Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome.

EEC syndrome is an autosomal dominant disorder characterized by ectrodactyly, ectodermal dysplasia, and facial clefts. We have mapped the genetic defect in several EEC syndrome families to a region of chromosome 3q27 previously implicated in the EEC-like disorder, limb mammary syndrome (LMS). Analysis of the p63 gene, a homolog of p53 located in the critical LMS/EEC interval, revealed heterozygous mutations in nine unrelated EEC families. Eight mutations result in amino acid substitutions that are predicted to abolish the DNA binding capacity of p63. The ninth is a frameshift mutation that affects the p63alpha, but not p63beta and p63gamma isotypes. Transactivation studies with these mutant p63 isotypes provide a molecular explanation for the dominant character of p63 mutations in EEC syndrome.     

SOX14 is a candidate gene for limb defects associated with BPES and Möbius syndrome

Members of the SOX gene family encode proteins with homology to the HMG box DNA-binding domain of SRY, the Y-linked testis-determining gene. SOX genes are expressed during embryogenesis and are involved in the development of a wide range of different tissues. Mutations in SRY, SOX9 and SOX10 have been shown to be responsible for XY sex reversal, campomelic dysplasia and Waardenburg-Hirschsprung disease, respectively. It is likely that mutations in other SOX genes are responsible for a variety of human genetic diseases. SOX14 has been identified from a human genomic library and the mouse and chicken sequences obtained by polymerase chain reaction amplification. The SOX14 amino acid sequence is highly conserved across these species, suggesting an important role for this protein in vertebrate development. SOX14 is expressed in the neural tube and apical ectodermal ridge of the developing chicken limb. This is the only SOX gene known to be expressed in the apical ectodermal ridge, a structure that directs outgrowth of the embryonic limb bud. Human SOX14 is localised to a 1.15-Mb yeast artificial chromosome on chromosome 3q23, close to loci for BPES (blepharophimosis, ptosis, epicanthus inversus syndrome) and Mobius syndrome. Although SOX14 maps outside these loci, its expression pattern and chromosomal localisation suggest that it is a candidate gene for the limb defects frequently associated with these syndromes.     

Mutations in SOX9, the Gene Responsible for Campomelic Dysplasia and Autosomal Sex Reversal

Campomelic dysplasia (CD) is a skeletal malformation syndrome frequently accompanied by 46,XY sex reversal. A mutation-screening strategy using SSCP was employed to identify mutations in SOX9, the chromosome 17q24 gene responsible for CD and autosomal sex reversal in man. We have screened seven CD patients with no cytologically detectable chromosomal aberrations and two CD patients with chromosome 17 rearrangements for mutations in the entire open reading frame of SOX9. Five different mutations have been identified in six CD patients: two missense mutations in the SOX9 putative DNA binding domain (high mobility group, or HMG, box); three frameshift mutations and a splice-acceptor mutation. An identical frameshift mutation is found in two unrelated 46,XY patients, one exhibiting a male phenotype and the other displaying a female phenotype (XY sex reversal). All mutations found affect a single allele, which is consistent with a dominant mode of inheritance. No mutations were found in the SOX9 open reading frame of two patients with chromosome 17q rearrangements, suggesting that the translocations affect SOX9 expression. These findings are consistent with the hypothesis that CD results from haploinsufficiency of SOX9.     

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.     

Analysis of a second family with hereditary non-chromaffin paragangliomas locates the underlying gene at the proximal region of chromosome 11q

The gene for autosomal, dominantly inherited, non-chromaffin paragangliomas has previously been mapped at 11q23-qter by linkage analysis of a single family. In the present study, we have used genetic markers from 11q for the analysis of two distantly related pedigrees with the same disorder. Linkage analysis and haplotyping indicate that the gene underlying the disorder in the present family is located on chromosome 11q proximal to the tyrosinase gene locus (11q14–q21). Closely linked markers are the human homologue of the murine INT2 protooncogene and the anonymous DNA marker D11S527. A maximum lod score of 5.4 (=0.0) has been obtained for linkage between the disorder and the chromosomal region defined by these markers. The human INT2 gene can be regarded as a candidate for the disorder on the basis of its expression pattern during embryogenesis in the mouse. However, haplotype analysis indicates that this gene is probably not the predisposing genetic factor in the present family.     

An expanded comparative map of bovine chromosome 27 targeting dairy form QTL regions

At present, the density of genes on the bovine maps is extremely limited and current resolution of the human-bovine comparative map is insufficient for selection of candidate genes controlling many economic traits of interest in dairy cattle. This study describes the chromosomal mapping of 10 selected gene-associated markers to bovine linkage and radiation hybrid maps to improve the breakpoint resolution in the human-bovine comparative map near two previously identified quantitative trait loci for the linear type trait, dairy form. Two regions of conserved synteny not previously described are reported between the telomeric region of bovine chromosome 27 (BTA27) and human chromosome 3 (HSA3) p24 region and between the HSA4q34.1 region and BTA8. These data increase the number of genes positioned on the bovine gene maps, refine the human-bovine comparative map, and should improve the efficiency of candidate gene selection for the dairy form trait in cattle.     

Position effects due to chromosome breakpoints that map approximately 900 Kb upstream and approximately 1.3 Mb downstream of SOX9 in two patients with campomelic dysplasia

Campomelic dysplasia (CD) is a semilethal skeletal malformation syndrome with or without XY sex reversal. In addition to the multiple mutations found within the sex-determining region Y-related high-mobility group box gene (SOX9) on 17q24.3, several chromosome anomalies (translocations, inversions, and deletions) with breakpoints scattered over 1 Mb upstream of SOX9 have been described. Here, we present a balanced translocation, t(4;17)(q28.3;q24.3), segregating in a family with a mild acampomelic CD with Robin sequence. Both chromosome breakpoints have been identified by fluorescence in situ hybridization and have been sequenced using a somatic cell hybrid. The 17q24.3 breakpoint maps approximately 900 kb upstream of SOX9, which is within the same bacterial artificial chromosome clone as the breakpoints of two other reported patients with mild CD. We also report a prenatal identification of acampomelic CD with male-to-female sex reversal in a fetus with a de novo balanced complex karyotype, 46,XY,t(4;7;8;17)(4qter-->4p15.1::17q25.1-->17qter;7qter-->7p15.3::4p15.1-->4pter;8pter-->8q12.1::7p15.3-->7pter;17pter-->17q25.1::8q12.1-->8qter). Surprisingly, the 17q breakpoint maps approximately 1.3 Mb downstream of SOX9, making this the longest-range position effect found in the field of human genetics and the first report of a patient with CD with the chromosome breakpoint mapping 3' of SOX9. By using the Regulatory Potential score in conjunction with analysis of the rearrangement breakpoints, we identified a candidate upstream cis-regulatory element, SOX9cre1. We provide evidence that this 1.1-kb evolutionarily conserved element and the downstream breakpoint region colocalize with SOX9 in the interphase nucleus, despite being located 1.1 Mb upstream and 1.3 Mb downstream of it, respectively. The potential molecular mechanism responsible for the position effect is discussed.     

A genomewide screen for autism-spectrum disorders: evidence for a major susceptibility locus on chromosome 3q25-27

To identify genetic loci for autism-spectrum disorders, we have performed a two-stage genomewide scan in 38 Finnish families. The detailed clinical examination of all family members revealed infantile autism, but also Asperger syndrome (AS) and developmental dysphasia, in the same set of families. The most significant evidence for linkage was found on chromosome 3q25-27, with a maximum two-point LOD score of 4.31 (Z(max )(dom)) for D3S3037, using infantile autism and AS as an affection status. Six markers flanking over a 5-cM region on 3q gave Z(max dom) >3, and a maximum parametric multipoint LOD score (MLS) of 4.81 was obtained in the vicinity of D3S3715 and D3S3037. Association, linkage disequilibrium, and haplotype analyses provided some evidence for shared ancestor alleles on this chromosomal region among affected individuals, especially in the regional subisolate. Additional potential susceptibility loci with two-point LOD scores >2 were observed on chromosomes 1q21-22 and 7q. The region on 1q21-22 overlaps with the previously reported candidate region for infantile autism and schizophrenia, whereas the region on chromosome 7q provided evidence for linkage 58 cM distally from the previously described autism susceptibility locus (AUTS1).     

Comparative genomics of the SOX9 region in human and Fugu rubripes: conservation of short regulatory sequence elements within large intergenic regions.

Campomelic dysplasia (CD), a human skeletal malformation syndrome with XY sex reversal, is caused by heterozygous mutations in and around the gene SOX9. SOX9 has an extended 5' control region, as indicated by CD translocation breakpoints scattered over 1 Mb proximal to SOX9 and by expression data from mice transgenic for human SOX9-spanning yeast artificial chromosomes. To identify long-range regulatory elements within the SOX9 5' control region, we compared approximately 3.7 Mb and 195 kb of sequence around human and Fugu rubripes SOX9, respectively. We identified only seven and five protein-coding genes in the human and F. rubripes sequences, respectively. Four of the F. rubripes genes have been mapped in humans; all reside on chromosome 17 but show extensive intrachromosomal gene shuffling compared with the gene order in F. rubripes. In both species, very large intergenic distances separate SOX9 from its directly flanking genes: 2 Mb and 500 kb on either side of SOX9 in humans, and 68 and 97 kb on either side of SOX9 in F. rubripes. Comparative sequence analysis of the intergenic regions revealed five conserved elements, E1-E5, up to 290 kb 5' to human SOX9 and up to 18 kb 5' to F. rubripes SOX9, and three such elements, E6-E8, 3' to SOX9. Where available, mouse sequences confirm conservation of the elements. From the yeast artificial chromosome transgenic data, elements E3-E5 are candidate enhancers for SOX9 expression in limb and vertebral column, and 8 of 10 CD translocation breakpoints separate these elements from SOX9.     

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.     

Cloning and chromosomal localization of the human BARX2 homeobox protein gene

The human BARX2 gene encodes a homeodomain-containing protein of 254 amino acids, which binds optimally to the DNA consensus sequence YYTAATGRTTTTY. BARX2 is highly expressed in adult salivary gland and is expressed at lower levels in other tissues, including mammary gland, kidney, and placenta. The BARX2 gene consists of four exons, and is located on human chromosome 11q25. This chromosomal location is within the minimal deletion region for Jacobsen syndrome, a syndrome including craniosynostosis and other developmental abnormalities. This chromosomal location, along with the reported expression of murine barx2 in craniofacial development, suggests that BARX2 may be causally involved in the craniofacial abnormalities in Jacobsen syndrome.     

Physical map of the chromosome 6q22 region containing the oculodentodigital dysplasia locus: analysis of thirteen candidate genes and identification of novel ESTs and DNA polymorphisms

Oculodentodigital dysplasia (ODDD) is an autosomal dominant condition with congenital anomalies of the craniofacial and limb regions and neurodegeneration. Genetic anticipation for the dysmorphic and neurologic features has been inferred in a few families. Our previous linkage studies have refined the ODDD candidate region to chromosome 6q22-->q23. In an attempt to clone the ODDD gene, we created a yeast artificial chromosome contig with 31 redundant clones spanning the region and identified and ordered candidate genes and markers. Fluorescent IN SITU hybridization mapped two of these YAC clones to chromosome 6q22.2 telomeric to a known 6q21 fragile site, excluding it as a possible cause of the suggested anticipation. We performed mutation analysis on thirteen candidate genes - GRIK2, HDAC2, COL10A1, PTD013, KPNA5, PIST, ROS1, BRD7, PLN, HSF2, PKIB, FABP7, and HEY2. Although no mutations were found, we identified 44 polymorphisms, including 28 single nucleotide polymorphisms. Direct cDNA selection was performed and fifty-five clones were found to contain sequences that were not previously reported as known genes or ESTs. These clones and polymorphisms will assist in the further characterization of this region and identification of disease genes.