Genetic Analyses of Enamel Hypoplasia in Multiethnic Cohorts.

Enamel hypoplasia causes reduction in the thickness of affected enamel and is one of the most common dental anomalies. This defect is caused by environmental and/or genetic factors that interfere with tooth formation, emphasizing the importance of investigating enamel hypoplasia on an epidemiological and genetic level. A genome-wide association of enamel hypoplasia was performed in multiple cohorts, overall comprising 7,159 individuals ranging in age from 7-82 years. Mixed-models were used to test for genetic association while simultaneously accounting for relatedness and genetic population structure. Meta-analysis was then performed. More than 5 million single-nucleotide polymorphisms were tested in individual cohorts. Analyses of the individual cohorts and meta-analysis identified association signals close to genome-wide significance (P < 510-8), and many suggestive association signals (510-8 < P < 510-6) near genes with plausible roles in tooth/enamel development. The strongest association signal (P = 1.5710-9) was observed near BMP2K in one of the individual cohorts. Additional suggestive signals were observed near genes with plausible roles in tooth development in the meta-analysis, such as SLC4A4 which can influence enamel hypoplasia. Additional human genetic studies are needed to replicate these results and functional studies in model systems are needed to validate our findings.

Genome-Wide Association Study (GWAS) of dental caries in diverse populations.

BACKGROUND: Dental caries is one of the most common chronic diseases and is influenced by a complex interplay of genetic and environmental factors. Most previous genetic studies of caries have focused on identifying genes that contribute to dental caries in specific ethnic groups, usually of European descent. METHODS: The aim of this study is to conduct a genome-wide association study (GWAS) to identify associations affecting susceptibility to caries in a large multiethnic population from Argentina, the Philippines, Guatemala, Hungary, and the USA, originally recruited for studies of orofacial clefts (POFC, N = 3686). Ages of the participants ranged from 2 to 12 years for analysis of the primary dentition, and 18-60 years for analysis of the permanent dentition. For each participant, dental caries was assessed by counts of decayed and filled teeth (dft/DFT) and genetic variants (single nucleotide polymorphisms, SNPs) were genotyped or imputed across the entire genome. Caries was analyzed separately for the primary and permanent dentitions, with age, gender, and presence/absence of any type of OFC treated as covariates. Efficient Mixed-Model Association eXpedited (EMMAX) was used to test genetic association, while simultaneously accounting for relatedness and stratification. RESULTS: We identified several suggestive loci (5 × 10-8 < P < 5 × 10-6) within or near genes with plausible biological roles for dental caries, including a cluster of taste receptor genes (TAS2R38, TAS2R3, TAS2R4, TASR25) on chromosome 7 for the permanent dentition analysis, and DLX3 and DLX4 on chromosome 17 for the primary dentition analysis. Genome-wide significant results were seen with SNPs in the primary dentition only; however, none of the identified genes near these variants have known roles in cariogenesis. CONCLUSION: The results of this study warrant further investigation and may lead to a better understanding of cariogenesis in diverse populations, and help to improve dental caries prediction, prevention, and/or treatment in future.

Association of low-frequency genetic variants in regulatory regions with nonsyndromic orofacial clefts.

Genome-wide scans have shown that common risk alleles for orofacial clefts (OFC) tend to be located in noncoding regulatory elements and cumulatively explain only part of the heritability of OFCs. Low-frequency variants may account for some of the "missing" heritability. Therefore, we scanned low-frequency variants located within putative craniofacial enhancers to identify novel OFC risk variants and implicate new regulatory elements in OFC pathogenesis. Analyses were performed in a multiethnic sample of 1,995 cases of cleft lip with or without cleft palate (CL/P), 221 cases with cleft palate (CP) only, and 1,576 unaffected controls. One hundred and nineteen putative craniofacial enhancers identified from ChIP-Seq studies in craniofacial tissues or cell lines contained multiple low-frequency (0.01-1%) variants, which we genotyped in participants using a custom Illumina panel. Two complementary statistical approaches, sequence kernel association test and combined multivariate and collapsing, were used to test association of the aggregated low-frequency variants across each enhancer region with CL/P and CP. We discovered a significant association between CP and a branchial arch enhancer near FOXP1 (mm60; p-value = .0002). Additionally, we observed a suggestive association between CL/P and a forebrain enhancer near FOXE1 (hs1717; p-value = .001). These findings suggest that low-frequency variants in craniofacial enhancer regions contribute to the complex etiology of nonsyndromic OFCs.

Identification of functional variants for cleft lip with or without cleft palate in or near PAX7, FGFR2, and NOG by targeted sequencing of GWAS loci.

Although genome-wide association studies (GWASs) for nonsyndromic orofacial clefts have identified multiple strongly associated regions, the causal variants are unknown. To address this, we selected 13 regions from GWASs and other studies, performed targeted sequencing in 1,409 Asian and European trios, and carried out a series of statistical and functional analyses. Within a cluster of strongly associated common variants near NOG, we found that one, rs227727, disrupts enhancer activity. We furthermore identified significant clusters of non-coding rare variants near NTN1 and NOG and found several rare coding variants likely to affect protein function, including four nonsense variants in ARHGAP29. We confirmed 48 de novo mutations and, based on best biological evidence available, chose two of these for functional assays. One mutation in PAX7 disrupted the DNA binding of the encoded transcription factor in an in vitro assay. The second, a non-coding mutation, disrupted the activity of a neural crest enhancer downstream of FGFR2 both in vitro and in vivo. This targeted sequencing study provides strong functional evidence implicating several specific variants as primary contributory risk alleles for nonsyndromic clefting in humans.

Soft tissue nasal asymmetry as an indicator of orofacial cleft predisposition.

The biological relatives of offspring with nonsyndromic orofacial clefts have been shown to exhibit distinctive facial features, including excess asymmetry, which are hypothesized to indicate the presence of genetic risk factors. The significance of excess soft tissue nasal asymmetry in at-risk relatives is unclear and was examined in the present study. Our sample included 164 unaffected parents from families with a history of orofacial clefting and 243 adult controls. Geometric morphometric methods were used to analyze the coordinates of 15 nasal landmarks collected from three-dimensional facial surface images. Following generalized Procrustes analysis, Procrustes ANOVA and MANOVA tests were applied to determine the type and magnitude of nasal asymmetry present in each group. Group differences in mean nasal asymmetry were also assessed via permutation testing. We found that nasal asymmetry in both parents and controls was directional in nature, although the magnitude of the asymmetry was greater in parents. This was confirmed with permutation testing, where the mean nasal asymmetry was significantly different (p < .0001) between parents and controls. The asymmetry was greatest for midline structures and the nostrils. When subsets of parents were subsequently analyzed and compared (parents with bilateral vs. unilateral offspring; parents with left vs. right unilateral offspring), each group showed a similar pattern of asymmetry and could not be distinguished statistically. Thus, the side of the unilateral cleft (right vs. left) in offspring was not associated with the direction of the nasal asymmetry in parents.

Genome-wide association studies in dogs and humans identify ADAMTS20 as a risk variant for cleft lip and palate.

Cleft lip with or without cleft palate (CL/P) is the most commonly occurring craniofacial birth defect. We provide insight into the genetic etiology of this birth defect by performing genome-wide association studies in two species: dogs and humans. In the dog, a genome-wide association study of 7 CL/P cases and 112 controls from the Nova Scotia Duck Tolling Retriever (NSDTR) breed identified a significantly associated region on canine chromosome 27 (unadjusted p=1.1 x 10(-13); adjusted p= 2.2 x 10(-3)). Further analysis in NSDTR families and additional full sibling cases identified a 1.44 Mb homozygous haplotype (chromosome 27: 9.29 - 10.73 Mb) segregating with a more complex phenotype of cleft lip, cleft palate, and syndactyly (CLPS) in 13 cases. Whole-genome sequencing of 3 CLPS cases and 4 controls at 15X coverage led to the discovery of a frameshift mutation within ADAMTS20 (c.1360_1361delAA (p.Lys453Ilefs*3)), which segregated concordant with the phenotype. In a parallel study in humans, a family-based association analysis (DFAM) of 125 CL/P cases, 420 unaffected relatives, and 392 controls from a Guatemalan cohort, identified a suggestive association (rs10785430; p =2.67 x 10-6) with the same gene, ADAMTS20. Sequencing of cases from the Guatemalan cohort was unable to identify a causative mutation within the coding region of ADAMTS20, but four coding variants were found in additional cases of CL/P. In summary, this study provides genetic evidence for a role of ADAMTS20 in CL/P development in dogs and as a candidate gene for CL/P development in humans.

Association studies of low-frequency coding variants in nonsyndromic cleft lip with or without cleft palate.

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a group of common human birth defects with complex etiology. Although genome-wide association studies have successfully identified a number of risk loci, these loci only account for about 20% of the heritability of orofacial clefts. The "missing" heritability may be found in rare variants, copy number variants, or interactions. In this study, we investigated the role of low-frequency variants genotyped in 1995 cases and 1626 controls on the Illumina HumanCore + Exome chip. We performed two statistical tests, Sequence Kernel Association Test (SKAT) and Combined Multivariate and Collapsing (CMC) method using two minor allele frequency cutoffs (1% and 5%). We found that a burden of low-frequency coding variants in N4BP2, CDSN, PRTG, and AHRR were associated with increased risk of NSCL/P. Low-frequency variants in other genes were associated with decreased risk of NSCL/P. These results demonstrate that low-frequency variants contribute to the genetic etiology of NSCL/P.

Hypertelorism and Orofacial Clefting Revisited: An Anthropometric Investigation.

OBJECTIVE: Since the 1960s, multiple studies have reported a tendency toward hypertelorism in individuals with nonsyndromic orofacial clefts (OFCs). However, the association between specific cleft types and increased interorbital distance has been inconsistent. Using three-dimensional (3D) surface imaging, we tested whether different forms of clefting showed evidence of increased interorbital distance. METHODS: Intercanthal and outercanthal distances and intercanthal indices were calculated from 3D facial surface images of 287 individuals with repaired OFCs. Raw measurements were converted to sex and age-normalized Z-scores. Mean Z-scores for individuals with cleft lip (CL), cleft lip and palate (CLP), and cleft palate (CP) were compared with reference normative values (controls) and one another directly using t tests and analysis of variance. RESULTS: The CLP group showed a significant increase in intercanthal width (P = .001) and intercanthal index (P < .001) compared with reference norms. The CP group showed a significant decrease (P < .001) in outercanthal width. The CL group showed no difference from reference norms. The proportion of clinically hyperteloric individuals was generally low but highest in the CLP group (7.4%). Cleft severity had little effect on interorbital spacing. CONCLUSIONS: Individuals with CLP exhibited on average a tendency toward mild hypertelorism, driven primarily by an increase in intercanthal distance. This tendency was not seen in CL or CP.

Ear Infection in Isolated Cleft Lip: Etiological Implications.

BACKGROUND AND HYPOTHESIS: Chronic ear infections are a common occurrence in children with orofacial clefts involving the secondary palate. Less is known about the middle ear status of individuals with isolated clefts of the lip, although several studies have reported elevated rates of ear infection in this group. The purpose of this retrospective study was to test the hypothesis that chronic ear infections occur more frequently in isolated cleft lip cases (n = 94) compared with controls (n = 183). METHODS: A questionnaire was used to obtain information on history of chronic ear infection. The association between ear infection status (present/absent) and cleft lip status (cleft lip case/control) was tested using both chi-square and logistic regression. RESULTS AND CONCLUSIONS: The reported occurrence of chronic ear infection was significantly greater in cleft lip cases (31%) compared with unaffected controls (11%). After adjusting for age and sex, having a cleft lip increased the odds of being positive for ear infection by a factor greater than 3 (odds ratio = 3.698; 95% confidence interval = 1.91 to 7.14). Within cleft lip cases, there was no difference in the occurrence of ear infection by defect laterality or by the type of clefting present in the family history. Although velopharyngeal insufficiency was present in 18.4% of our cleft lip sample, there was no statistical association between ear infection and abnormal speech patterns. These results may have potential implications both for the clinical management of isolated cleft lip cases and for understanding the etiology of orofacial clefting.

Evidence of olfactory deficits as part of the phenotypic spectrum of nonsyndromic orofacial clefting.

Improved understanding of the phenotypic spectrum associated with nonsyndromic orofacial clefting (OFC) has the potential to inform efforts to uncover the etiology of this complex trait. Prior studies report that individuals with OFC are characterized by impaired olfactory ability. In this study, we test whether olfactory dysfunction extends to the unaffected parents of children with OFC. The University of Pennsylvania Smell Identification Test was used to measure olfactory ability in a sample of 60 unaffected mothers and fathers with cleft-affected children. The proportion of deficit was compared with reference data obtained from published sex- and age-specific norms on more than 2700 individuals. The proportion of deficit was significantly higher in unaffected parents compared with baseline control subjects (41.7% vs 12.6%; P < 0.001). Of unaffected fathers, 41.7% displayed evidence of deficit compared with 15.1% of male control subjects (P = 0.001), whereas 41.7% of mothers exhibited deficits compared with 10.4% of female control subjects (P < 0.001). Olfactory deficits are present at a high proportion in the unaffected parents of individuals with OFC. This suggests that the deficits observed in affected cases may not simply be a secondary consequence of surgical repair and may instead be an informative phenotype reflecting underlying etiology.

The impact of congenital cardiovascular malformations on the assessment and surgical management of infants with cleft lip and/or palate.

OBJECTIVE: The purpose of this study was to assess the cardiac evaluation of cleft lip and/or palate patients, characterize their cardiovascular malformations, and determine the impact of cardiovascular malformations on surgical management. DESIGN: A single-institution retrospective study of 329 consecutive cleft patients was performed. Cardiovascular malformations were categorized according to involvement of cardiac septa, vasculature, and valves. Their impact on the need for cardiac surgery, timing of cleft repair, need for subacute bacterial endocarditis (SBE) prophylaxis, and the perioperative experience was evaluated. RESULTS: Ten percent (33/329) of cleft patients had a cardiovascular malformation, and 3% underwent cardiac surgery prior to cleft repair. Malformations of the septa, vasculature, and valves were present in 9%, 6%, and 2% of cleft infants, respectively. Murmur as a sign of structural cardiovascular disease was 79% sensitive and 97% specific. Cleft palate repair was delayed by 2 months in patients with a cardiovascular malformation (P = .001). Subacute bacterial endocarditis prophylaxis was recommended, not recommended, or not specified by cardiology in 18%, 33%, and 48% of cleft patients with a cardiovascular malformation, respectively. Postoperative stay and surgical complications were not associated with cardiovascular malformation. CONCLUSIONS: Even in the absence of a murmur, echocardiographic screening should be considered in infants with nonspecific signs of cardiovascular disease. Greater awareness of the guidelines for SBE prophylaxis is needed. Most cleft patients with a cardiovascular malformation do not require cardiac surgery and do not experience an increased rate of complications associated with cleft surgery.

Mutations in BMP4 are associated with subepithelial, microform, and overt cleft lip.

Cleft lip with or without cleft palate (CL/P) is a complex trait with evidence that the clinical spectrum includes both microform and subepithelial lip defects. We identified missense and nonsense mutations in the BMP4 gene in 1 of 30 cases of microform clefts, 2 of 87 cases with subepithelial defects in the orbicularis oris muscle (OOM), 5 of 968 cases of overt CL/P, and 0 of 529 controls. These results provide confirmation that microforms and subepithelial OOM defects are part of the spectrum of CL/P and should be considered during clinical evaluation of families with clefts. Furthermore, we suggest a role for BMP4 in wound healing.

Risk factors for death in elderly patients with facial fractures secondary to falls.

BACKGROUND: Craniomaxillofacial trauma is associated with severe injuries and disability. There are no recent reports analyzing the demographics of facial trauma in elderly patients. Our objective was to review the demographics, outcomes, and risk factors associated with death in elderly patients with facial fractures in a single-institution level I trauma center. METHODS: We performed a retrospective review of the University of Pittsburgh Medical Center Trauma Registry from January 1999 to December 2008 in an institutional review board-approved protocol. The search query included all types of craniofacial trauma using International Classification of Diseases, Ninth Revision coding. A χ test was used for analyzing categorical data. Continuous data were analyzed using unpaired t-test or analysis of variance. Logistic regressions were done to determine odds ratio (OR) for death. P = 0.05 was deemed significant. RESULTS: A total of 16,528 patients with facial trauma were identified. Of these, 4536 patients had falls. Among the falls, 1020 patients had facial fractures. Finally, 460 patients were younger than 65 years, and 560 were older than 65 years. The variable with the highest odds for mortality in patients with facial fractures was intensive care unit admission (OR, 13.1), followed by acute renal failure (OR, 10.3), medical history of myocardial infarction (OR, 5.1), coagulopathy (OR, 4.78), age older than 65 years (OR, 4.13), acute respiratory failure (OR, 3.55), Abbreviated Injury Scale score greater than 3 at the head (OR, 3.5), congestive heart failure (OR, 2.64), history of cardiac surgery (OR, 2.4), and vault skull fracture (OR, 2.35). CONCLUSIONS: Craniofacial trauma is a serious condition in the elderly. Associated injuries are demonstrated to have a deleterious effect on survival. Recognition of these risk factors is likely to promote the development of preventive measurements to decrease fatal outcomes.

Multivariate GWAS of Structural Dental Anomalies and Dental Caries in a Multi-Ethnic Cohort.

Odontogenesis is a complex process, where disruption can result in dental anomalies and/or increase the risk of developing dental caries. Based on previous studies, certain dental anomalies tend to co-occur in patients, suggesting that these traits may share common genetic and etiological components. The main goal of this study was to implement a multivariate genome-wide association study approach to identify genetic variants shared between correlated structural dental anomalies and dental caries. Our cohort (N = 3,579) was derived from the Pittsburgh Orofacial Clefts Study, where multiple dental traits were assessed in both the unaffected relatives of orofacial cleft (OFC) cases (n = 2,187) and unaffected controls (n = 1,392). We identified four multivariate patterns of correlated traits in this data: tooth agenesis, impaction, and rotation (AIR); enamel hypoplasia, displacement, and rotation (HDR); displacement, rotation, and mamelon (DRM); and dental caries, tooth agenesis and enamel hypoplasia (CAH). We analyzed each of these four models using genome-wide multivariate tests of association. No genome-wide statistically significant results were found, but we identified multiple suggestive association signals (P < 10-5) near genes with known biological roles during tooth development, including ADAMTS9 and PRICKLE2 associated with AIR; GLIS3, WDR72, and ROR2 associated with HDR and DRM; ROBO2 associated with DRM; BMP7 associated with HDR; and ROBO1, SMAD2, and MSX2 associated with CAH. This is the first study to investigate genetic associations for multivariate patterns of correlated dental anomalies and dental caries. Further studies are needed to replicate these results in independent cohorts.

Comparative microanatomy of the orbicularis oris muscle between chimpanzees and humans: evolutionary divergence of lip function.

The orbicularis oris muscle plays a role in the production of primate facial expressions and vocalizations, nutrient intake, and in some non-human primates it is used as a prehensile, manipulative tool. As the chimpanzee (Pan troglodytes) is the closest living relative of humans, a comparison of the orbicularis oris muscle between these species may increase our understanding of the morphological specializations related to the differing functional demands of their lips and the factors responsible for their divergent evolution. To this end, this study compares the microanatomy of the mid-line upper fibers of the orbicularis oris muscle between chimpanzees and humans. A mid-line portion of the orbicularis oris muscle was harvested from the upper lips of three chimpanzee and five human cadavers. The sampled blocks included the area between the lateral borders of the nasal alar cartilages in both species. Each sample was processed for paraffin histology, sectioned and stained with a variety of protocols. Sections were examined for fiber direction and relative thickness of muscle layers. Ratios of cross-sectional connective tissue area vs. cross-sectional muscle tissue area, muscle fiber diameter and relative dermal thickness were calculated for each species. In both species, a clear pars marginalis layer was recognized, contrary to previous reports that only humans possess this layer. In chimpanzees, the relative fiber diameter and relative amount of muscle tissue (i.e. based on ratio of connective tissue area : muscle tissue area) were significantly (P < 0.05) greater than in humans. In contrast, measurements of relative dermal thickness showed that humans have a greater average dermal thickness of the upper lip than chimpanzees. Taken together, these results suggest that both human and chimpanzee orbicularis oris muscle upper fibers meet the specific functional demands associated with their divergent vocal and facial display repertoires, the development of human speech, and the use of the upper lip as a prehensile tool in chimpanzees.

Ocular outcomes after gold weight placement and facial nerve resection.

OBJECTIVE: To assess the effectiveness of acute gold weight placement after facial nerve resection and to determine the role of concomitant lower eyelid procedures. STUDY DESIGN: Retrospective review. SUBJECTS AND METHODS: Twenty-two patients who received an upper eyelid gold weight at the time of parotidectomy and facial nerve resection were reviewed to assess ocular outcomes. RESULTS: After gold weight placement, twelve patients (12 of 22, 54.5%) subsequently presented with symptomatic ectropion (n = 9) and/or lagophthalmos (n = 5). Nine patients received a lower eyelid procedure (7 tarsal strips only, 1 tarsal strip combined with a lateral tarsorrhaphy, and 1 lateral tarsorrhaphy only). Six patients, in addition to a gold weight, also underwent a static sling to the midface at the time of facial nerve resection. None of these 6 received a subsequent lower eyelid procedure. Two patients required gold weight upsizing. Two patients required weight removal. CONCLUSIONS: Insertion of 1.2 gm upper eyelid weight with placement of midface sling is recommended at the time of facial nerve resection. Due to the need to tighten the lower eyelid in many of these patients, we now also consider performing a tarsal strip procedure at the time of facial nerve resection in any patient with pre-existing lower lid laxity.

Nasal tip complications of primary cleft lip nasoplasty.

BACKGROUND AND PURPOSE: Complications of primary nasoplasty, at the time of definitive primary cheiloplasty, are underreported in the literature. This study endeavors to examine the occurrences of these complications at our cleft-craniofacial center, in an effort to identify causative factors and management strategies. A case series of patients with postoperative nasal complications after primary cleft lip nasal surgery is presented. METHODS: A retrospective chart review of primary cleft lip nasal repairs was conducted at our cleft-craniofacial center between January 2003 and December 2007. Consecutive cases of 3 staff surgeons were evaluated. Specific data points included number and type of complications, subsequent required interventions, and relevant history, with particular attention paid to the details of the primary nasoplasty. RESULTS: Eighty-six primary cleft lip nasoplasties were completed between the years 2003 and 2007. Six complications (6.9%) related to the primary cleft lip nasoplasty were identified. Four patients (4.6%) experienced nasal tip infections; all 4 required surgical drainage. Twenty-four patients (27.9%) undergoing primary cleft lip and nose repair had postoperative nostril conformers placed, and 2 (8.3%) of them experienced complications deemed conformer related. CONCLUSIONS: Postoperative nasal complications of primary cheiloplasty occur and are likely underreported. In this series, complications resulted from infection, often occurring late, and secondary to the use of nostril conformers. Surgeon awareness and caregiver education, to identify the early signs of postoperative nasal complications, are critical to the successful treatment of these occurrences. Although this study did not intend on examining antibiotic use, the significance of nasal tip infections might support the regular use of antibiotics in this population, and the use of postoperative nostril conformers must be followed closely.

Rethinking isolated cleft palate: evidence of occult lip defects in a subset of cases.

Emerging research suggests that subepithelial defects of the upper lip musculature are part of the phenotypic spectrum of cleft lip and/or palate (CL/P) and may represent an occult, subclinical manifestation of the anomaly. The present study investigates whether similar occult lip defects are present in individuals affected with isolated cleft palate (CP). To this end, upper lip ultrasounds of 33 CP cases (12 males, 21 females) were evaluated retrospectively for the presence of discontinuities (i.e., breaks) within the orbicularis oris muscle (OOM). In four CP cases (2 males, 2 females), distinct discontinuities of the OOM were identified. Of the remaining CP individuals, 23 demonstrated normal lip morphology on ultrasound (7 males, 16 females), while, in 6 cases (3 males, 3 females), a definitive evaluation was not possible. As CP and CL/P are traditionally thought to be etiologically distinct, these findings raise the possibility that some CP cases may be misclassified. Such diagnostic errors could have important implications for recurrence risk estimation and studies aimed at discovering etiology. (c) 2008 Wiley-Liss, Inc.