Abstract:

Myelomeningocele (aka meningomyelocele, MM) is the most severe form of spina bifida, a neural tube defect (NTD) in humans and the most common CNS birth defect. MM is considered a genetically complex disease, and occurs in 3.72/10,000 live US birth, and is partly preventable with prenatal folate, but the genetic basis and the mechanisms by which folate work to reduce disease incidence remain obscure. MM is associated nearly uniformly with prenatal hydrocephalus and the Arnold-Chiari malformation, as well as paraplegia and lifelong neuromotor disability. The genes for several rare syndromic forms of NTDs are known, but the causes for the majority with sporadic MM remain unknown. Despite the importance of MM, most previous research has been limited to targeted sequencing and association studies of folate metabolism genes, or very small-scale exome sequencing. We hypothesize that de novo mutations (DNMs) produce likely gene disrupting (LGD) events that contribution to MM risk. Using conservative estimates of between 50-100 recurrently mutated discoverable genes contributing to risk, and our preliminary data demonstrating an excess of LGD DNMs in MM compared with control individuals, we estimate that with a cohort size of 1000 trios, we should uncover between 5-20 new recurrently mutated genes underlying MM, with minimal false-discovery. With this in mind, we formed the Spina Bifida Sequencing Consortium, and established a platform for data and sample sharing. Preliminary analysis of our first batch of 100 trios analyzed by WGS from GMKF suggests a wealth of important gene mutations. We have embarked on a new recruitment effort of an additional cohort of 400 new simplex MM trios, in collaboration with the US Spina Bifida Association, consented trios to allow for data sharing, and have performed detailed sample quality control. We also have preliminary data that use of dried bloodspot DNA from trios performs comparably to whole blood DNA, so will be happy to swap saliva for bloodspot recruitment at NIH’s preference. This cohort is now half-way assembled, with the remaining cohort to be ascertained in the next 6 months. We have established a workflow for de novo SNP/INDEL/SV detection from WGS and have ample computer storage and nodes to see the project to completion. We also plan to continue recruitment into the future with the goal of 2000 trios in the next 5 years. We propose a detailed bioinformatics workflow to identify gene mutations within a statistical framework, considering detailed scRNA expression profiling from developing mammalian neural tube, and have developed a robust functional validation workflow using Xenopus and mouse gene targeting. Our project has the potential to uncover a host of causes for this most common of the CNS birth defects, paving the way for future breakthroughs in detection, treatment, and prevention. PUBLIC HEALTH RELEVANCE: This work will identify new genetic disease genes predisposing to myelomeningocele, the most common pediatric structural brain disease.

Abstract:

Wilms Tumor is the most common renal tumor of childhood. Although cure rates approach 90% after initial therapy that includes a combination of surgery, chemotherapy and radiation therapy, our understanding of the biology of how children develop this cancer remains limited due to small patient cohorts. Prior studies have uncovered a number of important genetic alterations associated with Wilms Tumor. However these studies are based on small cohorts. Here, we propose to advance our prior studies with a multi-decade effort to obtain high quality samples from over 200 pediatric institutions through Children’s Oncology Group Renal Tumor studies. With samples from approximately 2,946 patients, we propose to assess the whole genome, methylome and transcriptome of the patient’s germline, normal adjacent kidney and tumor kidney. Given the large sample size, we will be powered for the detection of rare variant alleles and validation of prior studies. Just as importantly, our patient cohort represents the diversity of the United States. The multi-PI team along with senior leadership of the COG Renal Tumor studies have deep expertise in the analyses of epidemiology, genetics, epigenetics and transcriptomics in childhood cancers along with decades experience with the care of children with renal tumors. This proposed study provides a timely opportunity to aid our understanding of cancer risk in children with genitourinary congenital anomalies and more broadly, our understanding of Wilms Tumor, from a diverse population. These data will provide a critical resource for cancer germline risk, congenital anomalies, developmental biology and cancer biology. PUBLIC HEALTH RELEVANCE: Wilms Tumor is the most common kidney cancer in children. Although some predisposition syndromes have been associated with Wilms Tumor (e.g., Beckwith Wiedemann Syndrome, Denys Drash Syndrome, Hemihypertrophy, WAGR Syndrome), recent studies suggest many more children with Wilms Tumor may have an underlying predisposition syndrome. This study will explore how these germline changes relate to the developing kidney or structural birth defects in addition to the development of kidney cancer which may lead to prevention strategies or enhance risk stratification and therapeutic target identification.

Abstract:

Advances in sequencing have allowed identification of somatic variants, gene fusions, and copy number variants as potential therapeutic targets. Although myeloid disorders in children may show morphologic similarities to that seen in adults, TARGET AML initiative (Meshinchi, PI) clearly demonstrated that somatic genomic and transcriptome variants are highly distinct in children and young adults. In fact, there are a number of variants that are uniquely restricted to younger children, some with high therapeutic potential. In addition to identification of somatic variants, analysis of the germline data provided a glimpse into the constitutional make-up of patients with AML. The identification of numerous “function altering” variants may provide an insight into possible interactions between the host and the disease, where these germline variants might alter AML risk (predisposition), response to therapy (altering target expression, drug metabolism), susceptibilities to short and long-term complications (including infectious and cardiac complications), or modify risk of secondary malignancies. Armed with data from initial sequencing efforts in AML (including prior GMKF awards), we are poised to take full advantage of the available sequencing technologies to conduct the most comprehensive genome and transcriptome interrogation of myeloid disorders in children with specimens we have amassed over the last decade. To this end, we have put in place unparalleled specimen resources from children with de novo AML treated on prior Children’s Oncology Group trials, (AAML1031), to create the most comprehensive genome, transcriptome and epigenome profiling in AML. Our original X01 applications providing funding support for whole genome sequencing patients treated on AAML1031. Given that transcriptome (mRNA, miRNA, LncRNA), whole genome, and methylation data are available for the entire AAML1031 cohort, the addition of long-read sequencing would provide the most comprehensive profiling effort in this single trial cohort. Additionally, through an INCLUDE project award and private foundation funding, we have completed sequencing of whole genome, transcriptome, and epigenomic analysis of two COG trials (AAML1531 and AAML08B1) that investigate individuals with Down Syndrome who develop AML (DS-AML), as well as infants with Down Syndrome that have transient abnormal myelopoiesis (TAM) which often transitions to myeloid disease. Recent advances in long read sequencing provide an opportunity to identify structural alterations that are not amenable to detection by other methods. We propose to interrogate the genomes and transcriptomes of a cohort of affected children from AAML1031, AAML1531, and AAML08B1 from a variety of pediatric AML subtypes to discover cancer predisposition variants or structural alterations not detected by short-read sequencing. In addition, long read RNA-seq could provide detailed knowledge of the splice isoform variants that may be linked to AML pathogenesis, both as a route to better diagnostics and also for discovery of new therapeutic targets. PUBLIC HEALTH RELEVANCE: Clinical outcome in children with AML, DS-AML and TAM have remained poor in part due to lack of deep understanding of the genomic makeup of the disease as well as the host. Comprehensive studies of the host and disease may enable more informed therapies in order to optimize targeting the leukemia while minimizing short and long term toxicities, leading to improved survival with minimal morbidities.

Abstract:

Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) account for up to 50% of pediatric and 7% of adult end-stage kidney failure worldwide. The goal of this project is to apply genetic approaches to resolve the biological basis and clinical manifestations of CAKUT using three well-characterized cohorts with deep phenotypes and extensive longitudinal data. Here, we hypothesize that CAKUT is genetically heterogeneous, and caused by rare mutations with large effect on a background of polygenes with small effects that can be discovered by analysis of well phenotyped cohorts compared to genetically matched cohorts with WGS data available. We now propose to extend our prior studies by whole genome sequencing (WGS) in additional 500 trios with CAKUT as well as WES in 5,600 additional CAKUT singleton to achieve a total cohort of 1,160 WGS trios and 9,000 WES singletons. We expect that the proposed studies will provide new insight into urogenital development, clarify the clinical overlap with other syndromes and provide novel tools that can replace the current morphology-based diagnostic approaches. We will first perform annotation based on a standard ACMG guidelines to identify pathogenic variants diagnostic for known genetic disorders. In aim 2, we will perform comprehensive trios analysis of de novo mutations in coding and non-coding regions comparing data to the Simons Simplex Collection. In aim 3 we will combine all data in the largest sequencing effort to date by analysing WGS/WES in 10,160 independent CAKUT cases in order to provide a comprehensive catalogue of known and novel genetic variants for follow up clinical and functional studies. PUBLIC HEALTH RELEVANCE: Congenital defects of the kidney and urinary tract are a common cause of kidney failure in children and adults and elucidation of the genetics of these disorders will provide new opportunities for diagnosis, risk stratification and prevention of complications.

Abstract:

Orofacial cleft (OFC) birth defects are one of the most common structural birth defects in humans, and the most common craniofacial anomalies, with worldwide incidence of approximately 1 per 700 newborns. OFCs represent a major public health problem due to the associated morbidity, mortality, and significant medical care expenditures. Based on structures affected, OFCs have been categorized as three subtypes: clefts affecting the lip only (cleft lip, CL), clefts affecting both the lip and the palate (cleft lip and palate, CLP), and clefts affecting the palate only (cleft palate, CP). Historically, CL and CLP have been considered variations of the same malformation that differ in severity, whereas the developmental origins of the affected structures, epidemiology, and familial patterns suggest that CP has a separate etiology than CL and CLP. Both genetic and environmental factors play important roles in the development of OFCs, although understanding of these risk factors is incomplete. The proposed project aims to expand the Gabriella Miller Kids First (GMKF) resource by collecting data to investigate the role of DNA methylation – an epigenomic marker of gene activity – on the development of clefts. We propose to collect genome-wide DNA methylation assays in a large cohort of affected children as well as DNA methylation and transcriptomics assays in a subset of children with available discarded surgical tissue. Ultimately, these data will contribute new and complementary types of omics data to the GMKF resource for participants with already-available whole-genome sequencing data. This resource will allow us and others to perform analyses to identify the differentially methylated regions of the genome associated with OFCs and subtypes, and explore the functional roles of previously identified OFC-associated genetic loci. Successful completion of this project will expand and deepen our understanding of the genetic architecture and regulatory landscape of OFCs including identifying new risk loci and determining the mechanisms through which known risk loci influence the development of OFCs. PUBLIC HEALTH RELEVANCE: This project will expand the Gabriella Miller Kids First resource and deepen our understanding of the genetic architecture and regulatory landscape of non-syndromic orofacial clefts including identifying new risk loci and determining the mechanisms through which known risk loci influence the development of OFCs. This knowledge may ultimately be useful for applications such as recurrence prediction or personalized therapeutic interventions.

HD114131

Children's Hospital of Philadelphia

Genomic, somatic, transcriptional and epigenetic profiling of non-syndromic and syndromic craniosynostosis

Abstract:

Syndromic and non-syndromic craniosynostosis (CS) are complex malformations of the cranial vault that result from premature or anomalous fusion of cranial sutures. The mainstay of treatment remains complex surgical operations with significant morbidity and risks to a newborn. Meanwhile, advances in fundamental understanding of craniosynostosis genetics stand in stark contrast to the persistent gap in translation to clinical impact. This proposal tests the central hypothesis that by integrating genomic, biological and clinical data and approaches, we can correlate genetic diagnosis to clinical course and treatment outcomes. In order to realize this genomic translation strategy, we carried out a multi-year effort to clinically phenotype, catalog, and collect trios and affected tissue in every consenting patient that presents to our center. As a leading craniofacial clinical program, we were able to collect over 438 cases of syndromic and non-syndromic CS over 5 years. This biorepository is unique compared to previously sequenced craniosynostosis cohorts, as these cases include specimen of the pathologic tissue from the anomalous fused craniosynostotic suture and normal bone as control. In Aim 1, we propose to comprehensively analyze germline, somatic, transcriptional and epigenetic contribution to syndromic and non-syndromic CS by: 1A) Whole genome sequencing of syndromic and non-syndromic craniosynostosis from trio blood and saliva, 1B) Somatic and transcriptional profiling of non-syndromic and syndromic craniosynostosis across subtypes, and 1C) Epigenetic analysis of pathologic craniosynostotic bone and normal bone. In Aim 2, we will characterize the longitudinal natural history of disease and clinical functional outcomes in syndromic and non-syndromic CS. This will be achieved by: 2A) integrating WGS, EMR and phenotype data to map genetic diagnosis to clinical course of disease, and 2B) integrating WGS, EMR and post-operative data to map genetic diagnosis to treatment outcomes. Successful completion of this project will yield genomic, epigenetic and transcriptional data that are integrated with clinical presentation, longitudinal natural history of disease, treatment and other functional outcomes. This resource will enable downstream genomic, developmental and clinical studies to address translation gaps in craniofacial biology and treatment, with the goal of delivering clinically actionable data to advance diagnosis and treatment of syndromic and non-syndromic craniosynostosis. PUBLIC HEALTH RELEVANCE: Syndromic and non-syndromic craniosynostosis occur when the cranial sutures are aberrantly fused at birth, leading to constrained cerebral growth, irreversible brain damage, blindness and significant craniofacial deformity if left untreated. This study proposes to carry out germline, somatic, transcriptomic and epigenetic sequencing, and to integrate this multi-omic data to clinical natural history of disease and functional treatment outcomes.

David Teachey

Charles G. Mullighan

Abstract:

The outcome for children with relapsed T-cell acute lymphoblastic leukemia (T-ALL) is dismal. Thus, the primary goal in treatment is to prevent relapse, which requires accurate risk stratification. Prior attempts to identify genetic aberrations that are prognostic independent of treatment response have failed. We recently performed comprehensive genomic profiling (whole genome sequencing (WGS), whole exome sequencing (WES), and whole transcriptome profiling (WTS) of tumor; WGS of germline) from >1300 patients with T-ALL treated on the AALL0434 clinical trial through a Gabriella Miller Kids First X01 award (X01HD100702) and made several novel, practice changing observations. We found T-ALL can be classified into 15 distinct groups, many of which are novel. We found that leukemic drivers were in non-coding regions in 60% of cases, highlighting the importance of WGS. We identified multiple subtypes that were predictive of favorable and unfavorable outcome. The successor trial to AALL0434 was AALL1231. On AALL1231, several changes were made to the backbone to eliminate cranial radiation in most patients and these changes had prognostic implications. Before we can prospectively incorporate genetic aberrations into risk stratification, we need to validate our results in an independent cohort treated with current therapy and identify genomic variants that are reproducibly prognostic irrespective of therapeutic backbone. In addition, we found the prognostic impact of some variants differed based on genetic ancestry; some genetic variants that were associated with higher cure rates in children of European ancestry were not associated with higher cure rates in children of African ancestry. We need to increase the number of patients studied from different racial and ethnic groups to ensure equity in future risk stratification. Finally, we were unable to identify the genomic driver in a small percentage of cases (5%) and long-read sequencing may be able to overcome this gap. We hypothesize that comprehensive genomic profiling will identify recurrent genetic alterations that can be used prospectively to risk classify patients with T-ALL. Genomic profiling of a large cohort of patients treated on the AALL1231 trial will serve as a natural extension of our initial X01 award, providing the power to assess the impact of genomic variants on outcome across genetic ancestral groups. We will test our hypothesis with the following specific aims: (1) validate prognostic variants in an independent cohort of patients with T-ALL; (2) identify novel genomic structural variants using long-read sequencing; and (3) determine the association between genetic ancestry, tumor biology and outcomes. The goal of the Kids First Program is to improve understanding of genetic mechanisms of disease, leading to improved diagnostic capabilities and ultimately more targeted therapies. Genomic profiling across two of the largest clinical trials ever performed in children with T-ALL will clearly meet these important goals. This work will not only fundamentally transform the understanding of T-ALL disease biology but also allow us to risk stratify patients accurately and equitably understand differences in tumor biology based on genetic ancestry. PUBLIC HEALTH RELEVANCE: Modern genetic tests have helped find better ways to identify children with T-cell acute lymphoblastic leukemia (T-ALL) who are less likely to be cured. Before we can use these tests in the clinic, we need to show they are helpful regardless of therapy used to treat the leukemia