Human craniofacial development: Genes and their expression profiles

The pharyngeal arches are transient embryologic structures important in human craniofacial development. Abnormal development of the pharyngeal arches leads to craniofacial malformations. Few causative genes have been identified, such as TWIST. Saethre-Chotzen syndrome, an autosomal dominant craniosynostotic condition associated with pharyngeal arch defects, is caused by mutations in this gene that codes for a basic helix-loop-helix transcription factor. We devised a new strategy to screen for TWIST mutations: (1) PCR amplification with subsequent sequencing to identify point mutations, small insertions or deletions in the coding region, (2) real-time PCR-based gene dosage analysis to identify large deletions encompassing the gene because haploinsufficiency is the pathogenic mechanism for this disorder. Deletion size was refined using the gene dosage assays at multiple loci across the critical region. In 55 patients with features of Saethre-Chotzen syndrome, 11% were detected to have deletions by real-time gene dosage analysis. Of the 37 patients with classic features of Saethre-Chotzen syndrome, the overall detection rate for TWIST mutations was 68%.; To identify additional genes associated with pharyngeal arch development, we performed large scale gene expression profiling. We constructed 11 micro-cDNA libraries, generated 12 SAGE libraries, and utilized microarray analysis on 21 microdissected, normal human craniofacial structures, including pharyngeal arch 1 (PA1). Pairwise comparison between SAGE libraries revealed that 149 tags were upregulated, and 152 tags were downregulated from 4th to 5th week PA1 (p¡0.05). Many of the expression changes were confirmed by real-time RT-PCR in mouse. Mouse whole mount in situ hybridization revealed that the Set gene, which plays a role in cell proliferation, was highly expressed both in PA1 precursor cells at GD8.5 and in PA1 at GD9.5, while the expression was more restricted to the maxillary portion of PA1 at GD10.5, making it a candidate gene for cleft lip/palate. We then demonstrated that many genes are conserved in craniofacial development between human and mouse, and some of the same molecules are involved in both craniofacial and limb development. Identification of genes expressed in very early human development will advance research in genetics, development, and dysmorphology.