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No description available.

No description available.

No description available.

Cleft lip and/or palate (CL/P) is a group of congenital birth defect caused by the failure of the lip and/or palate to properly fuse during facial development. This defect occurs in approximately 1:700 live births and is the most second most common developmental defect. Twin studies and evaluation of family history reveals that risk for CL/P is influenced by genetics. However, to date less than half of the heritable risk for CL/P has been ascribed to specific genes. To identify new genes involved in CL/P, our colleagues, Dr. Manak and Dr. Murray, screened DNA from CL/P patients for rare copy number variants. A single copy deletion of Isthmin1 (ISM1) was identified in this screen. To test the hypothesis that this deletion contributed to the pathogenesis of CL/P we conducted functional tests of the zebrafish ortholog, isthmin1. The results indicated that Ism1 is necessary for development of the face in zebrafish, supporting the hypothesis. Together with Dr. Bassuk, we applied a similar approach to another structural birth defect, spina bifida. Dr. Manak discovered a de novo single copy deletion encompassing Glypican 5 (GPC5) and part of Glypican 6 (GPC6) in a spina bifida patient. Our functional tests in zebrafish support the notion that mutations in GPC5 cause spina bifida in some patients. To identify additional CL/P loci, we investigated two putative transcriptional targets of Interferon Regulatory Factor 6 (IRF6), a transcription factor encoded by a gene which is mutated in the majority of patients with Van der Woude orofacial clefting syndrome. The Cornell lab found evidence that Irf6 regulates expression of grhl3 and klf4 in zebrafish periderm. Partly in response to our findings, Jeff Murray's group sequenced the coding region GRHL3 gene in Van der Woude patients lacking IRF6 mutations and found 8 different coding mutations in GRHL3. We tested 5 of theses GRHL3 mutations in zebrafish-based functional studies, and found the 5 patient-derived GRHL3 variants had dominant negative effects. We conclude that GRHL3 is indeed a CL/P locus. Because all the patient derived variants were dominant negative (as opposed to null), and because such variants would be expected to block the function of the other copy of GRHL3 as well as other family members (GRHL1 and GRHl2), which are also cleft candidate genes. Multiple KLF4 coding mutations were also detected in patients with CL/P. We tested one of them but did not detect evidence of disruption in protein function. We conclude that this mutation does not seem to affect the function of KLF4, even though it was predicted to be damaging. This enforces the idea that conclusions from in silico studies should be examined in vivo. Finally, the protein structure of Irf6 has been examined to identify important residues within the C-terminus of the protein. Using constructs built by Dr. Mankad, we tested 5 different phosphor-mimetic amino acid substitutions. Of the variety of constructs, only Irf6 S447D in zebrafish Irf6 was able to cause ectopic expression of downstream Irf6 targets. This predicts that phosphorylation at S447 is sufficient to activate Irf6. All of these studies have expanded our understanding of the genetics behind CL/P, either by discovering new loci in human patients and testing them in Danio rerio, or from finding downstream targets of Irf6 in zebrafish, sequencing human patients for mutations in those genes, and then testing the functional changes of those variants. From our work with zebrafish, we can determine additional components of the IRF6 regulatory network.

Clefts of the lip and/or palate (CL/P) are one of the most common birth defects in the world occurring about every 1 in 700 live births. Individuals with non-syndromic clefting (NSCL/P) account for about 70% of all cleft cases and exhibit a cleft only whereas syndromic occurrences (SCL/P) include additional cognitive or structural abnormalities. Linkage, genome-wide association, candidate gene, animal model, sequencing and copy number variant (CNV) analyses have been used to study CL/P and have established that it is a heterogeneous, complex disorder. However, the impact of identified sequence variants on protein structure and the contribution of structural genetic variation to CL/P remains poorly understood. In our first analysis we reassessed the phenotype of a 30-year-old individual of SCL/P and noticed phenotypic overlap with Hartsfield syndrome, a rare syndrome resulting from sequence variants in Fibroblast growth factor 1 (FGFR1). We sequenced the coding region of FGFR1 and identified a novel, de novo variant. Due to the fact sequence variants in FGFR1 contribute to multiple syndromes encompassing a wide phenotypic spectrum, we performed an extensive literature search to record every published sequence variant of FGFR1 and mapped it to the protein structure by disease and phenotype. Although no statistically significant protein domain-phenotype correlations were identified, many regions neared significance. This work stresses the need for systematic, comprehensive phenotyping of patients and provides a method for assessing the impact of the location of sequence variants within the 3D structure of the protein. Although rare and common CNVs have been identified in individuals with CL/P, prior to our work no large-scale studies of rare CNVs for the identification of novel clefting genes had been performed. For our second set of analyses, we conducted two such studies, first focusing on a smaller cohort of 140 individuals with NSCL/P from the Philippines to establish our informatic and functional validation pipeline. We used whole-genome tiling arrays to assess rare deletions overlapping genes not previously implicated in clefting, and identified one deletion overlapping Isthmin1 (ISM1) and a deletion just 3’ of the gene in a second affected individual. Functional validation of Ism1 in Xenopus laevis showed strong expression in structures necessary for craniofacial development, and morpholino and CRISPR/Cas9 knockdown of Ism1 resulted in a median cleft lip in some embryos, establishing ISM1 as a novel craniofacial patterning gene. We then expanded our study and assessed genomic CNVs in 1021 individuals with NSCL/P and 81 individuals with SCL/P, finding no differences in CNV number, load or burden between these groups. We also identified 8 putative clefting genes overlapped by deletions in two or more individuals but at a rare (< 1% frequency) in the cohort. Functional validation of these genes using CRISPR/Cas9 in zebrafish and Xenopus tropicalis is currently underway. This work has identified a novel sequence variant leading to the diagnosis of Hartsfield syndrome in an individual with SCL/P, developed an innovative method for assessing the impact of sequence variation on protein structure, improved our understanding of the contribution of CNVs to SCL/P and NSCL/P and identified several putative novel clefting loci which may help explain a portion of the missing heritability of CL/P.

Orofacial clefts are among the most common congenital defects in the United States, affecting roughly 1 in 600 births annually. A majority of these cases are considered to be “isolated” clefts of the lip and/or palate (ICLP). However the term “isolated” is somewhat of a misnomer, as functional deficits frequently accompany ICLP. One of the most problematic yet understudied of these deficits involves the high prevalence of reading disabilities in this population. It has been estimated that as high as 46% of children with ICLP will be diagnosed with a reading disability, particularly dyslexia. Despite this high prevalence and the well-established neurological basis of dyslexia, relatively little attention has been paid to the role that brain development plays in the reading problems in ICLP. Previous studies from our lab have demonstrated significant changes in brain structure in children with ICLP (that have importantly correlated with functional measures). However we have yet to combine both a structural and functional neuroimaging study with an in-depth analysis of reading dysfunction in this population. The current study examined boys with ICLP, age 8-12 (boys have a higher prevalence of ICLP and show more significant reading problems that girls with ICLP) compared to healthy control boys. Measures of cognitive functioning were obtained with an emphasis on reading and language skills. In addition MRI scans were obtained which included volumetric measures, diffusion-weighted measures (DWI; white matter), and connectivity measures (resting-state fMRI). Even after controlling for the effect of socioeconomic status, boys with ICLP showed significant decreases in reading and language skills (particularly reading fluency). Boys with ICLP did not show significant differences on phonlogical measures (the primary cause of dyslexia). In addition, phonological measures were not predictive of reading fluency, while object naming tasks were predictive of reading fluency in boys with ICLP. For white matter integrity, measures of fractional anisotropy (FA) were found to be increased in the right occipital lobe for boys with ICLP indicating more organized white matter in this region. This increase in right occipital FA was also predictive of better reading outcomes, particularly reading fluency. For more specific white matter tracts, only the fornix and the tapetum (both associated with the temporal lobes) showed a significant difference with a decrease in FA for boys with ICLP. The decrease in FA in the tapetum was also predictive of better reading outcomes in ICLP. When looking at resting-state networks, boys with ICLP showed an increase in connectivity within posterior and subcortical regions when compared to healthy control boys, indicating stronger network connections within the posterior language regions of the brain. Taken together, these results point to differences in both structural and functional connectivity in the brains boys with ICLP. Furthermore, this pattern is different than that found in children with developmental dyslexia as there appears to be no disruption of the posterior reading systems. Cognitive measures also indicate normal phonological awareness in this group, further distinguishing them from dyslexic children. Boys with ICLP instead may be over-relying on these posterior, more visually oriented reading systems as a compensatory mechanism to overcome problems with the development of the typical “lexical route” of reading.

Background: Nonsyndromic cleft lip and palate (NSCL/P) is a complex trait caused by genetic and environmental factors that interact producing a wide spectrum of orofacial malformations, including dental anomalies. The underlying genetic etiology that accounts for phenotypic variation in affected families is poorly understood. Purpose: The purpose of this study is to utilize shape and microesthetic analysis to characterize the maxillary anterior dentition in unaffected parents of children with NSCL/P (cases) compared to control adults with no CL/P history to identify dental morphology features that are part of the NSCL/P phenotypic spectrum and can therefore be used in refining NSCL/P phenotypes and identifying genetic risk factors. Methods: Individuals were recruited from 5 sites including Iowa, Texas, Hungary, the Philippines, and Pittsburg, PA. From a total of 3202 individuals, 420 quailified after strict selective criteria. Digital photographs from 198 cases and 222 controls were analyzed using linear metrics and 2D-coordinate landmark-based geometric morphometrics (GM) to compare dental esthetics and deviations from golden proportions." Differences in central incisor and connector height proportions were evaluated using paired T-tests. Anterior tooth shapes were examined using GM techniques. Results: Three shape differences were found to be possible predictors of genetic risk. These included shorter maxillary anterior teeth overall, square shaped lateral incisors on the left side, as well as lateral incisors and canines with long axes angled inward toward the midline on the left side. Both the case and control groups were found to be significantly different than the proposed ideal values of tooth proportions. Conclusions: Significant differences in anterior dental morphology were found between cases and controls, with controls displaying a more ideal dental morphology than cases for most evaluated measures. The identification of these distinct dental features in carriers of NSCL/P genetic risk factors further characterizes the phenotypic spectrum of NSCL/P which can enhance the power of genetic studies.

Orofacial clefts are a heterogeneous group of craniofacial malformations that affect the lip and/or palate and represent the most common craniofacial birth defect in humans. In 30% of patients the cleft is accompanied by additional physical or cognitive abnormalities. Hundreds of these clefting syndromes have been described, many of which have Mendelian inheritance patterns. The most common of these is Van der Woude syndrome (VWS), caused by mutations in the transcription factor IRF6 (Kondo et al. 2002). The other 70% of patients lack additional features and are considered nonsyndromic. The etiology of nonsyndromic clefts is complex and involves the combined action of multiple genetic variants interacting with environmental factors. A common approach for identifying genetic risk factor for complex disorders such as nonsyndromic cleft lip with or without cleft palate (NSCL/P) is the genome wide association study (GWAS). We pursued a locus on 1p22 shown to be associated with NSCL/P by Beaty et al. (2010). Through a combination of expression studies in a mouse model and mutation screening in NSCL/P patients, we identified ARHGAP29 as a novel gene for NSCL/P and the likely etiologic gene at this locus. We identified eight rare variants in NSCL/P patients absent in controls including a nonsense and a frameshift mutation. These rare variants are reminiscent of previous resequencing studies that reported rare coding mutations in 20 different candidate genes for NSCL/P. We reviewed these variants and compared them with variants found in over 7000 exomes from the 1000 Genomes Project (1kGP) and NHLBI Exome Sequencing Project (ESP) to identify the variants and genes most likely to contain etiologic rare variants. We found good support for a role for rare variants in NSCL/P, particularly for MSX1 and genes of the FGF signaling pathway. We next performed several studies to understand the genetic architecture of syndromic forms of clefting, focusing on VWS and popliteal pterygium syndrome (PPS), which is allelic to VWS. We compiled all of the nearly 300 published IRF6 mutations and compared the distribution of these mutations with IRF6 variants obtained from the 1kGP and ESP exomes. We found that mutations causing VWS were significantly over-represented in the DNA-binding domain and for the most part were absent from control exomes, indicating that they are likely to be truly causative for VWS or PPS. These mutations in VWS and PPS only account for 70% of VWS and 97% of PPS. We next hypothesized that mutations in RIPK4, which causes an autosomal recessive pterygia syndrome, could underlie the remaining VWS and/or PPS cases. We found novel homozygous mutations in RIPK4 in two PPS patients. This result has significant clinical ramifications, as counseling of recurrence risk is very different for PPS patients whose disease is caused by dominant IRF6 mutations compared to recessive RIPK4 mutations. Finally, to understand the variable expressivity of VWS and PPS we performed an association study to identify genetic modifiers. We also looked for genotype-phenotype correlations between the type and location of IRF6 mutations. Although we did not find strong evidence that the candidate genes we selected from GWAS of NSCL/P or other clefting syndromes are modifiers of the VWS or PPS phenotypes, several marginal associations suggest that members of the IRF6 gene regulatory network could act as modifiers. Finally, we found evidence of a larger genotype-phenotype correlation by demonstrating that mutation-negative VWS families have a deficiency of cleft lip phenotypes. Together this work has advanced our understanding of the genetic basis of this diverse set of cleft lip and palate disorders, informing both the biology of craniofacial development and the clinical care of patients affected by these disorders.

Nonsyndromic cleft lip and/or palate (NS CL/P) is a common birth defect with estimated birth prevalence of 1/1000 worldwide. NS CL/P etiology may be explained by the action of as few as two or as many as 14 different genes (Schliekelman et al. 2002) as well as several environmental factors (e.g. smoking and folic acid) (Shi et al. 2008; Wehby et al. 2010). Convincing genetic and/or biologic evidence exists for the contribution of many genes: IRF6, 8q24, FGFR2, FOXE1, BMP4, TGFβ3, MSX1, MAFB , PAX7, ABCA4, and VAX1, to NS CL/P (Dixon et al. 2011). Clefts place substantial burdens on families and society with the estimated costs of cleft care and surgical repair around $100,000 in the United States (Jugessur et al. 2009). The health burden of NS CL/P does not end after the last surgery - impaired social development, surgical morbidity, and cancer are all reported in older children and adults with NS CL/P. Genes involved in clefting during development may be involved in other processes throughout life. We hypothesize that non-coding variation of the FGFR2 gene is associated with NS CL/P and these variants correlate with levels of gene expression. We investigate links between clefting, wound healing, and genes implicated in cancer in NS CL/P families. Analysis of family genotype data with the Transmission Disequilibrium Test (TDT), demonstrated suggestive association for two non-coding SNPs, rs2912770 with CL/P (p = 0.002 -USA) and rs2114684 with cleft palate (p = 0.002 - USA and all populations) but not for SNPs located within intron 2 - where genome wide association (GWA) signals overlap in both NS CL/P and breast cancer studies. Studies of foreskin tissue supported correlation of rs2912770 genotype with level of FGFR2 expression, though the magnitude of the effect was modest and did not remain significant after correction for multiple comparisons. Transcriptome array analysis suggested novel regions of regulated transcription, but their function as novel exons was not validated by real-time PCR (RT-PCR). As we expanded our focus from FGFR2, we examined association of cleft candidate genes with wound healing complications (WHC) with case-control and TDT analysis. Allelic and genotypic case-control analysis suggests association of rs2981582 with WHCs (p < 0.08). TDT analysis revealed WHC potential association with rs6478437 located upstream of the ( FOXE1) gene (p = 0.04). This study has medical implications, as the identification of children at high-risk for WHCs prior to surgical treatment and interventions may prevent post-surgical wound healing complications. We finally examined NS CL/P association with genes that had prior evidence for a role in clefting and cancer. In this analysis, we observed TDT association of rs4746409, located in intron 4 of the zinc finger 365 ( ZNF365) gene (p = 0.0003). Interestingly, this gene and SNP were tested due to suggestive association with breast cancer (Turnbull et al. 2010) and NS CL/P from GWA analyses (Beaty et al. 2010), which strengthens the connection between NSCL/P and breast cancer risk. Identification of NS CL/P patients at increased cancer risk could save lives and extend healthy life for current cleft patients as the field continues to seek ways to intervene and prevent cleft lip and palate from occurring.