X-chromosome inactivation patterns in monozygotic twins and sib pairs discordant for nonsyndromic cleft lip and/or palate

Nonsyndromic clefts of the lip and/or palate are common birth defects with a strong genetic component. Based on unequal gender ratios for clefting phenotypes, evidence for linkage to the X chromosome and the occurrence of several X-linked clefting syndromes, we investigated the role of skewed X chromosome inactivation (XCI) in orofacial clefts. Our samples consisted of female monozygotic (MZ) twins (n = 8) and sister pairs (n = 152) discordant for nonsyndromic clefting. We measured the XCI pattern in peripheral blood lymphocyte DNA using a methylation based androgen receptor gene assay. Skewing of XCI was defined as the deviation in inactivation pattern from a 50:50 ratio. Our analysis revealed no significant difference in the degree of skewing between twin pairs (P = 0.3). However, borderline significant differences were observed in the sister pairs (P = 0.02), with the cleft lip with cleft palate group showing the most significant result (P=0.01). We did not find evidence for involvement of skewed XCI in the discordance for clefting in our sample of female MZ twins. However, results from the paired sister study suggest the potential contribution of skewed XCI to orofacial clefting, particularly cleft lip and palate. (C) 2007 Wiley-Liss, Inc.

IRF6 is a Risk Factor for Nonsyndromic Cleft Lip in the Brazilian Population

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a complex disorder with a worldwide incidence estimated at 1:700. Among the putative susceptibility loci, the IRF6 gene and a region at 8q24.21 have been corroborated in different populations. To test the role of IRF6 in NSCL/P predisposition in the Brazilian population, we conducted a structured association study with the SNPs rs642961 and rs590223, respectively, located at 5' and 3' of the IRF6 gene and not in strong linkage disequilibrium (LD), in patients from five different Brazilian locations. We also evaluated the effect of these SNPs in IRF6 expression in mesenchymal stem cells (MSC). We observed association between rs642961 and cleft lip only (CLO) (P = 0.009; odds ratio (OR) for AA genotype = 1.83 [95% Confidence interval (CI), 0.64-5.31]; OR for AG genotype = 1.72 [95% CI, 1.03-2.84]). This association seems to be driven by the affected patients from Barbalha, a location which presents the highest heritability estimate (H-2 = 0.85), and the A allele at rs642961 is acting through a dominant model. No association was detected for the SNP rs590223. We did not find any correlation between expression levels and genotypes of the two loci, and it is possible that these SNPs have a functional role in some specific period of embryogenesis. (C) 2012 Wiley Periodicals, Inc.

Gene Discovery in Nonsyndromic Cleft Lip with or without Cleft Palate

Nonsyndromic cleft lip with or without cleft palate (NSCLP), a common, complex orofacial birth defect that affects approximately 4,000 newborns each year in the United States, is caused by both genetic and environmental factors. Orofacial clefts affect the mouth and nose, causing severe deformity of the face, which require medical, dental and speech therapies. Despite having substantial genetic liability, less than 25% of the genetic contribute to NSCLP has been identified. The studies described in this thesis were performed to identify genes that contribute to NSCLP and to demonstrate the role of these genes in normal craniofacial development. Using genome scan and candidate gene approaches, novel associations with NSCLP were identified. These include MYH9 (7 SNPs, 0.009≤p<0.05), Wnt3A (4 SNPs, 0.001≤p≤0.005), Wnt11 (2 SNPs, 0.001≤p≤0.01) and CRISPLD2 (4 SNPs, 0.001≤p<0.05). The most interesting findings were for CRISPLD2. This gene is expressed in the fused mouse palate at E17.5. In zebrafish, crispld2 localized to the craniofacial region by one day post fertilization. Morpholino knockdown of crispld2 resulted in a lower survival rates and altered neural crest cell (NCC) clustering. Because NCCs form the tissues that populate the craniofacies, this NCC abnormality resulted in cartilage abnormalities of the jaw including fewer ceratobranchial cartilages forming the lower jaw (three pairs compared to five) and broader craniofacies compared to wild-type zebrafish. These findings suggest that the CRISPLD2 gene plays an important role in normal craniofacial development and perturbation of this gene in humans contributes to orofacial clefting. Overall, these results are important because they contribute to our understanding of normal craniofacial development and orofacial clefting etiology, information that can be used to develop better methods to diagnose, counsel and potentially treat NSCLP patients.

Mutation analysis of the PVRL1 gene in caucasians with nonsyndromic cleft lip/palate.

Nonsyndromic cleft lip with or without cleft palate (nsCL/P, MIM 119530) is perhaps the most common major birth defect. Homozygous PVRL1 loss-of-function mutations result in an autosomal recessive CL/P syndrome, CLPED1, and a PVRL1 nonsense mutation is associated with sporadic nsCL/P in Northern Venezuela. To address the more general role of PVRL1 variation in risk of nsCL/P, we carried out mutation analysis of PVRL1 in North American and Australian nsCL/P cases and population-matched controls. We identified a total of 15 variants, 5 of which were seen in both populations and 1 of which, an in-frame insertion at Glu442, was more frequent in patients than in controls in both populations, though the difference was not statistically significant. Another variant, which is specific to the PVRL1 beta (HIgR) isoform, S447L, was marginally associated with nsCL/P in North American Caucasian patients, but not in Australian patients, and overall variants that affect the beta-isoform were significantly more frequent among North American patients. One Australian patient had a splice junction mutation of PVRL1. Our results suggest that PVRL1 may play a minor role in susceptibility to the occurrence of nsCL/P in some Caucasian populations, and that variation involving the beta (HIgR) isoform might have particular importance for risk of orofacial clefts. Nevertheless, these results underscore the need for studies that involve very large numbers when assessing the possible role of rare variants in risk of complex traits such as nsCL/P.

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

FZD6, MATN2 AND SLC25A32, POSSIBLE CANDIDATE GENES IN NONSYNDROMIC CLEFT LIP AND PALATE

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect with a multifactorial etiology. Despite decades of research, the genetic underpinnings of NSCLP still remain largely unexplained. A genome wide association study (GWAS) of a large NSCLP African American family with seven affected individuals across three generations found evidence for linkage at 8q21.3-24.12 (LOD = 2.98). This region contained three biologically relevant candidate genes: Frizzled-6 (FZD6) (LOD = 2.8), Matrilin-2 (MATN2) (LOD = 2.3), and Solute Carrier Family 25, Member 32 (SLC26A32) (LOD = 1.6). Sequencing of the coding regions and the 5’ and 3’ UTRs of these genes in two affected family members identified a rare intronic variant, rs138557689 (c.-153+432A>C), in FZD6. The rs138557689/C allele segregated with the NSCLP phenotype; in silico analysis predicted and EMSA analysis showed that the 138557689/C allele creates new DNA binding sites. FZD6 is part of the WNT pathway, which is involved in craniofacial development, including midface development and upper lip fusion. Our novel findings suggest that an alteration in FZD6 gene regulation may perturb this tightly controlled biological pathway and in turn contribute to the development of NSCLP in this family. Studies are underway to further define how the rs138557689/C variant affects expression of FZD6.

Epidemiological features of patients with nonsyndromic cleft lip and/or palate in Western Parana

Aim:To describe the clinical, demographic and environmental features associated with NSCL/P (nonsyndromic cleft lip and/or palate) patients born in western Parana state, Brazil. Methods: This cross-sectional, observational, retrospective study included 188 patients attended at the Association of Carriers of Cleft Lip and Palate - APOFILAB, Cascavel-Parana, between 2012 and 2014. Information on demographic characteristics, medical and dental histories and life style factors were obtained from records and personal interviews. Results: Among the 188 patients, cleft lip and palate (CLP) was the most frequent subtype (55.8%), followed by cleft lip only (CLO, 25.0%) and cleft palate only (CPO, 19.2%). Caucasian males were the most affected, although no differences among types of cleft were observed. The otorhinolaryngologic and respiratory alterations were the most frequent systemic alterations in NSCL/P patients, and more than 80% of the NSCL/P mothers reported no vitamin supplements during the first trimester of pregnancy. Conclusions: This study revealed that the prevalence of nonsyndromic oral cleft types in this cohort was quite similar to previously reported prevalence rates. Systemic alterations were identified among 23.4% of the patients and patients with CLP were the most affected. History of maternal exposition to environmental factors related to nonsyndromic oral clefts was frequent and most mothers reported no vitamin supplements during the pregnancy. This study highlights the importance of identifying systemic alterations and risk factors associated with NSCL/P in the Brazilian population for planning comprehensive strategies and integrated actions for the development of preventive programs and treatment.

Human Stem Cell Cultures from Cleft Lip/Palate Patients Show Enrichment of Transcripts Involved in Extracellular Matrix Modeling By Comparison to Controls

Nonsyndromic cleft lip and palate (NSCL/P) is a complex disease resulting from failure of fusion of facial primordia, a complex developmental process that includes the epithelial-mesenchymal transition (EMT). Detection of differential gene transcription between NSCL/P patients and control individuals offers an interesting alternative for investigating pathways involved in disease manifestation. Here we compared the transcriptome of 6 dental pulp stem cell (DPSC) cultures from NSCL/P patients and 6 controls. Eighty-seven differentially expressed genes (DEGs) were identified. The most significant putative gene network comprised 13 out of 87 DEGs of which 8 encode extracellular proteins: ACAN, COL4A1, COL4A2, GDF15, IGF2, MMP1, MMP3 and PDGFa. Through clustering analyses we also observed that MMP3, ACAN, COL4A1 and COL4A2 exhibit co-regulated expression. Interestingly, it is known that MMP3 cleavages a wide range of extracellular proteins, including the collagens IV, V, IX, X, proteoglycans, fibronectin and laminin. It is also capable of activating other MMPs. Moreover, MMP3 had previously been associated with NSCL/P. The same general pattern was observed in a further sample, confirming involvement of synchronized gene expression patterns which differed between NSCL/P patients and controls. These results show the robustness of our methodology for the detection of differentially expressed genes using the RankProd method. In conclusion, DPSCs from NSCL/P patients exhibit gene expression signatures involving genes associated with mechanisms of extracellular matrix modeling and palate EMT processes which differ from those observed in controls. This comparative approach should lead to a more rapid identification of gene networks predisposing to this complex malformation syndrome than conventional gene mapping technologies.

Longitudinal Study of Growth of Children With Unilateral Cleft-Lip Palate From Birth to Two Years of Age

Objective: To study the growth of children with complete unilateral cleft lip and palate (UCLP) from birth to 2 years of age and to construct specific UCLP growth curves. Design: Physical growth was a secondary outcome measure of a National Institutes of Health-sponsored longitudinal, prospective clinical trial involving the University of Florida (United States) and the University of Sao Paulo (Brazil). Patients: Six hundred twenty-seven children with UCLP, nonsyndromic, both genders. Methods: Length, weight, and head circumference were prospectively measured for a group of children enrolled in a clinical trial. Median growth curves for the three parameters (length, weight, head circumference) were performed and compared with the median for the National Center for Health Statistics (NCHS) curves. The median values for length, weight, and head circumference at birth and 6, 12, 18, and 24 months of age were plotted against NCHS median values and statistically compared at birth and 24 months. Setting: Hospital de Reabilitacao de Anomalias Craniofaciais, Universidade de Sao Paulo, Bauru, Brazil (HRAC-USP). Results: At birth, children of both genders with UCLP presented with smaller body dimensions in relation to NCHS median values, but the results suggest a catch-up growth for length, weight, and head circumference for girls and for weight (to some degree) and head circumference for boys. Conclusions: Weight was the most compromised parameter for both genders, followed by length and then head circumference. There was no evidence of short stature. This study established growth curves for children with UCLP.

Rehabilitative treatment of cleft lip and palate: experience of the Hospital for Rehabilitation of Craniofacial Anomalies/USP (HRAC/USP) - Part 1: overall aspects

C left lip and palate is the most common among craniofacial malformations and causes several esthetic and functional implications that require rehabilitation. This paper aims to generally describe the several aspects related to this complex pathology and the treatment protocol used by the Hospital for Rehabilitation of Craniofacial Anomalies, University of So Paulo (HRAC-USP) along 40 years of experience in the treatment of individuals with cleft lip and palate.

Nasolabial symmetry and esthetics in cleft lip and palate: analysis of 3D facial images

OBJECTIVES To determine the relationship between nasolabial symmetry and esthetics in subjects with orofacial clefts. MATERIAL AND METHODS Eighty-four subjects (mean age 10 years, standard deviation 1.5) with various types of nonsyndromic clefts were included: 11 had unilateral cleft lip (UCL); 30 had unilateral cleft lip and alveolus (UCLA); and 43 had unilateral cleft lip, alveolus, and palate (UCLAP). A 3D stereophotogrammetric image of the face was taken for each subject. Symmetry and esthetics were evaluated on cropped 3D facial images. The degree of asymmetry of the nasolabial area was calculated based on all 3D data points using a surface registration algorithm. Esthetic ratings of various elements of nasal morphology were performed by eight lay raters on a 100 mm visual analog scale. Statistical analysis included ANOVA tests and regression models. RESULTS Nasolabial asymmetry increased with growing severity of the cleft (p = 0.029). Overall, nasolabial appearance was affected by nasolabial asymmetry; subjects with more nasolabial asymmetry were judged as having a less esthetically pleasing nasolabial area (p < 0.001). However, the relationship between nasolabial symmetry and esthetics was relatively weak in subjects with UCLAP, in whom only vermilion border esthetics was associated with asymmetry. CONCLUSIONS Nasolabial symmetry assessed with 3D facial imaging can be used as an objective measure of treatment outcome in subjects with less severe cleft deformity. In subjects with more severe cleft types, other factors may play a decisive role. CLINICAL SIGNIFICANCE Assessment of nasolabial symmetry is a useful measure of treatment success in less severe cleft types.

Genome scan for teratogen-induced clefting susceptibility loci in the mouse: Evidence of both allelic and locus heterogeneity distinguishing cleft lip and cleft palate

Nonsyndromic clefting of the lip and palate in humans has a highly complex etiology, with both multiple genetic loci and exposure to teratogens influencing susceptibility. Previous studies using mouse models have examined only very small portions of the genome. Here we report the findings of a genome-wide search for susceptibility genes for teratogen-induced clefting in the AXB and BXA set of recombinant inbred mouse strains. We compare results obtained using phenytoin (which induces cleft lip) and 6-aminonicotinamide (which induces cleft palate). We use a new statistical approach based on logistic regression suitable for these categorical data to identify several chromosomal regions as possible locations of clefting susceptibility loci, and we review candidate genes located within each region. Because cleft lip and cleft palate do not frequently co-aggregate in human families and because these structures arise semi-independently during development, these disorders are usually considered to be distinct in etiology. Our data, however, implicate several of the same chromosomal regions for both forms of clefting when teratogen-induced. Furthermore, different parental strain alleles are usually associated with clefting of the lip versus that of the palate (i.e., allelic heterogeneity). Because several other chromosomal regions are associated with only one form of clefting, locus heterogeneity also appears to be involved. Our findings in this mouse model suggest several priority areas for evaluation in human epidemiological studies.

Self-Concept and School Achievement of Pupils with Cleft Lip, Cleft Palate or Both : A longitudinal study

The goal of this research was to survey the self-concept and school achievement of pupils with cleft lip, cleft palate or both from juvenile age to adolescence. Longitudinal researches of self-concept and school achievement among pupils with cleft lip, cleft palate or both are uncommon. This research was the first longitudinal research ever conducted in Finland among this population. This research can be considered to be a special educational study because of the target group involved. Self-concept consists of the person s entire personality. Personality is biological and deterministic. Self-concept includes concepts, attitudes and feelings that the person has about him or her qualities, abilities and relations to the environment. The individual associates experiences to this personality with earlier observations through the social interaction. The individual will have the consciousness of the person s existence and action. The target group in this study consisted of Finnish children with clefts, who were comprised of four different age groups. The questionnaire was sent to all subjects (N1 = 419) both times. A total of 74 % of children returned the questionnaire in 1988 (N2=305). 48 % of children returned the questionnaire in 1993 (N3=203). 42% of children returned the questionnaire both times (N4=175) . These 175 children formed the research subjects. The survey was conducted in 1988, and again in 1993. In 1988, the pupils surveyed were 9 to 12 years of age, while in 1993 they were between 14 and 17 years old. The data was collected through the use of a questionnaire, which consisted of common questions and a personality inventory test that was developed for Finnish students by professor Maija-Liisa Rauste-von Wright. Quantitative analysis methods were used to examine the structure of self-concept and school achievement. Structures found in this research were observed in relation to disorder, gender and maturation. According to these results, structures of self-concepts and school achievement are in fact stable. Basic self-concept elements are seen to be formed at an early age. The developmental aspects of self-concept following puberty are observed as the stability of self-concept and as the forming of a general self. The level of school achievement is stable, but the structure of school achievement changes. From these results, it is possible to state that the gender of the child has a statistical significance regarding self-concept and school achievement. However, the experienced disorder does not have statistical significance as regards to self-concept and school achievement. Results of self-concept support the research of self-concept conducted earlier in Finland.