Abstract: DESCRIPTION (provided by applicant): Craniosynostosis (CS), the premature fusion of one or more cranial sutures, is a common, major structural birth defect occurring in about 1 in 2,500 live births. About 85% of infants with CS present with nonsyndromic craniosynostosis (NCS) without associated birth defects or developmental delays. NCS is a heterogeneous condition with presumed multifactorial etiology and its causes remain largely unknown. Primary prevention strategies for NCS are limited. Our International Craniosynostosis Consortium (ICC) has advanced understanding of the genetic etiology for sagittal NCS (sNCS). Through our previous NIH-NIDCR funding (R01 DE016866), we successfully conducted the first genome-wide association study (GWAS) for sNCS and identified robust associations to loci near BMP2 and BBS9, both biologic plausible genes involved in skeletal development. A similar GWAS with 415 case-parent trios with metopic NCS (mNCS) is in progress, as is an additional GWAS of over 600 coronal NCS (cNCS) case-parent trios. Additionally, others reported that by whole exome sequencing (WES), SMAD6 mutations were found in 7% of probands in a cohort of sNCS, mNCS, or combined NCS cases. Importantly, among 17 NCS cases with SMAD6 mutations, 14 had T>C mutation (rs1884302) downstream of BMP2, suggesting a two-loci inheritance model. This discovery of an epistatic interaction between BMP2 and SMAD6 through use of GWAS and WES approaches explains only a small proportion of all NCS cases. Along with the data generated from the completed and ongoing GWAS’s, we believe that whole genome sequencing (WGS) is the next important step towards identifying causal variants in NCS cases, because it has the power to discover rare and common variants missed by other high- throughput technologies. We hypothesize that WGS will identify novel genetic factors beyond those identified with GWAS’s that contribute to the etiology of NCS. In this application, we propose to investigate 600 case- parent trios (200 cases each with sNCS, cNCS, and mNCS) and 20 multiplex families (11 with sNCS and 9 with mNCS) using WGS for discovery of all types of germline variants (de novo and inherited single nucleotide variants, insertions/deletions and structural variations). Somatic mutations contribute to the etiology of cancer and have been reported in some structural birth defects. Thus, we will perform WGS on 25 paired blood- derived and bone-derived DNA specimens obtained from sNCS probands for detection of somatic mutations. Our discovery specimen repository represents one of the largest collections compiled, and along with our extensive collection of independent specimens for future replication studies, represents an unparalleled resource for studying the genetic etiology of NCS. Given our past accomplishments, experienced interdisciplinary research team, and substantial resources, we are well-positioned to successfully complete the proposed research and provide critical insights into the multifactorial etiology of NCS. PUBLIC HEALTH RELEVANCE: Nonsyndromic craniosynostosis (NCS) is a common, major structural birth defect – due to the premature fusion of one or more cranial sutures – that requires extensive surgical correction and is associated with considerable ongoing medical problems and health care costs. Because little is known about the causes of NCS, whole genome sequencing will help advance knowledge of genetic factors contributing to the etiology of NCS. Sequencing data generated will lead to a better understanding of biological processes involved in the etiology of NCS and provide critical insights for development of early diagnostic tools and therapeutic strategies.

Abstract: DESCRIPTION (provided by applicant): PROJECT SUMMARY Principal Investigators: Dr Azeez Butali is a tenure-track Assistant Professor at the Iowa Institute for Oral Health Research, College of Dentistry, and the University of Iowa. His primary research focus is on the genetics and epidemiology of complex traits including orofacial clefts. Dr Terri Beaty is a Professor at the John Hopkins University. Her research focus is on genetic epidemiology studies of several chronic diseases with complex etiologies, where both genetic and environmental risk factors control risk of disease. Co-investigators: Dr Adebowale Adeyemo is Deputy Director at the National Human Genome Research Institute. His focus is on the genetics and genomics of complex traits in African population. Dr Marazita is a Professor at the University of Pittsburgh. She is an expert in statistical genetics application for complex traits and identification of sub-clinical cleft phenotypes. Dr Cao is an Assistant Professor at the University of Iowa. He uses bioinformatics tools to interrogate the human genome and for analyses of gene-regulatory networks. Dr Ruczinski is a Professor at the John Hopkins University. His expertise is in statistical genetics, genomics and proteomics of complex traits. Dr Taub is an Assistant Scientist at the John Hopkins University. Her area of expertise is in genomics and statistical genetics for gene expression data, genotyping data and DNA methylation data Environment: The University of Iowa is a leading institution with a strong reputation for excellence in teaching, research and healthcare. The John Hopkins University is one of the leaders in the research, teaching and healthcare in the US. Both institutions are consistently amongst centers supported by NIH grants Research Study: The focus of this study is to identify novel risk variants for OFC in Africa and Asian OFC case-parent triads through analysis of Whole Genome Sequencing data. PUBLIC HEALTH RELEVANCE: TITLE: Whole Genome Sequencing of African and Asian Orofacial Case-Parent Triads The long term goal of this study is to identify specific genomic variants through WGS of OFC case-parent triads from African and Asian populations. The knowledge gained from these WGS studies will drive future research on OFC and should eventually lead to more effective interventions to reduce the risk of OFC.

Abstract: DESCRIPTION (provided by applicant): Project Summary/Abstract Congenital diaphragmatic hernia (CDH) is defined as a defect in the muscular or tendinous portion of diaphragm that results in antenatal herniation of the abdominal contents into the thoracic cavity and pulmonary hypoplasia due to compression of the lungs. The incidence of CDH is 1 in 3000 live births, accounting for 1- 2% of infant mortality and 8% of all birth defects, making it one of the most common and lethal congenital anomalies. CDH is isolated in 50-60% of cases but is associated with other major anomalies, most commonly congenital heart disease or central nervous system malformations, in the remaining 40-50%. Historically CDH carried a grave prognosis with mortality of greater than 50%. However, with recent advances in the post-natal care of children with CDH, survival has improved significantly. However, with improved survival, many of the long term morbidities of CDH have been exposed including pulmonary hypertension, the leading cause of CDH morbidity and mortality. Many families and health care providers seek prognostic clinical information about other associated birth defects or genetic syndromes, but prognostic data are extremely limited unless a chromosomal anomaly is identified. The etiology of CDH is largely unknown. Evidence is accumulating that many congenital anomalies can result from copy number variants, de novo mutations, and inherited rare mutations, often unique to the family. We propose to elucidate the underlying genomic architecture of CDH and define new genes and conditions associated with CDH by performing whole genome sequencing on parent child trios and RNA sequencing of diaphragm tissue in a clinically well characterized cohort to identify rare de novo mutations and inherited variants. Our long-term goal is to define a set of genes important in the etiology of CDH and characterize new clinical syndromes associated with CDH. We believe this information will improve genetic diagnostic methods and provide more accurate clinical prognostic information to guide clinic decisions. PUBLIC HEALTH RELEVANCE: Congenital diaphragmatic hernia (CDH) is a serious birth defect accounting for 1-2% of infant mortality and 8% of all birth defects. We propose to elucidate the underlying genomic architecture of CDH by performing whole genome sequencing and RNA sequencing on diaphragm tissue to characterize new clinical syndromes associated with CDH to provide more accurate clinical prognostic information.

Abstract: DESCRIPTION (provided by applicant): Evidence of a connection between childhood cancers and birth defects comes from three major sources: clinical observations of syndromes, registry linkages, and case-control studies. These studies demonstrate that children with a variety of birth defects have a significantly increased risk of developing several types of childhood cancers. However, due to the sparsity of cases, few risk factors have been consistently confirmed for specific types of birth defects and childhood cancers, and the etiology of most of these entities remains unexplained. This proposal will leverage the unique resources of The Center for Applied Genomics (CAG) at The Children’s Hospital of Philadelphia (CHOP) which houses the largest genomic facility/pediatric biobank in the US. We have identified 1,205 pediatric cancer patients that were also diagnosed with a birth defect from the CAG biobank. All have banked DNA samples from peripheral blood that are ready for sequencing together with age, sex and ethnically matched controls. The patients are from diverse backgrounds and the majority of them authorize re-contact. This study will utilize two complementary analytical approaches to disease gene discovery. Patients with parental sequences will be analyzed as trios in a typical winnowing variant prioritization approach. We also propose to sequence matched controls for each of the cases allowing for powerful statistical case control approaches, namely burden tests, to be applied to the dataset. Two strengths of this study design are the large sample sizes for what are rare phenotypes and the combination of birth defects and childhood cancers in all cases which are more likely to be burdened with low frequency variants that confer risk and that more impactful variants are more likely to be discovered. PUBLIC HEALTH RELEVANCE: Birth defects and childhood cancer share biological pathways that are important for cell growth and division. We propose that sequencing pediatric patients suffering both conditions will allow us to discover the underlying genes and in turn advance our understanding of the causes of these devastating diseases.

Abstract: DESCRIPTION (provided by applicant): Craniofacial microsomia (CFM), also termed hemifacial microsomia or oculo-auricular-vertebral spectrum, is the third most common congenital craniofacial condition. CFM has an estimated birth prevalence in the US of 1 in 3,500-5,600, which is similar to conditions such as cystic fibrosis (1 in 3,700) and neurofibromatosis (1 in 4,200). CFM comprises a variable phenotype, and the most common features include malformations of the ear (i.e. microtia) and lower jaw (i.e. mandibular hypoplasia) on one or both sides. The etiology of CFM is largely unknown; however the presence of multiple cases within families, mouse models with CFM malformations and the increased risk of CFM in some ethnicities suggest that genetic variants contribute to its occurrence. Although chromosomal abnormalities have been associated with CFM, only three causative genes have been identified in few cases: HOXA2, FGF3, and MYT1. Our goal in this proposal is to identify coding and non- coding variants that are genetic risk factors to CFM by performing whole-genome sequencing (WGS) of case- parent trios with CFM. We propose to perform whole genome sequencing on DNAs from 105 trios (individuals with CFM and their parents or affected relatives in multi-affected families) to identify candidate genes with rare de novo and inherited variants. Our hypothesis is that CFM is caused by rare new and inherited DNA variation in gene(s) related to the craniofacial development. We will analyze the data on rare de novo coding and non- coding variants. Recognizing reduced penetrance in CFM, our analysis will include analyses for variants in a dominant inheritance with incomplete penetrance model. Our approach incorporates detailed phenotype, clinical characterization, and family history for each individual. We will also integrate the WGS data with our data on gene expression from murine embryonic pharyngeal arch and external ear human embryonic tissue to ascertain tissue specific expression at the relevant time of the development of tissues in CFM. Our statistical power by sampling patients with familial and severe disease who are most likely to have a high genetic loading. CFM represents an ideal condition in which to identify susceptibility variants because it is (1) relatively rare and represents more extreme selection under a liability threshold model, (2) distinctive, (3) stable, and (4) This study will be conducted by an interdisciplinary team with complementary expertise in clinical aspects of CFM, clinical genetics, genomics, and bioinformatics. Successful completion of this proposal will advance knowledge in the genetic architecture of susceptibility to CFM and will provide insight about the biological mechanisms underlying craniofacial development. The phenotypic and genomic data will be fully integrated into the Kids First Data Resource and available to all qualified investigators. The long-term goal of this project is to identify specific genetic risk factors to improve genetic counseling, enable tailored clinical care, and to provide more accurate prognosis. often familial (20-40% of cases). PUBLIC HEALTH RELEVANCE: Craniofacial microsomia (CFM) is the third most prevalent condition that affects craniofacial development; however, the cause of CFM is unknown for most affected individuals. We have established a cohort through previous studies and collected DNA to identify the genetic contributions to CFM, which could facilitate diagnosis, tailored treatment and guide prevention strategies. The results from the proposed study have potential to further research on the etiology of other craniofacial disorders, and the pathogenesis of typical and atypical craniofacial development.

Sequence and clinical data released in dbGap: Accession Number:phs002130

Abstract: DESCRIPTION (provided by applicant): Expanded Ewing sarcoma cohort for tumor genomics and association with DNA repair deficiencies, clinical presentation, and outcome Ewing sarcoma (ES) is the second most common bone tumor in children and adolescents, but is still relatively rare without much known about genetic risk and only small cohort studies linking tumor genomics to clinical features. Our group was awarded an initial Gabriella Miller Kids First (GMKF) Pediatric Research Program (X01) to study a cohort of germline DNA samples from the Project GENESIS cancer epidemiology study (Genetics of Ewing Sarcoma International Study, COG AEPI10N5), which included whole genome sequencing (WGS) on 329 ES trios (patient-mother-father) plus 123 individual ES patients, with 327 ES patients from the Children’s Oncology Group (COG). The GMKF WGS germline data are now in the process of being returned for analysis. For this expanded study to explore ES tumor genomics, we have assembled a team of leading biologists and clinical scientists in the field of ES along with leaders in the field of genomic sequence analyses and data storage. Utilizing the COG ES Biobank, we will request and submit for sequence analysis the available tumor pairs to the 185 COG ES germline trios plus an additional set of paired COG germline-tumor samples (N=315) to analyze the largest cohort ever assembled of 500 ES germline-tumor pairs for deep sequencing. Using our teams combined expertise, this ES expanded GMKF X01 will test the hypothesis that germline DNA repair deficiencies (as determined by DNA repair gene variants and germline rates of de novo alterations) will contribute to specific tumor genomic features including burden of genomic instability, translocation subtype, transcription profiles, and tumor subclonal heterogeneity (Aim 1). We also will test the hypothesis that these DNA repair deficiencies reflected in the germline will correlate with clinical features of presentation including patient age, sex, tumor site, and tumor stage, as well as test for any correlation between our measured tumor genomic features and clinical presentation (Aim 2). Finally, we will test the hypothesis that the same germline and tumor genomic features will correlate with clinical outcome of the ES patients as reflected in event-free survival and overall survival (Aim 3). Importantly, this study will allow us to determine the prevalence and clinical significance of ES-like tumors that previously were included in ES biological and clinical trials. This expanded GMKF X01 study including ES tumor genomics represents the largest and most comprehensive ES genomic analysis of its kind, and builds upon the successful sequencing of previous ES germline samples through the GMKF X01 program. PUBLIC HEALTH RELEVANCE: Ewing sarcoma (ES) is the second most common bone tumor in children and adolescents, but previous genomic studies have been limited due to this tumor’s overall rarity and difficulty linking to clinical features. Building upon our previous success with sending ES germline samples for whole genome sequencing (WGS) through the Gabriella Miller Kids First (GMKF) Pediatric Research Program (X01), we now will expand our cohort to include tumor samples from within the Children’s Oncology Group (COG) ES Biobank that have been linked to COG clinical trials. We will explore the association between ES tumor genomics and underlying germline DNA repair deficiencies, clinical presentation, and clinical outcome.

Abstract: DESCRIPTION (provided by applicant): Infantile hemangiomas are the most common benign vascular tumor in infants, affecting 4-5% of children. Thirty percent of segmental infantile hemangiomas on the face and scalp are associated with birth defects of multiple organs. This condition is known as PHACE, an acronym for posterior fossa brain malformations, segmental facial hemangiomas, arterial anomalies, cardiac defects, eye anomalies, and sternal clefting. There is high morbidity associated with PHACE including risk to vision, congenital heart disease often requiring surgery, risk of stroke, deafness and neurodevelopmental delays. The hemangioma is a vascular tumor that requires treatment in infancy to prevent functional complications and disfigurement, but later undergoes involution. Our strategy is to use this highly valuable PHACE cohort to discover critical genes related to structural birth defects which will be a valuable resource to link multiple different projects in the Kids First Program. We hypothesize that PHACE is caused by variants that occur very early during development resulting in birth defects of multiple organs and infantile hemangiomas. Aim 1: Use a custom bioinformatics pipeline to detect candidate variants for PHACE, Aim 2: Contribute the data generated in this project to the Kids First Data Resource and the National Center for Biotechnology Information's (NCBI) and Database of Genotypes and Phenotypes (dbGaP). PUBLIC HEALTH RELEVANCE: Genomic analysis of PHACE will inform treatment and expand knowledge about the causes of birth defects affecting the brain, arteries, heart, eye, midline development and hearing. The knowledge gained in this study will be used to drive strategies for prevention and provide critical targets for treatments for a range of birth defects and infantile hemangiomas.

Abstract: DESCRIPTION (provided by applicant): Chondrosarcoma is a malignant tumor that originates from cartilaginous cells. It is the third most common primary malignancy of bone after myeloma and osteosarcoma. It accounts for about 20% of bone tumors and is diagnosed in approximately 600 patients each year in the United States. Up to 40% of the chondrosarcomas arise from an enchondroma. Enchondromas are benign, intramedullary cartilaginous tumors of bone. They can be solitary or multiple and are present in >3% of the population. Enchondromatosis refers to a group of diseases characterized by multiple enchondromas including metachondromatosis (MC), Ollier disease (OD), and Maffucci syndrome (MS) among others. All have skeletal abnormalities with or without associated vascular anomalies that can cause severe limb deformities during early childhood. The risk for chondrosarcoma in OD is up to 45.8% and in MS up to 57.1%. Currently, the only treatment for patients with these disorders is surgical; there is no effective pharmacologic therapy. We identified heterozygous germline loss of function variants in PTPN11 (encoding a non-receptor protein tyrosine phosphatase SHP2) causing MC (Sobreira at al., 2010). In preliminary studies, we also identified the PTPN11 R138X variant in in a retiform hemangioendothelioma of a patient with MS and the germline PTPN11 L560F variant in a patient with OD. PTPN11 encodes SHP2, a cytosolic protein tyrosine phosphatase involved in an early step in RAS/MAPK signaling downstream of several receptor tyrosine kinases including EGFR and FGFR. Pansuriya et al. (2011) and Amary et al. (2011) identified heterozygous somatic variants of IDH1 (R132H, R132C, R132S) and IDH2 (R172S) in the tumors (enchondromas, chondrosarcoma, and hemangiomas) of a fraction of the patients with MS and OD. Neither variant was identified in the germline DNA of the affected individuals. On basis of these results, we hypothesize that OD and MS are tumor predisposition syndromes caused by germline variants. Moreover, these variants likely down-regulate the RAS/MAPK pathway or are in genes that interact with IDH1 or 2. Subsequent hits in the same or different genes such as IDH1 and IDH2 or other as yet identified genes are involved in the formation of enchondromas and chondrosarcomas. PUBLIC HEALTH RELEVANCE: : Ollier disease and Maffucci syndrome are characterized by multiple enchondromas that can cause multiple swellings on the extremity, deformity around the joints, Madelung deformity, angular deformity such as genu valgus, gene varum, cubitus valgus, coxa vara and coxa valga, limitations in joint mobility, scoliosis, bone shortening, leg-length discrepancy, gait disturbances, pain and loss of function, pathological fractures, facial asymmetry and cranial nerve palsies and the risk of developing a chondrosarcoma in patients with Ollier disease is up to 45.8% and up to 57.1% in patients with Maffucci syndrome. In addition, gliomas, acute myeloid leukemia, and juvenile granulosa cell tumors have been found in patients with OD and pancreatic and hepatic adenocarcinoma, mesenchymal ovarian tumors, brain tumors such as glioma and astrocytoma, and various kinds of sarcomas are observed in patients with MS (Verdegaal et al., 2011). The molecular basis of this two disorders is not completely understood and currently, there is no effective drug therapy for these disorders.