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MSX1 and TGFB3 Contribute to Clefting in South America
1 Departments of Pediatrics, 2613 JCP, Correspondence: *corresponding author, jeff-murray{at}uiowa.edu
MSX1 and TGFB3 have been proposed as genes in which mutations may contribute to non-syndromic forms of oral clefts; however, an interaction between these genes has not been described. The present study attempts to detect transmission distortion of MSX1 and TGFB3 in 217 South American children from their respective mothers. With transmission disequilibrium test analysis, cleft lip with/without cleft palate, cleft lip with palate plus cleft palate only, and all datasets combined showed evidence of association with MSX1 (p = 0.004, p = 0.037, and p = 0.001, respectively). With likelihood ratio test analysis, "cleft lip only" showed association with MSX1 (p = 0.04) and "cleft palate only" with TGFB3 (p = 0.02). A joint analysis of MSX1 and TGFB3 suggested that there may be an interaction between these two loci to increase cleft susceptibility. These results suggest that MSX1 and TGFB3 mutations make a contribution to clefts in South American populations.
Key Words: cleft lip and palate • cleft palate • MSX1 • TGFB3 • ECLAMC
Although oral-facial clefts are among the most common congenital defects, their etiology remains largely unknown, with only a few cases associated with identified rare syndromes or secondary to recognized teratogen exposure (Wyszynski et al., 1996). The first positive candidate gene result came when a Caucasian population showed an association between transforming growth factor alpha (TGFA) and oral clefts (Ardinger et al., 1989). Several subsequent studies have attempted replication, with some but not all showing significance (Mitchell, 1997; Ioannidis et al., 2001). Two other candidates, MSX1 and TGFB3, have been suggested by animal models (Satokata and Maas, 1994; Kaartinen et al., 1995; Proetzel et al., 1995), and association studies have shown positive results for both MSX1 and TGFB3 genes (Lidral et al., 1998; Beaty et al., 2001; Blanco et al., 2001). Using a population from South America, we measured the association of MSX1 and TGFB3 with oral-facial clefts.
From January, 1998, to June, 2000, the Latin American Collaborative Study of Congenital Malformations (ECLAMC) (Castilla and Orioli, 1983) collected blood spots on filter cards from patients with "cleft lip with or without cleft palate" and "cleft palate only" and their mothers from eight countries in Latin America (Table 1  ). Written informed consent was obtained from each mother, and this study has ECLAMC’s hospital Institutional Review Board approval. Only two mothers were affected, and 18% of the cases have a positive family history of clefting. Patients with unknown cleft type, a known syndrome, or other major or multiple minor defects (N = 19), as determined by record review, were excluded during data analysis.
DNA was extracted from filter card blood spots by modifications of published protocols (Lidral et al., 1997). Polymerase chain-reactions (PCR) were performed, and allelic variants of MSX1 (Padanilam et al., 1992) and TGFB3 (Lidral et al., 1998) were tested. Subjects were genotyped by means of single-strand conformational analysis, mutation-detection enhancement, and polyacrylamide gel electrophoresis techniques. Samples with known genotypes were included for each gel and PCR reaction. We used a silver-stain protocol to visualize the bands (Lidral et al., 1997). "Cleft lip only", "cleft lip with cleft palate", and "cleft palate only" cases were evaluated separately and then in combination [cleft lip with or without cleft palate, cleft lip with cleft palate plus cleft palate only, and all cases together]. We compared mother and proband genotypes to determine the transmitted alleles vs. the non-transmitted alleles. Transmission disequilibrium tests (TDT) were performed (Curtis and Sham, 1995; Spielman and Ewens, 1996). For a mother-child pair to be informative for the TDT analysis, it is necessary that (1) the mother be heterozygous for the particular genetic marker, and (2) the child be a heterozygote different from that of the mother. According to these criteria, only the MSX1-CA marker could be used for TDT, because the other marker studied has two alleles and only one possible type of heterozygote. Therefore, we also applied the likelihood ratio test (LRT) of Weinberg (1999) under the assumption that the distribution of paternal and maternal alleles is the same.
MSX1 For the TDT analyses of the 176 cases of cleft lip with or without cleft palate, there were 27 informative mothers (mother heterozygous and child being a heterozygote different from that of the mother). Both standard TDT (each allele vs. all others) and multi-allele TDT (all alleles simultaneously) showed significant association between cleft lip with or without cleft palate and the 169-base-pair allele of the MSX1-CA marker (p = 0.004). When cases of "cleft lip with palate" and "cleft palate only" were analyzed together, the results were significant (p = 0.037) for the same allele (Table 2 ). The same can be seen for all data analyzed together (p = 0.001).
In addition to the standard TDT, we applied the LRT of Weinberg (1999) to assess transmission distortion for each allele vs. all others. The LRT estimates transmission from the missing parent, assuming that there is the same allele distribution in the missing parents as in the sampled parents. Parameters R1 and R2 and model likelihoods were estimated. For each data subset, the addition of parameter R2 did not significantly improve the fit; therefore, Model II in Table 3 includes only the R1 parameter, and the hypothesis tests had 1 degree of freedom. None of the other MSX1 alleles had statistically significant evidence of increased transmission with clefting. The "cleft lip only" (p = 0.04) data subset showed transmission distortion with the 169-base-pair allele of MSX1 when the LRT method was used (Table 3).
TGFB3 The allelic variant for TGFB3 had only two alleles in this population; therefore, a standard TDT analysis was not possible (see previous section). Transmission distortion of TGFB3 was analyzed by the LRT of Weinberg (1999). Parameters R1 and R2 and model likelihoods were estimated. For each data subset, the addition of parameter R2 did not significantly improve the fit; therefore Model II includes only the R1 parameter, and the hypothesis tests had 1 degree of freedom. The 254-base-pair allele of the TGFB3 5'UTR.1 marker showed significant transmission distortion for the "cleft palate only" subset (p = 0.02) (Table 4 ).
MSX1 and TGFB3 We also performed a joint LRT analysis of MSX1 and TGFB3 in the total dataset (the data subsets were not large enough for joint analyses to be performed). To do so, we divided the families into two groups based on the MSX1 genotype of the proband, i.e., whether or not the proband had  1 169-base-pair MSX1 alleles. We then used the LRT to assess transmission distortion between clefting and TGFB3 in each group. In the families in which the probands had one or more 169-base-pair alleles, there was borderline evidence of transmission distortion with TGFB3 (p = 0.05), suggesting that there may be an interaction between the MSX1 and TGFB3 susceptibility alleles.
The TDT and LRT approaches allowed for the inclusion of cases of different ethnic origins and avoided the complications of mixed ancestry that can arise in case-control study designs. South America is, for the most part, a trihybrid of Native Indians, Caucasians, and Africans, but these three groups are not equally distributed across the continent. According to ECLAMC, the frequency of cleft lip with or without cleft palate is higher in a Bolivian population, which has a large proportion of Native Indians; however, this is not seen for cleft palate only (Castilla et al., 1995). ECLAMC efforts provided a robust and cost-effective way to collect biological samples on filter cards. The feasibility of this process is allowing ECLAMC to expand the sample collection for all major congenital defects. This study provided evidence of an association between genetic variation at two loci (MSX1 and TGFB3) and both "cleft lip with or without cleft palate" and "cleft palate only" in a South American population. Also, the results suggest a possible interaction of these two genes in the development of oral clefts in South Americans. For the identification of candidate genes involved in human clefting, information from linkage and linkage disequilibrium studies, as well as chromosomal re-arrangements, expression of the genes in culture cells, and animal models, is usually compiled. According to this, MSX1 and TGFB3 are the two strongest candidate genes for oral clefts in humans. MSX1 is a very strong candidate for clefting in humans, based on the mouse model (Satokata and Maas, 1994), linkage disequilibrium (Lidral et al., 1998; Beaty et al., 2001; Blanco et al., 2001), and the recent report of a family with autosomal-dominant cleft lip and palate, segregating with a nonsense mutation in MSX1 (van den Boogaard et al., 2000). In addition, MSX1 is commonly deleted in cases of 4p-/Wolf-Hirschhorn syndrome, in which cleft lip/palate is a common feature (Battaglia et al., 2001). Although most studies have carried out separate analyses for "cleft lip only" or "cleft lip with or without cleft palate" from isolated cleft palate, we did both separate and combined analyses. The recently published nonsense mutation in MSX1 (van den Boogaard et al., 2000) had both cleft lip and cleft palate in the same pedigree, and Van der Woude syndrome (Schutte and Murray, 1999), a single-gene Mendelian form of clefting, includes both isolated cleft lip and cleft palate, suggesting that at least some of the mechanisms for cleft lip may be shared by cleft palate.
The difference in the results observed for "cleft lip only" and "cleft lip with cleft palate" datasets (Tables 2, 3 TGFB3 is also a very strong candidate for clefting in humans, based on both the mouse model (Kaartinen et al., 1995; Proetzel et al., 1995) and on linkage disequilibrium (Maestri et al., 1997; Lidral et al., 1998; Beaty et al., 2001). In a Midwestern population (Lidral et al., 1998), it was "cleft lip with or without cleft palate" that was associated with TGFB3. In a population from the East coast (Beaty et al., 2001), both "cleft lip with or without cleft palate" and "cleft palate only" were associated with a marker close to TGFB3, but "cleft lip only" was not associated. For the South American population, under the LRT, "cleft palate only" cases showed significant transmission distortion for the TGFB3 marker studied. Analysis of all these data from humans suggests that TGFB3 is more related to the cleft palate phenotype, as seen in the animal models (Kaartinen et al., 1995; Proetzel et al., 1995). Furthermore, results from the current study provide suggestive evidence that there may be an interaction between susceptibility alleles at MSX1 and TGFB3. The study illustrates the power of sample collection tied to a birth defects registry activity and the ECLAMC collaboration and opens the door for more powerful studies involving even larger numbers of samples as well as studies of additional birth defects. The specific role for MSX1 and TGFB3 in this population can now be explored in more detail through direct mutation searches for sequence abnormalities as well as functional studies of these proteins or their regulation.
Thanks to Lora Muilenburg and Nancy Newkirk for administrative matters, Sandy Daack-Hirsch and Sarah O’Brien for technical advice, and Dr. Clarice Weinberg and Dr. Richard Morris for providing us with a freeware version of the LRT (Weinberg, 1999) program. Resources for this study were provided by NIH grants DE 08559-12, P60 DE 13076-02, and DK 25295, and by Brazilian grants CNPq 143178/1997-0 and CAPES BEX0927/99-6. Some of the results of this paper were obtained by use of the program package S.A.G.E., supported by a US Public Health Service Resource Grant (1-P41-RR03655) from the National Center for Research Resources. This paper is based on a thesis submitted to the graduate faculty, Federal University of Rio de Janeiro, in partial fulfillment of the requirements for the PhD degree (for A.R.V.). Received for publication February 11, 2002. Revision received January 15, 2003. Accepted for publication January 29, 2003.
Journal of Dental Research, Vol. 82, No. 4,
289-292 (2003) This article has been cited by other articles:
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