloticus.