Functional characterization of ATF1, GREM2 AND WNT10B variants associated with tooth agenesis.

OBJECTIVE: To determine the functional effects of ATF1, WNT10B and GREM2 gene variants identified in individuals with tooth agenesis (TA). SETTINGS AND SAMPLE POPULATION: Stem cells from human exfoliated deciduous teeth (SHED) were used as an in vitro model system to test the effect of TA-associated variants. MATERIALS AND METHODS: Plasmid constructs containing reference and mutant alleles for ATF1 rs11169552, WNT10B rs833843 and GREM2 rs1414655 variants were transfected into SHED for functional characterization of variants. Allele-specific changes in gene transcription activity, protein expression, cell migration and proliferation, and expression of additional tooth development genes (MSX1, PAX9 and AXIN2) were evaluated. Data analyses were performed using Student's t-test. P-values ≤ .05 were considered statistically significant. RESULTS: Mutant variants resulted in significantly decreased transcriptional activity of respective genes (P < 0.05), although no changes in protein localization were noted. Expression of MSX1 was significantly decreased in ATF1- and GREM2-mutant cells, whereas PAX9 or AXIN2 mRNA expression was not significantly altered. Mutant WNT10B had no significant effect on the expression of additional TA genes. ATF1- and GREM2-mutant cells presented increased cell migration. Cell proliferation was also affected with all three mutant alleles. CONCLUSIONS: Our results demonstrate that ATF1, WNT10B and GREM2 mutant alleles have modulatory effects on gene/protein function that may contribute to TA.

Ethnic and language influence on parents' perception of paediatric behaviour management techniques.

BACKGROUND: Parental preference for various behaviour management techniques (BMTs) used in paediatric dentistry has been shown to be influenced by many factors, including ethnicity. AIM: To measure parental acceptability of BMTs used in paediatric dentistry and how it is influenced by ethnicity and language. DESIGN: Parents of patients presenting to a paediatric dentistry residency clinic in Houston, Texas, USA or Medellín, Colombia watched ten video BMT vignettes and rated their acceptance on a visual analog scale (VAS). Participants were categorized into six groups based on language, ethnicity, and country of residence. RESULTS: Parental acceptance of BMTs was affected by language, ethnicity, and country of residence (P = 2.2 × 10-16 ). Ethnic groups in the USA had a mean overall acceptance rate of all BMTs. Colombians rated all BMTs less acceptable than the US cohorts (P < 0.05), with the exception of voice control, which Colombians rate less acceptable than English-speaking Caucasians and Spanish-speaking Hispanics in the USA (P < 0.05). The Colombian population were not accepting of conscious sedation, nitrous oxide, general anaesthesia, and protective stabilization. CONCLUSIONS: Parents from different ethnic groups express different preferences in BMTs. Parents continue to prefer noninvasive techniques over pharmacologic and advanced techniques, with the exception of voice control.

Fluoride Intake of Infants from Formula.

OBJECTIVE: This study aimed to assess fluoride intake in infants from formula reconstituted with water, with fluorosis risk in mind. STUDY DESIGN: Data on water source, formula brand/type, volume of formula consumption and infant weight were collected for infants at two-, four-, six-, nine- and twelve-month pediatrician well child visits. Identified formula brands and water types were reconstituted and analyzed for fluoride concentration. Patient body mass and volume consumed/day were used to estimate fluoride intake from reconstituted formula. Descriptive statistics, one-way analysis of variance and chi-square tests were utilized. RESULTS: All infants consumed formula reconstituted with minimally fluoridated water (0.0- 0.3 ppm). 4.4% of infants exceeded the recommended upper limit (UL) of 0.1mg/kg/day. Although mean daily fluoride consumption significantly differed among all groups, the proportion of infants at each visit milestone that exceeded daily fluoride intake of 0.1mg/kg/day was not statistically significantly different (p>0.05) for any age group. Predicted values calculated with optimally fluoridated water (0.7ppm) resulted in 36.8% of infants exceeding the UL. CONCLUSIONS: Optimally fluoridated water may increase fluorosis risk for patients younger than six months. Future investigation should include multiple sites and multi-year follow-up to assess actual fluorosis incidence.

Craniofacial abnormalities result from knock down of nonsyndromic clefting gene, crispld2, in zebrafish.

Nonsyndromic cleft lip and palate (NSCLP), a common birth defect, affects 4,000 newborns in the US each year. Previously, we described an association between CRISPLD2 and NSCLP and showed Crispld2 expression in the murine palate. These results suggested that a perturbation in CRISPLD2 activity affects craniofacial development. Here, we describe crispld2 expression and the phenotypic consequence of its loss of function in zebrafish. crispld2 was expressed at all stages of zebrafish morphogenesis examined and localized to the rostral end by 1-day postfertilization. Morpholino knockdown of crispld2 resulted in significant jaw and palatal abnormalities in a dose-dependent manner. Loss of crispld2 caused aberrant patterning of neural crest cells (NCC) suggesting that crispld2 is necessary for normal NCC formation. Altogether, we show that crispld2 plays a significant role in the development of the zebrafish craniofacies and alteration of normal protein levels disturbs palate and jaw formation. These data provide support for a role of CRISPLD2 in NSCLP.

The Prenatal Diagnosis and Consultation for Cleft Lip and Palate Prior to Presurgical Infant Orthopedics and Early Dental Care.

Children born with cleft lip and/or cleft palate (CL/P) have unique treatment needs and benefit from a team of health care providers, including surgeons, dentists, and allied health professionals. Presurgical infant orthopedic appliances can reduce the severity of the birth defect and can be provided to patients by a dental health care professional starting at birth. The dental needs of patients with CL/P are multifaceted and having an established dental home to monitor growth and development and help with disease prevention is key to improve oral health.

Cytotoxicity Analysis of Human Dental Pulp Stem Cells After Silver Diamine Fluoride Application.

Purpose: The purpose of this study was to evaluate the cytotoxicity of silver diamine fluoride (SDF) to human dental pulp stem cells (hDPSC). Methods: hDPSC were exposed to dilutions of 38 percent SDF ( 10-3, 10-4, and 10-5) and incubated for 24 hours. Cell viability was assessed with colorimetric detection assay at 24 hours. Fresh media was used as a negative control, and 0.1% sodium dodecyl sulfate was used as a positive control. Three independent experiments were performed in triplicate. Cell viability data were analyzed using analysis of variance and Tukey's multiple comparison test. Results: Cells exposed to dilution of 38 percent SDF 10-3 had an average cell viability of 17.0±3.5 (standard deviation) percent. Cells exposed to SDF 10-4 and 10-5 had an average cell viability of 101±2.5 percent and 94±4.4 percent, respectively. Dilution of 10-3 had significantly lower cell viability than the negative control (P<0.001). Dilution of 10-4 and 10-5 SDF had significantly higher cell viability than the positive control (P<0.001) and cells treated with a dilution of 10-3 (P<0.001). Conclusions: Thirty-eight percent silver diamine fluoride was cytotoxic to human dental pulp stem cells at a dilution of 10-3, but not at 10-4 and 10-5. In light of the cytotoxicity of SDF to hDPSC, this in vitro study supports the concern that exposure of the full concentration of 38 percent SDF to the pulp should be avoided.

Factors Associated with Prolonged Exposure to General Anesthesia During Dental Rehabilitation Under General Anesthesia In Patients Under Age Three Years.

Purpose: The purpose of this study was to determine the risk of prolonged general anesthesia (GA) for pediatric dental patients and understand factors that contribute to prolonged GA in patients under age three years in an academic hospital. Methods: A retrospective chart review for pediatric dental patients treated using GA collected data for patient age, treatment provided, other services involved in patient management, and case GA length. Further chart analysis was completed by a multidisciplinary team for cases of prolonged general anesthesia. Results: A total of 114 cases were evaluated. The incidence of prolonged GA exposure was 21.9 percent (N equals 25). Cohort data of cases younger than three years show that cases of prolonged GA exposure were more likely to be closer to age three, require longer non-throat pack time, require more restorative procedures, require longer procedure times, and utilize additional surgical services more often (P<0.05). Four common themes for prolonged exposure were identified (significant restorative needs, provider-level training, anesthesia complications, and utilization of other services), with most cases (88 percent) experiencing multiple themes as contributing factors. Few adverse effects were noted, and none had long-lasting effects. Conclusions: Dental rehabilitation cases in very young patients are at risk for prolonged exposure to GA. Providers should be aware of total anesthesia time while completing dental rehabilitation using GA and proactively attempt to reduce the risk of prolonged exposure.

Nonsyndromic cleft lip and palate: CRISPLD genes and the folate gene pathway connection.

BACKGROUND: Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect that has a multifactorial etiology. Despite having substantial genetic liability, <15% of the genetic contribution to NSCLP has been delineated. In our efforts to dissect the genetics of NSCLP, we found that variation in the CRISPLD2 (cysteine-rich secretory protein LCCL domain containing 2) gene is associated with NSCLP and that the protein is expressed in the developing murine craniofacies. In addition, we found suggestive linkage of NSCLP (LOD > 1.0) to the chromosomal region on 8q13.2-21.13 that contains the CRISPLD1 gene. The protein products of both CRISPLD1 and CRISPLD2 contain more cysteine residues than comparably sized proteins. Interestingly, the folic acid pathway produces endogenous cysteines, and variation in genes in this pathway is associated with NSCLP. Based on these observations, we hypothesized that variation in CRISPLD1 contributes to NSCLP and that both CRISPLD genes interact with each other and genes in the folic acid pathway. METHODS: Single nucleotide polymorphisms (SNPs) in CRISPLD1 were genotyped in our non-Hispanic white and Hispanic multiplex and simplex NSCLP families. RESULTS: There was little evidence for a role of variation for CRISPLD1 alone in NSCLP. However, interactions were detected between CRISPLD1/CRISPLD2 SNPs and variation in folate pathway genes. Altered transmission of one CRISPLD1 SNP was detected in the NHW simplex families. Importantly, interactions were detected between SNPs in CRISPLD1 and CRISPLD2 (15 interactions, 0.0031 ≤p < 0.05). CONCLUSION: These novel findings suggest that CRISPLD1 plays a role in NSCLP through the interaction with CRISPLD2 and folate pathway genes.

CRISPLD2 variants including a C471T silent mutation may contribute to nonsyndromic cleft lip with or without cleft palate.

OBJECTIVE: To assess the association between nonsyndromic (NS) cleft lip with or without cleft palate (CL(P)) and single-nucleotide polymorphisms (SNPs) within the CRISPLD2 gene (cysteine-rich secretory protein LCCL domain containing 2). DESIGN: Four SNPs within the CRISPLD2 gene domain (rs1546124, rs8061351, rs2326398, rs4783099) were genotyped to test for association via family-based association methods. PARTICIPANTS: A total of 5826 individuals from 1331 families in which one or more family member is affected with CL(P). RESULTS: Evidence of association was seen for SNP rs1546124 in U.S. (p  =  .02) and Brazilian (p  =  .04) Caucasian cohorts. We also found association of SNP rs1546124 with cleft palate alone (CP) in South Americans (Guatemala and ECLAMC) and combined Hispanics (Guatemala, ECLAMC, and Texas Hispanics; p  =  .03 for both comparisons) and with both cleft lip with cleft palate (CLP; p  =  .04) and CL(P) (p  =  .02) in North Americans. Strong evidence of association was found for SNP rs2326398 with CP in Asian populations (p  =  .003) and with CL(P) in Hispanics (p  =  .03) and also with bilateral CL(P) in Brazilians (p  =  .004). In Brazilians, SNP rs8061351 showed association with cleft subgroups incomplete CL(P) (p  =  .004) and unilateral incomplete CL(P) (p  =  .003). Prediction of SNP functionality revealed that the C allele in the C471T silent mutation (overrepresented in cases with CL(P) presents two putative exonic splicing enhancer motifs and creates a binding site AP-2 alpha, a transcription factor involved in craniofacial development. CONCLUSIONS: Our results support the hypothesis that variants in the CRISPLD2 gene may be involved in the etiology of NS CL(P).

Variation in WNT genes is associated with non-syndromic cleft lip with or without cleft palate.

Non-syndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect. Genetic and environmental factors have been causally implicated and studies have begun to delineate genetic contributions. The Wnt genes are involved in regulating mid-face development and upper lip fusion and are therefore strong candidates for an etiological role in NSCLP. Furthermore, the clf1 region in A/WyN clefting susceptible mice contains the Wnt3 and Wnt9B genes. To assess the role of the Wnt family of genes in NSCLP, we interrogated seven Wnt genes (Wnt3, Wnt3A, Wnt5A, Wnt7A, Wnt8A, Wnt9B and Wnt11) in our well-defined NSCLP dataset. Thirty-eight single nucleotide polymorphisms were genotyped in 132 multiplex NSCLP families and 354 simplex parent-child trios. In the entire dataset, single-nucleotide polymorphisms (SNPs) in three genes, Wnt3A (P = 0.006), Wnt 5A (P = 0.002) and Wnt11 (P = 0.0001) were significantly associated with NSCLP after correction for multiple testing. When stratified by ethnicity, the strongest associations were found for SNPs in Wnt3A (P = 0.0007), Wnt11 (P = 0.0012) and Wnt8A (P = 0.0013). Multiple haplotypes in Wnt genes were associated with NSCLP, and gene-gene interactions were observed between Wnt3A and both Wnt3 and Wnt5A (P = 0.004 and P = 0.039, respectively). This data suggests that alteration in Wnt gene function may perturb formation and/or fusion of the facial processes and predispose to 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.

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.

Influence of Ethnicity on Parental Preference for Pediatric Dental Behavioral Management Techniques.

Purpose: The purpose of this study was to determine how ethnicity influences parental acceptability of behavior management techniques (BMTs) used during dental treatment of children. This is the first known study to compare ethnic differences in acceptance levels of the BMTs. Methods: Parental acceptance of 10 BMTs (tell-show-do, voice control, non-verbal communication, positive reinforcement, distraction, parental presence/absence, nitrous oxide, protective stabilization, sedation, and general anesthesia) was rated using a visual analogue scale (VAS) after watching vignettes of each technique. Parental preferences were stratified by ethnicity and analyzed. Results: Among the 104 parents (21 Caucasians, 29 Hispanics, 30 Asians, and 24 African Americans) who qualified and completed the study, we observed that, overall, non-invasive techniques (positive reinforcement and tell-show-do) were most accepted by parents, while invasive techniques (voice control and protective stabilization) were least accepted (P<0.001). Within each ethnicity, there were significant differences between the BMTs (P<0.001). Additionally, conscious sedation was the only BMT to show a significant difference between the ethnic groups (P=0.047), with Asian parents having a lower mean score than Caucasian and Hispanic parents. Conclusions: Our results suggest that considering the ethnic/cultural differences of patients and their parents is an instrumental component for pediatric dentists to provide quality care to children patients.

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.

Dental Caries Experience in Texan Children with Cleft Lip and Palate.

PURPOSE: The purpose of this study was to assess the caries experience in the primary dentition of children born with cleft lip and palate (CLP). METHODS: A retrospective chart review was conducted on subjects between two and six years old recruited from a university-based pediatric dentistry residency clinic. The number of dental visits and professional fluoride applications, the plaque index and treatment modality, and the presence/location of caries, white spot lesions, and enamel hypoplastic lesions were compared between CLP patients and healthy age- and gender-matched controls. Descriptive statistics, Student's t test, Mann-Whitney U test, and regression analysis were completed. RESULTS: A total of 183 charts were reviewed. Compared to healthy children, CLP children had increases in number of dental visits (P<0.001), decayed-missing-filled surfaces (dmfs; P<0.001), decayed-missing-filled teeth (dmft; P<0.001), enamel hypoplastic lesions (P=0.003), treatment completed under general anesthesia (P<0.001), plaque score (P<0.001), and caries increment between baseline and most recent oral examination (P=0.003). Regression analysis revealed a positive association between age and dmft scores within the CLP group (P=0.018). The caries experience of unilateral and bilateral CLP cases was the same (P>0.05). CONCLUSIONS: Children with cleft lip and palate are at a greater risk of enamel hypoplasia and dental caries. No significant caries experience difference was found between unilateral or bilateral CLP cases.

Genomic screening identifies novel linkages and provides further evidence for a role of MYH9 in nonsyndromic cleft lip and palate.

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth anomaly that requires prolonged multidisciplinary rehabilitation. Although variation in several genes has been identified as contributing to NSCLP, most of the genetic susceptibility loci have yet to be defined. To identify additional contributory genes, a high-throughput genomic scan was performed using the Illumina Linkage IVb Panel platform. We genotyped 6008 SNPs in nine non-Hispanic white NSCLP multiplex families and a single large African-American NSCLP multiplex family. Fourteen chromosomal regions were identified with LOD>1.5, including six regions not previously reported. Analysis of the data from the African-American and non-Hispanic white families revealed two likely chromosomal regions: 8q21.3-24.12 and 22q12.2-12.3 with LOD scores of 2.98 and 2.66, respectively. On the basis of biological function, syndecan 2 (SDC2) and growth differentiation factor 6 (GDF6) in 8q21.3-24.12 and myosin heavy-chain 9, non-muscle (MYH9) in 22q12.2-12.3 were selected as candidate genes. Association analyses from these genes yielded marginally significant P-values for SNPs in SDC2 and GDF6 (0.01

CRISPLD2: a novel NSCLP candidate gene.

Non-syndromic cleft lip with or without cleft palate (NSCLP) results from the complex interaction between genes and environmental factors. Candidate gene analysis and genome scans have been employed to identify the genes contributing to NSCLP. In this study, we evaluated the 16q24.1 chromosomal region, which has been identified by multiple genome scans as an NSCLP region of interest. Two candidate genes were found in the region: interferon regulatory factor 8 (IRF8) and cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2). Initially, Caucasian and Hispanic NSCLP multiplex families and simplex parent-child trios were genotyped for single nucleotide polymorphisms (SNPs) in both IRF8 and CRISPLD2. CRISPLD2 was subsequently genotyped in a data set comprised of NSCLP families from Colombia, South America. Linkage disequilibrium analysis identified a significant association between CRISPLD2 and NSCLP in both our Caucasian and Hispanic NSCLP cohorts. SNP rs1546124 and haplotypes between rs1546124 and either rs4783099 or rs16974880 were significant in the Caucasian multiplex population (P=0.01, P=0.002 and P=0.001, respectively). An altered transmission of CRISPLD2 SNPs rs8061351 (P=0.02) and rs2326398 (P=0.06) was detected in the Hispanic population. No association was found between CRISPLD2 and our Colombian population or IRF8 and NSCLP. In situ hybridization showed that CRISPLD2 is expressed in the mandible, palate and nasopharynx regions during craniofacial development at E13.5-E17.5, respectively. Altogether, these data suggest that genetic variation in CRISPLD2 has a role in the etiology of NSCLP.