Exploring Subclinical Phenotypic Features in Twin Pairs Discordant for Cleft Lip and Palate.

OBJECTIVE: Monozygotic twins of an individual with an orofacial cleft have a significantly elevated risk for orofacial cleft compared with the general population, but still the concordance rate for orofacial cleft in monozygotic twins is about 40% to 50%. The goal of this study was to determine whether unaffected cotwins have an increased frequency of orbicularis oris muscle defects, a subclinical form of orofacial cleft. The presence of such defects may reduce the overall rate of discordance. METHOD: A total of 63 discordant monozygotic and dizygotic twin pairs, 262 unaffected nontwin siblings, and 543 controls with no history of orofacial clefts were assessed for orbicularis oris defects by high-resolution ultrasound. Frequencies were compared by the Fisher exact test. RESULTS: Unaffected cotwins from discordant monozygotic pairs had a higher frequency of defects (12.5%) than the other test groups (6.38% to 6.99%), but the difference was not statistically significant (P = .74). CONCLUSIONS: In this study, orbicularis oris defects were not statistically significantly more common among the unaffected twins from orofacial cleft discordant twin pairs. The trends in the results warrant future studies with larger sample sizes and additional subclinical phenotypes.

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.

Genetic Modifiers of Patent Ductus Arteriosus in Term Infants.

OBJECTIVE: To identify single-nucleotide polymorphisms (SNPs) in specific candidate genes associated with patent ductus arteriosus in term infants. STUDY DESIGN: We conducted an initial family-based, candidate gene study to analyze genotype data from DNA samples obtained from 171 term infants and their parents enrolled in the National Birth Defects Prevention Study (NBDPS). We performed transmission disequilibrium testing (TDT) using a panel of 55 SNPs in 17 genes. Replication of SNPs with P < .1 in the NBDPS trios was performed with a case-control strategy in an independent population. RESULTS: TDT analysis of the NBDPS trios resulted in 6 SNPs reaching the predetermined cutoff (P < .1) to be included in the replication study. These 6 SNPs were genotyped in the independent case-control population. A SNP in TGFBR2 was found to be associated with term patent ductus arteriosus in both populations after we corrected for multiple comparisons. (rs934328, TDT P = 2 × 10(-4), case-control P = 6.6 × 10(-5)). CONCLUSIONS: These findings confirm the importance of the transforming growth factor-beta pathway in the closure of the term ductus arteriosus and may suggest new therapeutic targets.

Aquaporin 5 Interacts with Fluoride and Possibly Protects against Caries.

Aquaporins (AQP) are water channel proteins and the genes coding for AQP2, AQP5, and AQP6 are clustered in 12q13. Since AQP5 is expressed in serous acinar cells of salivary glands, we investigated its involvement in caries. DNA samples from 1,383 individuals from six groups were studied. Genotypes of eight single nucleotide polymorphisms covering the aquaporin locus were tested for association with caries experience. Interaction with genes involved in enamel formation was tested. The association between enamel microhardness at baseline, after creation of artificial caries lesion, and after exposure to fluoride and the genetic markers in AQP5 was tested. Finally, AQP5 expression in human whole saliva, after exposure to fluoride in a mammary gland cell line, which is known to express AQP5, and in Wistar rats was also verified. Nominal associations were found between caries experience and markers in the AQP5 locus. Since these associations suggested that AQP5 may be inhibited by levels of fluoride in the drinking water that cause fluorosis, we showed that fluoride levels above optimal levels change AQP5 expression in humans, cell lines, and rats. We have shown that AQP5 is involved in the pathogenesis of caries and likely interacts with fluoride.

Oral Health in a Sample of Pregnant Women from Northern Appalachia (2011-2015).

Background. Chronic poor oral health has a high prevalence in Appalachia, a large region in the eastern USA. The Center for Oral Health Research in Appalachia (COHRA) has been enrolling pregnant women and their babies since 2011 in the COHRA2 study of genetic, microbial, and environmental factors involved in oral health in Northern Appalachia. Methods. The COHRA2 protocol is presented in detail, including inclusion criteria (healthy, adult, pregnant, US Caucasian, English speaking, and nonimmunocompromised women), recruiting (two sites: Pittsburgh, Pennsylvania, and West Virginia, USA), assessments (demographic, medical, dental, psychosocial/behavioral, and oral microbial samples and DNA), timelines (longitudinal from pregnancy to young childhood), quality control, and retention rates. Results. Preliminary oral health and demographic data are presented in 727 pregnant women, half from the greater Pittsburgh region and half from West Virginia. Despite similar tooth brushing and flossing habits, COHRA2 women in West Virginia have significantly worse oral health than the Pittsburgh sample. Women from Pittsburgh are older and more educated and have less unemployment than the West Virginia sample. Conclusions. We observed different prevalence of oral health and demographic variables between pregnant women from West Virginia (primarily rural) and Pittsburgh (primarily urban). These observations suggest site-specific differences within Northern Appalachia that warrant future studies.

Replication of 13q31.1 association in nonsyndromic cleft lip with cleft palate in Europeans.

Genome wide association (GWA) studies have successfully identified at least a dozen loci associated with orofacial clefts. However, these signals may be unique to specific populations and require replication to validate and extend findings as a prelude to etiologic SNP discovery. We attempted to replicate the findings of a recent meta-analysis of orofacial cleft GWA studies using four different ancestral populations. We studied 946 pedigrees (3,436 persons) of European (US white and Danish) and Asian (Japanese and Mongolian) origin. We genotyped six SNPs that represented the most significant P-value associations identified in published studies: rs742071 (1p36), rs7590268 (2p21), rs7632427 (3p11.1), rs12543318 (8q21.3), rs8001641 (13q31.1), and rs7179658 (15q22.2). We directly sequenced three non-coding conserved regions 200 kb downstream of SPRY2 in 713 cases, 438 controls, and 485 trios from the US, Mongolia, and the Philippines. We found rs8001641 to be significantly associated with nonsyndromic cleft lip with cleft palate (NSCLP) in Europeans (P-value = 4 × 10(-5), ORtransmission = 1.86 with 95% confidence interval: 1.38-2.52). We also found several novel sequence variants in the conserved regions in Asian and European samples, which may help to localize common variants contributing directly to the risk for NSCLP. This study confirms the prior association between rs8001641 and NSCLP in European populations.

Search for genetic modifiers of IRF6 and genotype-phenotype correlations in Van der Woude and popliteal pterygium syndromes.

Van der Woude syndrome is the most common form of syndromic orofacial clefting, accounting for 1-2% of all patients with cleft lip and/or cleft palate. Van der Woude and popliteal pterygium syndromes are caused by mutations in IRF6, but phenotypic variability within and among families with either syndrome suggests that other genetic factors contribute to the phenotypes. The aim of this study was to identify common variants acting as genetic modifiers of IRF6 as well as genotype-phenotype correlations based on mutation type and location. We identified an association between mutations in the DNA-binding domain of IRF6 and limb defects (including pterygia). Although we did not detect formally significant associations with the genes tested, borderline associations suggest several genes that could modify the VWS phenotype, including FOXE1, TGFB3, and TFAP2A. Some of these genes are hypothesized to be part of the IRF6 gene regulatory network and may suggest additional genes for future study when larger sample sizes are also available. We also show that families with the Van de Woude phenotype but in whom no mutations have been identified have a lower frequency of cleft lip, suggesting there may be locus and/or mutation class differences in Van de Woude syndrome.

Analysis of PAC1 receptor gene variants in Caucasian and African American infants dying of sudden infant death syndrome.

AIM: Stress peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), has been implicated in sudden infant death syndrome (SIDS). The aim of this exploratory study was to determine whether variants in the gene encoding the PACAP-specific receptor, PAC1, are associated with SIDS in Caucasian and African American infants. METHODS: Polymerase chain reaction and Sanger DNA sequencing was used to compare variants in the 5'-untranslated region, exons and intron-exon boundaries of the PAC1 gene in 96 SIDS cases and 96 race- and gender-matched controls. RESULTS: The intron 3 variant, A/G: rs758995 (variant 'h'), and the intron 6 variant, C/T: rs10081254 (variant 'n'), were significantly associated with SIDS in Caucasians and African Americans, respectively (p < 0.05). Also associated with SIDS were interactions between the variants rs2302475 (variant 'i') in PAC1 and rs8192597 and rs2856966 in PACAP among Caucasians (p < 0.02) and rs2267734 (variant 'q') in PAC1 and rs1893154 in PACAP among African Americans (p < 0.01). However, none of these differences survived post hoc analysis. CONCLUSION: Overall, this study does not support a strong association between variants in the PAC1 gene and SIDS; however, a number of potential associations between race-specific variants and SIDS were identified that warrant targeted investigations in future studies.

Sequence variants in oxytocin pathway genes and preterm birth: a candidate gene association study.

BACKGROUND: Preterm birth (PTB) is a complex disorder associated with significant neonatal mortality and morbidity and long-term adverse health consequences. Multiple lines of evidence suggest that genetic factors play an important role in its etiology. This study was designed to identify genetic variation associated with PTB in oxytocin pathway genes whose role in parturition is well known. METHODS: To identify common genetic variants predisposing to PTB, we genotyped 16 single nucleotide polymorphisms (SNPs) in the oxytocin (OXT), oxytocin receptor (OXTR), and leucyl/cystinyl aminopeptidase (LNPEP) genes in 651 case infants from the U.S. and one or both of their parents. In addition, we examined the role of rare genetic variation in susceptibility to PTB by conducting direct sequence analysis of OXTR in 1394 cases and 1112 controls from the U.S., Argentina, Denmark, and Finland. This study was further extended to maternal triads (maternal grandparents-mother of a case infant, N=309). We also performed in vitro analysis of selected rare OXTR missense variants to evaluate their functional importance. RESULTS: Maternal genetic effect analysis of the SNP genotype data revealed four SNPs in LNPEP that show significant association with prematurity. In our case-control sequence analysis, we detected fourteen coding variants in exon 3 of OXTR, all but four of which were found in cases only. Of the fourteen variants, three were previously unreported novel rare variants. When the sequence data from the maternal triads were analyzed using the transmission disequilibrium test, two common missense SNPs (rs4686302 and rs237902) in OXTR showed suggestive association for three gestational age subgroups. In vitro functional assays showed a significant difference in ligand binding between wild-type and two mutant receptors. CONCLUSIONS: Our study suggests an association between maternal common polymorphisms in LNPEP and susceptibility to PTB. Maternal OXTR missense SNPs rs4686302 and rs237902 may have gestational age-dependent effects on prematurity. Most of the OXTR rare variants identified do not appear to significantly contribute to the risk of PTB, but those shown to affect receptor function in our in vitro study warrant further investigation. Future studies with larger sample sizes are needed to confirm the findings of this study.

Role of TRAV locus in low caries experience.

Caries is the most common chronic, multifactorial disease in the world today; and little is still known about the genetic factors influencing susceptibility. Our previous genome-wide linkage scan has identified five loci related to caries susceptibility: 5q13.3, 13q31.1, 14q11.2, 14q 24.3, and Xq27. In the present study, we fine mapped the 14q11.2 locus to identify genetic contributors to caries susceptibility. Four hundred seventy-seven subjects from 72 pedigrees with similar cultural and behavioral habits and limited access to dental care living in the Philippines were studied. An additional 387 DNA samples from unrelated individuals were used to determine allele frequencies. For replication purposes, a total of 1,446 independent subjects from four different populations were analyzed based on their caries experience (low versus high). Forty-eight markers in 14q11.2 were genotyped using TaqMan chemistry. Transmission disequilibrium test was used to detect over transmission of alleles in the Filipino families, and Chi-square, Fisher's exact and logistic regression were used to test for association between low caries experience and variant alleles in the replication data sets. We finally assessed the mRNA expression of TRAV4 in the saliva of 143 study subjects. In the Filipino families, statistically significant associations were found between low caries experience and markers in TRAV4. We were able to replicate these results in the populations studied that were characteristically from underserved areas. Direct sequencing of 22 subjects carrying the associated alleles detects one missense mutation (Y30R) that is predicted to be probably damaging. Finally, we observed higher expression in children and teenagers with low caries experience, correlating with specific alleles in TRAV4. Our results suggest that TRAV4 may have a role in protecting against caries.

Replication of genome wide association identified candidate genes confirm the role of common and rare variants in PAX7 and VAX1 in the etiology of nonsyndromic CL(P).

Following recent genome wide association studies (GWAS), significant genetic associations have been identified for several genes with nonsyndromic cleft lip with or without cleft palate (CL(P)). To replicate two of these GWAS signals, we investigated the role of common and rare variants in the PAX7 and VAX1 genes. TaqMan genotyping was carried out for SNPs in VAX1 and PAX7 and transmission disequilibrium test (TDT) was performed to test for linkage and association in each population. Direct sequencing in and around the PAX7 and VAX1 genes in 1,326 individuals of European and Asian ancestry was done. The TDT analysis showed strong associations with markers in VAX1 (rs7078160, P = 2.7E-06 and rs475202, P = 0.0002) in a combined sample of Mongolian and Japanese CL(P) case-parent triads. Analyses using parent-of-origin effects showed significant excess transmission of the minor allele from both parents with the effect in the mothers (P = 6.5E-05, OR (transmission) = 1.91) more striking than in the fathers (P = 0.004, OR (transmission) = 1.67) for VAX1 marker rs7078160 in the combined Mongolian and Japanese samples when all cleft types were combined. The rs6659735 trinucleotide marker in PAX7 was significantly associated with all the US cleft groups combined (P = 0.007 in all clefts and P = 0.02 in CL(P)). Eight rare missense mutations found in PAX7 and two rare missense mutations in VAX1. Our study replicated previous GWAS findings for markers in VAX1 in the Asian population, and identified rare variants in PAX7 and VAX1 that may contribute to the etiology of CL(P). Determining the role of rare variants clearly warrants further investigation.

Candidate gene linkage approach to identify DNA variants that predispose to preterm birth.

BACKGROUND: The aim of this study was to identify genetic variants contributing to preterm birth (PTB) using a linkage candidate gene approach. METHODS: We studied 99 single-nucleotide polymorphisms (SNPs) for 33 genes in 257 families with PTBs segregating. Nonparametric and parametric analyses were used. Premature infants and mothers of premature infants were defined as affected cases in independent analyses. RESULTS: Analyses with the infant as the case identified two genes with evidence of linkage: CRHR1 (P = 0.0012) and CYP2E1 (P = 0.0011). Analyses with the mother as the case identified four genes with evidence of linkage: ENPP1 (P = 0.003), IGFBP3 (P = 0.006), DHCR7 (P = 0.009), and TRAF2 (P = 0.01). DNA sequence analysis of the coding exons and splice sites for CRHR1 and TRAF2 identified no new likely etiologic variants. CONCLUSION: These findings suggest the involvement of six genes acting through the infant and/or the mother in the etiology of PTB.

Vitamin D receptor (VDR) and group-specific component (GC, vitamin D-binding protein) polymorphisms in myopia.

PURPOSE: Epidemiologic evidence indicates that time outdoors reduces the risk of myopia, suggesting a possible role for vitamin D. This case-control study was conducted to determine whether single-nucleotide polymorphisms (SNPs) within VDR at 12q13.11 and GC at 4q12-13 are associated with myopia. METHODS: The primary analysis was conducted on 81 white adult control subjects between 18 and 50 years of age with a spherical equivalent refractive error between +0.50 and +2.00 D in both eyes and less than 1.50 D of astigmatism. Affected myopic subjects were 289 unrelated white adults at least 18 years of age with at least -0.75 D myopia in both principal meridians of both eyes. RESULTS: One SNP within VDR was significantly associated with myopia in the multivariate analysis of the primary sample (rs2853559: odds ratio = 1.99, P = 0.003). In a subsample of less severely myopic white subjects between -0.75 and -4.00 D, three SNPs within VDR were significantly associated in a multivariate model after adjustment for multiple comparisons (rs2239182: odds ratio = 2.17, P = 0.007; rs3819545: odds ratio = 2.34, P = 0.003; rs2853559: odds ratio = 2.14, P = 0.0035), accounting for 12% of model variance over age alone. CONCLUSIONS: Polymorphisms within VDR appear to be associated with low to moderate amounts of myopia in white subjects. Future studies should determine whether this finding can be replicated and should explore the biological significance of these variations with respect to myopia.

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).

A genome-wide association study of cleft lip with and without cleft palate identifies risk variants near MAFB and ABCA4.

Case-parent trios were used in a genome-wide association study of cleft lip with and without cleft palate. SNPs near two genes not previously associated with cleft lip with and without cleft palate (MAFB, most significant SNP rs13041247, with odds ratio (OR) per minor allele = 0.704, 95% CI 0.635-0.778, P = 1.44 x 10(-11); and ABCA4, most significant SNP rs560426, with OR = 1.432, 95% CI 1.292-1.587, P = 5.01 x 10(-12)) and two previously identified regions (at chromosome 8q24 and IRF6) attained genome-wide significance. Stratifying trios into European and Asian ancestry groups revealed differences in statistical significance, although estimated effect sizes remained similar. Replication studies from several populations showed confirming evidence, with families of European ancestry giving stronger evidence for markers in 8q24, whereas Asian families showed stronger evidence for association with MAFB and ABCA4. Expression studies support a role for MAFB in palatal development.

Maternal serum 25-hydroxyvitamin D concentrations are associated with small-for-gestational age births in white women.

Maternal vitamin D deficiency has been associated with numerous adverse health outcomes, but its association with fetal growth restriction remains uncertain. We sought to elucidate the association between maternal serum 25-hydroxyvitamin D [25(OH)D] concentrations in early pregnancy and the risk of small-for-gestational age birth (SGA) and explore the association between maternal single nucleotide polymorphisms (SNP) in the vitamin D receptor (VDR) gene and the risk of SGA. We conducted a nested case-control study of nulliparous pregnant women with singleton pregnancies who delivered SGA infants (n = 77 white and n = 34 black) or non-SGA infants (n = 196 white and n = 105 black). Women were followed from <16 wk gestation to delivery. Women's banked sera at <22 wk were newly measured for 25(OH)D and DNA extracted for VDR genotyping. SGA was defined as live-born infants that were <10th percentile of birth weight according to nomograms based on gender and gestational age. After confounder adjustment, there was a U-shaped relation between serum 25(OH)D and risk of SGA among white mothers, with the lowest risk from 60 to 80 nmol/L. Compared with serum 25(OH)D 37.5-75 nmol/L, SGA odds ratios (95% CI) for levels <37.5 and >75 nmol/L were 7.5 (1.8, 31.9) and 2.1 (1.2, 3.8), respectively. There was no relation between 25(OH)D and SGA risk among black mothers. One SNP in the VDR gene among white women and 3 SNP in black women were significantly associated with SGA. Our results suggest that vitamin D has a complex relation with fetal growth that may vary by race.

Whorl patterns on the lower lip are associated with nonsyndromic cleft lip with or without cleft palate.

Nonsyndromic cleft lip with or without cleft palate (CL/P) is a common birth defect due to both genetic and environmental factors. Whorl lip print patterns are circular grooves on the central upper lip and/or the left and right lower lip. To determine if whorls are more common in families with CL/P than in controls, the Pittsburgh Orofacial Cleft Study collected lip prints from over 450 subjects, that is, individuals with CL/P, their relatives, and unrelated controls-from the U.S., Argentina, and Hungary. Using a narrow definition of lower-lip whorl, the frequency of whorls in the U.S. sample was significantly elevated in cleft individuals and their family members, compared to unrelated controls (14.8% and 13.2% vs. 2.3%; P = 0.003 and 0.001, respectively). Whorls were more frequent in CL/P families from Argentina than in CL/P families from the U.S. or Hungary. If these results are confirmed, whorl lip print patterns could be part of an expanded phenotypic spectrum of nonsyndromic CL/P. As such, they may eventually be useful in a clinical setting, allowing recurrence risk calculations to incorporate individual phenotypic information in addition to family history data.