Craniofacial anomalies, and in particular cleft lip and palate, are major human birth defects with a worldwide frequency of 1 in 700 and substantial clinical impact. A wide range of studies in developmental biology has contributed to a better knowledge of how both genes and environmental exposures impact head organogenesis. Specific causes have now been identified for some forms of cleft lip and palate, and we are at the beginning of a time in which the common nonsyndromic forms may also have specific etiologies identified. Mouse models have an especially important role in disclosing cleft etiologies and providing models for environmental cotriggers or interventions. An overview of the gene-environment contributions to nonsyndromic forms of clefting and their implications for developmental biology and clinical counseling is presented.

MSX1 has been proposed as a gene in which mutations may contribute to non-syndromic forms of cleft lip and/or cleft palate. Support for this comes from human linkage and linkage disequilibrium studies, chromosomal deletions resulting in haploinsufficiency, a large family with a stop codon mutation that includes clefting as a phenotype, and the Msx1 phenotype in a knockout mouse. This report describes a population based scan for mutations encompassing the sense and antisense transcribed sequence of MSX1 (two exons, one intron). We compare the completed genomic sequence of MSX1 to the mouse Msx1 sequence to identify non-coding homology regions, and sequence highly conserved elements. The samples studied were drawn from a panethnic collection including people of European, Asian, and native South American ancestry. The gene was sequenced in 917 people and potentially aetiological mutations were identified in 16. These included missense mutations in conserved amino acids and point mutations in conserved regions not identified in any of 500 controls sequenced. Five different missense mutations in seven unrelated subjects with clefting are described. Evolutionary sequence comparisons of all known Msx1 orthologues placed the amino acid substitutions in context. Four rare mutations were found in non-coding regions that are highly conserved and disrupt probable regulatory regions. In addition, a panel of 18 population specific polymorphic variants were identified that will be useful in future haplotype analyses of MSX1. MSX1 mutations are found in 2% of cases of clefting and should be considered for genetic counselling implications, particularly in those families in which autosomal dominant inheritance patterns or dental anomalies appear to be cosegregating with the clefting phenotype.

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Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant form of muscular dystrophy. The FSHD locus has been linked to the most distal genetic markers on the long arm of chromosome 4. Recently, a probe was identified that detects an EcoRI fragment length polymorphism which segregates with the disease in most FSHD families. Within the EcoRI fragment lies a tandem array of 3.2 kb repeats. In several familial cases and four independent sporadic FSHD mutations, the variation in size of the EcoRI fragment was due to a decrease in copy number of the 3.2 kb repeats. To gain further insight into the relationship between the tandem array and FSHD, a single 3.2 kb repeat unit was characterized. Fluorescence in situ hybridization (FISH) demonstrates that the 3.2 kb repeat cross-hybridizes to several regions of heterochromatin in the human genome. In addition, DNA sequence analysis of the repeat reveals a region which is highly homologous to a previously identified family of heterochromatic repeats, LSau. FISH on interphase chromosomes demonstrates that the tandem array of 3.2 kb repeats lies within 215 kb of the 4q telomere. Together, these results suggest that the tandem array of 3.2 kb repeats, tightly linked to the FSHD locus, is contained in heterochromatin adjacent to the telomere. In addition, they are consistent with the hypothesis that the gene responsible for FSHD may be subjected to position effect variegation because of its proximity to telomeric heterochromatin.

Recently, two truncating mutations in the PHF8 (plant homeodomain finger protein 8) gene have been found to cause X-linked mental retardation associated with cleft lip/cleft palate (CL/P). One of the truncating mutations was found in the original family with Siderius-Hamel CL/P syndrome where only two of the three affected individuals had mental retardation (MR) with CL/P and one individual had mild MR. The second mutation was present in a family with four affected men, three of whom had MR and CL/P, while the fourth individual had mild MR without clefting. Here, we report a novel nonsense mutation (p.K177X) in a male patient who has MR associated with CL/P. The mutation results in a truncated PHF8 protein lacking the Jumonji-like C terminus domain and five nuclear localization signals. Our finding further supports the hypothesis that the PHF8 protein may play an important role in cognitive function and midline formation.