Association of MSX1 and TGFB3 with nonsyndromic clefting in humans.

Nonsyndromic cleft lip with or without cleft palate (CL/P) and nonsyndromic cleft palate only (CPO) are common congenital anomalies with significant medical, psychological, social, and economic ramifications. Both CL/P and CPO are examples of complex genetic traits. There exists sufficient evidence to hypothesize that disease loci for CL/P and CPO can be identified by a candidate-gene linkage-disequilibrium (LD) strategy. Candidate genes for clefting, including TGFA, BCL3, DLX2, MSX1, and TGFB3, were screened for LD with either CL/P or CPO in a predominantly Caucasian population, with both case-control- and nuclear-family-based approaches. Previously reported LD for TGFA with both CL/P and CPO could not be confirmed, except in CL/P patients with a positive family history. Also, in contrast to previous studies, no LD was found between BCL3 and either CL/P or CPO. Significant LD was found between CL/P and both MSX1 and TGFB3 and between CPO and MSX1, suggesting that these genes are involved in the pathogenesis of clefting. In addition, a mutation search in the genes DLX2, MSX1, and TGFB3 was performed in 69 CPO patients and in a subset of the CL/P patients. No common mutations were found in the coding regions of these genes; however, several rare variants of MSX1 and TGFB3 were found that may alter the latters' normal function. These results form the basis for future research, including (a) mutation searches in the MSX1 and TGFB3 genes in Caucasian CL/P patients and (b) extension of the search for MSX1 mutations in CPO patients to the noncoding regions.

Cloning, characterization, and mapping of the mouse homeobox gene Hmx1.

Homeobox-containing genes play an important role in development, including positional specification of the body plan and organogenesis. We previously isolated the human HMX1 (H6) gene, a novel homeobox-containing gene of the HMX family, from a human embryonic craniofacial cDNA library. The closely related mouse genes Hmx3 (Nkx5.1) and Hmx2 (Nkx5.2) are in the same class as the HMX1 gene and are expressed in the craniofacial region of the developing embryo. To provide a resource for further characterization of the human HMX1 gene, we isolated the mouse Hmx1 genomic clone. We show here the mouse Hmx1 genomic sequence, its gene mapping, and its expression pattern in the developing mouse embryo. Evidence is presented showing that the three known Hmx genes in the mouse likely play complementary roles in the development of the second arch, retina, sympathetic nerve ganglia, and cranial neural ganglia. Hmx1 may play an important role in the development of craniofacial structures and may interact with Hoxa-2 and Dlx-2 in the second branchial arch.

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.

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

Rieger syndrome (RIEG) 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 Rathke's 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.

Exclusion of epidermal growth factor and high-resolution physical mapping across the Rieger syndrome locus.

We have evaluated the 4q25-4q26 region where the autosomal dominant disorder Rieger syndrome has been previously mapped by linkage. We first excluded epidermal growth factor as a candidate gene by carrying out SSCP analysis of each of its 24 exons using a panel of seven unrelated individuals with Rieger syndrome. No evidence for etiologic mutations was detected in these individuals, although four polymorphic variants were identified, including three that resulted in amino acid changes. We next made use of two apparently balanced translocations, one familial and one sporadic, to identify a narrow physical localization likely to contain the gene or to be involved in regulation of gene function. Somatic cell hybrids were established from individuals with these balanced translocations, and these hybrids were used as a physical mapping resource for, first, preliminary mapping of the translocation breakpoints using known sequence tagged sites from chromosome 4 and then, after creating YAC and cosmids contigs encompassing the region, for fine mapping of those breakpoints. A cosmid contig spanning these breakpoints was identified and localized the gene to within approximately 150 kb of D4S193 on chromosome 4. The interval between the two independent translocations is approximately 50 kb in length and provides a powerful resource for gene identification.

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).

A detailed multipoint map of human chromosome 4 provides evidence for linkage heterogeneity and position-specific recombination rates.

Utilizing the CEPH reference panel and genotypic data for 53 markers, we have constructed a 20-locus multipoint genetic map of human chromosome 4. New RFLPs are reported for four loci. The map integrates a high-resolution genetic map of 4p16 into a continuous map extending to 4q31 and an unlinked cluster of three loci at 4q35. The 20 linked markers form a continuous linkage group of 152 cM in males and 202 cM in females. Likely genetic locations are provided for 25 polymorphic anonymous sequences and 28 gene-specific RFLPs. The map was constructed employing the LINKAGE and CRIMAP computational methodologies to build the multipoint map via a stepwise algorithm. A detailed 10-point map of the 4p16 region constructed from the CEPH panel provides evidence for heterogeneity in the linkage maps constructed from families segregating for Huntington disease (HD). It additionally provides evidence for position-specific recombination frequencies in the telomeric region of 4p.