Published: 2020-06-26

Role of ARHGAP29 nucleotide variants in the etiology of non-syndromic cleft lip with or without cleft palate.

Poznan University of Medical Sciences
Clinic of Craniofacial Anomalies, Poznan University of Medical Sciences, Poznan, Poland
Department of Jaw Orthopaedics, Medical University of Lublin, Lublin, Poland
Department of Biochemistry and Molecular Biology, Poznan University of Medical Sciences, Poznan, Poland
Department of Biochemistry and Molecular Biology, Poznan University of Medical Sciences, Poznan, Poland
nsCL/P ARHGAP2 SNVs

Abstract

Aim. Non-syndromic cleft lip with or without cleft palate (nsCL/P) is a common birth defect of complex and heterogeneous aetiology. Genome-wide association studies (GWAS) of nsCL/P have identified an association for the 1p22.1 chromosomal region, in which ARHGAP29 was suggested as a candidate gene. Thus, the current study aimed to determine the contribution of the common and rare ARHGAP29 nucleotide variants to the risk of nsCL/P in the Polish population.

Material and Methods. In total,197 common nucleotide variants (SNVs) and 22 missense variants located within the ARHGAP29 locus at chromosome 1p22.1 were genotyped by SNV microarray. The study was conducted in 269 individuals with nsCL/P and 569 healthy individuals.

Results. Statistical analysis revealed that 31 common nucleotide variants located at the ARHGAP29 locus were significantly associated with the increased risk of nsCL/P. The strongest individual SNV was rs2391467 with a p-value = 2.49E-06 (OR = 1.64, 95%CI: 1.34–2.02). Besides, one potentially deleterious missense variant (rs140877322, p. Arg348Leu) was identified in a single patient with nsCLP.

Conclusion. These findings confirm ARHGAP29 as a strong candidate gene for nsCL/P, with both common and rare nucleotide variants of this gene involved in the aetiology of nsCL/P in the Polish population.

Introduction

Non-syndromic cleft lip with or without cleft palate (nsCL/P) is one of the most common congenital defects in humans, affecting approximately 1/700 live-born children worldwide [1]. The prevalence of nsCLP varies by geographic location, ethnic/racial background, and socioeconomic status [2]. The complex aetiology of this congenital anomaly reflects the action of multiple genetic factors and environmental exposures, hence it has not been fully elucidated. NsCL/P can be divided into the cleft of the palate only (CPO), and those affecting the lip with or without the palate (CL/P) [3]. Over 500 human syndromes in which clefting is a common feature have been identified (https://www.ncbi.nlm.nih.gov/OMIM/), although most cases correspond to isolated non-syndromic clefts with the absence of other structural or cognitive abnormalities [2]. Previous studies revealed that nsCL/P might have unique aetiological features, including specific genetic associations [2,4], however, the genetic components of nsCL/P have remained elusive due to the influence of multiple environmental risk factors [5].

To date, a variety of research methods have been applied to identify genetic factors contributing to nsCL/P. Genome-wide association studies (GWAS) have been crucial in identifying 40 novel risk loci that show strong associations of single nucleotide variants (SNVs) with nsCL/P [6-10]. The most consistent results in multiple populations were observed for nucleotide variants in IRF6 (OMIM* 607199) gene, encoding a transcription factor critically involved in craniofacial development [2] and the gene-poor region of chromosome 8q24.21 [11-14]. Also, the chromosomal region consistently associated with nsCL/P is 1p22.1, initially implicating ABCB4(OMIM:*601691) as a candidate gene at this locus. However, ABCB4 was excluded due to its retinal expression and known role in a spectrum of retinal disorders [15,16]. Therefore, it has been hypothesised that a neighbouring gene, ARHGAP29 (OMIM:*610496), maybe the aetiologic gene within the same region. During craniofacial development in murine embryos, ARHGAP29 expression was detected in the frontonasal, lateral prominences and palatal shelves [17]. Moreover, there are reports that IRF6 regulates keratinocyte migration through ARHGAP29. Cells lackingtheIRF6 gene had lower levels of ARHGAP29 and hyperactive Rho GTPase activating protein (GAP), which is involved in crucial cellular functions essential for craniofacial development [12,18]. In addition, sequencing of ARHGAP29in patients with nsCL/P identified eight potentially deleterious and aetiological variants, including a frameshift and a nonsense variant [17]. Further functional studies identified a novel missense variant in ARHGAP29 (c.1654T>C, p.Ser552Pro), showing ARHGAP29 to be a regulatory protein affecting the development of the face [19]. These findings suggest that ARHGAP29 may play a role as the aetiologic gene at the 1p22.1 locus for nsCL/P [17]. Therefore, this study investigated whether common SNVs and rare missense variants at the ARHGAP29 locus contribute to the risk of nsCL/P in the Polish population.

Material and Methods

The present study was designed similar to our previous cleft association studies and conducted on the same study population [9,20].

Study population

Patients with a diagnosis of nsCL/P (58.0% of males) were recruited from several Polish medical centres. Among patients, 229 (85.1%) individuals had non-syndromic cleft lip and palate (nsCLP) and 40 (14.9%) individuals had non-syndromic cleft lip only (nsCLO). The control group was composed of 569 healthy individuals (49.6% males) without any developmental anomalies. Detailed characteristics of patients and controls enrolled in the study are presented in Table 1. All study participants were of Polish origin. DNA was isolated from peripheral blood lymphocytes by salt extraction. All experimental protocols were approved by the Institutional Review Board of Poznan University of Medical Sciences, Poland [21]. Written and oral consent was obtained from all participants or their legal guardians.

Common SNV selection and genotyping

Common single nucleotide polymorphisms (SNVs) located within the ARHGAP29 locus at chromosome 1p22.1 were genotyped with using the HumanOmniExpressExome-8 v1 array (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. After applying stringent quality control criteria (SNV call rate > 0.95, minor allele frequency, MAF, > 0.05, Hardy-Weinberg, HW, equilibrium p-value > 0.001 in controls), 197 common SNVs were subjected to statistical analysis.

Statistical analysis

The association of ARHGAP29 locus SNVs with nsCL/P was tested with the Cochran-Armitage trend test. Odds ratios (ORs) and corresponding 95% confidence intervals (95% CIs) were used to assess the strength of the association. ORs were calculated for the allelic model (a vs A; a is the risk allele). The Bonferroni correction was applied to account for multiple comparisons, and p-values < 2.54E-4 (0.05 / 197 SNVs) were considered as statistically significant. The pair-wise linkage disequilibrium (LD) between the top was evaluated using the Haploview 4.2 software (www.broadinstitute.org/haploview/haploview, Table 2). Separate statistical analyses were conducted for individuals with nsCLP and nsCLO to assess the subgroup-specific effects of the significantly associated SNVs. In addition, separate analyses were performed in male and female groups. The effects of genotype x gender interactions were evaluated by logistic regression approach.

In silico analysis of missense variants

The SNV microarray used in the present study allowed for the genotyping of 22 ARHGAP29 missense variants. The putative functional consequences of these missense SNVs were analysed in silico using SIFT (http://sift.jcvi.org/), PolyPhen (http://genetics.bwh.harvard.edu/pph2/), and Mutation Assessor (http://mutationassessor.org/r3/) tools. Additionally, for all these variants, the frequency of the minor allele was checked in the Exome Aggregation Consortium (ExAC) database (http://exac.broadinstitute.org/).

Results

Common SNVs

Statistical analysis of the 197 ARHGAP29 locus SNVs revealed that 31 were nominally associated (ptrend < 0.05) with the risk of nsCL/P (Figure 1, Table 7). Three SNVs, rs11165101, rs11165110 and rs2391467, were statistically significant even after applying the strict Bonferroni correction for multiple comparisons (ptrend = 1.71E-04, ptrend = 2.19E-04 and ptrend = 2.56E-06, respectively). These variants located within the same recombination region were in moderate LD with each other (the mean r2 = 0.76 and D’ = 0.99; Table 2). Two other SNVs, rs3789688 and rs2065971, were close to the study significance level (ptrend = 8.34E-04 and ptrend = 2.91E-04, respectively). The minor allele of the strongest individual SNV (rs2391467) located 14.7 kb upstream of ARHGAP29 was associated with a 1.64-fold increased risk of nsCL/P (95%CI: 1.34 – 2.02, p = 2.49E-06), with the allelic ORs for the other four top variants in the range of 1.43 to 1.52. For all of them, the major allele was the risk allele. Association results for tested variants are presented in Table 3 and Table 7. The sub-phenotype analysis revealed that the most significant SNVs identified in the present study were exclusively associated with the risk of nsCLP. No significant association was identified between them and the nsCLO risk (ptrend values > 0.05), however, the differences in ORs between nsCLP and nsCLO subgroups were not statistically significant (heterogeneity p-values >0.05). Besides, for all these variants, the trend test p-values for nsCLP were higher than the p-values for the overall phenotype (Table 4). Logistic regression analysis revealed that ARHGAP29 locus variants did not show evidence of gender-dependent association with the risk of nsCL/P. No significant sex genotype interactions were detected (Table 5).

Figure 1.Regional plot of association results within the ARHGAP29 locus

nsCL/P patients (n = 269)* Controls (n = 569)*
Cleft type
nsCLP 229 (85.1%)
nsCLO 40 (14.9%)
Gender
male 156 (58.0%) 282 (49.6%)
female 113 (42.0%) 287 (50.4%)
Table 1.Characteristics of study patients and controls*Final number of samples analysed in the present study after exclusion of individuals based on stringent quality control criteriansCL/P — non-syndromic cleft lip with or without cleft palate; nsCLP — non-syndromic cleft lip and palate; nsCLO — non-syndromic cleft lip only
rs3789688 rs11165101 rs11165110 rs2065971 rs2391467
rs3789688 - 0.78 0.78 0.71 0.83
rs11165101 0.46 - 1.00 0.98 0.98
rs11165110 0.46 1.00 - 0.98 0.98
rs2065971 0.41 0.89 0.89 - 1.00
rs2391467 0.61 0.64 0.64 0.71 -
Table 2.Linkage disequilibrium values D' and r2 for the most significant SNVs located at the ARHGAP locusNumbers denote D’ and r2 values expressed as a percentage of maximal value (1.0). D' values are presented above diagonal. A red-to-white gradient shows highest (1.0) to lowest (0.0) D’. r2 vales are presented below diagonal. A black-to-white gradient shows highest (1.0) to lowest (0.0) r2.
RAF
rs number Location (bp)a Consequence type Gene Allelesb ptrendc nsCL/P Controls OR (95%CI)d
rs3789688 chr1: 94691240 intronic ARHGAP29 T / C 8.34E-04 0.59 0.50 1.43 (1.17–1.77)
rs11165101 chr1: 94729088 intergenic ARHGAP29 / ABCD3 A / C 1.71E-04 0.67 0.57 1.52 (1.23–1.89)
rs11165110 chr1: 94752469 intergenic ARHGAP29 / ABCD3 A / G 2.19E-04 0.67 0.57 1.51 (1.22–1.87)
rs2065971 chr1: 94807102 intergenic ARHGAP29 / ABCD3 A / C 2.91E-04 0.65 0.55 1.48 (1.20–1.83)
rs2391467 chr1: 94850443 intergenic ARHGAP29 / ABCD3 A / G 2.56E-06 0.58 0.46 1.64 (1.34–2.02)
Table 3.Association results for the most significant variants at the ARHGAP29 locus (ptrend values < 1.00E-03)aNCBI build 37 / hg19.bUnderline denotes the risk allele (for all variants, exept rs2391467, the major allele is a risk allele).cThe ptrend values below 2.54E-04 (0.05 / 197 SNVs) were interpreted as statistically significant.dOdds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated for the allelic model (a vs A; a is the risk allele).Significant p-values are highlighted in bold font.RAF — risk allele frequency; nsCL/P — non-syndromic cleft lip with or without cleft palate.
RAF
rs number Allelesa ptrendb Patients Controls OR (95%CI)d
nsCLP
rs3789688 T / C 2.48E-03 0.59 0.50 1.41 (1.13–1.76)
rs11165101 A / C 2.62E-04 0.67 0.57 1.54 (1.23–1.93)
rs11165110 A / G 2.23E-04 0.67 0.57 1.55 (1.23–1.94)
rs2065971 A / C 3.25E-04 0.65 0.55 1.51 (1.21–1.89)
rs2391467 A / G 4.28E-06 0.58 0.46 1.67 (1.34–2.08)
nsCLO
rs3789688 T / C 5.68E-02 0.62 0.50 1.58 (0.99–2.54)
rs11165101 A / C 1.53E-01 0.65 0.57 1.43 (0.88–2.31)
rs11165110 A / G 2.51E-01 0.64 0.57 1.33 (0.83–2.12)
rs2065971 A / C 2.22E-01 0.63 0.55 1.34 (0.84–2.14)
rs2391467 A / G 7.03E-02 0.56 0.46 1.51 (0.95–2.38)
Table 4.Association results for oral cleft sub-phenotypesaThe ptrend-values below 2.54E-04 (0.05 / 197 SNVs) were interpreted statistically significant.For all tested SNVs, there was no heterogeneity betwen oral cleft sub-phenotypes (heterogeneity pvalues between 0.551 and 0.780). nsCLP — non-syndromic cleft lip and palate; nsCLO — non-syndromic cleft lip only.
SNV ORint(95%CI)a pd ORmales(95%CI)b pd ORfemales(95%CI)c pd
rs3789688 0.94 (0.62 - 1.43) 7.77E-01 1.44 (1.09 - 1.91) 1.16E-02 1.36 (1.00 - 1.84) 4.94E-02
rs11165101 0.89 (0.58 - 1.37) 5.91E-01 1.56 (1.16 - 2.10) 3.40E-03 1.39 (1.02 - 1.89) 3.63E-02
rs11165110 0.93 (0.61 - 1.43) 7.52E-01 1.51 (1.13 - 2.03) 6.03E-03 1.41 (1.04 - 1.92) 4.80E-02
rs2065971 0.88 (0.58 - 1.36) 5.73E-01 1.54 (1.14 - 2.07) 4.32E-03 1.36 (1.00 - 1.86) 3.03E-02
rs2391467 1.12 (0.73 - 1.71) 6.05E-01 1.72 (1.28 - 2.31) 3.24E-04 1.54 (1.13 - 2.09) 7.95E-03
Table 5.Gender-dependent interaction of the most significant variants at the ARHGAP29 locus and nsCL/Pa Odds ratio for the gene x gender interaction.b Odds ratio for the males.c Odds ratio for the females.d Based on logistic regression under the additive model.

Missense variants

Six out of twenty-two (27.3%) genotyped missense ARHGAP29 variants were identified in the tested samples. Four of them were found only in cleft patients, and one was found exclusively in controls (Table 6). One of the cleft specific variants (rs140877322, p. Arg348Leu) was predicted to be deleterious and functional by all prediction tools used in this study. In a non-Finnish European population of ExAC, the frequency of rs140877322 was 5.995E-05.

NsCL/P Cases Controls MAF
rs number Allelesa Amino acid changeb Genotypes MAF Genotypes MAF ExACc SIFTd PolyPhene Mutation Assessorf
rs1999272 T / C p.Gly1255Asp 0 / 1 / 268 0.002 0 / 0 / 569 0.000 0.0004 tolerated low confidence benign predicted non-functional (neutral)
rs143877998 T / G p.Gln893Pro 0 / 1 / 268 0.002 0 / 0 / 569 0.000 0.0002 tolerated benign predicted functional (medium)
rs147752270 T / C p.Val875Ile 0 / 1 / 268 0.002 0 / 2 / 567 0.002 0.0016 tolerated benign predicted non-functional (low)
rs41311172 T / C p.Arg798Gln 0 / 0 / 269 0.000 0 / 3 / 566 0.003 0.0025 tolerated probably damaging predicted non-functional (low)
rs140877322 A / C p.Arg348Leu 0 / 1 / 268 0.002 0 / 0 / 569 0.000 5.995E-05 deleterious probably damaging predicted functional (medium)
rs183410431 T / C p.Arg84His 0 / 2 / 267 0.004 0 / 0 / 569 0.000 4.505E-05 deleterious benign predicted non-functional (neutral)
Table 6.Missense variants identified in the ARHGAP29 gene with the use of SNV microarrayaUnderline denotes the minor allele.bENST00000260526.11. cExome Aggregation Consortium (ExAC), European (Non-Finnish).dhttp://genetics.bwh.harvard.edu/pph2/.ehttp://sift.jcvi.org/.fhttp://mutationassessor.org/r3/.

Discussion

Numerous genes and several polymorphic variants have been detected to confer an increased risk of nsCL/P [22,23]. Beaty et al. characterised four significant loci in GWAS for nsCL/P: IRF6, 8q24, MAFB(OMIM:*608968), and ABCA4 [11], with multiple follow-up studies in different populations successfully confirming these loci [24-31]. A critical role for IRF6 and the 8q24 region in craniofacial development has been previously identified, while the roles of the loci in the genes ABCA4 and MAFB remains largely unclear [32-34]. The most significant SNVs strongly associated with nsCL/P have been identified within the introns of ABCA4 [35]. However, ABCA4 is not a good candidate as the etiologic gene for nsCL/P at the 1p22 locus because of its lack of expression in the developing lip or palate in mice [11]. Additionally, there were no reported defects in the craniofacial structure in mice homozygous for targeted loss-of-function mutations inABCA4 [36]. Moreover, despite identifying several missense mutations in ABCA4 in humans, none of them showed suggestive evidence of causing craniofacial malformations [15,37]. However, a recent study suggests a role for ARHGAP29 (a neighbouring gene of ABCA4) in nsCL/P based on expression in craniofacial development using a murine model. The ARHGAP29 transcript was detected in the medial and lateral nasal processes, and expression was also observed in the mandibular and maxillary processes of developing mouse embryo at E10.5 and the shelves of the secondary palate at E13.5. [38]. Furthermore, its expression depends on IRF6 [38], one of the pivotal contributors to the underlying genetics of human nsCLP/P [41]. ARHGAP29 is located 47 kb centromeric to ABCA4 and encodes Rho GTPase activating protein (GAP) 29, which is involved in many functions related to cellular shape, movement, proliferation, all essential for craniofacial development [12]. Rho is downstream of Tgfb and Wnt signalling pathways [39,40], which have also been implicated in craniofacial development. These are suggestive evidence that ARHGAP29 is the etiologic gene at this locus and may play a role in nsCL/P. During the last few years, about twelve potentially pathogenic missense variants in ARHGAP29 have been reported in nsCL/P cases. [17,19,28,35,42]. However, it is not clear if these possibly pathogenic rare variants contribute to the phenotype. Therefore, the purpose of this study was to evaluate the association between common and rare missense SNVs located within the ARHGAP29 locus and the risk of nsCL/P in the Polish population. Patients with non-syndromic forms of cleft lip with cleft palate and cleft lip only were recruited, with patients with a diagnosis of non-syndromic cleft palate only were excluded from the molecular analyses due to the distinct aetiology of this subtype of oral clefts [43].

The findings showed that common nucleotide variants of the ARHGAP29 gene are significantly correlated with the risk of nsCL/P. Statistical analysis of the genotyping results revealed that three common SNVs represent a single cleft association signal since they are located within the same intergenic region of ARHGAP29/ABCD3 genes. These three risk variants rs11165101, rs11165110 and rs2391467 are strongly associated with the risk of this craniofacial anomaly in a tested group of patients. Our data also demonstrated that the minor allele carriers at rs2391467 have a 1.64-fold increased risk of nsCL/P. Moreover, all these results were statistically significant even after applying Bonferroni correction for multiple comparisons. Two other tested variants, rs3789688 and rs2065971, were close to reaching the study significance level. It is of note that the intronic SNV rs3789688 in families of non-Hispanic white ethnicities also showed a strong association with nsCL/P [29]. Although the intronic variants are unlikely to have effects on gene transcription or the final protein structure, multiple analysis revealed that non-coding variants have a significant role in the genetic causes of nsCL/P [3,35,44]. This suggests that the true casual variants implicated in the risk of nsCL/P might affect the ARHGAP29 gene expression level rather than the structure of an encoded protein or are in pair-wise LD with an unknown actual pathogenic variant.

We hypothesised that some genetic risk for nsCL/P in Polish populations lies in rare exonic markers, thus, our study also included an analysis of twenty-two missense variants. Our results showed six missense ARHGAP29 variants in the tested samples. One of the cleft specific variants (rs140877322, p.Arg348Leu) was predicted to be probably damaging and deleterious by multiple in silico tools. Furthermore, none of the unaffected individuals carried the variant. However, these results should be interpreted with caution because the functional impact of these variants is unknown. Therefore, functional studies in biological model systems are required to identify pathogenic variants and a possible mechanism contributing to the nsCL/P phenotype. Savastano at al. identified ten rare variants in the ARHGAP29 gene using next-generation sequencing, of these, five were missense changes and the remaining were predicted to be loss-of-function (LoF). These findings provide evidence that the LoF variants but not missense variants may be an important genetic factor and contribute to the aetiology of nsCL/P [45]. To take this idea further, new coding variants which confer risk to nsCL/P should be identified by sequencing, which is crucial for rare variant discovery.

There was no evidence of a gender-dependent association with any of the SNVs studied. However, Carlson at al. confer that the impact of genetic variants on nsCL/P risk differs for males and females. These results are not surprising because the incidence rates of nsCL/P vary by sex. Carlson et al. used a genome‐wide approach to identify the genetic contribution to this phenomenon and examined gene by sex interactions in a group of 2142 nsCL/P cases and 1700 controls recruited from different countries. Their analysis identified three loci that achieved genome-wide significance interaction effect, rs11142081, rs72804706, rs77590619 from the 9q22.1, 10q21.1, and 13q13.3 loci, respectively showed evidence of a higher risk of CL/P for females carrying the minor risk allele, while this trend was not present in males. It is worth noting that many biochemical mechanisms affect gene by sex interactions, hence they suggest that due to the diversity of possible mechanisms, it is challenging to explore or discuss each locus adequately [46].

Given the impact of rare variants as potential phenotypic modifiers diversity, which has been highlighted by Carlson et al. [47], we analysed cleft type-dependent association with the studied SNVs. However, our sub-phenotype analysis did not reveal any significant genotype-phenotype correlations. Nonetheless, these results should be interpreted with caution due to the small number of patients with nsCLO recruited to our study, therefore, insufficient power to detect significant cleft type differences.

The strength of this study lies in the homogenous study cohort recruited from a single ethnic group. Although the study is limited by a relatively small sample size, the risk variants with the lower allele frequencies may have been missed. Hence, statistical analyses were not well suited to draw reliable associations from low-frequency variants (MAF < 0.05), which may be important in explaining nsCL/P susceptibility. Another limitation of our study is that the association analysis focused only on the genetic factors without considering environmental factors that appear to contribute to the aetiology of nsCL/P, like maternal folic acid supplementation or maternal smoking [48,49].

Despite these limitations, the nsCL/P risk loci identified in our research are consistent with previous studies and biological mechanisms, thereby providing further evidence for the role of ARHGAP29 and new insight into the pathogenesis of the nsCL/P in the Polish population. Functional analyses are required to explore the mechanisms by which nucleotide variants of the ARHGAP29 gene might increase risk of nsCL/P

Acknowledgements

The study was supported by the Polish National Science Centre grant no. 2012/07/B/NZ2/00115. The authors would like to thank Prof. Margareta Budner, Prof. Kamil K. Hozyasz, and Prof. Piotr Wójcicki, for their help with the sample collection.

rs number Location (bp)b Gene Exonic variant Allelesc MAFd Ptrend-value
rs743117 chr1: 94441950 T / c 0.37 1.12E-01
rs743113 chr1: 94442739 T / c 0.41 2.20E-01
rs1889547 chr1: 94450131 A / g 0.44 1.31E-01
rs11165057 chr1: 94455420 t / G 0.08 1.72E-01
rs11165058 chr1: 94455721 t / G 0.08 1.42E-01
rs17110736 chr1: 94462769 ABCA4 a / G 0.07 7.88E-01
rs3789375 chr1: 94465132 ABCA4 T / g 0.11 4.69E-01
rs4147871 chr1: 94465461 ABCA4 A / g 0.06 7.72E-01
rs4147869 chr1: 94465677 ABCA4 t / C 0.06 5.75E-01
rs4147868 chr1: 94466066 ABCA4 g / C 0.06 6.62E-01
rs12070273 chr1: 94466088 ABCA4 A / g 0.06 6.62E-01
rs4147864 chr1: 94467238 ABCA4 a / G 0.06 5.70E-01
rs17110761 chr1: 94467407 ABCA4 t / C 0.06 6.62E-01
rs7537325 chr1: 94469631 ABCA4 T / c 0.23 9.20E-01
rs1762114 chr1: 94471075 ABCA4 Synonymous_I2023I a / G 0.07 9.80E-01
rs4147863 chr1: 94471154 ABCA4 t / C 0.18 6.29E-01
rs4147862 chr1: 94471519 ABCA4 t / C 0.18 8.63E-01
rs4147861 chr1: 94471948 ABCA4 a / C 0.06 5.86E-01
rs557026 chr1: 94472489 ABCA4 t / C 0.11 2.43E-01
rs3789379 chr1: 94472520 ABCA4 A / g 0.18 8.63E-01
rs7531001 chr1: 94472909 ABCA4 a / G 0.18 6.54E-01
rs2275029 chr1: 94473845 ABCA4 Synonymous_P1948P T / c 0.16 7.95E-01
rs2275031 chr1: 94473896 ABCA4 t / G 0.18 7.89E-01
rs1191234 chr1: 94474020 ABCA4 A / g 0.11 2.21E-01
rs2275032 chr1: 94474185 ABCA4 A / c 0.18 8.95E-01
rs4147857 chr1: 94474328 ABCA4 Synonymous_L1938L T / c 0.19 5.82E-01
rs4147856 chr1: 94474452 ABCA4 T / g 0.19 5.78E-01
rs17110808 chr1: 94474872 ABCA4 a / G 0.07 2.41E-01
rs2065712 chr1: 94476035 ABCA4 t / C 0.25 6.17E-01
rs11165062 chr1: 94477634 ABCA4 a / G 0.13 5.36E-01
rs945067 chr1: 94477893 ABCA4 t / C 0.20 9.69E-01
rs17391542 chr1: 94477935 ABCA4 a / G 0.11 6.99E-01
rs12085639 chr1: 94478293 ABCA4 a / G 0.16 9.93E-01
rs486879 chr1: 94478425 ABCA4 T / c 0.20 7.67E-01
rs567370 chr1: 94478573 ABCA4 T / c 0.33 9.75E-01
rs12082181 chr1: 94478595 ABCA4 T / c 0.33 8.37E-01
rs17391612 chr1: 94478847 ABCA4 a / G 0.12 5.87E-01
rs3789391 chr1: 94479338 ABCA4 t / C 0.06 6.29E-01
rs12049183 chr1: 94479468 ABCA4 t / C 0.06 5.89E-01
rs2275034 chr1: 94480439 ABCA4 t / C 0.40 5.85E-01
rs3818778 chr1: 94480529 ABCA4 a / C 0.48 1.52E-01
rs914958 chr1: 94481068 ABCA4 A / g 0.21 7.47E-01
rs915201 chr1: 94481596 ABCA4 A / g 0.25 9.13E-01
rs915200 chr1: 94481904 ABCA4 t / C 0.08 7.70E-01
rs915199 chr1: 94481929 ABCA4 t / C 0.23 8.72E-01
rs6681968 chr1: 94484705 ABCA4 t / C 0.42 4.56E-01
rs17110850 chr1: 94485491 ABCA4 A / g 0.11 6.34E-01
rs10493867 chr1: 94486406 ABCA4 T / c 0.25 8.25E-01
rs4147848 chr1: 94486587 ABCA4 t / G 0.29 4.45E-01
rs933073 chr1: 94486667 ABCA4 T / g 0.08 1.62E-01
rs472908 chr1: 94487354 ABCA4 A / g 0.43 1.59E-01
rs2282229 chr1: 94488326 ABCA4 a / T 0.09 1.96E-01
rs1932014 chr1: 94488497 ABCA4 A / g 0.44 3.67E-01
rs1889407 chr1: 94489553 ABCA4 T / g 0.44 3.49E-01
rs2151847 chr1: 94489975 ABCA4 a / C 0.43 4.86E-01
rs11165065 chr1: 94491468 ABCA4 a / G 0.32 5.83E-01
rs3945204 chr1: 94492773 ABCA4 t / C 0.44 5.79E-01
rs4147846 chr1: 94495407 ABCA4 T / c 0.49 6.68E-01
rs4147845 chr1: 94495417 ABCA4 T / c 0.49 6.69E-01
rs4147844 chr1: 94495487 ABCA4 A / g 0.49 6.69E-01
rs2297671 chr1: 94496253 ABCA4 A / g 0.50 6.78E-01
rs4147841 chr1: 94497178 ABCA4 A / g 0.44 4.42E-01
rs3789393 chr1: 94499133 ABCA4 t / C 0.44 4.39E-01
rs3789395 chr1: 94501594 ABCA4 a / C 0.43 3.33E-01
rs1320502 chr1: 94501799 ABCA4 T / c 0.43 3.89E-01
rs1889548 chr1: 94503197 ABCA4 a / C 0.31 7.80E-01
rs11165069 chr1: 94504545 ABCA4 t / C 0.21 8.74E-01
rs2297633 chr1: 94510673 ABCA4 t / G 0.34 6.51E-01
rs3789399 chr1: 94511717 ABCA4 c / G 0.48 2.89E-01
rs544830 chr1: 94512893 ABCA4 T / c 0.48 4.56E-01
rs4147836 chr1: 94516474 ABCA4 t / C 0.22 9.65E-02
rs1191231 chr1: 94516985 ABCA4 a / C 0.48 4.73E-01
rs497511 chr1: 94523113 ABCA4 A / g 0.46 5.19E-01
rs549848 chr1: 94524856 ABCA4 T / c 0.34 4.39E-01
rs521538 chr1: 94525623 ABCA4 a / G 0.22 5.97E-01
rs4147833 chr1: 94528363 ABCA4 t / C 0.21 5.94E-02
rs4847273 chr1: 94529743 ABCA4 A / g 0.26 7.51E-02
rs1007347 chr1: 94530518 ABCA4 T / c 0.26 7.36E-02
rs553608 chr1: 94531013 ABCA4 t / C 0.21 2.17E-01
rs1191232 chr1: 94531192 ABCA4 a / G 0.37 3.28E-01
rs3789405 chr1: 94531324 ABCA4 T / c 0.26 6.85E-02
rs3789407 chr1: 94531606 ABCA4 c / G 0.22 1.08E-01
rs4140392 chr1: 94532013 ABCA4 t / C 0.21 6.51E-02
rs1191228 chr1: 94532562 ABCA4 t / C 0.21 2.17E-01
rs1931575 chr1: 94533014 ABCA4 T / c 0.23 2.76E-01
rs549114 chr1: 94534354 ABCA4 a / G 0.33 7.45E-01
rs2151849 chr1: 94535174 ABCA4 A / g 0.24 3.28E-01
rs3789411 chr1: 94535546 ABCA4 a / G 0.33 8.01E-01
rs4612636 chr1: 94535689 ABCA4 A / c 0.06 7.91E-01
rs3789412 chr1: 94536067 ABCA4 t / C 0.24 2.06E-01
rs12758774 chr1: 94537295 ABCA4 t / C 0.20 9.00E-01
rs12759306 chr1: 94537642 ABCA4 a / C 0.20 9.17E-01
rs1761375 chr1: 94538011 ABCA4 a / G 0.31 9.04E-01
rs492220 chr1: 94542569 ABCA4 T / c 0.29 6.85E-01
rs3120133 chr1: 94542770 ABCA4 T / g 0.08 5.64E-01
rs4147831 chr1: 94544233 ABCA4 Synonymous_H423H a / G 0.07 3.45E-01
rs4147828 chr1: 94547889 ABCA4 A / g 0.18 8.39E-02
rs4147827 chr1: 94548080 ABCA4 G / c 0.23 1.50E-02
rs574741 chr1: 94549083 ABCA4 t / C 0.22 6.14E-01
rs546550 chr1: 94550555 ABCA4 A / g 0.29 7.35E-01
rs17461953 chr1: 94551450 ABCA4 A / c 0.23 1.89E-02
rs563429 chr1: 94553866 ABCA4 A / g 0.29 6.51E-01
rs4847196 chr1: 94554453 ABCA4 a / G 0.22 1.06E-01
rs1191238 chr1: 94556894 ABCA4 a / G 0.09 8.84E-01
rs554931 chr1: 94557357 ABCA4 t / C 0.23 7.85E-01
rs483904 chr1: 94557434 ABCA4 t / C 0.23 7.85E-01
rs952499 chr1: 94558425 ABCA4 T / c 0.47 2.95E-01
rs538880 chr1: 94558774 ABCA4 g / C 0.23 8.19E-01
rs2068334 chr1: 94559715 ABCA4 a / G 0.17 2.25E-01
rs4147825 chr1: 94560938 ABCA4 a / G 0.38 8.42E-01
rs4147823 chr1: 94561272 ABCA4 A / c 0.22 6.95E-01
rs4147820 chr1: 94562084 ABCA4 t / C 0.21 6.05E-01
rs12088309 chr1: 94563916 ABCA4 T / c 0.30 1.14E-01
rs3789421 chr1: 94565577 ABCA4 a / G 0.19 8.61E-01
rs950283 chr1: 94567223 ABCA4 T / c 0.36 6.74E-03
rs4147819 chr1: 94569504 ABCA4 a / G 0.08 2.29E-01
rs481931 chr1: 94570016 ABCA4 t / G 0.39 1.25E-03
rs570926 chr1: 94570218 ABCA4 T / c 0.39 1.02E-03
rs570878 chr1: 94570234 ABCA4 t / G 0.48 1.93E-02
rs1211213 chr1: 94571420 ABCA4 A / g 0.36 1.90E-03
rs4147816 chr1: 94574780 ABCA4 t / C 0.40 2.56E-03
rs4147812 chr1: 94575043 ABCA4 A / c 0.40 2.50E-03
rs3827712 chr1: 94575171 ABCA4 T / c 0.40 3.37E-03
rs3789433 chr1: 94575440 ABCA4 a / G 0.26 2.87E-01
rs3789434 chr1: 94575978 ABCA4 T / c 0.08 2.52E-01
rs3789435 chr1: 94576360 ABCA4 A / g 0.48 2.09E-02
rs3827713 chr1: 94576524 ABCA4 c / G 0.48 2.09E-02
rs4147810 chr1: 94576664 ABCA4 A / g 0.08 2.52E-01
rs2297635 chr1: 94576893 ABCA4 a / G 0.08 3.08E-01
rs2297634 chr1: 94576968 ABCA4 T / c 0.48 2.09E-02
rs1889405 chr1: 94577410 ABCA4 t / C 0.24 3.26E-01
rs1889404 chr1: 94577423 ABCA4 t / C 0.24 2.67E-01
rs3789438 chr1: 94577462 ABCA4 t / G 0.08 2.98E-01
rs4147807 chr1: 94579053 ABCA4 A / g 0.22 4.89E-01
rs3789439 chr1: 94579426 ABCA4 T / c 0.22 2.56E-01
rs10782976 chr1: 94581125 ABCA4 a / G 0.31 3.96E-01
rs3789441 chr1: 94581384 ABCA4 t / C 0.27 2.56E-01
rs3789442 chr1: 94581456 ABCA4 c / G 0.22 2.14E-01
rs3789443 chr1: 94581529 ABCA4 A / g 0.23 2.06E-01
rs3789444 chr1: 94581540 ABCA4 t / C 0.23 1.84E-01
rs7535005 chr1: 94581905 ABCA4 t / C 0.25 3.63E-01
rs3789445 chr1: 94582249 ABCA4 T / g 0.25 1.08E-01
rs4147803 chr1: 94582293 ABCA4 g / C 0.43 9.24E-02
rs4147798 chr1: 94585009 ABCA4 t / C 0.23 1.60E-01
rs3761906 chr1: 94587362 t / G 0.05 7.37E-01
rs2151846 chr1: 94587687 T / g 0.43 1.06E-01
rs1931572 chr1: 94588992 T / c 0.40 5.48E-02
rs10874835 chr1: 94591481 a / G 0.23 2.55E-01
rs1105123 chr1: 94592290 T / c 0.28 1.40E-01
rs12071152 chr1: 94593399 a / G 0.05 9.04E-01
rs11802196 chr1: 94594043 A / c 0.41 8.50E-02
rs11165081 chr1: 94594080 A / c 0.41 8.00E-02
rs17111122 chr1: 94596464 A / g 0.25 1.59E-01
rs6686599 chr1: 94596831 a / G 0.39 2.89E-02
rs1931565 chr1: 94596867 a / G 0.33 2.00E-01
rs11581939 chr1: 94607234 T / c 0.35 7.70E-02
rs2022378 chr1: 94607607 a / C 0.24 9.07E-01
rs4847286 chr1: 94607848 T / g 0.41 8.22E-02
rs871664 chr1: 94609478 t / C 0.49 3.78E-02
rs2774920 chr1: 94611300 A / g 0.09 5.60E-01
rs12742802 chr1: 94629643 a / C 0.25 5.39E-01
rs1411701 chr1: 94635028 ARHGAP29 a / G 0.38 2.75E-02
rs12044374 chr1: 94635986 ARHGAP29 t / C 0.43 6.87E-02
rs10874840 chr1: 94636836 ARHGAP29 A / g 0.36 1.63E-02
rs12752790 chr1: 94637058 ARHGAP29 T / c 0.43 6.87E-02
rs1048866 chr1: 94638711 ARHGAP29 t / C 0.37 2.91E-02
rs1048854 chr1: 94643531 ARHGAP29 Synonymous_Q891Q T / c 0.27 5.92E-02
rs11577575 chr1: 94646514 ARHGAP29 a / G 0.23 7.50E-01
rs4847294 chr1: 94657769 ARHGAP29 A / g 0.43 7.99E-02
rs1541098 chr1: 94667970 ARHGAP29 T / c 0.27 9.07E-02
rs2274788 chr1: 94674726 ARHGAP29 T / c 0.24 2.36E-01
rs3789689 chr1: 94685585 ARHGAP29 T / g 0.07 2.75E-01
rs6541343 chr1: 94689027 ARHGAP29 a / G 0.08 1.16E-01
rs12724116 chr1: 94689734 ARHGAP29 A / g 0.15 7.11E-01
rs3789688 chr1: 94691240 ARHGAP29 t / C 0.47 8.34E-04
rs6673491 chr1: 94693145 ARHGAP29 t / C 0.08 1.18E-01
rs12750249 chr1: 94721660 T / c 0.29 3.39E-03
rs11165101 chr1: 94729088 a / C 0.40 1.71E-04
rs17396055 chr1: 94730954 a / G 0.32 3.47E-03
rs2391472 chr1: 94737583 T / c 0.08 9.28E-02
rs11165110 chr1: 94752469 a / G 0.40 2.19E-04
rs1330855 chr1: 94760885 A / g 0.20 3.21E-02
rs11580391 chr1: 94769368 t / C 0.29 6.70E-03
rs16928 chr1: 94786514 t / C 0.08 1.14E-01
rs17111408 chr1: 94788623 t / C 0.08 1.28E-01
rs12027548 chr1: 94788768 A / g 0.08 1.07E-01
rs11584317 chr1: 94792449 t / C 0.15 1.93E-01
rs2065971 chr1: 94807102 A / c 0.42 2.91E-04
rs2391467 chr1: 94850443 A / g 0.50 2.56E-06
rs1572575 chr1: 94852474 A / g 0.06 1.59E-01
rs12037634 chr1: 94867056 T / g 0.07 3.36E-01
rs11165135 chr1: 94870535 T / g 0.48 1.20E-02
rs6681849 chr1: 94874521 t / G 0.48 1.14E-02
rs10399785 chr1: 94877801 T / c 0.48 1.21E-02
rs4148060 chr1: 94881143 A / g 0.44 1.03E-01
rs10493872 chr1: 94890418 ABCD3 t / G 0.28 6.61E-02
rs12750904 chr1: 94893928 ABCD3 A / g 0.36 1.55E-02
Table 7.Association results for common nucleotide variants at the ARHGAP29 locusaa ARHGAP + / - 200kb.b NCBI build 37 / hg19.c Lowercase letter denotes the minor allele.d MAF, minor allele frequency based on the entire sample frequencies. e The ptrend-values below 2.54E-04 (0.05 / 197 SNVs) were interpreted statistically significant. Significant p-values are highlighted in bold font.

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