Evidence for craniofacial enhancer variation underlying nonsyndromic cleft lip and palate.

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect for which only ~ 20% of the underlying genetic variation has been identified. Variants in noncoding regions have been increasingly suggested to contribute to the missing heritability. In this study, we investigated whether variation in craniofacial enhancers contributes to NSCLP. Candidate enhancers were identified using VISTA Enhancer Browser and previous publications. Prioritization was based on patterning defects in knockout mice, deletion/duplication of craniofacial genes in animal models and results of whole exome/whole genome sequencing studies. This resulted in 20 craniofacial enhancers to be investigated. Custom amplicon-based sequencing probes were designed and used for sequencing 380 NSCLP probands (from multiplex and simplex families of non-Hispanic white (NHW) and Hispanic ethnicities) using Illumina MiSeq. The frequencies of identified variants were compared to ethnically matched European (CEU) and Los Angeles Mexican (MXL) control genomes and used for association analyses. Variants in mm427/MSX1 and hs1582/SPRY1 showed genome-wide significant association with NSCLP (p ≤ 6.4 × 10-11). In silico analysis showed that these enhancer variants may disrupt important transcription factor binding sites. Haplotypes involving these enhancers and also mm435/ABCA4 were significantly associated with NSCLP, especially in NHW (p ≤ 6.3 × 10-7). Importantly, groupwise burden analysis showed several enhancer combinations significantly over-represented in NSCLP individuals, revealing novel NSCLP pathways and supporting a polygenic inheritance model. Our findings support the role of craniofacial enhancer sequence variation in the etiology of NSCLP.

Crispld2 is required for neural crest cell migration and cell viability during zebrafish craniofacial development.

The CAP superfamily member, CRISPLD2, has previously been shown to be associated with nonsyndromic cleft lip and palate (NSCLP) in human populations and to be essential for normal craniofacial development in the zebrafish. Additionally, in rodent models, CRISPLD2 has been shown to play a role in normal lung and kidney development. However, the specific role of CRISPLD2 during these developmental processes has yet to be determined. In this study, it was demonstrated that Crispld2 protein localizes to the orofacial region of the zebrafish embryo and knockdown of crispld2 resulted in abnormal migration of neural crest cells (NCCs) during both early and late time points. An increase in cell death after crispld2 knockdown as well as an increase in apoptotic marker genes was also shown. This data suggests that Crispld2 modulates the migration, differentiation, and/or survival of NCCs during early craniofacial development. These results indicate an important role for Crispld2 in NCC migration during craniofacial development and suggests involvement of Crispld2 in cell viability during formation of the orofacies.

Further evidence suggesting a role for variation in ARHGAP29 variants in nonsyndromic cleft lip/palate.

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect of complex etiology. Several genes have been implicated in the etiology of NSCL/P, although only a few have been replicated across datasets. METHODS: ARHGAP29 was suggested as a candidate gene for NSCL/P as it is located in close proximity to ABCA4 (1p22), a gene previously identified in a genome-wide association study of NSCL/P. RESULTS: Rare, potentially damaging, coding variants in ARHGAP29 were found in NSCL/P cases, and its expression was detected during murine craniofacial development. In this study, we investigated whether variations in ARHGAP29 were associated with NSCL/P in our family based dataset. Five single-nucleotide polymorphisms (SNPs) flanking and within ARHGAP29 were genotyped in our NSCL/P datasets consisting of simplex and multiplex families of non-Hispanic white (NHW, primarily European) and Hispanic ethnicities. Results showed strong association of three ARHGAP29 SNPs with NSCL/P in the NHW families. Two intronic SNPs (rs1541098 and rs3789688) showed strong association with NSCL/P in all NHW families (p = 0.0005 and p = 0.0002, respectively), and simplex NHW families (p = 0.003 for both SNPs). A SNP in the 3' untranslated region (rs1576593) also showed strong association with NSCL/P in all NHW families (p = 0.002), and the multiplex subset (p = 0.002). ARHGAP29 SNP haplotypes were also associated with NSCL/P. Evidence of gene-gene interaction was found between ARHGAP29 and additional cleft susceptibility genes. CONCLUSION: This study further supports ARHGAP29 as a candidate gene for human NSCL/P in families of Caucasian descent.

Facial analytics based on a coordinate extrapolation system (zFACE) for morphometric phenotyping of developing zebrafish.

Facial development requires a complex and coordinated series of cellular events that, when perturbed, can lead to structural birth defects. A quantitative approach to quickly assess morphological changes could address how genetic or environmental inputs lead to differences in facial shape and promote malformations. Here, we report on a method to rapidly analyze craniofacial development in zebrafish embryos using facial analytics based on a coordinate extrapolation system, termed zFACE. Confocal images capture facial structures and morphometric data are quantified based on anatomical landmarks present during development. The quantitative morphometric data can detect phenotypic variation and inform on changes in facial morphology. We applied this approach to show that loss of smarca4a in developing zebrafish leads to craniofacial anomalies, microcephaly and alterations in brain morphology. These changes are characteristic of Coffin-Siris syndrome, a rare human genetic disorder associated with mutations in SMARCA4. Multivariate analysis of zFACE data facilitated the classification of smarca4a mutants based on changes in specific phenotypic characteristics. Together, zFACE provides a way to rapidly and quantitatively assess the impact of genetic alterations on craniofacial development in zebrafish.

Disruption of fos causes craniofacial anomalies in developing zebrafish.

Craniofacial development is a complex and tightly regulated process and disruptions can lead to structural birth defects, the most common being nonsyndromic cleft lip and palate (NSCLP). Previously, we identified FOS as a candidate regulator of NSCLP through family-based association studies, yet its specific contributions to oral and palatal formation are poorly understood. This study investigated the role of fos during zebrafish craniofacial development through genetic disruption and knockdown approaches. Fos was expressed in the periderm, olfactory epithelium and other cell populations in the head. Genetic perturbation of fos produced an abnormal craniofacial phenotype with a hypoplastic oral cavity that showed significant changes in midface dimensions by quantitative facial morphometric analysis. Loss and knockdown of fos caused increased cell apoptosis in the head, followed by a significant reduction in cranial neural crest cells (CNCCs) populating the upper and lower jaws. These changes resulted in abnormalities of cartilage, bone and pharyngeal teeth formation. Periderm cells surrounding the oral cavity showed altered morphology and a subset of cells in the upper and lower lip showed disrupted Wnt/ß-catenin activation, consistent with modified inductive interactions between mesenchymal and epithelial cells. Taken together, these findings demonstrate that perturbation of fos has detrimental effects on oral epithelial and CNCC-derived tissues suggesting that it plays a critical role in zebrafish craniofacial development and a potential role in NSCLP.

PBX-WNT-P63-IRF6 pathway in nonsyndromic cleft lip and palate.

Nonsyndromic cleft lip and palate (NSCLP) is one of the most common craniofacial anomalies in humans, affecting more than 135,000 newborns worldwide. NSCLP has a multifactorial etiology with more than 50 genes postulated to play an etiologic role. The genetic pathway comprised of Pbx-Wnt-p63-Irf6 genes was shown to control facial morphogenesis in mice and proposed as a regulatory pathway for NSCLP. Based on these findings, we investigated whether variation in PBX1, PBX2, and TP63, and their proposed interactions were associated with NSCLP. Fourteen single nucleotide variants (SNVs) in/nearby PBX1, PBX2, and TP63 were genotyped in 780 NSCLP families of nonHispanic white (NHW) and Hispanic ethnicities. Family-based association tests were performed for individual SNVs stratified by ethnicity and family history of NSCLP. Gene-gene interactions were also tested. A significant association was found for PBX2 rs3131300 and NSCLP in combined Hispanic families (p = .003) while nominal association was found for TP63 rs9332461 in multiplex Hispanic families (p = .005). Significant haplotype associations were observed for PBX2 in NHW (p = .0002) and Hispanic families (p = .003), and for TP63 in multiplex Hispanic families (.003). An independent case-control group was used to validate findings, and significant associations were found with PBX1 rs6426870 (p = .007) and TP63 rs9332461 (p = .03). Gene-gene interactions were detected between PBX1/PBX2/TP63 with IRF6 in NHW families, and between PBX1 with WNT9B in both NHW and Hispanic families (p < .0018). This study provides the first evidence for a role of PBX1 and PBX2, additional evidence for the role of TP63, and support for the proposed PBX-WNT-TP63-IRF6 regulatory pathway in the etiology of NSCLP.

WNT gene polymorphisms and predisposition to apical periodontitis.

Single nucleotide polymorphisms (SNPs) in WNT genes may impact gene/protein function and contribute to individual predisposition to apical periodontitis (AP). Here, we investigated the association of SNPs in/nearby WNT3, WNT3A, WNT5A, WNT8A, WNT9B and WNT11 genes with AP using a case-control dataset. Cases were defined as individuals with deep caries and AP (n = 188); controls had deep caries and no AP (n = 230). Genotyping was performed using Taqman chemistry in real time PCR. Data analyses was performed using Fisher Exact tests assuming a Bonferroni correction threshold value of 0.005. Single-SNP association analysis revealed a trend for association with WNT3 rs9890413 genotypes (P = 0.009) under a dominant model and allelic association for WNT3A rs1745420 (P = 0.009). Haplotypes involving WNT3-WNT9B-WNT3A alleles were also significantly associated with AP (P ≤ 0.003). Luciferase reporter assays showed higher transcriptional activity (1.4-fold) with the alternate G allele in rs1745420. Expression of WNT3, WNT3A and WNT5A in AP tissues was significantly higher than in control tissues, and inversely correlated with the expression of SERPINB1, COL1A1 and TIMP1 (P < 0.05). Our results suggest that WNT genes have a role in modulating AP and polymorphisms in these genes may increase susceptibility to AP.

Association of IFT88 gene variants with nonsyndromic cleft lip with or without cleft palate.

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect with multifactorial etiology. Genetic studies have identified numerous gene variants in association with NSCLP. IFT88 (intraflagellar transport 88) has been suggested to play a major role in craniofacial development, as Ift88 mutant mice exhibit cleft palate and mutations in IFT88 were identified in individuals with NSCLP. OBJECTIVE: To investigate the association of IFT88 single nucleotide gene variants (SNVs) with NSCLP in a large family data set consisting of non-Hispanic white (NHW) and Hispanic families. METHODS: Nine SNVs in/nearby IFT88 were genotyped in 482 NHW families and 301 Hispanic NSCLP families. Genotyping was performed using TaqMan® chemistry. Single- and pairwise-SNV association analyses were performed for all families stratified by ethnicity and family history of NSCLP using the family-based association test (FBAT), and association in the presence of linkage (APL). Bonferroni correction was used to adjust for multiple testing and p values ≤.0055 were considered statistically significant. RESULTS: Significant association was found between IFT88 rs9509311 and rs2497490 and NSCLP in NHW all families (p = .004 and .005, respectively), while nominal associations were found for rs7998361 and rs9509307 (p < .05). Pairwise association analyses also showed nominal associations between NSCLP in both NHW and Hispanic data sets (p < .05). No association was found between individual variants in IFT88 and NSCLP in Hispanics. CONCLUSIONS: Our results suggest that variation in IFT88 may contribute to NSCLP risk, particularly in multiplex families from a non-Hispanic white population.

BRCA1 and BRCA2 gene variants and nonsyndromic cleft lip/palate.

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a debilitating condition that not only affects the individual, but the entire family. The purpose of this study was to investigate the association of BRCA1 and BRCA2 genes with NSCL/P. METHODS: Twelve polymorphisms in/nearby BRCA1 and BRCA2 were genotyped using Taqman chemistry. Our data set consisted of 3,473 individuals including 2,191 nonHispanic white (NHW) individuals (from 151 multiplex and 348 simplex families) and 1,282 Hispanic individuals (from 92 multiplex and 216 simplex families). Data analysis was performed using Family-Based Association Test (FBAT), stratified by ethnicity and family history of NSCL/P. RESULTS: Nominal associations were found between NSCL/P and BRCA1 in Hispanics and BRCA2 in NHW and Hispanics (p < .05). Significant haplotype associations were found between NSCL/P and both BRCA1 and BRCA2 (p ≤ .004). CONCLUSIONS: Our results suggest a modest association between BRCA1 and BRCA2 and NSCL/P. Further studies in additional populations and functional studies are needed to elucidate the role of these genes in developmental processes and signaling pathways contributing to NSCL/P.

Knockdown of Crispld2 in zebrafish identifies a novel network for nonsyndromic cleft lip with or without cleft palate candidate genes.

Orofacial development is a multifaceted process involving tightly regulated genetic signaling networks, that when perturbed, lead to orofacial abnormalities including cleft lip and/or cleft palate. We and others have shown an association between the cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2) gene and nonsyndromic cleft lip with or without cleft palate (NSCLP). Further, we demonstrated that knockdown of Crispld2 in zebrafish alters neural crest cell migration patterns resulting in abnormal jaw and palate development. In this study, we performed RNA profiling in zebrafish embryos and identified 249 differentially expressed genes following knockdown of Crispld2. In silico pathway analysis identified a network of seven genes previously implicated in orofacial development for which differential expression was validated in three of the seven genes (CASP8, FOS, and MMP2). Single nucleotide variant (SNV) genotyping of these three genes revealed significant associations between NSCLP and FOS/rs1046117 (GRCh38 chr14:g.75746690 T > C, p = 0.0005) in our nonHispanic white (NHW) families and MMP2/rs243836 (GRCh38 chr16:g.55534236 G > A; p = 0.002) in our Hispanic families. Nominal association was found between NSCLP and CASP8/rs3769825 (GRCh38 chr2:g.202111380 C > A; p < 0.007). Overtransmission of MMP2 haplotypes were identified in the Hispanic families (p < 0.002). Significant gene-gene interactions were identified for FOS-MMP2 in the NHW families and for CASP8-FOS in the NHW simplex family subgroup (p < 0.004). Additional in silico analysis revealed a novel gene regulatory network including five of these newly identified and 23 previously reported NSCLP genes. Our results demonstrate that animal models of orofacial clefting can be powerful tools to identify novel candidate genes and gene regulatory networks underlying NSCLP.

Role of WNT10A in failure of tooth development in humans and zebrafish.

BACKGROUND: Oligodontia is a severe form of tooth agenesis characterized by the absence of six or more permanent teeth. Oligodontia has complex etiology and variations in numerous genes have been suggested as causal for the condition. METHODS: We applied whole-exome sequencing (WES) to identify the cause of oligodontia in a 9-year-old girl missing 11 permanent teeth. Protein modeling and functional analysis in zebrafish were also performed to understand the impact of identified variants on the phenotype. RESULTS: We identified a novel compound heterozygous missense mutation in WNT10A (c.637G>A:p.Gly213Ser and c.1070C>T:p.Thr357Ile) as the likely cause of autosomal recessive oligodontia in the child. Affected residues are located in conserved regions and variants are predicted to be highly deleterious for potentially destabilizing the protein fold and inhibiting normal protein function. Functional studies in zebrafish embryos showed that wnt10a is expressed in the craniofacies at critical time points for tooth development, and that perturbations of wnt10a expression impaired normal tooth development and arrested tooth development at 5 days postfertilization (dpf). Furthermore, mRNA expression levels of additional tooth development genes were directly correlated with wnt10a expression; expression of msx1, dlx2b, eda, and axin2 was decreased upon wnt10a knockdown, and increased upon wnt10a overexpression. CONCLUSIONS: Our results reveal a novel compound heterozygous variant in WNT10A as pathogenic for oligodontia, and demonstrate that perturbations of wnt10a expression in zebrafish may directly and/or indirectly affect tooth development recapitulating the agenesis phenotype observed in humans.

Regulatory variant in FZD6 gene contributes to nonsyndromic cleft lip and palate in an African-American family.

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect affecting 135,000 newborns worldwide each year. While a multifactorial etiology has been suggested as the cause, despite decades of research, the genetic underpinnings of NSCLP remain largely unexplained. In our previous genome-wide linkage study of a large NSCLP African-American family, we identified a candidate locus at 8q21.3-24.12 (LOD = 2.98). This region contained four genes, Frizzled-6 (FZD6), Matrilin-2 (MATN2), Odd-skipped related 2 (OSR2) and Solute Carrier Family 25, Member 32 (SLC25A32). FZD6 was located under the maximum linkage peak. In this study, we sequenced the coding and noncoding regions of these genes in two affected family members, and identified a rare variant in intron 1 of FZD6 (rs138557689; c.-153 + 432A>C). The variant C allele segregated with NSCLP in this family, through affected and unaffected individuals, and was found in one other NSCLP African-American family. Functional assays showed that this allele creates an allele-specific protein-binding site and decreases promoter activity. We also observed that loss and gain of fzd6 in zebrafish contributes to craniofacial anomalies. FZD6 regulates the WNT signaling pathway, which is involved in craniofacial development, including midfacial formation and upper labial fusion. We hypothesize, therefore, that alteration in FZD6 expression contributes to NSCLP in this family by perturbing the WNT signaling pathway.

Forced Abstinence from Cocaine Self-Administration is Associated with DNA Methylation Changes in Myelin Genes in the Corpus Callosum: a Preliminary Study.

BACKGROUND: Human cocaine abuse is associated with alterations in white matter integrity revealed upon brain imaging, an observation that is recapitulated in an animal model of continuous cocaine exposure. The mechanism through which cocaine may affect white matter is unknown and the present study tested the hypothesis that cocaine self-administration results in changes in DNA methylation that could result in altered expression of several myelin genes that could contribute to the effects of cocaine on white matter integrity. METHODS: In the present study, we examined the impact of forced abstinence from cocaine self-administration on chromatin associated changes in white matter. To this end, rats were trained to self-administer cocaine (0.75 mg/kg/0.1 mL infusion) for 14 days followed by forced abstinence for 1 day (n = 6) or 30 days (n = 6) before sacrifice. Drug-free, sham surgery controls (n = 7) were paired with the experimental groups. Global DNA methylation and DNA methylation at specific CpG sites in the promoter regions ofmyelin basic protein (Mbp), proteolipid protein-1 (Plp1), and SRY-related HMG-box-10 (Sox10) genes were analyzed in DNA extracted from corpus callosum. RESULTS: Significant differences in the overall methylation patterns of the Sox10 promoter region were observed in the corpus callosum of rats at 30 days of forced abstinence from cocaine self-administration relative to sham controls; the -189, -142, -93, and -62 CpG sites were significantly hypomethylated point-wise at this time point. After correction for multiple comparisons, no differences in global methylation or the methylation patterns of Mbp or Plp1 were found. CONCLUSION: Forced abstinence from cocaine self-administration was associated with differences in DNA methylation at specific CpG sites in the promoter region of the Sox10 gene in corpus callosum. These changes may be related to reductions in normal age related changes in DNA methylation and could be a factor in white matter alterations seen after withdrawal from repeated cocaine self-administration. Further research is warranted examining the effects of cocaine on DNA methylation in white matter.