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. Author manuscript; available in PMC: 2009 Aug 30.
Published in final edited form as: Genet Med. 2008 Sep;10(9):668–674. doi: 10.1097/GIM.0b013e3181833793

Candidate Gene/Loci Studies in Cleft Lip/Palate and Dental Anomalies Finds Novel Susceptibility Genes for Clefts

Alexandre R Vieira 1,2,3,4, Toby G McHenry 1,3, Sandra Daack-Hirsch 6, Jeffrey C Murray 6, Mary L Marazita 1,3,4,5
PMCID: PMC2734954  NIHMSID: NIHMS127805  PMID: 18978678

Abstract

We revisited 42 families with two or more cleft affected siblings that participated in previous studies and collected complete dental information. Genotypes from 1489 single nucleotide polymorphism (SNP) markers located in 150 candidate genes/loci were reanalyzed. Two sets of association analyses were carried out. First we ran the analysis solely on the cleft status. Second we assigned affection to any cleft or dental anomaly (tooth agenesis, supernumerary teeth, and microdontia), and repeated the analysis. Significant over-transmission was seen for a SNP in ANKS6 (rs4742741, 9q22.33; p=0.0004) when a dental anomaly phenotype was included in the analysis. Significant over-transmission was also seen for a SNP in ERBB2 (rs1810132, 17q21.1; p=0.0006). In the clefts only data, the most significant result was also for ERBB2 (p=0.0006). Other markers with suggestive p-values included IRF6 and 6q21-q23 loci. In contrast to the above results, suggestive over-transmission of markers in GART, DPF3, and NRXN3 were seen only when the dental anomaly phenotype was included in the analysis. These findings support the hypothesis that some loci may contribute to both clefts and congenital dental anomalies. Thus, including dental anomalies information in the genetics analysis of cleft lip and palate will provide new opportunities to map susceptibility loci for clefts.

Introduction

Isolated or nonsyndromic cleft lip and palate (CL/P) is a complex disorder resulting from multiple genetic and environmental factors. CL/P is a common birth defect, and the source of substantial morbidity and mortality worldwide1. With an average birth prevalence of 1/700 live births, there is remarkable population to population variation2. In general, Asian populations have a higher birth prevalence of clefting (1/500 births), Caucasians are intermediate (1/1,100), and African populations have the lowest (1/2,500 births). However, the notion that Asians have a higher prevalence of clefts has been challenged based on the evidence that many published prevalence rates included all pregnancies (live and still births) and do not distinguish between syndromic and nonsyndromic clefts, or between cleft palate alone and cleft lip with or without cleft palate3.

An examination of familial recurrence patterns in CL/P indicated that there may be anywhere from 3 to 14 interacting loci involved in clefting4. This analysis indicates that very large sample sizes may be necessary to detect the loci involved in CL/P. For a complex genetic disorder such as CL/P, several experimental techniques may be used. These include breakpoint mapping, deletion mapping, direct sequencing of candidate genes/loci, linkage analysis, and linkage disequilibrium analysis5. A number of studies on populations with clefts from the Philippines have been productive, in part because of the common occurrence of isolated clefting, large average family sizes, and a motivated public health enterprise6. Studies with the Filipino population included MSX17–9, TGFA7–9, TGFB27, TGFB37,9, IRF610, FGF family of genes11, PVRL112, genes at 19q1313, genes at 8p11-2314, genes at 9q2115,16, and an additional 18 candidate genes9. Furthermore, a meta-analysis of seven genome scans15 that included Filipino family data revealed significant linkage signals at 9q21 (heterogeneity LOD score 6.6) and 6q23-25 (heterogeneity LOD score 3.55) among other regions. Even though these studies included as many as 403 families (ranging from 3 to 76 individuals in each), the results were, for the most part, modest. The only exceptions are IRF610 and MSX17–9. IRF6 has also been consistently associated with cleft lip and palate in a number of populations17–22. In the same way, MSX1 has been associated with cleft lip and palate in several independent studies23–37. We hypothesize that increasing the sophistication of the clinical description would allow reducing misclassification and improving ones ability to see associations that may have been otherwise masked by a larger more heterogeneous classification approach. We propose to use the presence of dental anomalies outside of the cleft area to subphenotype clefts. Preliminary analysis suggests dental anomalies are preferentially associated with clefts in some families38 and gene expression studies show that a number of genes co-localize to the developing tooth and palate39–42. In order to extend these earlier studies, we proposed to revisit the subset of the initially genotyped families with two or more siblings affected by CL/P and perform a dental examination to broaden the phenotypic description of the families.

Subjects and Methods

Dental assessments

Information on dental anomalies outside the cleft area was collected from the cases and all available relatives. Aside from tooth agenesis, which is the most common congenital anomaly in humans and the one we expected to see the most, other dental anomalies included supernumerary teeth, microdontia, macrodontia, missing cusps, and supernumerary cusps. In many instances, tooth agenesis needed confirmation by an X-ray exam for which we used a portable X-ray system (MinXray P200D MarkIII; Toshiba, Tokyo, Japan). In addition, missing teeth due to tooth decay (caries) needed to be distinguished from congenitally missing teeth. We conducted careful exams and collected comprehensive caries data (data not shown) to aid in the differential diagnosis.

The University of Iowa IRB (approval # 200507743) and University of Pittsburgh IRB (approval # 0511198) gave approval for the study in conjunction with local approval in the Philippines.

Despite local political issues, geographic locations, and weather conditions (thirteen typhoons and severe tropical storms hit the Philippines between May 23rd and December 19th, 2006), we were able to re-contact 46 families with two or more siblings affected with cleft lip with or without cleft palate (CL/P) out of the 70 families that we attempted to contact. Forty-two of the 46 families had available genotyping data. All 42 families had additional affected relatives beyond the two or more affected siblings. We collected data on approximately 500 individuals, including 100 unrelated control families that were used to calculate dental anomalies frequency in the general population for our power studies.

Candidate Gene Association Analysis

Forty-two families for which clinical dental information was available were genotypes for 1489 SNP polymorphisms. These SNPs included 727 SNPs in 150 candidate genes, 431 spanning 6q23-25, and 331 9q21, respectively. The complete list of the markers is presented in the appendix. Genotypes were performed by CIDR (Center for Inherited Disease Research) using the Illumina bead system. The design of using families with multiple affected individuals (and with additional sib cases of dental anomalies only added in by our study) allowed us to increase the statistical power of the linkage disequilibrium approaches. The candidate genes we have been studying (MSX1, IRF6, PAX9, and FGFR1) are represented in this collection of 500 markers, as well as other interesting regions. Among the 150 candidate genes are BMP2, BMP4, EGF and its receptor, DLX family members, FGF1, FGF8, FGF10, MSX2, PVR, PVRL family members, TGFA, TGFB family members and their receptors, SKI, SHH, PTCH, WNT family members, TBX family members, PITX2, and RARA.

The data for all SNPs were consistent with Hardy-Weinberg equilibrium in both the affected and unaffected individuals, as well as in a group of unrelated individuals. Alleles at each marker were tested for association twice under an additive model: (1) first only those individuals with CL/P were considered affected, (2) second, the affection status was broadened to include individuals with dental anomalies who were also assigned as affected. The Family Based Association Test implemented in the FBAT software package43,44 was used in these analyses.

Results

In the 42 families, there were 519 individuals total. One hundred and twenty-eight (128) people were born with CL/P, and genotyping data was available for 125 of them. The remaining 391 family members were not affected by CL/P, and genotyping data was available for 215 of them. Among the 391 unaffected relatives, 48 individuals had dental anomalies (and genotyping data was available for 43 of them).

Tooth agenesis was the most prevalent dental anomaly found in this study. Third molars were the most frequently affected tooth, followed by second premolars. Although other dental anomalies such as supernumerary teeth, microdontia, and supernumerary cusps were found in the families, the affected individuals usually had tooth agenesis as well, or these families always had other family members with tooth agenesis. Only nine probands did not have any relatives with dental anomalies (the other 33 probands had relatives with dental anomalies). However, four probands out of the nine did have dental anomalies outside the cleft area themselves. A total of 23 probands had concomitant dental anomalies outside the cleft area.

Table 1 presents all markers with p-values 0.05 or below (before multiple test correction) in each of the analyses. A SNP in ANKS6 (rs4742741, 9q22.33; p=0.0004) was significantly over-transmitted when the dental anomalies were added to the analysis. Another significantly over-transmitted SNP was seen in ERBB2 (rs1810132, 17q21.1; p=0.0006). In the clefts only analysis, a SNP in ERBB2 was significantly over-transmitted (p=0.0006). Other markers with interesting p-values included IRF6, CDH2, and 6q21-q23 loci (Table 1). Table 2 highlights the differences found between the two analyses performed. In summary, many of the over-transmitted SNPs were seen under both analysis (cleft only versus cleft plus dental anomalies), but notably the loci 14q24.3-q31.1 (DPF3 and NRXN3) and 21q22.11 (GART) showed evidence for over-transmission only with the addition of dental anomaly phenotypes in the analysis.

Table 1.

Most significant linkage disequilibrium results in the cleft lip and palate families with and without dental anomalies as an additional affection status.

Cleft Lip and Palate + Dental Anomalies
chromosome snp cM p-value Gene
1 rs2013162 206.3571 0.002 IRF6
1 rs2279455 91.89721 0.008 TGFBR3
1 rs674433 206.3533 0.01 IRF6
1 rs3738480 147.8039 0.02 PRUNE
1 rs786908 88.96538 0.04 PKN2
2 rs377122 70.67868 0.03 TGFA
2 rs7583130 202.7306 0.04 SUMO1 close to
3 rs9849690 185.8107 0.02 EPHB3 close to
3 rs1515490 191.0796 0.04 p63/TP73L
4 rs6841268 139.5262 0.04 SLC7A11
4 rs7677751 54.96539 0.04 PDGFRA
5 rs4559013 170.7842 0.04 FGF18
5 rs3934591 170.8002 0.05 FGF18
6 rs9320231 108.1769 0.005 SCML4
6 rs6921044 140.3791 0.006 BC039503
6 rs969282 134.2468 0.008 TCF21
6 rs971402 112.5946 0.008 LAMA4
6 rs2503791 153.7521 0.009 MTRF1L
6 rs7772821 132.9342 0.01 TAAR6
6 rs9206 151.7713 0.01 MTHFD1L
6 rs3757316 151.8665 0.01 Corf211
6 rs1555091 127.4783 0.02 AK127472
6 rs9491385 125.6747 0.02 IBRDC1 close to
6 rs1546943 116.6082 0.02 NT5DC1
6 rs485640 125.4073 0.03 IBRDC1
6 rs2503322 127.499 0.03 RSPO3
6 rs2811674 134.3728 0.03 SLC2A12
6 rs3800223 108.6792 0.03 SNX3
6 rs238590 115.4712 0.03 HS3ST5
6 rs6570847 148.7266 0.04 SASH1
6 rs1741820 122.7632 0.04 HSF2
6 rs1267948 122.8153 0.04 SERINC1
6 rs576247 122.7886 0.05 HSF2
6 rs2802288 109.0029 0.05 FOXO3A
6 rs6913898 151.474 0.05 MTHFD1L
6 rs911477 109.3696 0.05 ARMC2
9 rs4742741 98.61916 0.0004 ANKS6
9 rs843258 102.6709 0.007 CYLC2
9 rs1930135 98.49069 0.009 GABBR2
9 rs1020884 97.23761 0.01 FOXE1
9 rs4743088 97.23837 0.01 FOXE1
9 rs2636879 114.1241 0.01 COL27A1
9 rs4443717 107.0198 0.01 ZNF462
9 rs418919 99.11206 0.02 TGFBR1 close to
9 rs4129220 94.46965 0.03 FBP1
9 rs337572 98.5662 0.03 ANKS6
9 rs1555573 99.20898 0.03 DQ673940
9 rs773515 91.06249 0.03 AUH
9 rs3747496 97.1669 0.03 KIAA1529
9 rs4743077 97.17104 0.03 KIAA1529
9 rs2416682 118.5309 0.03 TLR4
9 rs3794486 105.5419 0.04 TMEM38B
9 rs1979993 105.6141 0.04 TMEM38B
9 rs3793524 109.299 0.04 PTPN3
9 rs1059273 97.92906 0.04 TRIM14
9 rs4743348 99.25561 0.05 TGFBR1 close to
9 rs2281732 97.92456 0.05 TRIM14
14 rs2536143 72.25479 0.03 DPF3
14 rs221430 79.13787 0.04 NRXN3
14 rs1018466 36.193 0.04 PAX9 close to
15 rs2879515 32.65859 0.01 SLC12A5 close to
15 rs878960 24.48003 0.02 GABRB3
15 rs690 56.62203 0.03 LIPC
15 rs1426223 24.50339 0.05 GABRB3
17 rs1810132 35.11953 0.0005 ERBB2
17 rs2015729 42.70949 0.002 ITGB3
17 rs2292699 42.71729 0.005 ITGB3
17 rs890397 45.45893 0.01 DLX3/DLX4
17 rs1905339 37.83582 0.01 STAT3 close to
17 rs8071740 22.54986 0.02 WSB1 close to
17 rs744166 37.76773 0.02 STAT3
17 rs2313430 35.18334 0.04 IKZF3
17 rs9906933 37.66357 0.04 STAT5B
18 rs2215502 24.03802 0.02 CDH2 close to
20 rs819133 32.33398 0.02 AHCY
20 rs6123674 55.19635 0.04 BMP7
21 rs4817579 33.83209 0.02 GART
Cleft Lip and Palate
chromosome snp cM p-value Gene
1 rs674433 206.3533 0.001 IRF6
1 rs2013162 206.3571 0.001 IRF6
1 rs513287 167.3959 0.003 PRRX1
1 rs2279455 91.89721 0.01 TGFBR3
1 rs4245660 78.25275 0.02 GIPC2
1 rs1051740 222.3264 0.03 EPHX1
1 rs786908 88.96538 0.03 PKN2
1 rs1007512 75.2837 0.05 LHX8 close to
2 rs7583130 202.7306 0.01 SUMO1 close to
2 rs4328603 9.611782 0.01 ADAM17
2 rs2280509 202.7229 0.03 FZD7
2 rs6705408 9.580829 0.03 ADAM17
2 rs2276338 9.596387 0.03 ADAM17
2 rs377122 70.67868 0.04 TGFA
2 rs512535 21.17943 0.04 OSR1 close to
3 rs9849690 185.8107 0.02 EPHB3 close to
5 rs7715062 7.959907 0.04 MTRR close to
6 rs2503791 153.7521 0.003 MTRF1L close to
6 rs971402 112.5946 0.003 LAMA4
6 rs7772821 132.9342 0.005 TAAR6
6 rs9320231 108.1769 0.01 SCML4
6 rs6921044 140.3791 0.01 BC039503
6 rs969282 134.2468 0.02 TCF21 close to
6 rs1546943 116.6082 0.02 NT5DC1
6 rs1983721 117.0122 0.02 RWDD1
6 rs485640 125.4073 0.02 IBRDC1
6 rs911477 109.3696 0.03 ARMC2
6 rs718174 108.4799 0.03 OSTM1
6 rs3734679 107.6221 0.03 PDSS2
6 rs2503322 127.499 0.03 RSPO3
6 rs3800229 109.1037 0.03 FOXO3A
6 rs2811674 134.3728 0.03 SLC2A12
6 rs3127657 107.2111 0.04 QRSL1
6 rs549332 116.5613 0.04 NT5DC1
6 rs1555091 127.4783 0.04 AK127472
6 rs9400504 112.213 0.04 FYN
8 rs6987534 38.41887 0.02 FGFR1
8 rs3925 38.40082 0.03 FGFR1
9 rs4742741 98.61916 0.001 ANKS6
9 rs418919 99.11206 0.005 TGFBR1 close to
9 rs1020884 97.23761 0.01 FOXE1 close to
9 rs4743088 97.23837 0.01 FOXE1 close to
9 rs2636879 114.1241 0.01 COL27A1
9 rs1930135 98.49069 0.01 GABBR2
9 rs4129220 94.46965 0.01 FBP1
9 rs2281732 97.92456 0.02 TRIM14
9 rs1059273 97.92906 0.02 TRIM14
9 rs843258 102.6709 0.02 CYLC2
9 rs1555573 99.20898 0.02 DQ673940
9 rs995294 109.8189 0.03 PALM2-AKAP2
9 rs3794486 105.5419 0.03 TMEM38B
9 rs3750396 88.85173 0.03 AK127258
9 rs4743348 99.25561 0.03 TGFBR1 close to
9 rs1320547 93.79928 0.04 BARX1 close to
9 rs1462090 97.24908 0.05 FOXE1 close to
9 rs773515 91.06249 0.05 AUH
11 rs10790332 119.0589 0.02 PVRL1
12 rs11065374 119.8629 0.01 TCF1 close to
12 rs1039302 119.699 0.02 UNQ1887
14 rs1018466 36.193 0.01 PAX9 close to
15 rs690 56.62203 0.02 LIPC
17 rs1810132 35.11953 0.0006 ERBB2
17 rs2015729 42.70949 0.001 ITGB3
17 rs2292699 42.71729 0.01 ITGB3
17 rs890397 45.45893 0.01 DLX3/DLX4 close to
17 rs2056131 42.68874 0.01 ITGB3
17 rs8071740 22.54986 0.01 WSB1 close to
17 rs4461115 43.15458 0.03 ITGB3 close to
18 rs2215502 24.03802 0.003 CDH2 close to
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Table 2.

Contrasting results between the two candidate genes/association analyses.

Locus Gene SNP CLP Data (p-value) CLP + Dental Anomalies Data (p-value)
Loci where association is present in both clefts and clefts + dental anomalies data
9q22.33 ANKS6 rs4742741 0.001 0.0004
CYLC2 rs843258 0.02 0.008
GABRB2 rs1930135 0.01 0.009
TGFBR1 rs418919 0.005 0.02
FOXE1 rs1020884 0.01 0.01
FOXE1 rs4743088 0.01 0.01
17q21.1 ERBB2 rs1810132 0.0006 0.0006
ITGB3 rs2015729 0.002 0.003
ITGB3 rs2292699 0.01 0.005
1q32.3-q41 IRF6 rs2013162 0.002 0.002
IRF6 rs674433 0.001 0.01
6q21-q23 SCML4 rs9320231 0.01 0.006
BC039503 rs6921044 0.01 0.006
TCF21 rs969282 0.02 0.008
LAMA4 rs971402 0.003 0.008
MTRFL1 rs2503791 0.003 0.009
TAAR6 rs7772821 0.005 0.01
2p13 TGFA rs377122 0.04 0.03
Loci where association is stronger in the clefts + dental anomalies than that in the clefts data
5q34 FGF18 rs4559013 0.09 0.05
FGF18 rs3934591 0.1 0.05
14q24.3-q31.1 DPF3 rs2536143 0.18 0.03
NRXN3 rs221430 0.32 0.04
15q11.2-q12 SLC12A5 rs2879515 0.06 0.01
GABRB3 rs878960 0.06 0.02
GABRB3 rs1426223 0.06 0.05
LIPC rs690 0.02 0.04
20q13 AHCY rs819133 0.06 0.02
BMP7 rs6123674 0.07 0.04
21q22.11 GART rs4817579 0.16 0.02
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Discussion

Our results from the candidate gene data suggest that dental anomalies are part of an extended cleft phenotype. In addition, some genes may contribute to clefts in association with dental anomalies. However, there are obvious limitations in our study. Although the Filipino families included in our study tend to have large sibships, it was not always possible to examine all potential subjects in all families. A number of reasons account for that, such as having a job in another city and not being available at the time of data collection, or choosing not to participate in the study. Another limitation is that this family dataset is probably not representative of the Filipino population. Although it is possible that this group of families may be representative of the Cebu province or even the Central Visayas region, the lack of official population-based records of birth defects in the Philippines does not allow us to make any assumptions regarding the Filipino population as a whole.

The association we found between families with clefts and IRF6 confirms our previous work10 with this same population. It is remarkable that the association is still evident with only 42 families, which corroborates that IRF6 is a major contributor to clefts in Filipinos. While concerned about multiple testing, we did not apply the strict Bonferroni correction as it would increase type II errors and a major focus of this study was to identify putative associations with the combined dental anomaly/cleft phenotype for further studies. For example, under the Bonferroni correction, we would have lowered the alpha to 0.00003 (0.05/1489) and the known association with IRF6 (interferon regulatory factor 6; p = 0.001) would have been missed. Therefore we report here all results with p-values below 0.05. However, our data must be carefully interpreted since it is expected that some of the p-values below 0.05 can be due to chance.

Analyses under both the narrow and broad affection statuses resulted in significant evidence of over-transmission for markers in 6q21-q23.2, 9q21, and 17q12. The 6q21-q23.2 and 9q21 regions previously showed linkage to clefts in a meta-analysis of genome wide scan data from seven populations15. In the current study, markers in 6q21-q23.2 yielded p-values between 0.009 and 0.003, and those in 9q21 yielded p-values between 0.009 and 0.0004. The most significantly over-transmitted marker in 9q21 was rs4742741 in ANKS6 (ankyrin repeat and sterile alpha motif domain containing 6) located at 9q22.33 (p = 0.001 for clefts only, and p = 0.0004 for clefts and dental anomalies). Adrenomedulin, a vasodilator peptide, prevents the suppression of the inhibitory SMAD6 (SMAD, mother against DPP homolog 6) protein by TGFB1 (transforming growth factor beta 1) and restores SMAD2-ANKS6 complex formation in human renal tubular epithelial cell lines45. TGFB/BMP (bone morphogenetic protein) signals rely on SMAD-dependent pathways in the ectomesenchyme to mediate epithelial-mesenchymal interactions that control the first branchial arch patterning and tooth development46.

The rs1810132 marker in ERBB2 (receptor tyrosine-protein kinase erbB-2, precursor), located in 17q12, yielded p-values of 0.0006. Previous work has suggested that RARA (retinoic acid receptor alpha), located at 17q21.1, is associated with isolated cleft lip and palate47,48. ERBB2 is 642,088 base pairs upstream from RARA. Since they are relatively near to each other, the previous association suggested for RARA could actually be due to variation in ERBB2. ERBB2 is an essential component of a neuregulin-receptor complex but it is not activated by EGF (ectodermal growth factor) or TGFA (transforming growth factor alpha). Erbb2-deficient mice die at birth and display defects in pre-synaptic development49. Ethanol consumption during pregnancy affects the expression of Erbb2 and induces a delay in murine fetal dental morphogenesis50. ERBB2 has not been previously considered as a candidate gene for clefts.

In contrast to the above results, suggestive over-transmission of markers in GART (phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase), DPF3 (D4, zinc and double PHD fingers, family 3), and NRXN3 (neurexin 3) were seen only when the dental anomaly phenotype was included in the analysis. These genes have not been shown to be expressed during tooth development and their function is still largely unknown. According to the Entrez database, GART is required for de novo purine biosynthesis, NRXN3 functions in the vertebrate nervous system as cell adhesion molecules and receptors, and DPF3 is probably involved in RNA transcription.

In summary, our results support the hypothesis that increasing the complexity of the clinical description by adding dental anomalies information will provide new opportunities to map susceptibility loci for clefts. Here we report, for the first time, an extensive candidate gene analysis for cleft susceptibility loci using dental anomalies to subphenotype clefts. This approach appears to be a promising one and may help in the identification of genetic variants that increase cleft susceptibility, which would be a crucial step that may allow better estimates of recurrence risks for individual families.

Acknowledgments

The authors are indebted with the families that participated in this study. The Phenomics Group of the Philippines, Inc. facilitated the field work. Andrew Lidral was instrumental during the process that allowed the access to the genotype data. This work was supported by the NIH grants R21-DE016718, R37-DE08559, R01-DE016148, P50-DE016215, and CIDR NIH contract N01-HG-65403 (genotyping).

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