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Research Article
Revised

A genome-wide scan of cleft lip triads identifies parent-of-origin interaction effects between ANK3 and maternal smoking, and between ARHGEF10 and alcohol consumption

[version 2; peer review: 2 approved]
PUBLISHED 19 Jul 2019
Author details Author details
OPEN PEER REVIEW
REVIEWER STATUS

Abstract

Background: Although both genetic and environmental factors have been reported to influence the risk of isolated cleft lip with or without cleft palate (CL/P), the exact mechanisms behind CL/P are still largely unaccounted for. We recently developed new methods to identify parent-of-origin (PoO) interactions with environmental exposures (PoOxE) and now apply them to data from a genome-wide association study (GWAS) of families with children born with isolated CL/P.
Methods: Genotypes from 1594 complete triads and 314 dyads (1908 nuclear families in total) with CL/P were available for the current analyses. Of these families, 1024 were Asian, 825 were European and 59 had other ancestries. After quality control, 341,191 SNPs remained from the original 569,244. The exposures were maternal cigarette smoking, use of alcohol, and use of vitamin supplements in the periconceptional period. Our new methodology detects if PoO effects are different across environmental strata and is implemented in the R-package Haplin.
Results: Among Europeans, there was evidence of a PoOxSmoke effect for ANK3 with three SNPs (rs3793861, q=0.20, p=2.6e-6; rs7087489, q=0.20, p=3.1e-6; rs4310561, q=0.67, p=4.0e-5) and a PoOxAlcohol effect for ARHGEF10 with two SNPs (rs2294035, q=0.32, p=2.9e-6; rs4876274, q=0.76, p=1.3e-5).
Conclusion: Our results indicate that the detected PoOxE effects have a plausible biological basis, and thus warrant replication in other independent cleft samples. Our demonstration of the feasibility of identifying complex interactions between relevant environmental exposures and PoO effects offers new avenues for future research aimed at unravelling the complex etiology of cleft lip defects.

Keywords

Orofacial cleft, cleft lip with or without cleft palate, case-parent triads, gene-environment interaction, parent-of-origin, PoOxE, Haplin

Revised Amendments from Version 1

In this version we have considered the remarks of Dr. Evie Stergiakouli. We have addressed some limitations that were pointed out and generally edited the manuscript for clarity.

Abstract:
Removed reference to previously published work and added information about the methodology.

Introduction:
Rephrased sentence that overemphasized how intriguing the problem of missing heritability is in clefting.

Methods:
Clarified that 59 individuals of other ethnicities than Asian or European were included in the pooled sample and added a sentence about the American Statistical Association’s negative attitudes towards using a fixed p-value as a threshold for statistical significance.

Results:
Revised titles of Tables 3, 4 and 7 to include information about which analyses were conducted.

Discussion:
Added a paragraph about genomic imprinting as a possible mechanism for PoO effects, clarified that our findings should be treated with caution until they have been replicated, and addressed the limitation that we have not imputed genotypes.

In general:
Corrected minor errors and slightly altered text to improve flow and precision.

See the authors' detailed response to the review by Evie Stergiakouli

Introduction

Cleft lip with or without cleft palate (CL/P) appears in approximately 3.4 to 22.9 per 10,000 live births1. Based on the severity of the cleft, patients undergo varying degrees of medical, dental, speech and psychosocial interventions over the first two decades of their lives, a long-term multidisciplinary treatment that not only imposes a heavy burden on patients and their families2,3, but also accounts for a substantial outlay in national healthcare budgets4,5.

Multiple genetic and environmental factors have been reported to influence the risk of CL/P, individually and through complex interactions in relevant biological pathways610. Major advances in high-throughput genotyping technologies, coupled with a boost in international collaborations, have led to substantial progress in gene-mapping for orofacial clefts, and the first wave of genome-wide association studies (GWAS) identified and replicated several key genes and loci associated with clefting1116. Despite this success, the genetic variants identified so far collectively explain only a minor fraction of the total variance attributable to additive genetic effects, even though the heritability of CL/P among Europeans is more than 70%1720. This has spurred renewed interest in investigating disease mechanisms other than fetal or maternal effects21. One example is parent-of-origin (PoO), where the effect of a particular allele in the offspring differs according to its parental origin2224, and another is gene-environment interaction (GxE), where fetal effects differ across strata of environmental exposures25. Identifying GxE effects may not only provide new insights into the causes of CL/P, but may also provide an opportunity to intervene on environmental risk factors alone, particularly in subgroups of the population that are genetically more susceptible to these environmental effects.

Recently, we went one step further and developed new methods for a genome-wide screening for PoO interactions with environmental exposures (i.e., PoOxE) in the case-parent triad setting22. We applied the new methodology, implemented in the R-package Haplin26, to isolated cleft palate only (CPO)27, using genotypes and exposure data from the largest published GWAS dataset on case-parent triads of orofacial clefts11. Epidemiological and embryological findings have previously shown that CL/P and CPO may have distinct etiologies. Therefore, we used the same GWAS dataset and methodology to perform a genome-wide scan for PoOxE effects in the larger sample of isolated CL/P.

Methods

Study participants

The study participants were mainly of Asian or European origin and were recruited as part of an international cleft collaboration11. Information was available on genotypes as well as maternal vitamin use, cigarette smoking and alcohol consumption in the periconceptional period (three months before and three months after pregnancy). The information on environmental exposures was based on interviews and questionnaires. More detailed characteristics of the study participants can be found in our recent work28.

Table 1 shows the distribution of the CL/P families according to ethnicity, triad completeness and maternal exposure. There were 1908 families in the pooled sample (5424 individuals in total), which included all the participants. Of these, 825 families were in the European sample, 1024 families were in the Asian sample, and 59 families were in the sample consisting of other ethnicities (Table 1). We performed three main sets of analyses on the following samples: All participants (denoted as “pooled analysis”), only Asians (“Asian analysis”), and only Europeans (“European analysis”). The 59 families with other ethnicities were not analyzed as a group due to the small sample size, but they were included in the pooled analysis. In the pooled and European analyses, we examined all exposures. As cigarette smoking and alcohol consumption were rare among Asian mothers, we were only able to conduct PoOxVitamin analyses for this ethnicity.

Table 1. Number of isolated cleft lip with or without cleft palate families according to ethnicity, triad completeness and maternal exposure to alcohol, smoking, and vitamin.

Complete + incomplete triadsTotalMother exposed (missing)
EthnicityIndividualsFamiliesIndividualsFamiliesAlcoholSmokingVitamin
European2024+310670+1552334825325 (8)249 (6)462 (98)
Asiana2670+268 890+13429381024--142 (155)
Otherb102+ 5034+ 2515259---
Pooled4796+6281594+31454241908350 (22)284 (9)638 (255)

aNo analyses of parent-of-origin interactions with alcohol (PoOxAlcohol) or parent-of-origin interactions with smoking (PoOxSmoke) were conducted for this group because of a lack of observations for these exposures. bOwing to the small sample size, no analysis of parent-of-origin interactions with environmental exposures (PoOxE) was conducted for this group. Note that a subset of the complete triads included more than one offspring. Incomplete triads are parent-offspring dyads.

Quality control for excluding single-nucleotide polymorphisms (SNPs) and individuals were conducted as described in Haaland et al. (2017)27. That is, we included SNPs with a missing call rate less than 5%, a minor allele frequency (MAF) greater than 5%, a p-value of less than 0.001 for the test for Hardy-Weinberg equilibrium presented by Wigginton et al. (2005)29, and a Mendelian error rate greater than 10%. Further, if two or more SNPs were in perfect linkage disequilibrium (r2=1) with each other, we only included one in the analyses. After applying these same criteria here, 341,191 were left for the current analyses from a total of 569,244 SNPs (Table 2).

Table 2. Quality control.

Total number of single-nucleotide polymorphisms (SNPs)569,244
Criteria:
     Failed HWE test (p<0.001)173,955
     More than 5% missing calls1934
     MAF less than 5%61,167
     r2=1 with flanking SNPs2880
     Mendelian errors detected (>1%)349
Number of SNPs remaining after quality controla341,191

aSome SNPs failed several criteria. Hence, the remaining number of SNPs (341,191) plus the ones that failed the different criteria do not add up to the total number of SNPs (569,244). HWE, Hardy-Weinberg equilibrium; MAF, minor allele frequency.

Statistical analysis

For statistical analysis, we used the statistical software Haplin26, which is written in the R statistical programming language30. Haplin is based on log-linear modeling in a maximum likelihood framework and is well-suited for the analysis of offspring-parent triads. Because Haplin uses the expectation-maximization (EM) algorithm to account for missing parental genotypes26, we were able to include the 314 case-parent dyads in the analyses beside the complete triads (Table 1). Haplin also uses the EM algorithm to reconstruct haplotypes, which enabled haplotype analyses for different combinations of SNPs in the genes that showed a plausible PoOxE effect.

A detailed description of the method for PoOxE analysis has been provided in our previous works22,27,31. Briefly, PoOxE effects were calculated as follows:

  • 1) Calculate the relative risk (RR) for an allele inherited from the mother (RRmat) and do the same for the father (RRpat).

  • 2) Calculate the relative risk ratio (RRRPoO=RRmat/RRpat) between the RRs in (1). RRRPoO is thus an estimate of the parent-of-origin (PoO) effect.

  • 3) Calculate RRRPoOxE as RRRPoO(Exposed)/RRRPoO(Unexposed), where RRRPoO(Exposed) and RRRPoO(Unexposed) are RRRPoO among triads with exposed and unexposed mothers.

Haplin uses a Wald test to test the null hypothesis of RRRPoOxE=1.

In order to control for multiple testing (one test for each of 341,191 SNPs), we obtained q-values using the false discovery rate (FDR) method described by Storey & Tibshirani (2003)32. Specifically, the q-values were calculated from the p-values with the R-function qvalue()33. A q-value of 0.2 corresponds to an FDR of 20%, which means that at least 80% of SNPs with a q-value less than 0.2 would be expected to be truly associated with the outcome. As in our previous work on isolated CPO27, we identified the top 20 SNPs for each of the analyses performed (see Results for details) and calculated relative risk ratios (RRRs) with 95% confidence intervals (95% CI). We paid more attention to a given gene if SNPs in that gene showed up multiple times in one set or across different sets of analyses. In accordance with recent recommendations by the American Statistical Association and others34,35, we did not consider a fixed p-value as a threshold for statistical significance.

To illustrate the general ability of the PoOxE analyses to detect true associations, power analyses for a wide range of PoOxE scenarios were performed using the Haplin function hapPowerAsymp(), as described in our recent works22,36.

We focused on the regions flanking SNPs in the most interesting genes and constructed regional plots based on R-scripts developed by the Diabetes Genetics Initiative of Broad Institute of Harvard and MIT, Lund University and Novartis Institutes of BioMedical Research37. Such plots capture the extent of linkage disequilibrium between a lead SNP and neighboring SNPs, while also providing information on recombination patterns and the position of genes.

R-scripts used to conduct the statistical analyses and create figures are available (see Software availability)38.

Bioinformatics analyses

To contextualize the findings, we searched for connections among a selection of genes in the STRING database39, as well as for enrichment of these genes in expression patterns using ExpressionAtlas40 and BGee (R package BgeeDB_2.10.0)41. Further, using Hetionet (Ver.1.0)42, we searched for indirect links between the genes highlighted by our analyses, the exposures and the phenotype (“cleft lip”). Hetionet is a heterogeneous network of various relationships among various data types, such as interactions between genes, or regulation of gene expression between a drug and a gene. The data used in Hetionet were carefully curated from 29 publicly available databases. To simplify the query output, the number of relationships between any two of the input query nodes (i.e., exposure, cleft lip, and the genes) was set to at most two. The exact queries together with their output are available (see Software availability)38.

Ethical statement

The individual institutional review boards of the members of the International Cleft Consortium provided ethical approval, which can be found in the online supplementary material of the original publication43. Written informed consent was provided by all participating families. Please refer to the dbGaP database for more information.

Results

For clarity, this section is structured as follows: We present the results of the PoOxE analyses of the pooled sample first (Table 3), followed by those of the European (Table 4) and Asian (Table 5) samples. We used the integrative database GeneCards and the gene-centric links therein to collate information on the genes in these tables. The 1000 Genomes browser was used to determine the chromosomal band location of a SNP. In the following sections, we focus on q-values, but all the corresponding p-values can also be found in Table 3Table 5. Table 6 provides a reference for the full names of all the genes mentioned in Table 3Table 5. Table 7 shows the results of the haplotype analyses of SNPs in the most interesting genes. Figure 1 and Figure 2 present visualizations of the results from the bioinformatics analyses, and regional plots for the most important regions from Table 3Table 5 are shown in Figure 3 and Figure 4. Figure 5 illustrates power calculations to detect different PoOxE effects in single-SNP analyses under different parameters, such as different sample sizes and minor allele frequencies. Quantile-quantile (QQ) plots for each set of analyses are shown in Figure 6Figure 8.

Table 3. The top 20 single-nucleotide polymorphisms (SNPs) sorted by p-value in the pooled PoOxE analysis.

ExposureSNPChromosomal
band locationa
P-valueQ-valueRRR (95% CI)Gene symbolbSharedc
ALCOHOLrs796447412p13.317.4e-060.990.34 (0.22-0.55)ANO2
rs99978316q23.3-q24.11.8e-050.992.63 (1.69-4.10)MBTPS1
rs498261914q11.22.1e-050.992.44 (1.62-3.68)TRA
rs794555011p132.1e-050.992.46 (1.62-3.72)EHFEurope
rs8808132p122.5e-050.992.36 (1.58-3.51)CTNNA2
rs228002516q23.3-q24.12.7e-050.992.59 (1.66-4.03)MBTPS1
rs115845061q42.13.4e-050.990.39 (0.25-0.61)NC
rs1089706611q12.23.8e-050.992.29 (1.54-3.40)~MS4A5 and
MS4A1
rs203244214q11.23.9e-050.992.37 (1.57-3.59)TRA
rs16368412q14.1-q14.24.2e-050.993.23 (1.84-5.65)PPM1H
rs802576315q26.35.6e-050.992.31 (1.54-3.47)NC
rs130080962p156.1e-050.992.26 (1.52-3.36)NC
rs46992284q246.2e-050.992.80 (1.69-4.63)NC
rs27230574q246.2e-050.992.76 (1.68-4.54)NC
rs720165916p12.36.5e-050.990.43 (0.29-0.65)XYLT1
rs21512259q21.36.8e-050.992.54 (1.61-4.02)NC
rs719747616p12.36.9e-050.990.44 (0.29-0.66)XYLT1
rs23672839q21.37.0e-050.990.42 (0.28-0.65)GPR98
rs291435419q13.427.7e-050.990.47 (0.32-0.68)~VN1R4
rs720965217p128.7e-050.990.45 (0.30-0.67)LINC00670
SMOKErs100973868q22.12.6e-060.572.86 (1.85-4.43)NC
rs23831629p21.38.5e-060.572.73 (1.75-4.24)FOCAD
rs107385719p21.31.3e-050.572.67 (1.72-4.16)FOCAD
rs74192011q431.4e-050.573.21 (1.90-5.44)NCEurope
rs75415371q431.4e-050.572.52 (1.66-3.81)NCEurope
rs70421929p21.31.5e-050.572.70 (1.72-4.22)FOCAD
rs49778489p21.31.5e-050.572.71 (1.72-4.24)FOCAD
rs792008810p141.6e-050.572.94 (1.80-4.81)SFMBT2
rs127408261q25.21.9e-050.570.35 (0.22-0.57)NPHS2
rs131737415q14.12.5e-050.572.51 (1.63-3.84)NC
rs107571689p21.32.5e-050.572.60 (1.67-4.05)FOCAD
rs818154311q22.32.6e-050.570.34 (0.21-0.56)PDGFDEurope
rs1682834q21.212.7e-050.570.36 (0.22-0.58)FRAS1
rs174086031p31.12.7e-050.573.19 (1.86-5.49)NC
rs1162438014q22.32.8e-050.570.37 (0.23-0.59)PELI2
rs21779718p21.22.8e-050.573.43 (1.93-6.11)NCEurope
rs794340111q22.32.9e-050.570.34 (0.20-0.56)PDGFD
rs379386110q21.23.0e-050.572.71 (1.70-4.34)ANK3Europe
rs43946821p36.133.4e-050.580.34 (0.20-0.57)~CAPZB
rs708748910q21.23.5e-050.582.69 (1.68-4.30)ANK3Europe
VITAMINrs230230419p13.31.3e-060.463.12 (1.97-4.94)TJP3
rs26891281q434.2e-060.713.28 (1.98-5.43)NCEurope
rs957225013q21.337.8e-060.880.44 (0.31-0.63)KLHL1
rs48753988p23.21.4e-050.992.08 (1.49-2.89)CSMD1
rs390955113q21.331.7e-050.990.46 (0.32-0.65)KLHL1
rs93714946q25.12.4e-050.992.23 (1.54-3.24)MTHFD1L
rs810198119p13.122.9e-050.990.48 (0.34-0.68)LINC00905Europe
rs793997511.p123.6e-050.992.08 (1.47-2.94)NC
rs104957672p23.23.6e-050.992.28 (1.54-3.36)NC
rs116738842q36.34.2e-050.990.51 (0.37-0.70)~SLC19A3
rs648963012p13.314.6e-050.992.23 (1.52-3.28)NTF3
rs381531117p125.3e-050.993.19 (1.82-5.59)ARHGAP44Europe
rs3580173p21.1-p14.35.4e-050.992.25 (1.52-3.34)~CACNA2D3
rs708228610q21.15.8e-050.994.03 (2.04-7.96)NC
rs92174310p136.0e-050.992.18 (1.49-3.19)RSU1
rs1076403710p12.316.3e-050.990.50 (0.36-0.70)MALRD1
rs811225619p13.116.8e-050.992.13 (1.47-3.10)FAM129C
rs45695212q21.18.1e-050.990.42 (0.27-0.65)ARHGEF4
rs68305094q288.7e-050.991.96 (1.40-2.73)NC
rs95031556p25.38.8e-050.990.49 (0.34-0.70)GMDS-AS1

aThe 1000 Genomes browser was used to determine the chromosomal band location of a SNP. bIf a SNP is located within a gene itself, the gene symbol is provided (the full names of the genes are provided in Table 6). SNPs located within 40 kb of a gene have the prefix ‘~’, and those not located within a 40 kb-distance of a gene are denoted as NC (for ‘not close’). Note that pseudogenes and non-coding RNAs are excluded. cShared: Also featured in Table 4 or Table 5. SNP, single-nucleotide polymorphism; RRR, relative risk ratio; CI, confidence interval; NC, not close.

Table 4. The top 20 single-nucleotide polymorphisms (SNPs) sorted by p-value in the European PoOxE analysis.

ExposureSNPChromosomal
band locationa
P-valueQ-valueRRR (95% CI)Gene symbolbSharedc
ALCOHOLrs104964102q127.5e-070.156.04 (2.96-12.32)NC
rs75799262q129.3e-070.155.95 (2.92-12.13)NC
rs22940358p23.32.9e-060.320.31 (0.19-0.51)ARHGEF10
rs69756507q331.1e-050.760.31 (0.19-0.52)NC
rs48762748p23.31.3e-050.762.99 (1.83-4.90)ARHGEF10
rs224522512q141.4e-050.763.46 (1.98-6.05)NC
rs9273189p24.22.0e-050.760.36 (0.22-0.57)GLIS3
rs1073533712q23.12.0e-050.760.36 (0.23-0.58)CCDC38
rs64272471q242.1e-050.762.87 (1.77-4.67)NC
rs126694937p21.12.4e-050.793.11 (1.84-5.26)LRRC72
rs132555618p23.33.6e-050.880.30 (0.17-0.53)DLGAP2
rs1224253510q21.23.9e-050.883.94 (2.05-7.57)NC
rs94388114q32.24.3e-050.880.36 (0.22-0.59)CYP46A1
rs104913275q344.4e-050.880.28 (0.15-0.52)NC
rs794555011p134.5e-050.882.82 (1.71-4.64)EHFPooled
rs723249218p11.315.3e-050.880.27 (0.15-0.51)DLGAP1
rs112422135q31.15.4e-050.884.71 (2.22-10.00)UBE2B
rs343522125q346.0e-050.880.32 (0.18-0.55)NC
rs199018517q246.1e-050.883.16 (1.80-5.54)NC
rs52141917p126.5e-050.882.80 (1.69-4.64)NC
SMOKErs1076370710p12.1-p11.231.5e-060.204.08 (2.30-7.23)LYZL1
rs75415371q432.0e-060.203.31 (2.02-5.43)NCPooled
rs74192011q432.1e-060.204.77 (2.50-9.11)NCPooled
rs379386110q21.22.6e-060.203.67 (2.13-6.32)ANK3Pooled
rs708748910q21.23.1e-060.203.63 (2.11-6.25)ANK3Pooled
rs81451819q13.24.5e-060.253.35 (2.00-5.62)SHKBP1
rs46931424q21.36.4e-060.300.26 (0.15-0.47)MAPK10
rs445461610p149.2e-060.383.06 (1.86-5.00)NC
rs29040964q21.31.2e-050.400.27 (0.15-0.49)MAPK10
rs229068219q13.21.3e-050.403.22 (1.90-5.44)SHKBP1
rs65320134q221.4e-050.403.04 (1.84-5.02)NC
rs18683688q24.22.2e-050.610.29 (0.17-0.52)NC
rs21779718p21.22.6e-050.644.02 (2.10-7.68)NCPooled
rs68075223q22.13.4e-050.672.91 (1.75-4.81)TMEM108
rs1760455015q25.33.6e-050.670.33 (0.20-0.56)AGBL1
rs1288377614q22.33.7e-050.670.34 (0.20-0.57)PELI2
rs723478718q21.13.8e-050.670.22 (0.11-0.45)ZBTB7C
rs818154311q22.33.8e-050.670.28 (0.16-0.52)PDGFD Pooled
rs431056110q21.24.0e-050.672.90 (1.75-4.83)ANK3
rs38000366p25.34.1e-050.670.35 (0.22-0.58)GMDS
VITAMINrs26891281q432.2e-060.444.82 (2.52-9.25)NCPooled
rs22373607p15.14.0e-060.440.29 (0.18-0.50)CREB5
rs77930507p214.0e-060.443.94 (2.20-7.05)RPA3-AS1
rs77661066q22.336.4e-060.530.31 (0.19-0.52)RSPO3
rs28099641p36.111.2e-050.653.05 (1.85-5.03)~RCAN3, NCMAP
and RPL26P8
rs385912116q12.11.2e-050.650.16 (0.07-0.37)N4BP1
rs10927333p262.4e-050.870.32 (0.19-0.54)NC
rs75596782q11.22.7e-050.870.35 (0.22-0.57)VWA3B
rs23668375p13.23.0e-050.870.35 (0.21-0.57)NC
rs100848524q28.33.0e-050.877.26 (2.86-18.45)PCDH10
rs64463894p16.24.2e-050.873.38 (1.89-6.06)EVC2
rs224290921q22.14.2e-050.872.92 (1.75-4.87)NC
rs5955361q42.24.4e-050.873.21 (1.83-5.60)~SIPA1L2
rs67265272q37.14.5e-050.870.24 (0.12-0.48)~SP140 and SP140L
rs127330191p32.14.8e-050.870.24 (0.12-0.47)NC
rs810198119p13.124.8e-050.870.34 (0.21-0.58)LINC00905Pooled
rs177931458p224.9e-050.873.39 (1.88-6.12)DLC1
rs49733102q37.15.1e-050.870.24 (0.12-0.48)~SP140 and SP140L
rs381531117p125.5e-050.874.57 (2.18-9.57)ARHGAP44Pooled
rs807288517q25.35.5e-050.870.22 (0.10-0.46)RBFOX3

aThe 1000 Genomes browser was used to determine the chromosomal band location of a SNP. bIf a SNP is located within a gene itself, the gene symbol is provided (the full names of the genes are provided in Table 6). SNPs located within 40 kb of a gene have the prefix ‘~’, and those not located within a 40 kb-distance of a gene are denoted as NC (for ‘not close’). Note that pseudogenes and non-coding RNAs are excluded. cShared: Also featured in Table 3 or Table 5. SNP, single-nucleotide polymorphism; RRR, relative risk ratio; CI, confidence interval; NC, not close.

Table 5. The top 20 SNPs sorted by p-value in the Asian parent-of-origin interactions with vitamins (PoOxVitamin) analysis.

SNPChromosomal band locationaP-valueQ-valueRRR (95% CI)Gene symbola
rs18899761q25.38.8e-060.863.88 (2.13-7.05)SWT1
rs2593956q24.31.1e-050.860.23 (0.12-0.45)ADGB
rs107980041q25.31.5e-050.863.70 (2.04-6.68)~IVNS1ABP and SWT1
rs1243148414q11.22.2e-050.860.24 (0.12-0.46)TRA
rs1051898115q15.3-q21.12.3e-050.860.22 (0.11-0.45)~CTDSPL2 and EIF3J-AS1 and EIF3J
rs194069811q23.22.4e-050.860.21 (0.10-0.43)NCAM1
rs17147721q212.5e-050.860.23 (0.12-0.46)C21orf91-OT1
rs98628663p14.13.1e-050.860.24 (0.12-0.47)~RPL21P41
rs8655856p21.13.6e-050.860.19 (0.09-0.42)NC
rs1759173211q23.23.8e-050.860.22 (0.11-0.45)NCAM1
rs126301063q13.15.6e-050.863.56 (1.92-6.61)NC
rs731635012q156.0e-050.860.22 (0.10-0.46)NC
rs733629613q316.1e-050.863.39 (1.87-6.17)NC
rs14999162q226.4e-050.860.22 (0.10-0.46)NC
rs715357414q11.26.5e-050.860.26 (0.13-0.50)TRA
rs64397723q227.0e-050.860.26 (0.14-0.51)NC
rs13485643q227.1e-050.860.27 (0.14-0.52)NC
rs236083811p15.47.3e-050.863.37 (1.85-6.14)~OR10A3 and NLRP10 and OR10A6
rs122048086q14.17.3e-050.864.63 (2.17-9.87)IMPG1
rs14075551q25.37.5e-050.863.30 (1.83-5.96)TRMT1L

aThe 1000 Genomes browser was used to determine the chromosomal band location of a SNP. bIf a SNP is located within a gene itself, the gene symbol is provided (the full names of the genes are provided in Table 6). SNPs located within 40 kb of a gene have the prefix ‘~’, and those not located within a 40 kb-distance of a gene are denoted as NC (for ‘not close’). Note that pseudogenes and non-coding RNAs (ncRNA) are excluded. There is no column for “shared” here, as none of these SNPs featured among those listed in Table 3 or Table 4. SNP, single-nucleotide polymorphism; RRR, relative risk ratio; CI, confidence interval; NC, not close.

Table 6. Full names of all the genes and loci mentioned in Table 3Table 5.

Gene symbolFull gene/locus name
ADGBAndroglobin
AGBL1ATP/GTP binding protein like 1
ANK3Ankyrin 3, node of Ranvier (ankyrin G)
ANO2Anoctamin 2
ARHGAP44Rho GTPase activating protein 44
ARHGEF10Rho guanine nucleotide exchange factor 10
ARHGEF4Rho guanine nucleotide exchange factor (GEF) 4
C21orf91-OT1NA
CACNA2D3Calcium channel, voltage-dependent, alpha 2/delta subunit 3
CAPZBCapping protein (actin filament) muscle Z-line, beta
CCDC38coiled-coil domain containing 38
CREB5cAMP responsive element binding protein 5
CSMD1CUB and Sushi multiple domains 1
CTDSPL2CTD small phosphatase like 2
CTNNA2Catenin (cadherin-associated protein), alpha 2
CYP46A1cytochrome P450 family 46 subfamily A member 1
DLC1DLC1 Rho GTPase activating protein
DLGAP1DLG associated protein 1
DLGAP2DLG associated protein 2
EHFEts homologous factor
EIF3Jeukaryotic translation initiation factor 3 subunit J
EIF3J-AS1EIF3J divergent transcript
EVC2EvC ciliary complex subunit 2
FAM129CFamily with sequence similarity 129, member C
FOCADFocadhesin
FRAS1Fraser syndrome 1
GLIS3GLIS family zinc finger 3
GMDS GDP-mannose 4,6-dehydratase
GMDS-AS1GMDS antisense RNA 1 (head to head)
GPR98G protein-coupled receptor 98
IMPG1interphotoreceptor matrix proteoglycan 1
IVNS1ABPinfluenza virus NS1A binding protein
KLHL1Kelch-like family member 1
LINC00670Long intergenic non-protein coding RNA 670
LINC00905Long intergenic non-protein coding RNA 905
LRRC72leucine rich repeat containing 72
LYZL1 Lysozyme like 1
MAPK10 Mitogen-activated protein kinase 10
MALRD1MAM and LDL receptor class A domain containing 1
MBTPS1Membrane-bound transcription factor peptidase, site 1
MS4A1Membrane-spanning 4-domains, subfamily A, member 1
MS4A5Membrane-spanning 4-domains, subfamily A, member 5
MTHFD1LMethylenetetrahydrofolate dehydrogenase (NADP+ dependent)
1-like
N4BP1NEDD4 binding protein 1
NCAM1neural cell adhesion molecule 1
NCMAPnon-compact myelin associated protein
NLRP10NLR family pyrin domain containing 10
NPHS2Nephrosis 2, idiopathic, steroid-resistant (podocin)
NTF3Neurotrophin 3
OR10A3olfactory receptor family 10 subfamily A member 3
OR10A6olfactory receptor family 10 subfamily A member 6 (gene/
pseudogene)
PCDH10protocadherin 10
PDGFDPlatelet derived growth factor D
PELI2Pellino E3 ubiquitin protein ligase family member 2
PPM1HProtein phosphatase, Mg2+/Mn2+ dependent, 1H
RBFOX3RNA binding fox-1 homolog 3
RCAN3RCAN family member 3
RPL21P41ribosomal protein L21 pseudogene 41
RPL26P8ribosomal protein L26 pseudogene 8
RSPO3R-spondin 3
RSU1Ras suppressor protein 1
SFMBT2Scm-like with four mbt domains 2
SHKBP1 SH3KBP1 binding protein 1
SIPA1L2signal induced proliferation associated 1 like 2
SLC19A3Solute carrier family 19 (thiamine transporter), member 3
SP140SP140 nuclear body protein
SP140LSP140 nuclear body protein like
SWT1SWT1, RNA endoribonuclease homolog
TJP3Tight junction protein 3
TMEM108 Transmembrane protein 108
TRAT cell receptor alpha locus
TRMT1LtRNA methyltransferase 1 like
UBE2Bubiquitin conjugating enzyme E2 B
UMAD1UBAP1-MVB12-associated (UMA) domain containing 1
VN1R4Vomeronasal 1 receptor 4
VWA3Bvon Willebrand factor A domain containing 3B
XYLT1Xylosyltransferase I
ZBTB7Czinc finger and BTB domain containing 7C

Table 7. Stratified analyses of the top single-nucleotide polymorphisms (SNPs) and haplotypes in ANK3 (PoOxSmoke) and ARHGEF10 (PoOxAlcohol).

Gene nameSNP/haplotypeaTarget allele/ReferencebFrequencyEffect typecRRR (95% CI)p-value
ANK3rs3793861c/G0.30Child1.06 (0.91-1.24)0.45
GxSmoke1.20 (0.83-1.60)0.41
PoO1.39 (1.09-1.76)0.007
PoOxSmoke3.67 (2.13-6.32)2.6e-6
rs7087489t/A0.30Child1.06 (0.91-1.24)0.44
GxSmoke1.10 (0.82-1.60)0.42
PoO1.40 (1.10-1.77)0.006
PoOxSmoke3.63 (2.11-6.25)3.1e-6
rs4310561a/T0.34Child1.12 (0.97-1.24)0.13
GxSmoke1.20 (0.87-1.60)0.27
PoO1.31 (1.03-1.64)0.02
PoOxSmoke2.90 (1.75-4.83)4.0e-5
rs3793861- rs7087489c-t/G-A0.30Child1.07 (0.92-1.24)0.39
GxSmoke1.10 (0.81-1.60)0.45
PoO1.38 (1.08-1.75)0.008
PoOxSmoke3.71 (2.16-6.39)2.2e-6
rs7087489- rs4310561A-a/A-T0.04Child1.32 (0.95-1.83)0.10
GxSmoke1.50 (0.71-3.10)0.29
PoO0.93 (0.60-1.45)0.74
PoOxSmoke1.57 (0.62-3.96)0.34
t-a/A-T0.30Child1.09 (0.94-1.28)0.26
GxSmoke1.20 (0.83-1.60)0.37
PoO1.34 (1.06-1.70)0.02
PoOxSmoke3.65 (2.13-6.28)2.7e-6
rs3793861- rs7087489-
rs4310561
G-A-a/G-A-T0.04Child1.32 (0.95-1.83)0.10
GxSmoke1.50 (0.71-3.10)0.29
PoO0.93 (0.60-1.45)0.75
PoOxSmoke1.56 (0.62-3.94)0.35
c-t-a/G-A-T0.30Child1.09 (0.94-1.28)0.26
GxSmoke1.20 (0.83-1.60)0.37
PoO1.35 (1.06-1.71)0.01
PoOxSmoke3.62 (2.10-6.21)3.3e-6
ARHGEF10rs2294035a/T0.49Child0.94 (0.82-1.08)0.38
GxAlcohol1.20 (0.87-1.50)0.32
PoO0.95 (0.75-1.20)0.67
PoOxAlcohol0.32 (0.19-0.51)2.9e-6
rs4876274t/A0. 47Child1.04 (0.90-1.20)0.57
GxAlcohol0.90 (0.67-1.20)0.47
PoO1.02 (0.80-1.29)0.90
PoOxAlcohol2.99 (1.83-4.90)1.3e-5
rs2294035-rs4876274T-A/a-A0.04Child1.15 (0.80-1.68)0.44
GxAlcohol0.73 (0.33-1.60)0.45
PoO1.41 (0.85-2.37)0.19
PoOxAlcohol1.56 (0.49-4.93)0.45
T-t/a-A0.47Child1.04 (0.90-1.20)0.63
GxAlcohol0.90 (0.67-1.20)0.46
PoO1.00 (0.80-1.27)0.98
PoOxAlcohol3.20 (1.97-5.21)2.8e-6

aEffect allele or haplotype against the reference. Lowercase indicates the minor allele at the SNP. bMinor allele frequency for a given SNP. In haplotype analyses, this corresponds to the frequencies of haplotypes other than the reference. cRR for child effects; RRR for GxSmoke or GxAlcohol, PoO and PoOxSmoke or PoOxAlcohol. All p-values <0.05 are highlighted in bold. Note that in a two-SNP-haplotype, there are four possible combinations, and in a three-SNP-haplotype there are eight. However, only two or three of these combinations were actually observed in the data. SNP, single nucleotide polymorphism; RRR, relative risk ratio; RR, risk ratio; CI, confidence interval; PoO, parent-of-origin; GxSmoke, gene-smoking interaction; GxAlcohol, gene-alcohol interaction; PoOxAlcohol, parent-of-origin interactions with alcohol; PoOxSmoke, parent-of-origin interactions with smoking.

018ed9cc-5a82-4d08-babc-323d669675d9_figure1.gif

Figure 1. Indirect relationships between ANK3 and nicotine dependence, and between ANK3 and cleft lip.

The brown nodes represent diseases, blue nodes show genes/proteins, and green nodes represent organs (anatomy). Each arrow represents a specific relationship between nodes: “LOCALIZES_DiA” = disease was found to be localized in an anatomy (organ); “EXPRESSES_AuG”, “UPREGULATES_AuG”, “DOWNREGULATES_AuG” mean that the gene is expressed, upregulated, or downregulated in the anatomy (organ), respectively; “INTERACTS_GiG” means that the two genes were found to interact with each other (physically, as proteins); “ASSOCIATES_DaG” means that the gene was found to be associated with the disease; “RESEMBLES_DrD” means that the two diseases were found to occur significantly more often together in MEDLINE articles than would be expected by chance alone. Note that in this setting, the term "disease" includes any adverse medical condition, like syndromes, mental disorders, congenital anomalies, and so on.

018ed9cc-5a82-4d08-babc-323d669675d9_figure2.gif

Figure 2. Indirect relationships between ARHGEF10 and alcohol dependence, and between ARHGEF10 and cleft lip.

The brown nodes represent diseases, blue nodes show genes/proteins, and green nodes represent organs (anatomy). Each arrow represents a specific relationship between nodes: “LOCALIZES_DiA” = disease was found to be localized in an anatomy (organ); “EXPRESSES_AuG”, “UPREGULATES_AuG”, “DOWNREGULATES_AuG” mean that the gene is expressed, upregulated, or downregulated in the anatomy (organ), respectively; “INTERACTS_GiG” means that the two genes were found to interact with each other (physically, as proteins); “ASSOCIATES_DaG” means that the gene was found to be associated with the disease; “RESEMBLES_DrD” means that the two diseases were found to occur significantly more often together in MEDLINE articles than would be expected by chance alone. Note that in this setting, the term "disease" includes any adverse medical condition, like syndromes, mental disorders, congenital anomalies, and so on.

018ed9cc-5a82-4d08-babc-323d669675d9_figure3.gif

Figure 3. Regional association plot for rs3793861 in ANK3.

The plot provides information on the recombination rate and linkage disequilibrium between the lead SNP (blue diamond) and other SNPs in the region.

018ed9cc-5a82-4d08-babc-323d669675d9_figure4.gif

Figure 4. Regional association plot for rs2294035 in ANK3.

The plot provides information on the recombination rate and linkage disequilibrium between the lead SNP (blue diamond) and other SNPs in the region.

018ed9cc-5a82-4d08-babc-323d669675d9_figure5.gif

Figure 5. Power vs. RRR.

Left panel: Setting the minor allele frequency to 0.2 while varying the number of unexposed and exposed triads (unexposed-exposed). Right panel: Setting the number of unexposed and exposed triads to 1100 and 500, respectively, while varying the minor allele frequency. In all analyses, the significance level was 0.05. We varied the maternal RR in exposed triads, so that RRR=RRmat(Exposed). The black curve is the same in both panels because of shared parameters. RRR, relative risk ratio; RRmat, relative risk for an allele inherited from the mother; MAF, minor allele frequency.

018ed9cc-5a82-4d08-babc-323d669675d9_figure6.gif

Figure 6. Pooled analyses.

Q-Q plots for PoOxSmoke (left) PoOxAlcohol (middle) and PoOxVitamin (right) with 95% pointwise confidence bands. Q-Q, quantile-quantile; PoOxAlcohol, parent-of-origin interactions with alcohol; PoOxSmoke, parent-of-origin interactions with smoking; PoOxVitamin, parent-of-origin interactions with vitamins.

018ed9cc-5a82-4d08-babc-323d669675d9_figure7.gif

Figure 7. European analyses.

Q-Q plots for PoOxSmoke (left) PoOxAlcohol (middle) and PoOxVitamin (right) with 95% pointwise confidence bands. Q-Q, quantile-quantile; PoOxAlcohol, parent-of-origin interactions with alcohol; PoOxSmoke, parent-of-origin interactions with smoking; PoOxVitamin, parent-of-origin interactions with vitamins.

018ed9cc-5a82-4d08-babc-323d669675d9_figure8.gif

Figure 8. Asian analyses.

Q-Q plots for PoOxVitamin with 95% pointwise confidence bands. Q-Q, quantile-quantile; PoOxVitamin, parent-of-origin interactions with vitamins.

Pooled sample

All the top 20 SNPs in the PoOxAlcohol analysis had the same q-value of 0.99 and are therefore not considered here as they are probably false positives (Table 3). All the SNPs in the PoOxSmoke analysis had q-values of around 0.6. Even though these q-values are still quite large, they indicate that around 40% of the SNPs are potentially true PoOxE associations. Among the top 20 SNPs in the PoOxSmoke analysis, two are in the gene for ‘Focadhesin’ (FOCAD), two are in ‘Platelet derived growth factor D’ (PDGFD), two are in ‘Ankyrin 3’ (ANK3), and one is in ‘Fraser syndrome 1’ (FRAS1). Note that associations with PDGFD and ANK3 were also detected in the European analyses (see below). In the PoOxVitamin analysis, only three SNPs had q-values below 0.99, and none of the genes linked to these SNPs have previously been associated with orofacial clefts.

European sample

Among the SNPs with the lowest q-values in the PoOxAlcohol analysis, rs2294035 (q=0.32, p=2.9e-6) and rs4876274 (q=0.76, p=1.3e-5) are in ‘Rho guanine nucleotide exchange factor 10’ (ARHGEF10; GeneCards identifier [GCID]: GC08P001823) (Table 4). The remaining SNPs had q-values above 0.76 and are not considered any further. ARHGEF10 has not previously been linked with orofacial clefts. In the PoOxSmoke analysis, three of the SNPs were in ANK3 (rs3793861: q=0.20, p=2.6e-6; rs7087489: q=0.20, p=3.1e-6; and rs4310561: q=0.67, p=4.0e-5). PoOxE effects in ANK3 were also detected in the analysis of the pooled sample above. To our knowledge, ANK3 has not previously been linked with orofacial clefts, and the same applies to SNP rs10763707 in ‘Lysosome like 1’ (LYZL1; GCID: GC10P029297), which had a q-value of 0.20. In the PoOxVitamin analysis, several of the SNPs shared the same q-value of 0.87 and are not considered any further. The top six SNPs had q-values of 0.44-0.65. Again, none of these genes appear to have any previous connections to clefting. For example, ‘cAMP responsive element binding protein 5’ (CREB5; GCID: GC07P028305) and its network of genes are involved in colorectal cancer44, while ‘R-Spondin 3’ (RSPO3; GCID: GC06P127118) is implicated in tumor development. That said, Park and co-workers reported that RSPO3 acts as an agonist in the canonical Wnt/β-catenin signaling45, a pathway known to be implicated in a wide range of developmental processes, including craniofacial development and homeostasis4649.

Asian sample

In the only analysis possible for this ethnic group (PoOxVitamin), all the SNPs had the same q-value of 0.86 (Table 5). They are thus most likely to be false positives and will not be considered any further.

Haplotype analysis of SNPs in ANK3 and ARHGEF10

We chose to focus here on the PoOxE effects detected with SNPs in ANK3 and ARHGEF10. As mentioned above, ANK3 showed up several times among the top PoOxSmoke hits both in the pooled and European analyses, and strong signals for SNPs in ARHGEF10 were detected twice in the European analysis of PoOxAlcohol. We conducted stratified analyses of the effect of the child’s allele, GxE effects, PoO effects, and PoOxE effects for each SNP and haplotype (with haplotypes analyzed both in two-SNP and three-SNP combinations) in these two genes (Table 7). Specifically, we analyzed rs3793861, rs7087489 and rs4310561 in ANK3 that showed PoOxSmoke effects in the European sample, and rs2294035 and rs4876274 in ARHGEF10 that showed PoOxAlcohol effects in the same sample (Table 4). The results did not show any child effects or GxSmoke effects for single SNPs in ANK3. By contrast, the p-values were low for all three SNPs in the PoO analyses or PoOxSmoke analyses. This was also the case with the ‘t-a’ allele in the two-SNP combination rs3793861-rs7087489 and the ‘c-t-a’ allele in the three-SNP-combination rs3793861-rs7087489-rs4310561. The other alleles were only associated with PoOxSmoke effects.

For ARHGEF10, we analyzed the two SNPs that showed PoOxAlcohol effects in the European sample (rs2294035 and rs4876274) but did not discover any effects in either single-SNP or haplotype analyses.

Bioinformatics analysis

Because of their low q-values, the genes appearing in the PoOxSmoke and PoOxAlcohol analyses in Table 4 were selected for further analyses using the STRING database, ExpressionAtlas and BGee. However, none of the searches for direct links among the genes yielded any evidence to explain why those genes appeared in our results together.

Regarding the indirect relationships, these are visualized in Figure 1 for relationships between ANK3 and cleft lip (Disease Ontology ID [DOID]: 9296), and, simultaneously, between ANK3 and nicotine dependence (DOID: 0050742). As Hetionet does not include information about smoking, we chose “nicotine dependence” as a proxy. ANK3 is connected to nicotine dependence through several nodes, two of which are particularly noteworthy. First, ANK3 has been reported to be strongly associated with attention-deficit/hyperactivity disorder (ADHD)50, and a connection between ADHD and nicotine dependence has been reported. The connectiom was calculated based on articles listed in MEDLINE, where this pair of conditions co-occured significantly more frequently than would be expected by chance51,52. The second path goes through the gene ‘CRK Like Proto-Oncogene, Adaptor Protein’ (CRKL). It interacts with ANK3 and is downregulated in nicotine dependence. Furthermore, ANK3 is expressed in the telencephalon (the most highly developed part of the forebrain), the embryo, and the head all of which are all relevant to CL/P.

Figure 2 shows the indirect relationships between cleft lip, ARHGEF10 and alcohol dependence. Like nicotine dependence in the above analyses, alcohol dependence (DOID: 0050741) was used here as a proxy for maternal alcohol consumption. The only relationships found between ARHGEF10 and cleft lip are the expression of ARHGEF10 in the head and telencephalon. By contrast, there were twelve different relationships between ARHGEF10 and alcohol dependence. However, there were no shared paths connecting cleft lip and alcohol dependence via any of the 12 organs.

Regional plots

The regional plot for rs3793861 (Figure 3) shows that several SNPs in ANK3 that were not in linkage disequilibrium with rs3793861 had p-values in the range 10-4 to 10-3, which lends support to either ANK3 itself or genes in its vicinity influencing the risk of clefting. However, we did not observe a similar pattern in the regional plot for rs2294035 (Figure 4).

Power analysis

Figure 5 shows that the power does not increase appreciably when the minor allele frequency increases beyond 0.2. However, there is a lot to be gained by increasing the sample size from 500 unexposed and 300 exposed (European, smoke/alcohol) to 1400-600 (pooled, vitamin). Further, the RRRs in the plots are based on changing only the effect of the maternal allele in the exposed triads. The same RRRs could have been achieved in a number of ways, which complicates the interpretation of the RRRs in Table 3Table 5. Still, if a strong effect is detected with a SNP in a gene, this strengthens the case for its contribution to clefting.

Discussion

The main aim of this paper was to identify genome-wide PoOxE effects in the larger sample of isolated CL/P, based on the same methodology and GWAS dataset we had previously used in a similar analysis of the smaller sample of isolated CPO27. As with the CPO study, the current analyses benefitted from being based on the largest available GWAS dataset of case-parent triads of orofacial clefts to date. Moreover, data were available for two major ethnicities, European and Asian, which is useful in assessing the generalizability of the findings across different ethnic groups. In the current dataset, however, very few of the Asian mothers reported smoking cigarettes or consuming alcohol during the periconceptional period, thus preventing a comparison of PoOxE effects for these exposures across these two ethnic groups. This is a common impediment to GxE studies, where the number of exposed individuals needs to be large enough for a meaningful analysis53.

A possible mechanism for a PoO effect is genomic imprinting21. This occurs when DNA methylation in the germline causes the expression of alleles to be silenced depending on their parental origin. Maternal environmental exposures that affect methylation patterns may also affect paternally and maternally inherited alleles differently. Furthermore, a PoO effect that is not affected by an environmental exposure will not be detected in our PoOxE analysis. Hence, a detected PoOxE effect may have a better chance than a PoO or GxE effect of uncovering a true causal relationship involving genomic imprinting.

Relying on the q-values for assessing the false positive rate, our analyses detected possible PoOxSmoke effects with SNPs in LYZL1, ANK3, PDGFD, FOCAD and FRAS1, and possible PoOxAlcohol effects with two SNPs in ARHGEF10. Without formal validation in a comparable and independent replication cohort, it would be premature to accept these associations as true PoOxE effects. Not having previously been linked with orofacial clefts does not necessarily imply that the identified gene is not relevant for clefting. This applies to several genes in our analyses; for example, SHKBP1 and MAPK10 in the PoOxSmoke analysis of the European sample and TJP3 in the PoOxVitamin analysis of the pooled sample. The current study was primed to explore new hypotheses for disease mechanisms and to provide as many of the results as possible so that other researchers with access to similar GWAS datasets would be able to validate the findings presented here. To avoid being overly stringent, we thus presented all the results for the top 20 SNPs in Table 3Table 5.

Despite an exhaustive literature search, we were unable to find any obvious evidence linking ANK3 and orofacial clefts. The Hetionet results confirmed this lack of a direct connection (Figure 1). ANK3 encodes a member of the Ankyrin family of proteins, whose function is to bind the integral membrane proteins to the spectrin-actin cytoskeleton. This is important for cell motility, activation, proliferation, contact and the maintenance of specialized membrane domains; cellular activities that are also relevant for the proper development of craniofacial structures. For example, Stankewich and colleagues54 showed that the spectrin–ankyrin scaffold is important for cell migration, tissue patterning and organogenesis. Homozygous deletion of the gene encoding αII-spectrin in mice (Spna2) resulted in craniofacial, neural tube and cardiac anomalies, in addition to retarded intrauterine growth. Figure 3 indicates that several SNPs in ANK3 are potentially associated with clefting.

Like ANK3, ARHGEF10 has not previously been associated with orofacial clefts, and the Hetionet results are consistent with this observation (Figure 2). ARHGEF10 encodes a Rho guanine nucleotide exchange factor that may be involved in neural morphogenesis55. LYZL1 belongs to the family of lysozyme-like proteins that are implicated in sperm function and innate immunity56. According to GeneCards (GCID: GC09P020659), FOCAD encodes a tumor suppressor gene that is highly expressed in the brain. It has also been linked to Alzheimer's disease57. Furthermore, germline deletions in FOCAD are associated with polyposis and colorectal cancer58. Again, as with ANK3 and ARHGEF10 above, there do not seem to be any obvious connections between LYZL1 or FOCAD with clefting.

In contrast to the above genes, FRAS1 and several members of the platelet-derived growth factor (PDGF) gene family are known to be implicated in orofacial clefts. PDGFD is a member of the PDGF gene family and plays a central role in the PDFG receptor-alpha (PDGFR-α) signaling pathway. More specifically, disruption of Pdgf signaling results in clefting of the palate59. FRAS1 (GCID: GC04P078056) encodes an extracellular matrix protein that plays a critical role in epithelial-mesenchymal interactions during embryonic development60. Loss-of-function mutations in FRAS1 underlie Fraser syndrome, which is characterized by craniofacial, urogenital and respiratory system abnormalities61. Both of these genes are therefore worthy of further investigations in other isolated orofacial cleft cohorts.

A limitation of this study is that genotypes were not imputed. To avoid Mendelian inconsistencies, the imputation procedure would have had to account for the full triads, as opposed to imputing each sample independently, which has not been done for our data. Instead we conducted post hoc haplotype analyses for combinations of top SNPs located close to each other. This is akin to imputation, in that such an analysis takes into account information about a whole area of DNA, instead of just one SNP. Table 7 shows that in the PoOxSmoke analysis of the two-SNP combination rs3793861-rs7087489 in ANK3, the p-value was slightly lower and the RRR slightly higher than in the corresponding analyses of each individual SNP. A similar pattern was observed in the PoOxAlcohol analyses of the rs2294035-rs4876274 haplotype in ARHGEF10. This indicates that the two-SNP combinations may be driving the effects observed with the individual SNPs.

The genes PDGFD, CSMD1 and RSUI detected here had previously showed up in a study focusing on identifing GxE effects in the same CL/P triads28. In that study, a possible GxVitamin effect was detected with PDGFD and RSUI, and a possible GxAlcohol effect was detected with CSMD1. In the current study, a PoOxSmoke effect was detected with PDGFD and PoOxVitamin effects were detected with RSUI and CSMD1. In other words, only RSUI had the same exposure (vitamin) across the studies. RSUI stands for ‘Ras suppressor protein 1’ and is localized to chromosome 10p13. Its protein product is found at cell–extracellular matrix adhesion sites and has been reported to be involved in supressing v-Ras transformation in the Ras signal transduction pathway62. CSMD1 stands for ‘CUB and Sushi multiple domains 1’ and is localized to chromosome 8p23.2 (GCID: GC08M002953). It is involved in tumor suppression, as it has frequently been found to be deleted in many types of cancers63,64. Again, there does not seem to be any obvious connections to clefting.

None of the top SNPs identified in our previous study focusing on PoOxE effects in CPO triads overlapped with SNPs identified in this study of CL/P27. This is consistent with the observation that CPO and CL/P are etiologically distinct, so that the lead SNPs may be subtype-specific and differ between the two conditions. However, we detected associations with the ‘cytochrome P450 family 4 subfamily F member 3’ gene (CYP4F3 on chr 19p13.12) in the previous CPO analyses, and with the ‘cytochrome P450 family 46 subfamily A member 1’ gene (CYP46A1 on chr 14q32.2) in the present study. These two genes are members of the cytochrome P450 superfamily of enzymes that are primarily found in liver cells and whose function is to catalyze many reactions involved in the biotransformation of xeno- and endobiotics, and the biosynthesis of cholesterol and lipids, among others65. It is therefore not surprising that these genes would appear in an analysis focusing on smoking, alcohol and vitamin intake.

We searched Hetionet for indirect links between ANK3 or ARHGEF10 and cleft lip, as well as between ANK3 or ARHGEF10 and nicotine or alcohol dependence, respectively. This approach has several limitations. First, using “nicotine dependence” and “alcohol dependence” in lieu of the actual smoking and alcohol consumption status may introduce some bias. Second, Hetionet is built from a curated set of database information, which means that not all the information, especially the newest, would be available. However, when interpreting our results, we used the source databases to make sure that the connections between the nodes are reliable.

To conclude, our search for an interaction between a PoO-effect and an environmental exposure for CL/P identified possible relationships between SNPs in ANK3 and maternal smoking, and SNPs in ARHGEF10 and maternal intake of alcohol. There is a possibility that these interactions have a biological basis, although without replication they remain speculative. Our demonstration of the feasibility of identifying complex interactions between relevant environmental exposures and PoO-effects opens new possibilities in the search for the genetic etiology of CL/P.

Data availability

Underlying data

The GWAS data are available in the dbGaP database. Additional information regarding the inclusion/exclusion criteria of the study, the ethics statements, data variables, study history, publications, and other documentation related to the study is provided on the dbGaP website.

The dbGaP database at the National Center for Biotechnology Information, U.S. National Library of Science (NCBI/NLM) provides an extensive overview of the cleft dataset used in this study. Entering the dbGaP accession number phs000094.v1.p1 provides access to information regarding the variables, study documents, and datasets. For example, detailed information about the mother’s exposure to alcohol, vitamins, and smoke is provided under the header “Variable Selection”. Information on study questionnaires, institutional review boards and consent forms from each participating cohort can be found under the header “Documents”.

Controlled-access data from dbGaP is available only through the dbGaP authorized access portal. There are separate procedures for accessing individual data, depending on whether the researcher is NIH-affiliated (intramural) or not (extramural). In addition, other restrictions apply; e.g., the principal investigator from the applying institution needs to be a permanently employed professor, senior scientist, or equivalent, to submit a data access request. A valid eRA Commons account for logging in to the dbGaP system is also mandatory.

Software availability

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Haaland ØA, Romanowska J, Gjerdevik M et al. A genome-wide scan of cleft lip triads identifies parent-of-origin interaction effects between ANK3 and maternal smoking, and between ARHGEF10 and alcohol consumption [version 2; peer review: 2 approved]. F1000Research 2019, 8:960 (https://doi.org/10.12688/f1000research.19571.2)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Current Reviewer Status: ?
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 2
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PUBLISHED 19 Jul 2019
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Reviewer Report 26 Jul 2019
Evie Stergiakouli, School of Oral and Dental Sciences, University of Bristol, Bristol, UK;  MRC Integrative Epidemiology at the University of Bristol, Bristol, UK 
Approved
VIEWS 7
The authors have improved the manuscript substantially, although not having imputed their data is ... Continue reading
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Stergiakouli E. Reviewer Report For: A genome-wide scan of cleft lip triads identifies parent-of-origin interaction effects between ANK3 and maternal smoking, and between ARHGEF10 and alcohol consumption [version 2; peer review: 2 approved]. F1000Research 2019, 8:960 (https://doi.org/10.5256/f1000research.21886.r51378)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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PUBLISHED 24 Jun 2019
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Reviewer Report 25 Jul 2019
Yongchu Pan, Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University (NMU), Nanjing, China 
Approved
VIEWS 9
The authors conducted a genome-wide interaction analysis with smoking, alcohol, and vitamin on cleft lip triads. The analysis was based on 1,908 nuclear families from dbGaP database with both genotyping and environmental exposure status. Their results suggested that three SNPs ... Continue reading
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CITE
HOW TO CITE THIS REPORT
Pan Y. Reviewer Report For: A genome-wide scan of cleft lip triads identifies parent-of-origin interaction effects between ANK3 and maternal smoking, and between ARHGEF10 and alcohol consumption [version 2; peer review: 2 approved]. F1000Research 2019, 8:960 (https://doi.org/10.5256/f1000research.21459.r50328)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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22
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Reviewer Report 04 Jul 2019
Evie Stergiakouli, School of Oral and Dental Sciences, University of Bristol, Bristol, UK;  MRC Integrative Epidemiology at the University of Bristol, Bristol, UK 
Approved with Reservations
VIEWS 22
This is a very interesting paper focusing on identifying parent of origin interaction effects in CL/P using a genome-wide approach. The environmental factors selected are biologically plausible to be involved in cleft and the sample, although modest in size, involved ... Continue reading
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CITE
HOW TO CITE THIS REPORT
Stergiakouli E. Reviewer Report For: A genome-wide scan of cleft lip triads identifies parent-of-origin interaction effects between ANK3 and maternal smoking, and between ARHGEF10 and alcohol consumption [version 2; peer review: 2 approved]. F1000Research 2019, 8:960 (https://doi.org/10.5256/f1000research.21459.r50329)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 10 Jul 2019
    Øystein Ariansen Haaland, Department of Global Public Health and Primary Care, University of Bergen, Bergen, N-5020, Norway
    10 Jul 2019
    Author Response
    We would like to thank Dr. Stergiakouli for her insightful comments, and have addressed her concerns below. We have also made minor changes throughout the manuscript to correct grammatical errors ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 10 Jul 2019
    Øystein Ariansen Haaland, Department of Global Public Health and Primary Care, University of Bergen, Bergen, N-5020, Norway
    10 Jul 2019
    Author Response
    We would like to thank Dr. Stergiakouli for her insightful comments, and have addressed her concerns below. We have also made minor changes throughout the manuscript to correct grammatical errors ... Continue reading

Comments on this article Comments (0)

Version 2
VERSION 2 PUBLISHED 24 Jun 2019
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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