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. 2018 Nov 19;28(6):1038–1051. doi: 10.1093/hmg/ddy402

Genomic analyses in African populations identify novel risk loci for cleft palate

Azeez Butali 1,✉, Peter A Mossey 2, Wasiu L Adeyemo 3, Mekonen A Eshete 4, Lord J J Gowans 5, Tamara D Busch 1, Deepti Jain 6, Wenjie Yu 7, Liu Huan 8, Cecelia A Laurie 6, Cathy C Laurie 6, Sarah Nelson 6, Mary Li 1, Pedro A Sanchez-Lara 9, William P Magee III 10, Kathleen S Magee 11, Allyn Auslander 10, Frederick Brindopke 10, Denise M Kay 12, Michele Caggana 12, Paul A Romitti 13, James L Mills 14, Rosemary Audu 15, Chika Onwuamah 15, Ganiyu O Oseni 16, Arwa Owais 17, Olutayo James 3, Peter B Olaitan 16, Babatunde S Aregbesola 18, Ramat O Braimah 19, Fadekemi O Oginni 18, Ayodeji O Oladele 18, Saidu A Bello 20, Jennifer Rhodes 21, Rita Shiang 21, Peter Donkor 5, Solomon Obiri-Yeboah 5, Fareed Kow Nanse Arthur 5, Peter Twumasi 5, Pius Agbenorku 5, Gyikua Plange-Rhule 5, Alexander Acheampong Oti 5, Olugbenga M Ogunlewe 3, Afisu A Oladega 3, Adegbayi A Adekunle 3, Akinwunmi O Erinoso 3, Olatunbosun O Adamson 3, Abosede A Elufowoju 3, Oluwanifemi I Ayelomi 3, Taiye Hailu 4, Abiye Hailu 4, Yohannes Demissie 4, Miliard Derebew 4, Steve Eliason 7, Miguel Romero-Bustillous 7, Cynthia Lo 1, James Park 1, Shaan Desai 1, Muiawa Mohammed 1, Firke Abate 4, Lukman O Abdur-Rahman 22, Deepti Anand 23, Irfaan Saadi 24, Abimibola V Oladugba 25, Salil A Lachke 23, Brad A Amendt 7, Charles N Rotimi 26, Mary L Marazita 27, Robert A Cornell 7, Jeffrey C Murray 28, Adebowale A Adeyemo 26,✉
PMCID: PMC6400042  PMID: 30452639

Abstract

Orofacial clefts are common developmental disorders that pose significant clinical, economical and psychological problems. We conducted genome-wide association analyses for cleft palate only (CPO) and cleft lip with or without palate (CL/P) with ~17 million markers in sub-Saharan Africans. After replication and combined analyses, we identified novel loci for CPO at or near genome-wide significance on chromosomes 2 (near CTNNA2) and 19 (near SULT2A1). In situ hybridization of Sult2a1 in mice showed expression of SULT2A1 in mesenchymal cells in palate, palatal rugae and palatal epithelium in the fused palate. The previously reported 8q24 was the most significant locus for CL/P in our study, and we replicated several previously reported loci including PAX7 and VAX1.

Introduction

Orofacial clefts (OFCs) are the most common birth defects in the head and neck region, affecting 1 out of every 700 live births worldwide (1). These defects lead to significant financial, educational, medical, psychological and cultural problems for affected individuals and their families. Management of these disorders requires a multidisciplinary team of experts to restore aesthetics and function. Such expertise is often lacking in many parts of the world resulting in significant inequities in OFC care (2,3). A total of 70% of the OFCs are classified as non-syndromic with no visible recognizable structural defects other than clefts. Syndromic clefts account for 30% of the OFCs, where there is a consistently defined structural anomaly in addition to clefts. In terms of etiology, OFCs are complex traits, with genetic, environmental and stochastic factors contributing to the phenotypic expression (4). To date, 6 genome-wide association studies (GWASs) and 3 meta-analysis for cleft lip with or without cleft palate (CL/P) and 3 GWASs for cleft palate only (CPO) have been conducted, and over 40 risk loci have been identified (5–16). All of these studies have been conducted in individuals of European and Asian ancestry, with this study representing the first GWAS in Africans.

African populations represent novel and richly productive populations for genetic and environmental exposure studies for OFC because they have the greatest genetic diversity of any continental population (17,18) while residing in widely different environments. In this study involving individuals of African ancestry from Ghana, Nigeria and Ethiopia, we identified novel loci associated with CPO using data from 3178 participants (814 CL/P cases, 205 CPO cases, 2159 controls). Two of the identified novel loci were genome-wide significant after combined analysis with an independent replication sample. We also confirmed previously reported loci from GWAS of OFC in other populations, including populations of European and Asian ancestry.

Results

Novel loci identified for CPO

The discovery analysis for CPO revealed a chromosome 2 locus with genome-wide significance (lead single nucleotide polymorphism (SNP) rs80004662, near CTNNA2; P = 7.41 × 10−9; Fig. 1). Other loci on chromosomes 7, 9 and 19 showed suggestive genome-wide significance (5 × 10−7>P > 5 × 10−8) on discovery analysis (Table 1; Supplementary Material, Table S1). On meta-analysis with an independent replication sample, the chromosome 2 locus remained genome-wide significant (P = 7.29 × 10−9; Table 2; Supplementary Material, Table S2). Genes within the same topologically associated domains (TADs), as the GWAS SNP, are potential GWAS candidates. The TAD that includes the genome-wide significant SNPs contains just three genes—CTNNA2, LRRTM1 and SUCLG1 (Fig. 2). Among these genes, CTNNA2 is the best candidate as the chick ortholog has been implicated in control of cranial neural crest (19). Ctnna2 has been reported to be expressed in the oral structures of the mouse embryo at E14.5 (Fig. 2).

Figure 1.

Figure 1

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Manhattan plots of association statistics for CPO (A) and CL/P (B) in sub-Saharan Africa.

Table 1.

Top hits for discovery association analysis for CPO and cleft lip/palate (CL/P)

SNP Chr BP (base pairs) Effect allele Non-effect allele Effect allele frequency OR (odds ratio) 95% CI (confidence intervals) (OR) P-value
CPO
rs80004662 2 82025185 A G 0.013 7.5 3.45–16.28 7.41E-09
rs113691307 2 82028390 C T 0.013 7.5 3.45–16.28 7.41E-09
rs62529857 19 48386473 T C 0.023 3.5 2.16–5.68 7.84E-08
rs117381175 9 98403220 C T 0.012 7.45 3.16–17.55 1.52E-07
rs143238378 7 119266270 G A 0.015 4.26 2.35–7.71 1.64E-07
rs188681640 7 119146159 A G 0.011 4.82 2.52–9.24 2.15E-07
rs150382487 7 119140602 T A 0.011 4.81 2.52–9.21 2.16E-07
rs189675673 19 48383400 G A 0.02 3.51 2.12–5.82 2.38E-07
rs3858092 9 98291448 A C 0.396 1.72 1.39–2.11 2.62E-07
rs182830500 7 119161353 T C 0.01 4.94 2.54–9.63 2.71E-07
CL/P
rs72728755 8 129990382 T A 0.097 1.62 1.33–1.97 1.52E-06
rs1474306 3 145361479 T C 0.942 0.57 0.45–0.72 3.16E-06
rs6768171 3 145361918 T G 0.942 0.57 0.45–0.73 3.76E-06
rs55658222 8 129976136 G A 0.098 1.58 1.30–1.92 4.20E-06
rs151084002 5 172805743 C A 0.048 1.81 1.39–2.35 7.02E-06
rs112640811 1 150097784 G A 0.21 1.4 1.21–1.62 7.06E-06
rs13274247 8 129981468 G A 0.42 1.32 1.17–1.50 7.06E-06
rs12090508 1 150107793 A G 0.211 1.4 1.21–1.62 7.33E-06
rs7517537 1 150114083 C T 0.211 1.4 1.21–1.62 7.47E-06
rs744835 8 129982547 C T 0.478 1.32 1.17–1.48 8.36E-06
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BP is base pairs; OR is odds ratio; CI is confidence intervals.

Table 2.

Variants near or at genome-wide significance on combined analysis for CPO and with consistency of direction of effect

Discovery sample Replication sample Combined analysis
SNP Gene Chr BP Score P-value Z score P-value Z score P-value Direction
rs80004662 CTNNA2 2 82025185 9.383 7.41E-09 0.199 0.842 5.784 7.29E-09 ++
rs113691307 CTNNA2 2 82028390 9.384 7.41E-09 −0.147 0.883 −5.783 7.33E-09 −−
rs62529857 SULT2A1 19 48386473 15.421 7.84E-08 0.289 0.773 5.376 7.63E-08 ++
rs2325377 DACH1 13 71895298 15.524 3.62E-07 0.904 0.366 5.105 3.31E-07 ++
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Figure 2.

Figure 2

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(A) Regional association plot in the chromosome 2 locus for CPO. (B) TAD around the chromosome 2 locus for CPO. (C) Ctnna2 expression in mouse embryo at 14.5 days post fertilization (dpf) (Eurexpress–A Transcriptome Atlas of the Mouse Embryo, http://www.eurexpress.org/).

SULT2A1 is expressed in the palate at E12.5 and E14.5

The chromosome 19 locus was near genome-wide significance (lead SNP rs62529857, SULT2A1; P = 7.63 × 10−8). We studied the expression of the ortholog of the chromosome 19 locus for CPO (Sult2a1) in mice. In situ hybridization of Sult2a1 in mice showed expression of SULT2A1 in mesenchymal cells in palate, palatal rugae and palatal epithelium in the fused palate (Fig. 3). We also observed expression in the tongue, mandible, maxilla and heart. SysFACE analysis also showed that SULT2A1 is expressed at low levels in the neural plate, mandible and maxilla (Supplementary Material, Table S3). The expression of SULT2A1 in the palate and other craniofacial tissues provides a biological rationale for its role in orofacial clefting.

Figure 3.

Figure 3

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In situ hybridization of Sult2a1 in E12.5 and E14.5 embryos. Blue asterisks show mesenchymal cells in palate; black asterisks, palatal rugae with Sult2a1 expression; red asterisk, palatal epithelium. Tg, tongue; Md, mandible; Mx, maxilla; Ht, heart. Scale bar, 200 μm.

The 8q24 region is the most significant locus for CL/P in African populations

While the analysis for CL/P showed no genome-wide significant loci (Fig. 1; Supplementary Material, Tables S4 and S5), the most significant hit was on chromosome 8 (lead SNP, rs72728755; P = 1.52 × 10−6). This locus is in the 8q24 region that has been previously reported to be associated with CL/P in other populations (5–9,11). The lead SNP in our study is also one of the top-scoring SNPs in the 8q24 region in the largest meta-analysis of OFC to date (14).

Fine-mapping of the 8q24 locus for CL/P

We fine-mapped the 8q24 locus for CL/P using a number of methods. We examined haplotypes around the lead SNPs in our African sample and did a comparison with European and Asian ancestry samples from the 1000 Genomes (1KG) Project. As expected, the African sample had smaller haplotypes and finer-grained linkage disequilibrium (LD) patterns in the region (Fig. 4). Specifically, the haplotype around the lead SNP (rs72728577) is 4.084 kb in the continental African sample in contrast to 13.345 kb in European (1KG EUR), 13.477 kb in East Asian (1KG EAS) and 12.104 kb in South Asian (1KG SAS) populations (Fig. 4). Clumping analysis revealed a single clump of SNPs around the lead SNP (data not shown). Fine-mapping using a shotgun stochastic search algorithm (20) showed that the most likely configuration is a single causal variant in the region (Supplementary Material, Fig. S1).

Figure 4.

Figure 4

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(A) Regional association plot in the chromosome 8q24 locus for CL/P. (B) Haplotype block sizes around the 8q24 lead SNP rs72728755 for CL/P. (C) LD patterns around the 8q24 locus for European (EUR), East Asian (EAS), South Asian (SAS) and continental African (AFR*) ancestries.

Given that the lead SNP in the 8q24 region in our study (rs72728755) is different from the lead SNP (rs987525) reported by the most previous GWAS, we investigated this region further. SNP rs987525 is in low LD with rs72728755 (r2 = 0.004) in our study. Reciprocal conditional analysis revealed that conditioning on rs987525 had a small effect on rs72728755 (P-value decreased to 1.451 × 10−5 from 1.52 × 10−6), but conditioning in the other direction abolished the nominal significance of rs987525 (P-value decreased to 0.231 from 3.296×10−2), suggesting that rs72728755 is driving the association in our study. We note that this finding does not exclude the possibility of more than one causal variant in the 8q24 region, given that the two SNPs are in different haplotype blocks in all 1KG Project continental ancestry populations (Supplementary Material, Fig. S2).

Characterization of chr8q.24 SNPs for enhancer elements that are active in palate formation

The 8q24 SNPs that are most strongly associated with CL/P may themselves be directly pathological (i.e. functional), or instead they may be in LD with those that are functional. We selected the lead SNP in the region (rs72728755) and two SNPs that are most strongly associated with CL/P and are in strong LD with the lead SNPs (rs17242358 and rs55658222) for further studies. To test whether these non-coding SNPs are functional by virtue of altering the function of a regulatory element, we examined the chromatin state model at each SNP based on chromatin-marked evidence from 128 cell lines from the Roadmap Epigenomics Consortium. None of the SNPs lie in chromatin-marked regions as any type of regulatory element (Fig. 5). We amplified ~1 kb of DNA centered on each SNP, engineered the elements with either the non-risk- or risk-associated allele of the SNP (introduced by site-directed mutagenesis) into a standard firefly luciferase reporter vector and electroporated the reporters (separately) into a human fetal oral epithelial cell line (GMSM-K) (21) or primary human embryonic palate mesenchymal (HEPM) cell line (22). In both cell lines, none of the elements, whether harboring the risk or non-risk SNP variant, induced luciferase expression more than 2-fold above that in control cells electroporated with an empty firefly luciferase vector (Fig. 5). In summary, we did not find evidence that rs72728755, rs17242358 or rs55658222 reside within enhancers active in two cell types that are relevant to palate formation. It is still possible they reside in enhancers active in a cell type that is not represented by the cell lines we tested or by those at the Roadmap Epigenomics Consortium (http://www.roadmapepigenomics.org/). Other possibilities are that one or more of the SNPs alter the sequence, and, thereby, the functions of an unknown long non-coding RNA or the SNPs are in LD with the actual untyped functional SNPs.

Figure 5.

Figure 5

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(A) Overlay of the three SNPs against chromatin marked as a regulatory element, (B) reporter assay in human fetal oral epithelial cell line (GMSM-K) and (C) primary HEPM.

Novel variants identified in known GWAS-associated genes for CL/P

We identified two novel variants (p.Gly739Ser in DACH1 and p.Leu187Pro in ACVR2A) following Sanger sequencing (Table 3). These variants have not been previously reported in any genomic databases, including the gnomAD, Exome Aggregate Consortium (ExAC) and 1KG. The DACH1 novel variant (p.Gly739Ser) was predicted to be benign and tolerated by Polymorphism Phenotyping (PolyPhen) and Sorting Intolerant from Tolerant (SIFT). However, structural analysis using the Have Your Protein Explained (HOPE) server reveals that the variant amino acid is larger than the wild type and a change in size could lead to bumps in protein folding. There may also be a loss of flexibility and torsion angles when the flexible amino acid glycine is substituted with the non-flexible serine (Supplementary Material, Fig. S3). The missense variant (p.Leu187Pro) in ACVR2A was predicted to be benign and tolerated by PolyPhen and SIFT.

Table 3.

Novel variants in GWAS-identified candidate genes following Sanger sequencing

Gene HGVc HGVp Type Ghana Nigeria 1KG EVS ExAC P S
ACVR2A p.Leu187Pro Missense 0 1 0 0 0
DACH1 p.Gly739Ser Missense 1 0 0 0 0 B T
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EVS, Exome Variant Server; P, PolyPhen; S SIFT; PS, PROVEAN score; B, benign; T, tolerated; PD, probably damaging; D, deleterious. c. refers to coding sequence position.

Some previously reported OFC loci are replicated in African populations

To investigate how many previously reported loci for OFC show evidence of association in our study, we extracted all association records for terms related to ‘orofacial clefts’, ‘cleft lip/palate’, ‘cleft lip’ and ‘cleft palate’ in the National Human Genome Research Institute - European Bioinformatics Institute (NHGRI-EBI) GWAS Catalog. There were a total of 139 unique SNPs of which 121 were in our data set. However, only 39 of these SNPs (all for CL/P and/or all clefts) were genome-wide significant (P < 5 × 10−8) and were reported along with effect sizes. Of this subset, six variants showed significant association, i.e. P < 0.05, of which four SNPs also showed consistency of direction of effect for CL/P including SNPs in the chr8q24 region and in the genes PAX7, VAX1 and SOX5P1 (Table 4; Supplementary Material, Table S6). The effect sizes estimated in the present study (as indicated by the associated odds ratios) were remarkably similar to the observations in previous studies (Table 4). For CPO, only three SNPs have previously been reported to be genome-wide significant (12). These SNPs were monomorphic or near monomorphic in our data set, as they also are in other African ancestry populations in the 1KG or gnomAD databases. We also checked the association statistics for CPO in our study for the 48 SNPs and found that only 2 SNPs had a P < 0.05 but neither of the SNPs had consistent direction of effect with previous studies (Supplementary Material, Table 7). Given that African populations exhibit lower LD and smaller haplotype block sizes across the genome, we investigated the possibility of fine-mapping the replicated SNPs for CL/P to smaller regions that were observed in the original reports. For most of the replicated signals, African ancestry populations had the smallest haplotype blocks around the lead SNP (Fig. 6A). Fine-mapping indicated that the evidence supported one causal variant at each locus (Fig. 6B; Supplementary Material, Table S8) with the exception of one locus, rs987525 (an SNP in the 8q24 region fine-mapped above), where there was support for up to two causal variants. This finding further supports the notion that there are at least two causal variants in the 8q24 region. Clumping analysis in our study sample revealed that each of the leading association signals was consisted of a single clump of SNPs (i.e. it was unlikely that there were two or more variants explaining the association at any of the loci examined) with the exception of rs987525, which is consistent with the Finemapping (FINEMAP) analysis.

Table 4.

Variants reported for CL/P from previous studies in NHGRI-EBI GWAS Catalog that were replicated in the present study

Present study Previous studies
SNP Effect allele P value OR OR 95% CI SNP-allele P-value OR OR 95% CI Reported gene Mapped gene Authors PubMed ID
rs742071 T 1.25E-03 1.22 1.08–1.37 rs742071-T 7.00E-09 1.32 1.126-1.537 PAX7 PAX7 Ludwig et al., 2012 22863734
rs6585429 A 8.86E-03 0.83 0.73–0.96 rs6585429-A 7.00E-13 1.23 NR VAX1 VAX1 Yu et al., 2017 28232668
rs7017252 A 2.00E-02 1.18 1.03–1.35 rs7017252-A 8.00E-16 1.6 MYC; LOC728724 LINC00824; LINC00977 Yu et al., 2017 28232668
rs12543318 C 2.22E-02 0.85 0.74–0.98 rs12543318-C 9.00E-12 1.23 NR DCAF4L2 SOX5P1; LOC100419762 Yu et al., 2017 28232668
rs987525 A 3.50E-02 1.14 1.01–1.29 rs987525-A 5.00E-35 1.92 1.66-2.218 NR LINC00824; LINC00977 Ludwig et al., 2012 22863734
rs7078160 A 3.54E-02 1.16 1.01–1.33 rs7078160-A 4.00E-11 1.38 1.213-1.576 NR KIAA1598 Ludwig et al., 2012 22863734
rs6129653 A 5.85E-02 1.16 0.99–1.36 rs6129653-A 9.00E-12 1.23 MAFB LOC102724968; LOC105372620 Yu et al., 2017 28232668
rs6495117 A 7.37E-02 1.12 0.99–1.26 rs6495117-A 6.00E-11 1.2 NR NR LOC102723750; CLK3 Yu et al., 2017 28232668
rs7552 G 7.75E-02 1.13 0.99–1.29 rs7552-G 6.00E-22 1.37 NR FAM49A FAM49A Yu et al., 2017 28232668
rs1838105 A 9.60E-02 0.9 0.79–1.02 rs1838105-A 1.00E-11 1.22 GOSR2 GOSR2 Yu et al., 2017 28232668
rs2283487 A 1.21E-01 0.91 0.80–1.03 rs2283487-A 1.00E-10 1.2 NR CREBBP; ADCY9 CREBBP; LOC102724927 Yu et al., 2017 28232668
rs861020 A 1.32E-01 0.88 0.75–1.04 rs861020-A 3.00E-12 1.44 1.273-1.635 IRF6 IRF6 Ludwig et al., 2012 22863734
rs8001641 A 1.39E-01 1.14 0.96–1.35 rs8001641-A 9.00E-11 1.35 1.141-1.607 SPRY2 LOC105370275 Ludwig et al., 2012 22863734
rs9545308 A 1.40E-01 1.29 0.92–1.80 rs9545308-A 2.00E-09 1.29 SPRY2 LOC101927216 Yu et al., 2017 28232668
rs2289187 G 1.50E-01 1.09 0.97–1.24 rs2289187-G 4.00E-11 1.21 NR UBL7 Yu et al., 2017 28232668
rs560426 G 1.87E-01 0.92 0.82–1.04 rs560426-G 3.00E-12 1.42 1.243-1.623 NR ABCA4 Ludwig et al., 2012 22863734
rs8049367 C 2.49E-01 1.08 0.95–1.23 rs8049367-C 9.00E-12 1.35 1.25-1.47 CREBBP; ADCY9 CREBBP; LOC102724927 Sun et al., 2015 25775280
rs7148069 A 2.66E-01 1.08 0.94–1.25 rs7148069-A 2.00E-08 1.22 LOC283553 LINC00640; LOC105370496 Yu et al., 2017 28232668
rs227731 C 3.29E-01 0.93 0.82–1.07 rs227731-C 9.00E-09 1.19 NOG; C17orf67 NOG; C17orf67 Yu et al., 2017 28232668
rs908822 A 3.76E-01 1.2 0.80–1.81 rs908822-A 4.00E-08 1.31 LOC285419 LINC01091; LOC105377407 Yu et al., 2017 28232668
rs2304269 A 3.82E-01 1.28 0.73–2.24 rs2304269-A 1.00E-12 1.23 NR TMEM19 TMEM19 Yu et al., 2017 28232668
rs13317 A 3.83E-01 1.07 0.92–1.24 rs13317-A 4.00E-08 1.18 NR FGFR1 FGFR1 Yu et al., 2017 28232668
rs287982 A 3.95E-01 0.95 0.84–1.07 rs287982-A 6.00E-09 1.22 NR TAF1B LOC105373421; TAF1B Yu et al., 2017 28232668
rs3741442 G 4.18E-01 0.92 0.76–1.12 rs3741442-G 4.00E-12 1.22 KRT18 KRT18; EIF4B Yu et al., 2017 28232668
rs2872615 A 4.73E-01 1.06 0.90–1.26 rs2872615-A 9.00E-12 1.22 NR NTN1 LOC101928235; NTN1 Yu et al., 2017 28232668
rs7871395 A 4.82E-01 1.05 0.92–1.20 rs7871395-A 6.00E-09 1.21 GADD45G LOC105376137; LOC105376139 Yu et al., 2017 28232668
rs957448 A 4.96E-01 0.96 0.84–1.09 rs957448-A 1.00E-12 1.23 NR KIAA1429 KIAA1429 Yu et al., 2017 28232668
rs2064163 C 5.92E-01 0.97 0.85–1.10 rs2064163-C 9.00E-19 1.3 NR IRF6; DIEXF DIEXF; SYT14 Yu et al., 2017 28232668
rs4791774 G 6.38E-01 1.03 0.91–1.16 rs4791774-G 5.00E-19 1.56 1.42-1.72 NTN1 NTN1 Sun et al., 2015 25775280
rs12681366 A 6.45E-01 1.03 0.91–1.17 rs12681366-A 2.00E-10 1.2 NR RAD54B RAD54B Yu et al., 2017 28232668
rs1243572 G 6.45E-01 1.04 0.88–1.22 rs1243572-G 4.00E-10 1.2 GSC LOC107984693; LOC107984639 Yu et al., 2017 28232668
rs9381107 G 6.92E-01 0.97 0.83–1.13 rs9381107-G 3.00E-09 1.2 NR LOC100506207 LOC107986562; LOC107986563 Yu et al., 2017 28232668
rs7590268 G 7.54E-01 1.02 0.88–1.18 rs7590268-G 1.00E-08 1.41 1.225-1.636 THADA THADA Ludwig et al., 2012 22863734
rs12229892 G 7.58E-01 1.09 0.62–1.92 rs12229892-G 2.00E-10 1.2 NR NR PTPN11 Yu et al., 2017 28232668
rs10512248 A 7.67E-01 1.02 0.90–1.15 rs10512248-A 5.00E-10 1.22 NR PTCH1 PTCH1 Yu et al., 2017 28232668
rs705704 A 7.84E-01 1.03 0.85–1.24 rs705704-A 1.00E-09 1.22 RPS26 LOC105369780 Yu et al., 2017 28232668
rs481931 C 8.32E-01 0.98 0.80–1.19 rs481931-C 1.00E-12 1.25 NR ABCA4 ABCA4 Yu et al., 2017 28232668
rs1907989 G 8.84E-01 1.01 0.89–1.15 rs1907989-G 2.00E-08 1.18 NR MSX1 LOC101928279; LINC01396 Yu et al., 2017 28232668
rs13041247 T 9.60E-01 1.00 0.87–1.15 rs13041247-T 2.00E-11 1.32 1.20-1.41 MAFB LOC102724968 Sun et al., 2015 25775280
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*Data from previous studies extracted from NHGRI-EBI GWAS Catalog (version 2018-09-30). ‘NR’ indicates ‘not reported’. Effect sizes were reported with respect to the same allele across studies. Where more than one study reported the same genome-wide significant SNP, the study with the smallest P-value is presented in the table.

Figure 6.

Figure 6

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Haplotype blocks around the lead SNPs from previous GWAS that were replicated in the present study.

Discussion

Genomic studies of diverse populations have the potential to enrich our knowledge of the genetic architecture of many complex disorders. Here, we conducted a case-control GWAS for two OFC phenotypes, CPO and CL/P, in individuals enrolled from Ghana, Ethiopia and Nigeria. We identified two functionally plausible novel loci for CPO on chromosome 2 near CTNNA2 and on chromosome 19 in SULT2A1.

CTNNA2 encodes the alpha-catenin protein that is involved in cell–cell adhesion by acting as a linker protein between cadherins and actin-containing filaments of the cytoskeleton (23). Although the role of CTNNA2 in clefting is currently unknown, several studies have reported an association between E-cadherin and clefting (24–27). A recent GWAS for CL/P also identified a significant association near a gene involved in actin cytoskeleton (11). A recent exome sequencing study for Mendelian non-syndromic CL/P identified mutations in the epithelial cadherin-p120-catenin complex that includes CTNND1 (28). Studies in the chick embryo show that ctnna2 is expressed in neural crest cells (19), and expression studies in the mouse embryo also demonstrate its expression in oral structures. SULT2A1 encodes the enzyme sulfotransferase 2A1. While the gene has not previously been reported in relation to OFC, our in situ hybridization experiments show an expression of this gene in the palate. Knockout experiments for this gene in model organisms would further clarify its role in clefting.

Four loci showed suggestive association (P < 5 × 10−7) for CPO. They are near ACVR2A on chromosome 2, SHH on chromosome 7, OPALIN on chromosome 10 and DACH1 on chromosome 13. ACVR2A encodes activin A type II receptor protein and is a member of the TGFB superfamily of structurally related signaling proteins (29). The ACVR2A mouse knockout has micrognathia and associated defects, such as cleft palate and no incisors (30). These defects are similar to the features of Pierre Robin sequence where the small mandible leads to the limited space for the tongue to descend into the mouth causing cleft palate (31). ACVR2A is expressed in human fetal palate suggesting that activin signaling plays a role in the development of the palate (32). DACH1, mouse homolog of Drosophila dachshund, is a transcription factor involved in the regulation of organ formation. It inhibits TGFB signaling by binding to SMAD4 and NCOR1 (33). DACH1 is required for eye, leg and brain development. Homozygous mutants die shortly after birth due to failure to suckle, cyanosis and respiratory distress (34). The mouse Dach2 has similar expression pattern as mouse Dach1, suggesting that there may be redundancy in the functions of these genes (34). Missense variations in DACH2 have been reported in Allan–Herndon–Dudley syndrome (OMIM: 300523), Miles–Carpenter syndrome, X-linked cleft palate and/or megalocornea (35–38). These reports support a role for the missense variation (p.Gly739Ser) that we found in an individual with CL/P. OPALIN encodes the Opalin protein and has never been reported to play a role in clefting. SHH encodes the sonic hedgehog protein and it plays a role in cell division and embryogenesis. Mutations in SHH have been implicated in holoprosencephaly (39,40). A few studies have suggested a role for SHH in non-syndromic CL/P (41,42). We are the first to report an association with SHH for isolated CPO from GWAS.

For CL/P, our most significant locus is in the 8q24 region that has been previously reported in several other studies (5–9,11) in European populations. The lead SNP in our study is different from previous reports. Our analyses suggest that the two SNPs represent distinct signals for CL/P within the 8q24 region. While the evidence in our study suggests that the lead SNP represents a single causal variant, our transfection experiments were unable to determine which of the three tightly linked lead SNPs was the causal variant. The identification of significant SNPs in the 8q24 locus in multiple populations strongly supports its role in CL/P and suggests the possibility of more than one causal locus within this region.

Our study replicated several SNPs that are previously reported to be associated with OFC. Of note is the chromosome 9 locus near PTCH1. PTCH1 encodes the patched homolog 1 protein, a member of the patched family that is mutated in Gorlin syndrome (whose features include OFC) (43). It is a receptor for sonic hedgehog and is involved in cell proliferation, formation of structures during embryogenesis and tumor formation (44–46). Rare and common variants in PTCH1 have been implicated in non-syndromic CL/P (16,47,48).

This study has some limitations. There is a lack of strong evidence in the replication cohort, which is likely due to the fact that it is small in size and has limited power to detect significant associations. Other potential reasons for this observation include differences in LD, allele frequency differences and other sources of heterogeneity between population groups. Therefore, there is a need for further replication of the novel signals in larger African cohorts. Additional replication in other populations is also warranted for the new significant signals on chromosomes 2 and 19. The present study considered only common and low-frequency variants but did not consider rare variants because the genotyping tool was a GWAS SNP array with the yield boosted by imputation. A more comprehensive analysis done with whole-genome sequencing would provide a more complete association study that includes all classes of variants (including rare variants). We also noted that most of the association P-values in the replication sample were not small (P < 0.05), and those that were small often displayed inconsistency of direction of effect. For this reason, we limited the SNPs of interest to those that showed consistency of direction of effect in the replication sample in addition to being genome-wide significant in the discovery and combined analysis.

In conclusion, this first GWAS of OFC in sub-Saharan Africans identified novel loci for CPO and confirmed several findings previously reported from other ancestral populations. These findings add to the growing evidence about genetic risk factors for OFC and provide new candidate genes for functional studies.

Materials and Methods

Study population and sample information

Ethical approval was obtained from the Institutional Review Boards (IRBs) at the Lagos University Teaching Hospital (ADM/DCST/HREC/VOL.XV/321), Obafemi Awolowo University Teaching Hospital (ERC/2011/12/01), Kwame Nkrumah University of Science and Technology (CHRPE/RC/018/13), Addis Ababa University (003/10/surg), New York State Department of Health (IRB 07-007) and the NIH Office of Human Subjects Research (OHSRP 11631). We have previously reported the recruitment and sample used for the discovery study (49). In summary, eligible subjects are individuals with non-syndromic OFC and with families born to Ghanaian, Ethiopian and Nigerian parents. Births from Caucasians and Asians are excluded.

We identified eligible cases after IRB approvals through various free OFC surgical repair projects, most of which participate in the Pan-African Association of Cleft Lip and Palate network for treatment of OFC in Africa. This network is supported by cleft charities, and all use a common standardized protocol for phenotyping. For all the enrolled cases, the surgeons carried out standardized physical examinations, took clinical photographs and provided full description of OFC phenotypes and other recognizable malformations in a clinical database. We used our access to echocardiogram results to rule out cardiac defects. For both the discovery and replication samples (Supplementary Material, Table S9), controls were apparently healthy individuals without clefts enrolled at the same sites as cases. Both related (usually the mother) and unrelated controls were included in the analysis. In Nigeria, Ghana and Ethiopia, unrelated controls were recruited at infant welfare/immunization clinics at the site of the same medical centers where the cases were enrolled and were matched for gender, age and geographical location. In the Democratic Republic of the Congo and the US sites, controls were recruited from the same medical centers as cases. Signed informed consent was obtained from all families that participated in the study. Every family recruited into the study was assigned a unique identifier (UNID) number. Data from all recruited families were remotely entered from all the centers in Africa into a secured REDCap database (50). Deidentified samples were shipped from sites in Africa to the United States.

DNA extraction and preliminary quality control

Saliva samples were labeled at the Butali laboratory in Iowa and assigned a UNID number prior to DNA extraction. The DNA extraction was done at the Butali laboratory using the Murray laboratory protocol (genetics@uiowa.edu). Every sample was quantified using Qubit (http://www.invitrogen.com/site/us/en/home/brands/Product-Brand/Qubit.html; Thermo Fisher Scientific, Grand Island, NY) and separated into a stock and several working aliquots for downstream applications. We confirmed the sex reported in the REDCap database using TaqMan XY genotyping. These were done as part of our quality control (QC) process in the laboratory to prevent sample mislabeling. We then shipped 25 μl aliquot of consented samples with confirmed genetic sex and DNA concentration of ≥50 ng/μl to the Center for Inherited Disease Research for Multi-Ethnic Genotyping Array (MEGA) genotyping.

Genotyping

The expanded Illumina MEGA v2 15070954 A2 (genome build 37) that contains over 2 million SNPs and over 60 000 rare variants selected from populations of African origin was used for genotyping. We successfully conducted genotyping on 3347 samples, which included 3198 unique samples and 70 duplicates. HapMap controls (70 unique samples and 9 duplicates) were also genotyped as part of the QC process.

Data cleaning

A detailed description of this process was recently published (51). Briefly, we checked for sex chromosome anomalies, missing call rates, batch effects, identification of large chromosomal anomalies, confirmation of relatedness (i.e. identity by descent) and establishment of continental ancestry with respect to HapMap samples using methods described in Laurie et.al (2010) (52) and implemented using R packages GWAS Tools (53), SNPRelate (54) and GENESIS (55). This process allowed the use of a high-quality genotype data set for identifying significant genotype associations with non-syndromic OFC.

Imputation and association analyses

As is usual for GWAS to conduct imputation, we did both preimputation and postimputation QC (a full report is available in dbGaP, and we present a summary here). Briefly, for preimputation genotypes, after applying technical filters, we filtered for missing call rates ≥2%, >1 discordant call in 70 study duplicates, >1 Mendelian errors in 890 duos and trios, an Hardy Weinberg equilibrium (HWE) of P < 10−3 and a minor allele frequency (MAF) of <0.01, among others (Supplementary Material, Table S10). For the imputed SNPs, we only retained variants with a MAF of ≥0.01 and a quality metric (INFO, score is an estimated quality measurement of imputation) of ≥0.3, with the latter chosen based on the balance between stringency and inclusivity as recommended by (56). In the present study, choosing a threshold of 0.3 retained 69.5% of all imputed variants for downstream analyses, while more stringent thresholds of 0.5 and 0.8 would retain 63.5% and 49.0% of imputed variants, respectively.

Imputation was carried out using IMPUTE2 (a genotype imputation and phasing program) into the 1KG Phase 3 reference imputation panel (57). The final data set that passed QC was consisted of 3178 (1133 male and 2045 female) participants enrolled from Ethiopia (30%), Ghana (43%) and Nigeria (27%). The data set included 814 cases of CL/P, 205 cases of isolated CPO and 2159 related and unrelated controls.

The imputation yield was ~45 million SNPs of which ~17 million passed our QC filter and were included in the final analyses. Given the known differences in the developmental and genetic basis of isolated CL/P versus CPO, we conducted two separate GWAS analyses (one for each phenotype). Single-variant association tests were done for imputed dosage data filtered for an imputed allelic dosage frequency of <0.01 and an INFO of <0.3 using logistic mixed models as implemented in the GMAAT package (58). This approach enabled us to obtain valid association tests while adjusting for population structure (the first seven eigenvectors of the genotypes), relationships between participants (using the computed genetic relatedness matrix) and covariates (sex and study site). The Q–Q plot of the distribution of P-values did not show any residual stratification (Supplementary Material, Figure 5).

Replication

For the replication study, we included an independent sample of OFC cases and controls (300 CL/P cases, 179 CPO cases and 2523 controls) from Ghana, Nigeria, Ethiopia, Democratic Republic of Congo and African-American samples from New York and Virginia, USA. (Supplementary Material, Table S9). DNA extracted from deidentified residual dried blood spots was genotyped for NY cases (identified from the New York State Congenital Malformations Registry) and controls (identified from birth records). We selected for genotyping GWAS-significant SNPs and SNPs in LD with index SNP for a total of 48 SNPs using Fluidigm Corporation (San Francisco, CA), which allowed simultaneous genotyping of variants in samples in a multiplex, high-throughput format. Data were analyzed using PLINK2 (https://www.cog-genomics.org/plink2). For high-quality SNPs (an SNP success rate of ≥97%), association with CPO and CL/P was tested under an additive genetic model. Combined analysis of discovery and replication studies for the 48 SNPs was done as implemented in METAL (a tool for the meta-analysis of genome-wide association studies) (59). Variants that had P < 5 × 10−8 and had the same direction of effect in both studies were considered genome-wide significant.

Fine-mapping

Haplotypes were constructed using the confidence interval method of Gabriel et al (2002) (60). Clumping analysis of association statistics was done with PLINK (61) (Purcell et al., 2007) using default parameters. Fine-mapping was done using a shotgun stochastic search algorithm as implemented in FINEMAP (20). Reciprocal conditional analysis was done with genome-wide complex trait analysis (GCTA) (62).

Identification of GWAS candidate genes with a TAD

GWAS signals that affect enhancers most likely influence the expression of genes within the same TAD. Each region was visualized in the human reference genome (hg19) by searching for interaction domain for the index SNP ID (http://promoter.bx.psu.edu/hi-c/view.php).

Sanger sequencing

We used methods that we reported previously (49). We optimized primers for the amplification of exons in the ACVR2A1 and DACH1 genes. These genes where chosen based on their expression in the craniofacial region and the presence of mouse knockouts with cleft palate (http://www.informatics.jax.org/). A DNA concentration of 4 ng/μl of in a 10 μl reaction for the polymerase chain reaction were used. Two Yoruba HapMap samples and two water samples were added to the 96-well plates as template and non-template controls, respectively. Details of primers used and annealing temperatures are available from the Butali laboratory upon request. A total of 270 cases from Ghana, Ethiopia and Nigeria were sequenced. We sent the amplified DNA products for sequencing at Functional Biosciences, Inc., Madison, WI (http://order.functionalbio.com/seq/index).

We compared the identified novel variations with variations in the 1KG database (http://www.1000genomes.org/), Exome Variant Server database (http://snp.gs.washington.edu/EVS/) and ExAC database (http://exac.broadinstitute.org/). The variants were also compared to over 5200 African and African-American control exomes in these databases. We also sequenced population-matched controls for each novel variant in order to validate novel variants. We predicted the functional effects of novel variants using bioinformatics tools such as PolyPhen (http://genetics.bwh.harvard.edu/pph2/) (63), SIFT (http://sift.jcvi.org/) (64) and HOPE (http://www.cmbi.ru.nl/hope) (65). Segregation analyses were performed to determine if variants are de novo or inherited by sequencing samples from parents, when available.

In situ hybridization of Sult2a1 in mice at E12.5 and E14.5

The in situ hybridization method used in this study was adapted from our Sox2 paper (66). In summary, we used formalin-fixed paraffin embedded tissue sections for in situ hybridization. Mouse palatal samples were processed following the typical paraffin embedding process. Sagittal sections were cut in 8 μm, and we used the standard in situ hybridization method listed in Gregorieff's protocol (67). Digoxigenin-labeled probe was made from DIG RNA Labeling Kit (Roche Diabetes Care, Inc., Indianapolis, IN, USA #11175025910). Primers used for Sult2a1 are the following: Sult2a1-F: 5′-ATGATGTCAGACTATAATTGGTT-3′, and Sult2a1-SP6-R: 5′-ATTTAGGTGACACTATAGTTATTCCCATGGGAAAATCCCTGGG-3′.

Luciferase experiments to determine the functional role of SNPs at the 8q24 locus

Plasmid Construct

We used RP11-976D7 as a template to clone all three candidate elements in the 8q24 locus. The entire products were cloned into pENTR/D-TOPO plasmid (Life Technologies, Carlsbad, CA) for validation using Sanger sequencing. Site-directed mutagenesis was employed to get either non-risk or risk allele into the elements. We then shuttled all the candidate elements into cFos-FFLuc plasmid for in vitro luciferase assay.

Cell culture, electroporation and dual luciferase assay GMSM-K human embryonic oral epithelial cell line 6 (a kind gift from Dr Daniel Grenier) were maintained in keratinocyte serum-free medium (Life Technologies) supplemented with epithelial growth factor (EGF) and bovine pituitary extract (Life Technologies). All cells were incubated at 37°C in 5% CO2. HEPM 7 were purchased from American Type Culture Collection (ATCC) (ATCC®, Manassas, VA, USA, CRL-1486™) and maintained in ATCC-formulated Eagle's Minimum Essential Medium (ATCC) supplemented with 10% fetal bovine serum (Life Technologies) and 1% antibiotic-antimycotic (Life Technologies). For dual luciferase activity assay, each reporter construct was cotransfected with Renilla luciferase plasmid for three biological replicates. Briefly, plasmids were electroporated into GMSM-K cells with AmaxaTM Cell Line Nucleofector® Kit V (Lonza, Cologne, Germany) using NucleofectorTM II (Lonza) (program: X-005), and plasmids were electroporated into HEPM cells with AmaxaTM Basic NucleofectorTM Kit for primary mammalian fibroblasts (Lonza) using NucleofectorTM II (Lonza) (program: U-020). The Dual-Luciferase Reporter Assay System (Promega, Madison, WI) and 20/20n Luminometer (Turner BioSystems, Sunnyvale, CA) were employed to evaluate the luciferase activity 72 h posttransfection. Relative luciferase activities were calculated by the ratio between the value for firefly and Renilla luciferase activities. Three measurements were made for the lysate from each transfection group. All quantified results are presented as mean ± scanned electron microscope (SEM). Student's t-test was used to determine statistical significance.

Supplementary Material

Supplementary Data
Click here for additional data file. (434.6KB, zip)

Acknowledgements

We owe a lot of gratitude to families in Ghana, Ethiopia and Nigeria who participated in this research. The Center for Research on Genomics and Global Health (CRGGH) (1ZIAHG200362) is supported by the National Human Genome Research Institute, the National Institute of Diabetes and Digestive and Kidney Diseases, the Center for Information Technology and the Office of the Director at the National Institutes of Health.

Conflict of Interest statement. None declared.

Funding

National Institute of Dental and Craniofacial Research (NIDCR) K99/R00 (DE022378); Robert Wood Johnson Foundation (72429 to A.B.), National Institute of Health (NIH) (R01 DE023575 to R.A.C.); R37 (DE-08559 and DE-016148 to J.C.M.); Ghana Cleft Foundation (G001 to L.J.J.G.); Intramural Research Program of the National Institutes of Health in the CRGGH; Intramural Research Program of the National Institutes of Health; Eunice Kennedy Shriver National Institute of Child Health and Human Development (HHSN275201100001I and HHSN27500005).

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