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. Author manuscript; available in PMC: 2019 Jul 17.
Published in final edited form as: Birth Defects Res. 2018 Jun 19;110(12):1043–1048. doi: 10.1002/bdr2.1346

BRCA1 and BRCA2 Gene Variants and Nonsyndromic Cleft Lip/Palate

Nicholas Rodriguez 1, Lorena Maili 3,4, Brett T Chiquet 1,2,5, Susan H Blanton 6, Jacqueline T Hecht 1,4,5, Ariadne Letra 1,3,5
PMCID: PMC6105370  NIHMSID: NIHMS962480  PMID: 29921024

Abstract

Background

Nonsyndromic cleft lip with or without cleft palate is a debilitating condition that not only affects the individual, but the entire family. The purpose of this study was to investigate the association of BRCA1 and BRCA2 genes with NSCL/P.

Methods

Twelve polymorphisms in/nearby BRCA1 and BRCA2 were genotyped using Taqman chemistry. Our data set consisted of 3,473 individuals including 2,191 nonHispanic white (NHW) individuals (from 151 multiplex and 348 simplex families) and 1,282 Hispanic individuals (from 92 multiplex and 216 simplex families). Data analysis was performed using Family-Based Association Test (FBAT), stratified by ethnicity and family history of NSCL/P.

Results

Nominal associations were found for BRCA1 in Hispanics and for BRCA2 in NHW and Hispanics (P<0.05). Significant haplotype associations were found for both BRCA1 and BRCA2 (P≤0.004).

Conclusions

Our results suggest a modest association between BRCA1 and BRCA2 and NSCL/P. Further studies in additional populations and functional studies are needed to elucidate the role of these genes in developmental processes and signaling pathways contributing to NSCL/P.

Keywords: association, family-based, BRCA1, BRCA2, cleft lip/palate

Introduction

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect with genetic and environmental factors playing etiologic roles. While numerous gene variants have been suggested to confer an increased risk of NSCL/P, it is estimated that only ~20% of the underlying etiology of NSCL/P has been identified, leaving additional causal genes to be identified (Dixon et al. 2011). This is supported by the fact that most of the NSCL/P cases reflect isolated forms, and when familial, the inheritance models are not always clear. Additionally, genetic factors of modest effects may have different contributions depending on the presence of specific environmental factors, thereby complicating the discovery of true etiologic variants. Moreover, in genome-wide association studies of NSCL/P, variants in IRF6, and in the chromosome regions 8q24 and 10q25 (VAX1), were suggested as strongly associated with NSCL/P, however the association signals were seemed to be more population-specific (Dixon et al. 2011).

Studies have demonstrated that the consequences of being born with NSCL/P extend beyond functional and esthetic complications. Individuals with NSCL/P were reported to have shorter lifespan and increased risk for all major causes of death, particularly cancer, when compared with individuals born without clefts (Christensen et al. 2004; Steinwachs et al. 2000; Vieira 2008). Studies in different populations also suggest that families with recurrent NSCL/P have increased susceptibility to cancer (Bille et al. 2005; Menezes et al. 2009; Zhu et al. 2002). A large epidemiological study in Denmark showed an increased occurrence of breast and brain cancer among adult females born with NSCL/P, and of primary lung cancer among adult males with NSCL/P (Bille et al. 2005). While additional studies did not confirm a general increase in the risk of breast cancer among female subjects in NSCL/P families (Dietz et al. 2012; Martelli et al. 2014), a higher frequency in family history of cancer (colon, brain, leukemia, breast, prostate, skin, lung, and liver) was found in NSCL/P families when compared to control families (Menezes et al. 2009). Interestingly, dysregulation of genes in networks associated with DNA double-strand break repair and cell cycle control, including BRCA1 and BRCA2, was observed in dental pulp stem cells of NSCL/P patients, meanwhile the expression of BRCA1 and BRCA2 genes was significantly downregulated in NSCL/P dental pulp cells compared to controls (Kobayashi et al. 2013). These observations led us to investigate whether variations in BRCA1 and/or BRCA2 genes were associated with NSCL/P, since the association between NSCL/P and cancer raises interesting possibilities to identify risk markers for cancer. In this study, we tested the association of BRCA1 and BRCA2 genes with nonsyndromic NSCL/P.

Study population

This study was approved by the University of Texas Health Science Center Committee for Protection of Human Subjects. Our study population consisted of a total of 3,473 individuals including 2,191 nonHispanic white (NHW) individuals (from 151 multiplex and 348 simplex families) and 1,282 Hispanic individuals (from 92 multiplex and 216 simplex families). Families were recruited at the University of Texas Health Science Center Craniofacial Clinics. Families were ascertained through probands, and additional relatives were recruited. Multiplex families are families with 2 or more individuals affected with NSCL/P. Simplex families (trios) are families in which the only the proband is affected with NSCL/P and parents are unaffected. Individuals presenting with syndromic clefts, cleft palate only, or unknown cleft types were excluded. Complete demographic and medical history, and DNA samples from peripheral blood were available for all individuals in the study.

Selection of single nucleotide polymorphisms and genotyping

Twelve single-nucleotide polymorphisms (SNPs) in/nearby BRCA1 and BRCA2 genes (Table 1) were selected for genotyping using a tag-SNP approach, considering heterozygosity values, gene structure, and the linkage disequilibrium (LD) block surrounding each gene, as previously described (Carlson et al. 2004) (Supplementary Figure 1). This approach allows for selection of a minimum number of SNPs ensuring maximum coverage within a LD block. Genotyping was performed using Taqman chemistry (Ranade et al. 2001) in a ViiA7 RealTime PCR System (Applied Biosystems, Foster City, CA).

Table 1.

Details of SNPs genotyped in BRCA1 and BRCA2 genes.

Gene dbSNP Id* Base position* Location Alleles§ MAF
NHW		 MAF
Hisp
BRCA1 rs799906 chr. 17:43126099 5’ UTR C/T 0.34 0.35
rs799917 chr. 17:43092919 Missense A/G 0.33 0.32
rs16941 chr. 17:43092418 Missense A/G 0.32 0.27
rs8176194 chr. 17:43079204 Intron G/T 0.36 0.32
rs8070179 chr. 17:43050671 Intron A/G 0.33 0.25
rs8176318 chr. 17:43045257 3’ UTR G/T 0.32 0.19
BRCA2 rs206115 chr. 13:32314336 5’ near gene A/G 0.42 0.29
rs144848 chr.13:32332592 Missense G/T 0.29 0.27
rs206079 chr. 13:32346481 Intron A/G 0.46 0.44
rs9943888 chr. 13:32354065 Intron A/G 0.28 0.27
rs9534342 chr. 13:32381260 Intron C/T 0.48 0.46
rs11571836 chr. 13:32399302 3’ UTR A/G 0.21 0.21
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*

According to NCBI dbSNP build 147

§

Ancestral allele listed first

MAF, minor allele frequency

Statistical analyses

Family-based single SNP association tests were performed using Family-Based Association Test (FBAT), with the ‘–e’ option for extended families. Analyses were performed for all families stratified by ethnicity (NHW or Hispanic) and family history of NSCL/P (multiplex and simplex). Pairwise haplotype analyses were performed using the ‘hbat’ function in FBAT. Bonferroni correction was used to adjust for multiple testing and P-values ≤0.004, considering the number of SNPs tested (0.05/12), were considered statistically significant.

In Silico Prediction of SNP Function

In silico analysis of SNP function was performed using miRdSNP v.11.03 (Bruno et al. 2012) for BRCA1 rs8176318, and using PATCH v.1.0 (Wingender et al. 2000), and PROMO v.3.0 (Messeguer et al. 2002) for BRCA2 rs206115.

Results

No SNPs met the Bonferroni correction, however, nominal associations (p≤0.05) were found between NSCL/P and individual SNPs in BRCA1 and BRCA2. While SNPs in BRCA2 showed nominal associations in NHW and Hispanic datasets, SNPs in BRCA1 were associated with NSCL/P in Hispanics only. BRCA1 SNPs rs16941 and rs8176318, a missense variant and a 3’ UTR variant, respectively, showed modest association in Hispanics (p=0.02 and p=0.04, respectively) (Table 2). For BRCA2, the promoter variant rs206115 was also modestly associated with NSCL/P in both NHW and Hispanic multiplex families (p=0.01). Additional BRCA2 SNPs (rs144848 and rs9534342) showed borderline associations with NSCL/P (p ≤ 0.05) (Table 2).

Table 2.

Results of single SNP association analyses.

BRCA1
SNPs		 NHW Hispanic
All
(n=499)		 Multiplex
(n=151)		 Simplex
(n=348)		 All
(n=308)		 Multiplex
(n=92)		 Simplex
(n=216)
FBAT FBAT-e FBAT FBAT-e FBAT FBAT FBAT-e FBAT FBAT-e FBAT
rs799906 0.64 0.65 0.64 0.67 0.32 0.56 0.57 0.09 0.10 0.54
rs799917 0.68 0.70 0.64 0.67 0.38 0.99 0.99 0.18 0.17 0.24
rs16941 0.62 0.64 0.77 0.79 0.38 0.12 0.09 0.04 0.02 0.73
rs8176194 0.62 0.62 0.26 0.27 0.76 0.26 0.24 0.08 0.06 1.00
rs8070179 0.54 0.54 0.82 0.83 0.53 0.35 0.35 0.05 0.06 0.73
rs8176318 0.68 0.69 0.45 0.49 0.26 0.05 0.04 0.07 0.08 0.27
BRCA2 SNPs NHW Hispanic
All (n=499) Multiplex (n=151) Simplex (n=348) All (n=308) Multiplex (n=92) Simplex (n=216)
FBAT FBAT-e FBAT FBAT-e FBAT FBAT FBAT-e FBAT FBAT-e FBAT
rs206115 0.11 0.13 0.01 0.02 0.94 0.15 0.15 0.01 0.02 0.51
rs144848 0.42 0.44 0.21 0.24 0.05 0.89 0.88 0.21 0.24 0.92
rs206079 0.29 0.32 0.10 0.15 0.94 0.42 0.42 0.10 0.15 0.48
rs9943888 0.78 0.78 0.53 0.52 0.38 0.91 0.91 0.53 0.52 0.97
rs9534342 0.54 0.54 0.05 0.06 0.50 0.60 0.60 0.05 0.06 0.85
rs11571836 0.79 0.80 0.31 0.34 0.21 0.55 0.55 0.31 0.36 0.53
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FBAT, Family Based Association Test; FBAT –e, extended pedigree option

NHW, nonHispanic white

All = all families combined (multiplex + simplex)

Significant if P≤0.004.

Haplotype analyses showed a few significant associations in 2-, 3- and 4-SNP haplotype windows for both BRCA1 and BRCA2, and suggest that the combination of specific alleles in an additive model may contribute to NSCL/P in those families (p ≤ 0.004) (Supplementary Table 1).

Of note, although the individual SNP associations did not reach the strict Bonferroni criteria significance threshold, the nominal associations and the specific haplotype associations found for BRCA1 and BRCA2 variants in our NSCL/P families warrant discussions of the potential biological implications of these variants in NSCL/P phenotypes. For example, BRCA1 rs8176318 and BRCA2 rs206115 are located in gene regulatory regions and harbor allele-specific microRNA or transcription factor binding sites, respectively, that may have an impact on gene expression (data not shown). Moreover, BRCA1 rs8176318 is predicted to bind to hsa-miR-205, known to be involved in epithelial to mesenchymal transformation (EMT) and tumor invasion. During palatogenesis, EMT is an important event contributing to the disappearance of the medial edge epithelia (MEE) and continuous mesenchymal confluence required for palatal fusion (Warner et al. 2015). Interestingly, the expression of miR-205 was found to be significantly downregulated (6-fold) in isolated MEE from wild type murine fetuses on gestational day 13.5 to 14.5 (prior to and during epithelial fusion of the palatal processes, respectively) (Warner et al. 2015). While these are intriguing findings, additional studies elucidating the biological roles of BRCA1 and BRCA2 during embryonic development might provide valuable insights into the underlying etiology of birth defects including NSCL/P.

NSCL/P is a complex disorder in which genes and environmental factors may have individual and interactive roles (Dixon et al. 2011). Disruptions of early developmental processes such as cell migration, proliferation, transdifferentiation and apoptosis due to variations in developmental genes have been shown to be closely related to the occurrence of NSCL/P (Greene and Pisano 2010) and cancer as well (Vieira 2008). Furthermore, variations in genes with critical roles in DNA damage repair, including BRCA1, have also been assessed and implicated in increased susceptibility to NSCLP and reinforced the hypothesis of etiological overlap between this common birth defect and cancer (Kobayashi et al. 2013; Mostowska et al. 2014). It is intriguing, however, that the available GWAS on NSCL/P (Birnbaum et al. 2009; Grant et al. 2009; Mangold et al. 2010) have not identified variants in BRCA1 or BRCA2 meanwhile numerous association studies in independent populations have reported the association of cancer-related genes with NSCL/P (Han et al. 2014; Letra et al. 2012; Letra et al. 2009; Menezes et al. 2009; Suzuki et al. 2009; Vieira 2008). Discrepancies among the results of previous studies may reflect genetic/allelic heterogeneity as well as differences in study design (family-based or case-control), sample sizes, and selection of SNPs to be studied (or specific genotyping arrays used in GWAS). A major GWAS finding is that the majority of risk variants for complex traits map to noncoding regions. For NSCLP, GWAS have identified around 40 risk loci, the majority of which are located in noncoding regions (Beaty et al. 2010; Birnbaum et al. 2009; Grant et al. 2009; Ludwig et al. 2012; Mangold et al. 2010; Yu et al. 2017), and for which the functional effects of on NSCLP phenotypes remain to be elucidated.

Recently, studies using whole exome sequencing of NSCLP families have also identified rare mutations in developmental and cancer-related genes as likely contributing to the cleft phenotypes (Fu et al. 2017; Liu et al. 2015). A frameshift variant caused by a 2bp insertion mutation (c.819_820dupCC; p.Gln274fs) in TP63, was found in a father-proband duo with NSCLP. Further, mRNA studies demonstrated that the mutant allele underwent nonsense-mediated-mRNA decay (NMD) as only the wild-type allele observed in mRNA obtained from the patient’s lymphocytes and lymphoblasts (Basha et al. 2018). TP63 encodes a transcription factor and a master regulator of epithelial lineage commitment during embryonic development, and was previously implicated in syndromic and NSCLP (Leslie et al. 2017; Scapoli et al. 2008). Interestingly, exome sequencing has also identified variations in TP63 in patients and tumor tissues of oral squamous cell carcinoma (Al-Hebshi et al. 2016; Stransky et al. 2011). Additional large-scale next-generation sequencing studies have the ability to reveal fundamental mechanisms underlying developmental and tumorigenic events and potential overlaps in disease pathways. These studies also have the potential to elucidate the full spectrum of genetic variation contributing to NSCLP. Corroborating previous findings, our study highlights the need to conduct a thorough medical and family history questionnaire, to identify families showing both NSCLP and cancer appearing together, and for which a same genetic variant may be contributing to the phenotype.

In summary, our results suggest a modest role for BRCA1 and BRCA2 in NSCL/P susceptibility. While the findings of our study may represent private mutations within private families, they continue to support the notion that genes involved at early stages of embryonic development may also have roles in cancer development later in life. Additional studies in other populations and functional studies are necessary to elucidate the role of BRCA1 and BRCA2 genes in the developmental processes and signaling pathways contributing to NSCL/P. Nonetheless it is possible that these genes may act through more than one signaling pathway to elicit numerous cellular responses that are cell- and tissue-dependent to contribute to birth defects and cancer later in life.

Supplementary Material

supp info

Acknowledgments

We thank the study families. This study was supported by NIH/NIDCR R01-DE011931 (to JTH). NR was supported by UTSD Summer Research Program.

Footnotes

The authors have no conflict of interest to declare.

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