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.

Nonsyndromic or isolated cleft lip with or without cleft palate (CL/P) occurs in wide geographic distribution with an average birth prevalence of 1/700. We used direct sequencing as an approach to study candidate genes for CL/P. We report here the results of sequencing on 20 candidate genes for clefts in 184 cases with CL/P selected with an emphasis on severity and positive family history. Genes were selected based on expression patterns, animal models, and/or role in known human clefting syndromes. For seven genes with identified coding mutations that are potentially etiologic, we performed linkage disequilibrium studies as well in 501 family triads (affected child/mother/father). The recently reported MSX1 P147Q mutation was also studied in an additional 1,098 cleft cases. Selected missense mutations were screened in 1,064 controls from unrelated individuals on the Centre d'Étude du Polymorphisme Humain (CEPH) diversity cell line panel. Our aggregate data suggest that point mutations in these candidate genes are likely to contribute to 6% of isolated clefts, particularly those with more severe phenotypes (bilateral cleft of the lip with cleft palate). Additional cases, possibly due to microdeletions or isodisomy, were also detected and may contribute to clefts as well. Sequence analysis alone suggests that point mutations in FOXE1, GLI2, JAG2, LHX8, MSX1, MSX2, SATB2, SKI, SPRY2, and TBX10 may be rare causes of isolated cleft lip with or without cleft palate, and the linkage disequilibrium data support a larger, as yet unspecified, role for variants in or near MSX2, JAG2, and SKI. This study also illustrates the need to test large numbers of controls to distinguish rare polymorphic variants and prioritize functional studies for rare point mutations.

Previous studies suggest that the relationship between genes and nonsyndromic cleft lip ± cleft palate (CLP) or cleft palate only (CP) may be modified by the environment. Using data from a population‐based case‐control study, we examined allelic variants for three genes, i.e., transforming growth factor alpha (TGFA), transforming growth factor beta 3 (TGFB3), and Msh (Drosophila) homeobox homolog 1 (MSX1), and their interactions with two exposures during pregnancy (maternal cigarette smoking and alcohol consumption) as risk factors for CLP and CP. For each cleft phenotype, risk estimates associated with most allelic variants tended to be near unity. Risk estimates for maternal smoking (≥10 cigarettes/day) were significantly elevated for CP and were most elevated among infants with allelic variants at the TGFB3 or MSX1 sites. By comparison, risk estimates for maternal alcohol consumption (≥4 drinks/month) were significantly elevated for CLP and were most elevated among infants with allelic variants at the MSX1 site. Our results suggest that development of CLP and CP may be influenced independently by maternal exposures but more significantly by interaction of such exposures and specific allelic variants. Teratology 59:39–50, 1999. © 1999 Wiley‐Liss, Inc.

Clinical and epidemiologic studies of defined geographic populations can serve as a means of establishing data important for genetic counseling and as a first step in identifying strategies best suited for identification of causes. Under the sponsorship of Operation Smile International, clinical, genetic, and epidemiologic studies were carried out at six sites within the Philippines between 1989 and 1996. Patients who were being evaluated for surgical repair of craniofacial anomalies (primarily clefts of the lip and palate) were briefly examined for the presence of associated anomalies, and a family history was obtained to look for the frequency of cleft lip and palate in siblings. Birth records of 47,969 newborns over an 8-year period at one hospital in Bacolod City in the province of Negros Occidental were reviewed. Medical records of infants born with clefts of the lip and/or palate and other major anomalies were reviewed and birth prevalence rates calculated. Findings include a birth prevalence of 1.94 per 1000 live births for cleft lip with/without palate in the Philippines. Recurrence rates in siblings for nonsyndromic clefts of the lip and palate were 23 per 1000 for cleft lip with or without cleft palate, and 14 per 1000 for cleft palate only. The percentage of clefts associated with multiple anomalies was 21% at birth and 6% for individuals examined during the screening process, providing evidence for a high postnatal death rate. These data provide groundwork for additional etiologic studies including segregation analysis and molecular genetic studies involving linkage or association, as well as for studies of environmental contributions to clefting such as vitamin deficiencies. Preliminary molecular analysis using an association approach is reported in a companion paper. The findings suggest a high incidence of cleft lip and palate in native-born Filipinos.

Maternal alcohol use during pregnancy is a known cause of birth defects associated with the fetal alcohol syndrome, but its role in more common, isolated, craniofacial birth defects is not well understood. A population‐based, case‐control study of orofacial clefts was conducted in Iowa using births during 1987–1991. Cases were identified by the Iowa Birth Defects Registry and classified as having a cleft lip with or without cleft palate (CLP) or cleft palate only (CP) and whether the cleft was isolated or occurred with other birth defects. Controls were selected from normal Iowa births. Maternal alcohol use during pregnancy was classified according to self‐reported drinks consumed per month. Results are based on 302 controls and the following numbers in each case group: 118 isolated CLP, 56 isolated CP, 51 CLP with multiple defects, and 62 CP with multiple defects. Compared to women who did not drink alcohol during pregnancy, the relative odds of isolated CLP rose with increasing level of maternal drinking as follows: 1–3 drinks per months, 1.5; 4–10 drinks per month, 3.1; more than 10 drinks per month, 4.7 (chi‐square test for trend, P = 0.003). Adjustment for maternal smoking, vitamin use, education, and household income did not substantially alter these results. No significant association was found between alcohol use and isolated cleft palate or clefts in children with multiple birth defects. Alcohol use during pregnancy may be a cause of isolated cleft lip with or without cleft palate. © 1996 Wiley‐Liss, Inc.