To replicate genetic associations with respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) in genes related to surfactant deficiency, inflammation and infection and the renin-angiotensin system.
We examined eight candidate genes for associations with RDS and BPD in 433 preterm (PTB - <37 weeks) infants (251 with RDS and 134 with BPD). Both case-control and family-based analyses were performed in preterm (<37 weeks) and very preterm (VPTB - <32 weeks) infants.
We replicated a previous finding that rs1923537, a marker downstream of surfactant protein D (SFTPD) is associated with RDS in VPTB infants in that the T allele was over-transmitted from parents to offspring with RDS (p=8.4×10−3). We also observed the A allele of rs4351 in the angiotensin-converting enzyme (ACE) gene was over-transmitted from parents to VPTB offspring with BPD (p=9.8×10−3).
These results give further insight into the genetic risk factors for complex neonatal respiratory diseases and provide more evidence of the importance of SFTPD and ACE in the etiology of RDS and BPD, respectively.
bronchopulmonary dysplasia; respiratory distress syndrome; single nucleotide polymorphism
Elevations or deficits in thyroid hormone levels are responsible for a wide range of neonatal and adult phenotypes. Several genome-wide, candidate gene and meta-analysis studies have examined thyroid hormones in adults; however, to our knowledge no genetic association studies have been performed with neonatal thyroid levels.
A population of Iowa neonates; term (n=827) and preterm (n=815), were genotyped for 45 single nucleotide polymorphisms. Thyroid stimulating hormone (TSH) values were obtained from the Iowa Neonatal Metabolic Screening Program. Analysis of variance was performed to identify genetic associations with TSH concentrations.
The strongest association was rs4704397 in the PDE8B gene (p=1.3×10−4), followed by rs965513 (p=6.4×10−4) on chromosome 9 upstream of the FOXE1 gene. Both of these SNPs met statistical significance after correction for multiple testing. Six other SNPs were marginally significant (p<0.05).
We demonstrated for the first time two genetic associations with neonatal TSH levels that replicate findings with adult TSH levels. These SNPs should be considered as early predictors of risk for adult diseases and conditions associated with thyroid hormone levels. Furthermore, this provides a better understanding of the thyroid profile and potential risk for thyroid disorders in newborns.
Genetic variation in the transcription factor Interferon Regulatory Factor 6 (IRF6) causes and contributes risk for oral clefting disorders. We hypothesized that genes regulated by IRF6 are also involved in oral clefting disorders. We used five criteria to identify potential IRF6 target genes; differential gene expression in skin taken from wild type and Irf6-deficient murine embryos, localization to the Van der Woude syndrome 2 (VWS2) locus at 1p36–1p32, overlapping expression with Irf6, presence of a conserved predicted binding site in the promoter region, and a mutant murine phenotype that was similar to the Irf6 mutant mouse. Previously, we observed altered expression for 573 genes; 13 were located in the murine region syntenic to the VWS2 locus. Two of these genes, Wdr65 and Stratifin, met four of five criteria. Wdr65 was a novel gene that encoded a predicted protein of 1250 amino acids with two WD domains. As potential targets for Irf6 regulation, we hypothesized that disease-causing mutations will be found in WDR65 and Stratifin in individuals with VWS or VWS-like syndromes. We identified a potentially etiologic missense mutation in WDR65 in a person with VWS who does not have an exonic mutation in IRF6. The expression and mutation data were consistent with the hypothesis that WDR65 was a novel gene involved in oral clefting.
cleft lip and palate; mutation; gene expression; syndrome; genomic; microvilli; WD domain; transcription factor
Epidermolytic ichthyosis (EI) is a rare skin disorder characterized by generalized erythroderma and cutaneous blistering at birth, which is substituted by hyperkeratosis later in life. It is caused by autosomal dominant mutations in highly conserved regions of KRT1 and KRT10. To date, only 4 mutations with autosomal recessive inheritance of EI have been described in consanguineous families. All of them affect the 2B domain of KRT10. In the present study we describe four patients with EI (including one lethal case) born from unaffected parents in a consanguineous family of a native Venezuelan community. The objective of this study was to characterize the clinical, genetic and morphological aspects of the disease in this family, as well as understand its functional implications. Genomic DNA was sequenced for KRT10 and KRT1. Immunofluoresence for keratin expression was performed on cutaneous biopsies. After examination of cutaneous biopsies histology, our results showed hyperkeratosis and acantholysis with an expanded granular layer. Sequencing of KRT10 demonstrated a non-sense mutation (p.Tyr282Ter.) corresponding to the 1B domain of the protein in patients and a heterozygous pattern in other family members, resulting in complete absence of K10. The loss of K10 was compensated by upregulation of K14 and K17. In conclusion, this novel mutation in KRT10 is the first recessive genetic variation that is not located in the so called “hot spot” for recessive EI, suggesting that other areas of the gene are also susceptible for such mutations.
Epidermolytic Ichthyosis; Epidermolytic Hyperkeratosis; Bullous Congenital Ichthyosiform Erythroderma; Non-sense mediated mRNA Decay; Keratin 10; Keratin; Genetics; Mutations
To identify genetic variants contributing to preterm birth using a linkage candidate gene approach.
We studied 99 single nucleotide polymorphisms for 33 genes in 257 families with preterm births segregating. Nonparametric and parametric analyses were used. Premature infants and mothers of premature infants were defined as affected cases in independent analyses.
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.
These findings suggest the involvement of six genes acting through the infant and/or the mother in the etiology of preterm birth.
Intraventricular hemorrhage (IVH) is a significant morbidity seen in very low birth weight infants. Genes related to the inflammation, infection, complement or coagulation pathways have been implicated as risk factors for IVH. We examined ten candidate genes for associations with IVH in 271 preterm infants (64 with IVH grade I-IV and 207 without IVH) weighing less than 1,500 grams. The heterozygous genotype (odds ratio (OR)=8.1, confidence interval (CI)=2.5–26.0, p=4×10−4) and the A allele (OR=7.3, CI=2.4–22.5, p=1×10−4) of the coagulation factor V (FV) Leiden mutation (rs6025) were associated with an increased risk of developing IVH grade I or II but not grades III or IV after correction for multiple testing with Bonferroni. Lack of association in the severe grades of IVH may be a result of lack of power to detect an effect given the small sample size (n=8). However, this result is consistent with previous research that demonstrates that the heterozygous genotype of the FV mutation is associated with increased risk for the development of IVH but a decreased risk for the progression or extension to more severe grades of IVH.
A major concern for all copy number variation (CNV) detection algorithms is their reliability and repeatability. However, it is difficult to evaluate the reliability of CNV calling strategies due to the lack of gold standard data that would tell us which CNVs are real. We propose that if CNVs are called in duplicate samples, or inherited from parent to child, then these can be considered validated CNVs. We used two large family-based Genome-Wide Association Study (GWAS) datasets from the GENEVA consortium to look at concordance rates of CNV calls between duplicate samples, parent-child pairs, and unrelated pairs. Our goal was to make recommendations for ways to filter and use CNV calls in GWAS datasets that do not include family data. We used PennCNV as our primary CNV-calling algorithm, and tested CNV calls using different datasets and marker sets, and with various filters on CNVs and samples. Using the Illumina core HumanHap550 SNP (single nucleotide polymorphism) set, we saw duplicate concordance rates of approximately 55% and parent-child transmission rates of approximately 28% in our datasets. GC model adjustment and sample quality filtering had little effect on these reliability measures. Stratification on CNV size and DNA sample type did have some effect. Overall, our results show that it is probably not possible to find a CNV calling strategy (including filtering and algorithm) that will give us a set of “reliable” CNV calls using current chip technologies. But if we understand the error process, we can still use CNV calls appropriately in genetic association studies.
evaluation; CNV calling strategies; family-based GWAS
Genome-wide association studies are now used routinely to identify genes implicated in complex traits. The panels used for such analyses can detect single nucleotide polymorphisms and copy number variants, both of which may help to identify small deleted regions of the genome that may contribute to a particular disease
We performed a candidate gene analysis involving 1221 SNPs in 333 candidate genes for orofacial clefting using 2823 samples from 725 two- and three-generation families with a proband with clefts of the lip and/or palate. We used SNP genotyping, DNA sequencing, high-resolution DNA microarray analysis and long-range PCR to confirm and characterize the deletion events
This dataset had a high duplicate reproducibility rate (99.98%), high Mendelian consistency rate (99.93%), and low missing data rate (0.55%), which provided a powerful opportunity for deletion detection. Apparent Mendelian inconsistencies between parents and child suggested deletion events in 15 individuals in 11 genomic regions. We confirmed deletions involving CYP1B1, FGF10, SP8, SUMO1, TBX1, TFAP2A, and UGT7A1, including both de novo and familial cases. Deletions of SUMO1, TBX1, and TFAP2A are likely to be etiologic
These deletions suggest the potential roles of genes or regulatory elements contained within deleted regions in the etiology of clefting. Our analysis took advantage of genotypes from a candidate-gene-based SNP survey and proved to be an efficient analytical approach to interrogate genes potentially involved in clefting. This can serve as a model to find genes playing a role in complex traits in general.
cleft lip; cleft palate; microdeletion; SNPs; CNV; candidate genes
Despite important recent work, US public attitudes toward specific biobank consent models are not well understood. Public opinion data can help shape efforts to develop ethically sound and publicly trusted mechanisms for informing and consenting prospective biobank donors. The purpose of this study was to explore public perspectives toward a range of consent models currently being used or considered for use among comprehensive US biobanks.
The study used an exploratory mixed-methods design, using focus groups and telephone surveys. Eligible participants were English-speaking residents in the catchment area of a comprehensive biobank being developed at the University of Iowa.
Forty-eight participants in seven focus groups and 751 survey participants were recruited. Biobanks were unfamiliar to almost all study participants but were seen as valuable resources. Most focus group (63%) and survey (67%) participants preferred a prospective opt-in over an opt-out consent approach. Broad, research-unspecific consent was preferred over categorical and study-specific consent models for purposes of approving future research use.
Many individuals may want to make an active and informed choice at the point of being approached for biobank participation but are prepared to consent broadly to future research use and to forego additional choices as a result.
biobank; consent models; public perspectives
Hyperglycemia has recently been described as a risk factor for the development of retinopathy of prematurity (ROP), a proliferative vascular disease of the retina that primarily affects premature infants. This study was to evaluate the relationship of hyperglycemia and the development of ROP in premature infants less than 32 weeks gestation.
This was a retrospective cohort study of all infants less than 32 weeks gestation from 2003–2007 who survived to discharge in our NICU. Demographic data including birthweight, gestational age, Apgar scores, method of delivery, antenatal steroid use, neonatal steroid use, and size for gestational age was collected for each infant. Episodes of sepsis, grade of intraventricular hemorrhage, presence of a patent ductus arteriosus, number of days on the ventilator, and stage of necrotizing enterocolitis were assessed as well as days of hyperglycemia, defined as number of days with whole blood glucose > 150 mg/dl. In addition, the highest stage of ROP was recorded for each infant. A Student’s two tailed t-test or Fisher’s exact test was performed to identify significant clinical risk factors associated with the development of ROP. From this univariate analysis, a multiple logistic regression was performed to determine the effect of hyperglycemia on the development of ROP, adjusting for significant clinical risk factors. Statistical analysis was performed using SAS v.9.2.
Univariate analysis demonstrated that infants with ROP were of lower birthweight and gestational age, and were affected by a patent ductus arteriosus, neonatal sepsis, intraventricular hemorrhage, have significant lung disease and received postnatal glucocorticoid therapy. Infants with ROP experienced more days with hyperglycemia (7 vs. 2, p = < 0.0001). Using multiple logistic regression analysis to compare no ROP vs. all stages of ROP, gestational age (OR 0.745, 95% CI [0.634, 0.877], p = 0.0004), mean days of hyperglycemia (OR 1.073, 95% CI [1.004, 1.146], p = 0.04), and mean days receiving mechanical ventilation (OR 1.012, 95% CI [1.000, 1.025], p = 0.05) remained significantly associated with ROP after adjusting for other risk factors.
Our data suggests that hyperglycemia is associated with the development of ROP in premature infants.
Prematurity; Retinopathy of prematurity; Hyperglycemia
Reports of birth defects rates may focus on defects observed in the newborn period or include defects diagnosed at older ages. However, little information is available on the rates of additional anomalies detected after birth or on the ages at which such anomalies are diagnosed. The aims of this work were to describe the initial diagnoses of oral clefts, isolated or associated with other defects, in newborn infants ascertained in hospitals of the ECLAMC network, and diagnostic changes that occurred due to detection of additional defects during a one-year follow-up period. Seven hundred ten liveborn infants with cleft lip only (CLO), cleft lip with cleft palate (CLP), or cleft palate (CP) were ascertained between 2003 and 2005. Prevalence estimates of isolated and associated clefts, diagnoses in infants with associated clefts, and the percentage of isolated clefts that were reclassified as associated were established. Birth prevalence estimates (per 1,000) were as follows: Total: 1.7; CLP: 0.94 (ASO=23.5%); CP: 0.46 (ASO=42.3%); CLO: 0.28 (ASO=7.6%). Initial diagnoses in infants with associated clefts included 38 infants with chromosomal abnormalities, 33 with non-chromosomal syndromes, 16 with malformation sequences, and 98 with multiple anomalies of unknown etiology. Seven percent of newborns initially classified as isolated were later reclassified as associated. Ten infants without associated defects or clinically suspected syndromes were diagnosed as syndromic only through laboratory findings or family history, illustrating the difference between the terms associated vs. isolated, which refers to presence or absence of associated anomalies, and syndromic vs. non-syndromic, which refers to etiology.
oral clefts; isolated; associated; follow-up; syndromic
We performed a genome wide association analysis of maternally-mediated genetic effects and parent-of-origin effects on risk of orofacial clefting using over 2,000 case-parent triads collected through an international cleft consortium. We used log-linear regression models to test individual SNPs. For SNPs with a p-value <10−5 for maternal genotypic effects, we also applied a haplotype-based method, TRIMM, to extract potential information from clusters of correlated SNPs. None of the SNPs were significant at the genome wide level. Our results suggest neither maternal genome nor parent of origin effects play major roles in the etiology of orofacial clefting in our sample. This finding is consistent with previous genetic studies and recent population-based cohort studies in Norway and Denmark, which showed no apparent difference between mother-to-offspring and father-to-offspring recurrence of clefting. We, however, cannot completely rule out maternal genome or parent of origin effects as risk factors because very small effects might not be detectable with our sample size, they may influence risk through interactions with environmental exposures or may act through a more complex network of interacting genes. Thus the most promising SNPs identified by this study may still be worth further investigation.
GWAS; CL/P; CP; maternal genes; parent-of-origin; family-based study; association study
Several studies have evaluated whether the high and rising obesity rates over the past three decades may be due to the declining smoking rates. There is mixed evidence across studies – some find negative smoking effects and positive cigarette cost effects on body weight, while others find opposite effects. This study applies a unique approach to identify the smoking effects on body weight and to evaluate the heterogeneity in these effects across the body mass index (BMI) distribution by utilizing genetic instruments for smoking. Using a data sample of 1,057 mothers from Norway, the study finds heterogeneous effects of cigarette smoking on BMI – smoking increases BMI at low/moderate BMI levels and decreases BMI at high BMI levels. The study highlights the potential advantages and challenges of employing genetic instrumental variables to identify behavior effects including the importance of qualifying the instruments and the need for large samples.
Smoking; obesity; body mass index; genetic instrumental variables; quantile regression; mendelian randomization
The objective of this study was to use array-CGH to detect causal microdeletions in samples of subjects with cleft lip and palate.
We analyzed DNA samples from a male patient and parents that was seen during surgical screening for an Operation Smile medical mission in the Philippines.
We used Affymetrix Genome Wide Human SNP Array 6.0 followed by sequencing and quantitative PCR using SYBR Green I dye.
We report the second case of 3q29 microdeletion syndrome including cleft lip with or without cleft palate and the first case of this microdeletion syndrome inherited from a phenotypically normal mosaic parent.
Our findings confirm the utility of aCGH to detect causal microdeletions; indicate that parental somatic mosaicism should be considered in healthy parents for genetic counseling of the families and discuss important ethical implications of sharing health impact results from research studies with the participant families.
microdeletion; cleft; 3q29; mosaicism; ethical
The amnion is a specialized tissue in contact with the amniotic fluid, which is in a constantly changing state. To investigate the importance of epigenetic events in this tissue in the physiology and pathophysiology of pregnancy, we performed genome-wide DNA methylation profiling of human amnion from term (with and without labor) and preterm deliveries. Using the Illumina Infinium HumanMethylation27 BeadChip, we identified genes exhibiting differential methylation associated with normal labor and preterm birth. Functional analysis of the differentially methylated genes revealed biologically relevant enriched gene sets. Bisulfite sequencing analysis of the promoter region of the oxytocin receptor (OXTR) gene detected two CpG dinucleotides showing significant methylation differences among the three groups of samples. Hypermethylation of the CpG island of the solute carrier family 30 member 3 (SLC30A3) gene in preterm amnion was confirmed by methylation-specific PCR. This work provides preliminary evidence that DNA methylation changes in the amnion may be at least partially involved in the physiological process of labor and the etiology of preterm birth and suggests that DNA methylation profiles, in combination with other biological data, may provide valuable insight into the mechanisms underlying normal and pathological pregnancies.
Objectives: To evaluate the effects of folic acid supplementation on isolated oral cleft recurrence and fetal growth. Patients and Methods: The study included 2,508 women who were at-risk for oral cleft recurrence and randomized into two folic acid supplementation groups: 0.4 and 4 mg per day before pregnancy and throughout the first trimester. The infant outcome data were based on 234 live births. In addition to oral cleft recurrence, several secondary outcomes were compared between the two folic acid groups. Cleft recurrence rates were also compared to historic recurrence rates. Results: The oral cleft recurrence rates were 2.9% and 2.5% in the 0.4 and 4 mg groups, respectively. The recurrence rates in the two folic acid groups both separately and combined were significantly different from the 6.3% historic recurrence rate post the folic acid fortification program for this population (p = 0.0009 when combining the two folic acid groups). The rate of cleft lip with palate recurrence was 2.9% in the 0.4 mg group and 0.8% in the 4 mg group. There were no elevated fetal growth complications in the 4 mg group compared to the 0.4 mg group. Conclusions: The study is the first double-blinded randomized clinical trial (RCT) to study the effect of high dosage folic acid supplementation on isolated oral cleft recurrence. The recurrence rates were similar between the two folic acid groups. However, the results are suggestive of a decrease in oral cleft recurrence compared to the historic recurrence rate. A RCT is still needed to identify the effect of folic acid on oral cleft recurrence given these suggestive results and the supportive results from previous interventional and observational studies, and the study offers suggestions for such future studies. The results also suggest that high dosage folic acid does not compromise fetal growth.
oral clefts; cleft lip; cleft palate; birth defects; folic acid; vitamins; prevention; pregnancy; nutrition; Brazil; NCT00397917
Nationally newborn screening programs use 17-hydroxyprogesterone (17-OHP) as the biomarker to detect the rare but potentially fatal inherited disease, congenital adrenal hyperplasia (CAH). However, this biomarker is highly variable with a high false positive rate of detection, particularly in neonates born preterm. Several studies have examined various clinical and genetic factors to explain the variability of 17-OHP in preterm infants. The purpose of this study was to replicate previous clinical and genetic associations with 17-OHP in a well-characterized cohort of 762 preterm infants. We replicated previous findings that respiratory distress syndrome (P = 2×10−3) is associated with higher 17-OHP. Higher 17-OHP and false positives were significantly associated with lower gestational age and birth weight, as previously reported. Incorporating gestational age and birth weight together decreases the false positive rate.
Mutations in the human GLI2 gene were first reported in association with defective anterior pituitary formation, pan-hypopituitarism, and forebrain anomalies represented by typical holoprosencephaly (HPE) and holoprosencephaly-like (HPE-L) phenotypes and postaxial polydactyly. Subsequent, anophthalmia plus orbital anomalies, heminasal aplasia, branchial arch anomalies and polydactyly have also been incorporated into the general phenotype. Here we described six Brazilian patients with phenotypic manifestations that range from isolated cleft lip/palate with polydactyly, branchial arch anomalies to semi lobar holoprosencephaly. Novel sequence variants were found in the GLI2 gene in patients with marked involvement of the temporomandibular joint (TMJ), a new clinical finding observed with mutations of this gene. Clinical, molecular and genetic aspects are discussed.
branchial arch anomalies; cleft lip and palate; GLI2; HPE; HPE-L; polydactyly; SHH signaling pathway; temporomandibular joint
To test whether females in families with cleft lip and/or palate (CL/P) have increased breast cancer risk
Using the Danish Facial Cleft Registry, females with CL/P, mothers of children with CL/P, and sisters to CL/P cases were identified for the Danish birth cohorts 1911 to 1975. These females were compared to a 5% random sample of these cohorts regarding the incidence and age of onset for breast cancer registered in the Danish Hospital Discharge Register 1977–2005.
Examining 48,404 person-years for 1,809 female CL/P cases (49 breast cancer cases) and 212,795 person-years for 7935 female relatives (188 breast cancer cases) we found no increased breast cancer risk for either CL/P cases (hazard ratio (HR) = 1.23, 95% confidence interval (CI): 0.92–1.63), mothers of children with CL/P (HR = 0.93, 95%, CI: 0.80–1.08), or sisters of CL/P cases (HR = 0.94, 95% CI: 0.55–1.60). Neither were there any significant differences in age of onset.
Both epidemiological and genetic studies have suggested common etiological factors for breast cancer and cleft lip and/or palate (CL/P). However, this population-based study was not able to confirm a general increase in breast cancer risk among females in families with CL/P.
Cleft lip; cleft palate; breast cancer; recurrence; family study
Analysis of cell-free fetal DNA in maternal plasma holds great promise for the development of non-invasive prenatal genetic diagnostics. However, previous studies have been restricted to detection of fetal trisomies (1, 2) or specific, paternally inherited mutations (3), or to genotyping common polymorphisms using invasively sampled material (4). Here, we combine genome sequencing of two parents, genome-wide maternal haplotyping (5), and deep sequencing of maternal plasma to non-invasively determine the genome sequence of a human fetus at 18.5 weeks gestation. Inheritance was predicted at 2.8×106 parentally heterozygous sites with 98.1% accuracy. Furthermore, 39 of 44 de novo point mutations in the fetal genome were detected, albeit with limited specificity. Subsampling these data and analyzing a second family trio by the same approach indicate that ~300 kilobase parental haplotype blocks combined with shallow sequencing of maternal plasma are sufficient to substantially determine the inherited complement of a fetal genome. However, ultra-deep sequencing of maternal plasma is necessary for the practical detection of fetal de novo mutations genome-wide. Although technical and analytical challenges remain, we anticipate that non-invasive analysis of inherited variation and de novo mutations in fetal genomes will facilitate the comprehensive prenatal diagnosis of both recessive and dominant Mendelian disorders.
To evaluate the extent of racial gaps in child health insurance coverage in South America and study the contribution of wealth, human capital and other household characteristics to accounting for racial disparities in insurance coverage.
Data sources/study setting
Primary data collected between 2005 and 2006 in 30 pediatric practices in Argentina, Brazil, Ecuador and Chile.
Country-specific regression models are used to assess differences in insurance coverage by race. A decomposition model is used to quantify the extent to which wealth, human capital and other household characteristics account for racial disparities in insurance coverage.
Data collection/extraction methods
In-person interviews were conducted with the mothers of 2,365 children.
The majority of children have no insurance coverage except in Chile. Large racial disparities in insurance coverage are observed. Household wealth is the single most important household-level factor accounting for racial disparities in coverage and is significantly and positively associated with coverage, followed by maternal education and employment/occupational status. Geographic differences account for the largest part of racial disparities in insurance coverage in Argentina and Ecuador.
Increasing the coverage of children in less affluent families is important for reducing racial gaps in health insurance coverage in the study countries.
Health insurance; racial disparities; socioeconomic disparities; child health; South America
Non-syndromic cleft lip and palate (NS CLP) is a complex birth defect resulting from multiple genetic and environmental factors. We have previously reported the sequencing of the coding region of genes in the fibroblast growth factor (FGF) signaling pathway, in which missense and non-sense mutations contribute to approximately 5%–6% NS CLP cases. In this article we report the sequencing of conserved non-coding elements (CNEs) in and around 11 of the FGF and FGFR genes, which identified 55 novel variants. Seven of variants are highly conserved among ≥8 species and 31 variants alter transcription factor binding sites, 8 of which are important for craniofacial development. Additionally, 15 NS CLP patients had a combination of coding mutations and CNE variants, suggesting that an accumulation of variants in the FGF signaling pathway may contribute to clefting.
cleft lip and palate; fibroblast growth factor; conserved non-coding elements; sequencing