The Journal of the American Dental Association
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J Am Dent Assoc, Vol 140, No 1, 80-84.
© 2009 American Dental Association
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RESEARCH

AXIS inhibition protein 2, orofacial clefts and a family history of cancer

Renato Menezes, DDS, MS, PhD, Mary Louise Marazita, PhD, Toby Goldstein McHenry, BS, Margaret E. Cooper, MSIS, MEd, MS, Kathleen Bardi, BS, Carla Brandon, MS, Ariadne Letra, DDS, MS, PhD, Rick A. Martin, PhD and Alexandre Rezende Vieira, DDS, MS, PhD


   ABSTRACT
TOP
 ABSTRACT
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Background. Cancer and congenital malformations occasionally may have acommon etiology. The authors investigated whether families with one or more members affected by orofacial clefts (that is, families segregating orofacial clefts) had an increased cancer incidence when compared with control families.

Methods. The authors assessed 75 white families with nonsyndromic cleft lip with or without cleft palate (CL/P) and 93 white control families regarding a history of cancer. They used {chi}2 and Fisher exact tests to determine significant differences. They then performed molecular studies with genes in which mutations have been independently associated with both cancer and craniofacial anomalies in a total of 111 families with CL/P.

Results. The families with CL/P reported a family history of cancer more often than did control families (P <.001), and they had higher rates of specific cancer types: colon (P <.001), brain (P = .003), leukemia (P = .005), breast (P = .009), prostate (P = .01), skin (P = .01), lung (P = .02) and liver (P = .02). The authors detected overtransmission of AXIS inhibition protein 2 (AXIN2) in CL/P probands (P = .003).

Conclusion. Families segregating CL/P may have an increased susceptibility to cancer, notably colon cancer. Furthermore, AXIN2, a gene that when mutated increases susceptibility to colon cancer, also is associated with CL/P.

Clinical Implications. People who are at a higher risk of developing disease need to adopt a healthier lifestyle, including avoiding exposure to risk factors that may interact with their genotypes.

Key Words: Orofacial clefts; family history of cancer; AXIN2

Abbreviations: AXIN2: AXIS inhibition protein 2. • CDH1: Epithelial cadherin. • CL/P: Cleft lip with or without cleft palate. • FBAT: Family based association test. • FGF: Fibroblast growth factor. • FGFR: FGF receptor. • IRB: Institutional review board. • SNP: Single nucleotide polymorphism. • 3'UTR: Three prime untranslated region.

Nonsyndromic oral clefts are considered multifactorial in origin, with the possibility of genetic and environmental components interacting.1 Several authors have proposed that cancer and congenital malformations such as cleft lip and palate occasionally may have a common etiology.2–5 The underlying concept is that the same genes can act in normal development, as well as in malignant development. People born with orofacial clefts have a shorter life span, and investigators have suggested that cancer may be one of the factors responsible for reducing the life expectancy of those born with a facial cleft.2–5 Zhu and colleagues6 reported a higher cancer risk in parents of children born with oral clefts, and a large population-based study7 demonstrated an increased occurrence of cancer in people born with both cleft lip and cleft palate.

Recent evidence from genetic studies also has supported the hypothesis that some genes are simultaneously associated with cancer and craniofacial disorders. Fibroblast growth factor (FGF) signaling pathway genes have been associated with various types of cancer8–11 and might contribute to approximately 3 percent of cases of nonsyndromic cleft lip with or without cleft palate (CL/P).12 Frebourg and colleagues13 reported mutations in epithelial cadherin (CDH1), a cell-cell adhesive molecule expressed in epithelial cell types, in two families in which two or more members had hereditary diffuse gastric cancer and oral clefts (that is, families segregating gastric cancer and oral clefts). In addition, Lammi and colleagues14 reported that mutations in AXIS inhibition protein 2 (AXIN2) caused oligodontia (that is, absence of six or more permanent teeth) and increased susceptibility to colorectal cancer.

With the goal of investigating whether families segregating isolated CL/P are at an increased risk of developing cancer, we compared the incidence of cancer in families with CL/P with that in control families without a history of clefts. Furthermore, we performed candidate gene studies, selecting genes that were independently associated with both cancer and craniofacial anomalies.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
After receiving institutional review board (IRB) approval (University of Pittsburgh, IRB No. 0607057), we collected self-reported data regarding family history of cancer via a structured questionnaire from 168 families (75 families with CL/P and 93 control families) of white ancestry (that is, people who did not report Native American, African or Asian ancestry) in Pittsburgh. We recruited the families with CL/P from a registry maintained by the Cleft-Craniofacial Center, Children’s Hospital of Pittsburgh of UPMC.

The questionnaire asked participants to describe the precise relationship with the reported affected relative and the type of cancer he or she had. Of the 75 families with at least one member affected by CL/P, 66 reported that they had two or more affected members. A total of 558 participants (309 female, including 223 adults and 86 children, and 249 male, including 163 adults and 86 children) completed the questionnaire. The age range for females was 4 months to 85 years (mean age, 30 years) and for males was 6 months to 86 years (mean age, 28 years). Parents were responsible for answering the questions for their children. The analysis did not account for incomplete information about cancer history. We used {chi}2 and Fisher exact tests to determine statistically significant differences between families with CL/P and control families, with an {alpha} of .05.

We also investigated the role of genes in which mutations have been associated, independently or in the same study, with both cancer and CL/P or other craniofacial anomalies. In addition to the 75 families with CL/P participating in this study, we included 36 white families from Pittsburgh and St. Louis (recruited by R.A.M.) to improve the statistical power. Three of us (K.B., C.B., R.A.M.) obtained genomic DNA samples from saliva, blood, mouthrinse or buccal swabs from each member of the 111 families with CL/P (including the 75 families with CL/P who answered the questionnaires), a total of 427 people, 131 of whom were affected by CL/P.

We performed genotyping via the TaqMan method,15 by using an automatic instrument and predesigned probes (7900HT Fast Real-Time PCR System, Applied Biosystems, Foster City, Calif). We used the family based association test16 to detect transmission distortions in the families segregating CL/P. For AXIN2 and CDH1 genes, we used the approach proposed by Carlson and colleagues17 to select a subset of single nucleotide polymorphisms (SNPs) that maximally represent the linkage disequilibrium structure of a given region (HapMap European-derived block structures, International HapMap Project).18 We derived all FGF and FGF receptor SNPs from previous studies that showed an association with clefts, cancer or both.9,10,12 Table 1Go9,10,17–22 summarizes the genes and SNPs used in this study.


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  		TABLE 1 Summary of candidate genes and SNPs* studied and results observed.

 

   RESULTS
TOP
 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
On the basis of a questionnaire administered to participants of white ethnicity (75 families with CL/P and 93 control families), we observed that families with CL/P more often reported a family history of cancer than did families without a history of oral clefts (P <.001). The expected number of families with cancer in the control group was 75.3. However, the actual number was 66. Among the families with CL/P, the expected number of those with cancer was 60.7; the actual number was 70 (Table 2Go).


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  		TABLE 2 Families with history of cancer among families with CL/P* and control families.

 
In addition, families segregating CL/P more commonly reported a history of multiple types of cancer (three or more) compared with the control families (P <.001) (Table 3Go). Families with CL/P reported increased rates of colon cancer (P <.001), brain cancer (P = .003), leukemia (P = .005), female breast cancer (P = .009), prostate cancer (P = .01), skin cancer (P = .01), lung cancer (P = .02) and liver cancer (P = .02) in comparison with control families.


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  		TABLE 3 Occurrences of cancer among families with CL/P.*

 
We also observed an association between AXIN2 and CL/P (P = .003) (Table 1Go).


   DISCUSSION
TOP
 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
Childhood cancers accompany many congenital malformations. Within that context, it is important to investigate the relationship between malformations and malignancies, because investigators speculate that they might have common causes.2–5

In our study, families segregating CL/P reported an increased familial history of cancer compared with families without a history of oral clefts. These observations provided us with the starting point to investigate genes in which mutations have been associated (independently or in the same study) with both cancer and clefts or other craniofacial anomalies—namely, AXIN2, CDH1 and members of the FGF gene family. Some of these genes are effectors of cell-cell adhesion and cell motility functions and/or play critical roles during embryonic development; in turn, they may lead us to believe that variations in these genes could contribute to the development of craniofacial disorders (such as CL/P, microtia, profound congenital deafness, tooth agenesis, microdontia) and cancer.

We observed an association between AXIN2 and CL/P (P = .003). The protein product of AXIN2 is a negative regulator of the Wnt signaling pathway.23,24 Moreover, evidence has shown that germline mutations in one of the Wnt pathway components (AXIN2) have been associated with tooth agenesis–colorectal cancer syndrome.14 Because tooth agenesis is a common finding in people affected by oral clefts, it is interesting to find mutations in AXIN2 associated with tooth agenesis and cancer. Letra and colleagues20 recently proposed that tooth agenesis be used to subphenotype clefts. Furthermore, the involvement of Wnt signaling genes in carcinogenesis is well-established (regulating cell growth, motility and differentiation), although relatively little is known about the connection between these genes and congenital malformations in humans. Wnt signaling has been implicated in the regulation of diverse developmental events (such as axis and mesoderm formation), as well as in aberrations of cell homeostasis that may lead to cancer,21,22,25 which might explain, in part, the results observed here.

In our study sample, we did not observe associations between CDH1 or FGF pathway genes and CL/P; however, other investigators12,13 found a relationship between these genes and CL/P. Future studies should consider them as candidate genes for oral clefts, because they are responsible for cell-cell adhesion and various developmental steps.

We compared self-reported family history of cancer in families segregating CL/P with that in families without a history of CL/P; however, the cancer types reported may not all have been related to the same causes. Furthermore, we did not have access to specific data regarding family members’ age at onset of cancer, which makes it impossible to determine if the family members of a person born with CL/P developed cancer at younger ages than those of people in the general population. In addition, only three people in our study who were born with CL/P also developed cancer (one child had leukemia, one adult had colon cancer and one adult had skin cancer). Although we do not have enough information to determine whether people born with CL/P have an increased susceptibility to cancer themselves, the results of a previous population-based study7 suggest that this is the case. We are increasing our sample size to replicate our findings and test for any association between genetic variants and specific types of cancer segregating in families.


   CONCLUSION
TOP
 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
The results of this study indicate that families segregating CL/P may have an increased susceptibility to cancer, notably colon cancer. Furthermore, AXIN2, a gene that when utated increases the susceptibility to colon cancer, also is associated with CL/P.


   FOOTNOTES
 

Dr. Menezes is a postdoctoral associate, Department of Oral Biology and Center for Craniofacial and Dental Genetics, School of Dental Medicine, University of Pittsburgh.


Dr. Marazita is the associate dean for research and director, Center for Craniofacial and Dental Genetics, and a professor and chair, Department of Oral Biology, School of Dental Medicine, University of Pittsburgh.


Ms. Goldstein McHenry is a member of the research staff, Department of Oral Biology and Center for Craniofacial and Dental Genetics, School of Dental Medicine, University of Pittsburgh.


Ms. Cooper is a research assistant professor, Department of Oral Biology and Center for Craniofacial and Dental Genetics, School of Dental Medicine, University of Pittsburgh.


Ms. Bardi is a member of the research staff, Department of Oral Biology and Center for Craniofacial and Dental Genetics, School of Dental Medicine, University of Pittsburgh.


Ms. Brandon is a member of the research staff, Department of Oral Biology and Center for Craniofacial and Dental Genetics, School of Dental Medicine, University of Pittsburgh.


Dr. Letra is a postdoctoral associate, Department of Oral Biology and Center for Craniofacial and Dental Genetics, School of Dental Medicine, University of Pittsburgh.


Dr. Martin is an adjunct professor, Department of Pediatrics, Division of Medical Genetics, St. Louis University School of Medicine, St. Louis.


Dr. Vieira is an assistant professor, Department of Oral Biology, Center for Craniofacial and Dental Genetics, School of Dental Medicine, University of Pittsburgh, 3501 Terrace St., 614 Salk Hall, Pittsburgh, Pa. 15261, e-mail "arv11{at}dental.pitt.edu". Address reprint requests to Dr. Vieira.


Disclosure. The authors did not report any disclosures.


This research was supported by National Institutes of Health grants K99-DE018413, R21-DE016718, R01-DE016148 and P50-DE016215.


The authors are indebted to the participants in the study.


   REFERENCES
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 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 

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