Nancy J. Leysens

Cloning and characterization of a novel bicoid-related homeobox transcription factor gene, RIEG, involved in Rieger syndrome

Rieger syndrome (REG) is an autosomal–dominant human disorder that includes anomalies of the anterior chamber of the eye, dental hypoplasia and a protuberant umbilicus. We report the human cDNA and genomic characterization of a new homeobox gene, RIEG, causing this disorder. Six mutations in RIEG were found in individuals with the disorder. The cDNA sequence of Rieg, the murine homologue of RIEG, has also been isolated and shows strong homology with the human sequence. In mouse embryos Rieg mRNA localized in the periocular mesenchyme, maxillary and mandibular epithelia, and umbilicus, all consistent with RIEG abnormalities. The gene is also expressed in Rathkes pouch, vitelline vessels and the limb mesenchyme. RIEG characterization provides opportunities for understanding ocular, dental and umbilical development and the pleiotropic interactions of pituitary and limb morphogenesis.

Linkage of Inflammatory Bowel Disease to Human Chromosome 6p

Inflammatory bowel disease (IBD) is characterized by a chronic relapsing intestinal inflammation. IBD is subdivided into Crohn disease and ulcerative colitis phenotypes. Given the immunologic dysregulation in IBD, the human-leukocyte-antigen region on chromosome 6p is of significant interest. Previous association and linkage analysis has provided conflicting evidence as to the existence of an IBD-susceptibility locus in this region. Here we report on a two-stage linkage and association analysis of both a basic population of 353 affected sibling pairs (ASPs) and an extension of this population to 428 white ASPs of northern European extraction. Twenty-eight microsatellite markers on chromosome 6 were genotyped. A peak multipoint LOD score of 4.2 was observed, at D6S461, for the IBD phenotype. A transmission/disequilibrium test (TDT) result of P=.006 was detected for D6S426 in the basic population and was confirmed in the extended cohort (P=.004; 97 vs. 56 transmissions). The subphenotypes of Crohn disease, ulcerative colitis, and mixed IBD contributed equally to this linkage, suggesting a general role for the chromosome 6 locus in IBD. Analysis of five single-nucleotide polymorphisms in the TNFA and LTA genes did not reveal evidence for association of these important candidate genes with IBD. In summary, we provide firm linkage evidence for an IBD-susceptibility locus on chromosome 6p and demonstrate that TNFA and LTA are unlikely to be susceptibility loci for IBD.

Phylogenetic conservation and physical mapping of members of the H6 homeobox gene family.

Homeobox genes represent a class of transcription factors that play key roles in the regulation of embryogenesis and development. Here we report the identification of a homeobox-containing gene family that is highly conserved at both the nucleotide and amino acid levels in a diverse number of species. These species encompass both vertebrate and invertebrate phylogenies, ranging from Homo sapiens to Drosophila melanogaster. In humans, at least two homeobox sequences from this family were identified representing a previously reported member of this family as well as a novel homeobox sequence that we physically mapped to the 10q25.2–q26.3 region of human Chromosome (Chr) 10. Multiple members of this family were also detected in three additional vertebrate species including Equus caballus (horse), Gallus gallus (Chicken), and Mus musculus (mouse), whereas only single members were detected in Tripneustes gratilla (sea urchin), Petromyzon marinus (lamprey), Salmo salar (salmon), Ovis aries (sheep), and D. melanogaster (fruit fly).

Genomic structure, sequence, and mapping of human FGF8 with no evidence for its role in craniosynostosis/limb defect syndromes

Fibroblast growth factor-8 (Fgf8) is a recently identified growth factor that stimulates the androgen-dependent growth of mouse mammary carcinoma cells. Evidence from mouse development also shows that Fgf8 may play an important role in growth and patterning of limbs, face, and the central nervous system. We describe here the human FGF8 genomic sequence and demonstrate conservation between the human and mouse sequences, including alternatively spliced exons in the mouse. Mapping of FGF8 by FISH using an FGF8-containing bacterial artificial chromosome and by genetic linkage using a SSCP variant identified in this study is also reported and refines the FGF8 map location to 10q24. Since FGF8 maps to the same chromosomal region as FGFR2, has indeed been shown to be a ligand for FGFR2, and has an expression pattern consistent with limb and craniofacial anomalies, we have screened two kindreds with Pfeiffer syndrome that were previously linked to markers from 10q24-25 and a large number of individuals with craniosynostosis and limb anomalies for mutations in the coding sequence of FGF8. While no such mutations were identified, a rare polymorphic variant, consisting of an 18-base-pair (six-amino-acid) duplication in exon 1c, is reported that apparently has no clinical effect. Our exclusionary data suggest that mutations in FGF8 would be, at best, an infrequent cause of such disorders.

Exclusion of Ifa and Ifb as the Lps gene and mapping of three markers near the Lps locus.

The lipopolysaccharide ( Lps) locus in mice controls the biological response to endotoxin, the lipopolysaccharide (LPS) from the cell wall of Gram-negative bacteria. The Lps locus has been shown to be a single Mendelian gene that maps to Chromosome (Chr) 4 (Watson et al. 1978). A LPS hyporesponsive mouse arose spontaneously and was first identified in the C3H/HeJ strain (Heppner and Weiss 1965; Sultzer 1968). C3H/HeJ strain mice are profoundly hyporesponsive to LPS with an LPS LD 50 at least 20 times that observed in A/HeJ mice (normal responders; Sultzer 1968). Both biological and genetic data suggest that interferona (Ifa) and interferonb (Ifb) are candidates for the Lpsgene. Physiologically, C3H/HeJ mice fail to produce normal levels of type 1 interferons, and treatment of cells derived from C3H/HeJ mice with either IFN-a or IFN-b partially normalizes the biological response to LPS (Akagawa et al. 1987; Vogel et al. 1983; Vogel and Fertsch 1987). These results suggest that If or Ifb may be altered in the C3H/HeJ mice. In addition to the possible biological roles interferons play in the LPS response, genetic studies place If and Ifb near theLps locus. Backcross linkage analysis has suggested that theIfa andIfb structural genes (a total of eleven genes) are located in the same region as the Lps gene on Chr 4 (DeMaeyer and Dandoy 1987; Watson et al. 1978; Dandoy et al. 1985). The results of one linkage study, however, suggested that the Lps locus was distinct from that ofIfa (Fultz and Vogel 1989). This conclusion was based on a poorly reproducible restriction fragment length polymorphism, and no other studies have repeated this finding (Fultz and Vogel 1989). Although there is disagreement regarding the relationship of theLps locus toIfa andIfb, the biological data suggest that these genes are exceptional candidates for the Lps gene. In this study, theIfa and Ifb genes were investigated as candidates for theLpsgene. We compared the sequences of the Ifa and Ifb genes in the hyporesponsive C3H/HeJ strain of mice and the ostensibly related C3H/OuJ strain (normal responders) of mice. C3H/OuJ strain mice were investigated because they are the progenitor of the C3H/HeJ strain of mice and would be expected to differ only at theLps locus. All of the genes were analyzed for sequence variants by single strand conformational polymorphism (SSCP) analysis and direct sequencing. In addition, several new Chr 4 markers were mapped in 96 progeny of a backcross of hyporesponsive mice with responsive CastEi/C3H/HeJ mice along with a recently identified simple sequence length polymorphism (SSLP) located in theIfa cluster. Our results definitively exclude Ifa and Ifb as candidates for the Lps gene. All of the Ifa andIfb genes show considerable sequence similarity and are each composed of one exon approximately 540 bp in length (DeMaeyer et al. 1987; Dandoy et al. 1985). Unique primers derived from sequences obtained from Genome Data Base (GDB; http://www.ncbi.nlm.nih.gov/) were developed to specifically amplify 300 bp overlapping segments of each of the eleven known Ifa and Ifb genes. Each gene segment was amplified from DNA isolated from C3H/HeJ mice and LPS-sensitive C3H/OuJ mice by the polymerase chain reaction (PCR). These strains are closely related; any differences identified would most likely represent significant changes and not polymorphisms. All gene fragments were investigated for sequence variation by SSCP analysis. SSCP has been shown to detect approximately 60% of all single base substitutions in fragments approximately 300 bp in size (Sheffield et al. 1993). No variants were identified (data not shown). Approximately 40% of single base substitutions in the gene fragments may have gone undetected owing to the relatively low sensitivity of SSCP for DNA fragments of this size. Consequently we sequenced all of the gene fragments from DNA isolated from C3H/HeJ strain mice and LPS-sensitive C3H/OuJ mice to identify