The Center for Applied Genomics at The Children’s Hospital of Philadelphia – Pediatric Perspectives on Genomic Medicine

by John J Connolly, Joseph T Glessner, Dong Li, Patrick MA Sleiman and Hakon Hakonarson

Pediatric Perspectives on Genomic Medicine

The Center for Applied Genomics (CAG) is one of the Centers of Emphasis at The Children’s Hospital of Philadelphia (CHOP). Founded in 2006, the CAG has grown to become one of the world’s largest biobanking and genotyping/sequencing facilities, hosting hundreds of thousands of biosamples, ranging from blood samples to DNA, RNA, plasma, protein and peripheral blood mononuclear cells (PBMCs). With a genotyping and sequencing throughput of several thousand samples per week, and a mature drug-development program that has taken several products to clinical trial, the Center is actively working on over 60 disease areas, including many with significant unmet medical needs. Its mission is to identify novel risk variants and biomarkers for rare and complex diseases and to translate discoveries into novel diagnostic products and innovative therapies. Deep electronic health records are available for all CHOP patients, and samples banked in an automated biorepository. Plasma samples are used for biomarker validation, and PBMCs are used to make lymphoblastoid cell lines (LCLs) and iPSCs that can be differentiated into organ‑specific tissues tailored towards target diseases. Against the backdrop of this burgeoning translational program, this review focuses on some of the unique aspects of pediatric genomic medicine, and how this is leading to early interventions and better outcomes in the long term.

Introduction and Motivation

Children occupy a relatively early stage in the developmental trajectory. As a result, research focused on health in children is uniquely placed to shape public health outcomes. Where early intervention is possible, it is, for virtually every human disease, an indicator of improved health outcome. To examine how we may realize this objective, we first look at Pediatrics in the context of Genomic Medicine, where we specifically consider the unique benefits and challenges of the pediatric approach. Second, we focus specifically on the leading role of the CAG team, its facility, and the components necessary to building a collaborative bench-to-bedside translational program. Finally, we address how researchers have successfully leveraged these resources to drive new discoveries across a range of phenotypes.

[Editor’s Note: An outline of the paper describing the CAG program is provided below – essentially a blueprint for how such programs have been (and other programs should be) developed. The highlight of this paper, however, is described in Part C where all the pieces come together in the ARAF- Trametinib Story – an incredible tale of how the CAG team went from family history and genetic data to identify an effective therapy for the unmet medical needs of a 12-year old boy. A precision medicine tour de force. Read on.]

Sections Outline

PART A. Pediatrics in the Context of Genomic Medicine

1. Clinical Infrastructure
2. Development Windows and Rich Data
3. Family Data and Convenience Recruiting
4. Consent
5. Funding & Policy

PART B. Building a Pediatric Research Infrastructure

1. Recruitment
2. Biobanking (blood/saliva)
3. CLIA Accreditation
4. Hardware and Throughput
5. Linked Reads
6. Computing: Cloud-based Bioinformatics Pipeline
7. Clinical Knowledgebases
8. Clinical Decision Support
9. Data Legacy

PART C. Putting the Pieces Together – A Translational Blueprint

Step 1. Recruitment from an enriched cohort
Step 2. Deep phenotyping and Clinical Expertise
Step 3. Analysis Infrastructure and Patient Families
Step 4. The Trametinib Story: Generating Functional Data from Gene to Treatment
Step 5. Personalized and Precise Treatment – The Results!
Step 6. Program Acceleration
Step 7. The Translational Model

PART D. Other Representative CAG Rare Disease Programs

1. Rare Multiple Congenital Anomaly (MCA)
2. Re-evaluation of ‘Negative’ Clinical Exome
• Ashkenazi Founder LS Mutation
• Causal USP9X Variant Missed by Disease Program (UDP)
• Intellectual Disability, Novel Cause & Translational Impact
• Causal Gene for Hoffman Syndrome

PART A. Pediatrics in the Context of Genomic Medicine

For many complex diseases, pediatric patients are often enriched for genomic risk factors, in part because environmental exposures are comparatively limited. Pediatric cases have had less time to accumulate potential confounders; thereby increasing the probability that a factor associated with early-onset disease is gene‑causative. Importantly, even in diseases where phenotypic and etiological differences for pediatric/adult types are well-established, the same genomic risk factors are nevertheless often common to both. Researchers at our group have confirmed this hypothesis in numerous discovery studies, including but not limited to inflammatory bowel disease (IBD),1-3 type-1 diabetes (T1D),4-7 asthma,8,9 and obesity.10,11 We delve into the former by way of brief illustration.

Early-onset IBD has been shown to have distinct features in terms of phenotype and severity.12,13 Using genome-wide association analysis, we previously demonstrated that the large majority of loci associated with the IBD subtypes (Crohn’s disease and ulcerative colitis) were also evident in our pediatric cohort, but also included several novel variants.2,3 These discoveries were enabled by the existence of genomic enrichment in pediatric sets – a family history of Crohn’s disease and ulcerative colitis is significantly higher for pediatric cases (<17 years), 16% and 13%, respectively), versus adult cases (7% and 3%, respectively).14,15 Because early-onset IBD has a stronger familial component than the adult type, studies targeting this subgroup provide additional power to identify genes of modest effects. The impact of this phenomenon goes beyond simply substantiating a catalog of disease-associated loci. We are now in the era of polygenic risk scores derived from clinically‑relevant disease risk calculations, which, in turn, are based on thousands of variants contributing to the pathogenesis of common complex diseases. Deriving these critical risk scores makes it a priority to address the biological pathways and gene networks involved, thereby guiding when and how intervention can be undertaken. Similarly, autosomal recessive mutations for very-early onset IBD have been identified in the IL-10 receptor in infants born to consanguineous parents.16,17 While this may not necessarily be relevant as a risk locus for the majority of patients, it nevertheless suggests an alternative pathway to treatment for the broader phenotype. Numerous examples exist in the pediatric literature to illustrate this point. For example, the T1D Genetics Consortium (T1DGC) has identified and exome-sequenced numerous families where two or more children have non-autoimmune insulinopenic diabetes. Rare variant analyses highlighted several families with compound heterozygotic mutations leading to altered protein structure at WFS1,18,19 including a proband* sibling pair with a pathogenic HNF1A mutation and a family with three children carrying a mutation in KCNJ11 (p. E227K) known to cause neonatal diabetes and reported in association with rare cases of non-neonatal diabetes onset20,21 [*in general, a proband refers to an individual affected with a disorder who is the first subject in a study due to, e.g., an aberrant or expressed genetic trait in a family lineage]. These risk factors for the onset and pathogenesis of atypical diabetes have broad implications. They represent potentially important biomarkers and approaches for screening, diagnosis, and management of diabetes phenotypes in general. Thus, while type 2 diabetes is predominantly a late‑onset condition – sometimes described as an “epidemic” in adult disease22 – pediatric‑driven studies have been a major stimulus behind efforts to develop novel diagnostic and treatment strategies. Pediatric-onset diabetes is a case in point of the archetypal phenomenon discussed above, an example than can be extended to diseases across the medical spectrum, even where onset is more typical in adulthood.

1. Clinical Infrastructure

In the US and Europe, the majority of states/countries have at least rudimentary requirements for newborn screening for rare diseases, many of which are decades old (dating back to 1963 with blood spot tests for phenylketonuria23). In 2006 the American College of Medical Genetics (ACMG) recommended screening for 29 primary and 25 secondary conditions,24 and remains highly active in advocacy and support.25 Although these do not begin to cover the total number of rare diseases that could potentially be screened, it has long been the case that healthcare providers at pediatric institutions have comparatively mature resources for testing and returning genetic results (adult patients account for only ~12% of patients seen be geneticist);26 consequently, pediatric healthcare providers are more accustomed to interacting with genomic data. Clinical newborn screening (NBS) is the area perhaps most amendable to mass-scaling via next generation sequencing. Many pediatric institutes are now poised to adopt massive whole exome or whole genome sequencing to extend the scope of NBS.27 Such initiatives are necessarily reliant on policies to address complex issues such as data legacy and custody; data maintenance and revision; return of variants of uncertain significance (VUS); cascade screening; education; and a broad spectrum of ethical, legal, and social issues.28 While a deep dive into these issues is beyond the scope of this review, we foresee that with few exceptions (see below), similar policy and oversight will be required to screen adult patients. Pediatric sites have a head start in provider readiness, since the systems and personnel needed to support large-scale screening are well-established. As healthcare systems transition to increasingly personalized health management, this experience will be increasingly valuable for screening patients of all ages. As outlined below, our team has leveraged our phenomenal experience to foster local research collaborations with clinicians across CHOP.

2. Developmental Windows and Rich Data

Human biological systems go through developmental stages from in utero through childhood; these systems are susceptible to environmental triggers and epigenetic imprinting during key developmental windows when genetic factors specific to particular stages are expressed (growth factors being an obvious example29). Similarly, numerous studies have highlighted critical windows for neurodevelopment, where the central nervous system (CNS) is particularly susceptible to toxicity and other environmental factors.30,31 Toxins, including heavy metals, organics and air pollution affect development, and particularly neurologic development.32-39 The psychosocial environment is also a critical component of developmental models, and stress has long been shown to relate to a wide range of congenital conditions, as well as asthma, obesity, behavioral disorders, and growth.40-43 In addition, stress significantly impacts immunologic development and can alter metabolic function through its actions on the immune system.44-47 The effects of prenatal stress on emotional behavioral and cognitive outcomes is also well studied.48-50 Gene-environment models are widely used to capture these interactions, and large datasets can define interactions between variables and have greater power to define the magnitude of effects in a given cohort.51 For most biological insults, there is inherent variability in outcomes based on differential combinations of environmental exposure and genetic influence. These developmental windows, when such data are available for capture and robust modeling, are uniquely accessible to pediatric institutions. Critically, pediatric sites have another major advantage in addressing the “gene x environment” paradigm, which offers a wealth of information that is often unavailable in the adult context. Because variant discovery projects typically rely upon mining massive quantities of health data derived from electronic health records (EHRs), completeness of EHRs is a critical factor in avoiding errors of omission. Children have far more frequent contact with health services, where well-child visits are recommended 6-times in the first year, three times in the second year, twice in the third, and annually thereafter. This equates to an extraordinarily deep EHR,and is particularly advantageous in the epigenetic field to drive our understanding of disease-associated DNA methylation and hypomethylation and histone posttranslational modifications. For virtually every developmental disorder and a large proportion of autoimmune and allergic disorders, extensive data has been linked to alterations in epigenetic states, particularly DNA methylation,52 where further interventional opportunities exist to innovate epigenetic editing.

3. Family Data and Convenience Recruiting

Pediatric repositories benefit enormously from access to families as opposed to single patients/probands. For example, for the vast majority of patient visits catalogued at CAG, the patient is accompanied by at least one family member, which provides a unique recruitment opportunity. At CAG, we have established an extensive repository of family members, all of whom complete a short medical history. Of >100,000 pediatric patients in CAG, approximately one-half have either a parent or sibling also recruited. To date, we have collected extensive phenotype data from both parents for thousands of CAG patients, importantly contributing to family-based analyses such as transmission disequilibrium testing (TDT).

These large numbers afford massive statistical power in terms of risk variant discovery, especially in cases of sporadic disease with a negative family history, which may account for~97% of human genetic disability cases.53 The number of de novo germline mutations is estimated to be in the region of 40–70 per individual with paternal ages of approximately 21–50 years at conception,54-56 a relatively manageable (although non-trivial) total in terms of variant discovery. Aggregating the enhanced power of trio-derived exome data with the deep phenotypes and family histories greatly enhances our abilities to identify the causes of genetic diseases, which we have been able to use to great effect as outlined below in Part C.

4. Consent

In genomic research, pediatric and adult approaches are perhaps most distinctive in terms of the consent process. Research involving children must consider the viewpoint of both parent and child as well as establish and maintain a process that considers the developing autonomy of the child. Furthermore, while a parent’s decision must account for the best interests of the child, this decision cannot be made in a vacuum; such decisions must also consider the future needs of both the family and the autonomy of the child.57,58 This point is particularly germane to (broad) biorepository-based research, where the consent process and subsequent genomic studies can be years apart. To develop practical guidance on informed consent in pediatric cohorts, the Electronic Medical Records and Genomics Network (eMERGE), of which CAG is a member, addressed these issues in a 2014 paper,59 which remain relevant and are summarized in brief here:

  • Permission from one parent is adequate for a child’s participation in a biorepository: A requirement of permission from both parents would not be expected to improve the quality of the informed consent process but would likely impede enrollment.
  •  Developmentally appropriate explanations about biorepository participation are recommended for all children. At a developmentally appropriate age (e.g. 7-12 years), children should also be asked to provide assent. It may be advisable to engage more mature adolescents in a “co-consent” process rather than an assent process. From an ethical perspective, a developmentally appropriate explanation about the research process is apposite for any biobank participant. While adolescents in most jurisdictions are not legally authorized to provide consent for research participation, they are often sufficiently mature to discuss the process. In addition to respecting the child’s autonomy, this conversation could yield information on preferences when they reach age of majority.
  •  Sharing de-identified data is appropriate for pediatric biobanks; (potential) participants should be provided with an explanation of relevant risks and benefits: The majority of research biorepositories share de-identified data, which is an important catalyst for discovery science. While genomic information per se does not contain (immediately) identifiable data, there is a risk of identification similar to that of adult data.
  • Pediatric biorepositories should develop a policy for managing data and samples once a pediatric participant reaches the age of majority: Several models can be considered here, ranging from destruction of data/biosamples failing re-contact to no re-contact requirement. At CAG, we retain data and samples indefinitely, but require re-contact in order to continue mining EHRs.
  • It is appropriate for a biorepository to either return results or not; biobanks choosing to return results should take individual participant preferences into account: Different types of results (e.g. pharmacogenomics, disease risk) are likely to yield different preferences and the relevant implications of each should be addressed.

5. Funding & Policy

It has become something of a mantra in pediatric circles to state that “children are not just small adults”,60 and there are many examples of childhood diseases or sub-types that are not directly analogous to the adult type. (e.g. 61,62). An unwelcome consequence of this phenomenon, however, may be its aberrant effect on the research paradigm – because children constitute a “special” population, they are often excluded from clinical research and trials. While the intention of such exclusions is clearly well-meaning (i.e., safety and protection), they may have yielded a comparative under-investment in pediatric clinical trials.27.

Policymakers in the US (Pediatric Research Equity Act (PREA) and Europe have recently taken steps to address this imbalance, implementing legislation to promote clinical research in children.63 While these initiatives seem to be impactful, two studies since legislation in the US found off-label drug rates of 34%64 and 21%65 for pediatric inpatients and outpatients respectively, while off-label drug use rate was between 33.2-46.5% in pediatric inpatients and between 3.3-13.5% in pediatric outpatients across several EU nations.63 In short, pediatriciansstill rely on evidence that has been generated in adult populations, and unlicensed and off-label drug use in children, remains a pertinent clinical issue.

These data make a strong case for further investment in pediatric research and underline a broader issue in medical research – an adult-centric focus in the research model. We can see examples of this in flagship programs in the US (e.g. All of Us which, in spite of suggestions to the contrary has not expanded to the children (, and in Europe (e.g. the UK Biobank). As discussed, there are specific challenges in working with pediatric cohorts, but we would argue strongly that just because something is complex, does not mean it should be avoided.

PART B. Building a Pediatric Research Infrastructure

As noted in Part A, CAG has developed a highly-scalable infrastructure to store and track biological specimens. In Part B, we describe the infrastructure of our pediatric biorepository, which has grown to become the largest in the world. To date, we have recruited samples from >100,000 children at CHOP and approximately 50% of them have additional family members, with ongoing efforts yielding ~15,000 new participants per year. CAG is also home to an additional >400,000 samples established through various collaborations, covering a similarly broad range of rare and complex diseases. The biorepository is the primary driver of CAG’s genomic medicine programs, which are dedicated to translating basic science discoveries into the novel therapeutic and diagnostic innovations.

1. Recruitment

We approach patients during in-/out-patient visits at over 20 core clinical sites, including emergency, ambulatory, surgical, general, and specialty pediatric practices. We are approved to recruit from any of CHOP’s ~50 satellite clinics. We recruit patients in the age range of 0-21 years who obtain healthcare at CHOP. Parental consent is obtained for individuals aged 0-17 years and assent is obtained in subjects aged 7-17 years. Whenever possible, parents/siblings are also asked to enroll, sign informed consent, and provide a sample.

Our recruiters are additionally certified to obtain vital signs and collect a standardized two-page intake survey of primary medical problem (or healthy control status), medication, ancestry, and family history. Broad consent allows samples to be analyzed using any technology and to address any research question. Parents can opt-in to permit regular updates of their child’s medical data and to re-contact for future study. Over 95% consent to EHR updating (every 3 months), and >85% to re-contact. If either are permitted, then links between biosamples, genetic data, and the child’s identity are maintained on the clinical side of a firewall, with stringent safeguards to protect confidentiality.

Minority and underserved populations have been an important focus at CAG for more than a decade. We are the largest biorepository in the United States in which the majority of samples are from racial or ethnic minorities, primarily African Americans (AAs). A cornerstone of our mission is to address the growing inequities created by the under-representation of AAs in genomics and healthcare. Out of >800 relevant peer-reviewed publications to date, our scientific output includes >300 peer‑reviewed publications on genomic risk factors in African Americans.

2. Biobanking

Biosampling is taken from blood and/or saliva, from which DNA is extracted. For blood samples, portions of the sample are typically set aside to collect plasma and PBMCs). All pediatric samples to date have been genotyped. CAG has made numerous discoveries in this dataset and provides genetic/genomic information enriched for quality control measures and scientific annotations to end users who are collaborating with CAG. Under the standard workflow, every sample entering the biobank is extracted for DNA, which is then genotyped in a dense (>500K for all samples; currently >2M) genome-wide SNP array from Illumina or Affymetrix. The high-throughput genotyping workflow is optimized for workloads on the order of thousands of samples per week.

3. CLIA Accreditation

CAG’s DNA extraction, genotyping and sequencing laboratories are CLIA certified since 2016. We have an in-house Compliance Manager who works closely with the Medical Director (board certified clinical molecular geneticist) and the CAG laboratories to ensure continuous quality assurance of all the laboratory workflows including genotyping, sequencing, informatics, analysis, interpretation, and reporting. The CAG Quality Assurance (QA) program was established to monitor, assess, evaluate and when indicated, correct problems identified in the pre-analytical, analytical and post analytical systems.

Reports are written by board certified genetic counselors and signed-out by board certified molecular geneticists. When appropriate, we may recommend genetic counseling or a medical consultation to assist patients in understanding test results and making informed decisions. Prior to sign out, each report and supporting data including the quality control data is reviewed by a board-certified molecular geneticist at CAG.

4. Hardware and throughput:

For the past 14 years, CAG has operated one of the largest genotyping facilities in academia. In that time, the Center has engaged in leading-edge research and analytical activities thereby creating an expertise base that spans molecular genetics, innovative genetic and genomic technologies, and bioinformatics. We developed the PennCNV(66) and ANNOVAR(67) genome analysis tools; we have genotyped >500,000 samples genome wide; and we currently process an average of 400 exomes per week through our custom sequencing pipeline (average mean depth clean coverage, ~70x).

CAG technology operators are experienced with distinct genotyping techniques such as Illumina microarrays, Fluidigm, Sanger, and many others. CAG has the ability to process different specimen types from tissue, cell culture, DNA and RNA extraction for different applications. Our sequencing team is experienced, and implementation of any new targeted or exome enrichment method poses no major challenge, as the components and steps are similar to steps already automated.

CAG has built a significant infrastructure for sequencing with state-of-the-art automation and sequencing capacities including Illumina’s sequencing systems for next-generation sequencing (NGS) studies:

  • Novaseq6000
  • Hiseqs250
  • MiSeq

Our library prep protocols are optimized to minimize loss of genomic DNA during processing by increasing efficiency of enzymatic reactions, adjusting the molarity of the sequencing adaptors during ligation as well as reducing the number of steps required to generate a pre-capture library (e.g. wash steps).

Genome-Wide Association Studies (GWAS) have been performed on >250,000 individuals (~100,000 children and ~150,000 parents/adults – including ~50,000 African American children) using different Affymetrix and Illumina Infinium array platforms (starting with 550HH in 2006 and currently using OMNI-2.5M for Illumina and Axiom for Affymetrix) across over 60 common childhood diseases including IBD, asthma, Type 1 and 2 diabetes, JIA, ADHD, autism, and obesity. In addition, GWAS on a large cohort of transplant patients (currently more than 20,000) has been performed on an in-house developed Axiom Chip

Since 2007, we have published >800 peer-reviewed papers, including multiple first- and last-authored Nature and Nature Genetics papers (>60 in total) together with several papers in Nat Medicine, Science, Cell, and NEJM.

5. Linked Reads

Genome analysis typically involves sequencing an individual genome with short reads and expecting to align to a haploid consensus reference assembly. While this approach can call single nucleotide variants across most of the genome, haplotype information is not retained, and reconstruction of long-range haplotypes is challenging due to:

1. large structural variants, particularly balanced events such as inversions and translocations, cannot be called and;
2. due to the prevalence of high identity repeats and paralogs, entire genomic regions are inaccessible.

Chromium™ driven 10x linked-reads offer a powerful solution and will be part of our standard workflow. Chromium™ partitions and barcodes large numbers of DNA samples using microfluidics, producing sequencing-ready libraries of >1 million barcodes; these libraries are compatible with most Illumina sequencing systems, including Novaseq. We have recently implemented an enhanced linked-read alternative library protocol that allows us to generate long-read sequence information from short-reads sequencing methods for all exome capture methods, including Agilent SureSelect and WGS reads.

6. Computing: Cloud-based Bioinformatics Pipeline

We have developed a scalable bioinformatics pipeline built on the HIPAA-compliant Amazon Web Services (AWS) to store, process, and share the NGS sequencing data and analysis results. To maximize our chances to identify novel causal genes for genetic diseases, we adopt the following strategy:

1. identify those patients with unknown Mendelian diseases (MDs);
2. use the most proven target enrichment solution today;
3. follow the best practice of variant calling methods to generate high-sensitive and low false positive variant call files;
4. complement the WES with the WGS to identify missed coding variants, non-coding regulatory variants and structural variants.

Currently, the molecular basis of a large number of OMIM entries with Mendelian or suspected Mendelian diseases (MDs) is unknown. These many undiscovered Mendelian/suspected diseases are either very rare or extremely difficult to diagnose clinically. The key to success lies in our dual strengths of a large population set, and our ability to integrate clinical and genomic data from multiple centers to identify patients with comparable phenotypes carrying rare variants in the same gene or same pathway.

To identify patients with similar phenotypes, the clinical phenotypes for each patient collected from multiple centers is annotated using a standard measure by the Human Phenotype Ontology project (HPO), which provide annotations to diseases listed in the Online Mendelian Inheritance in Man (OMIM) database, Orphanet, and DECIPHER. All MDs are clustered on the basis of HPO clinical phenotypes to identify patients with identical/similar clinical phenotypes

A subset of families are selected from those associated with unknown MDs for exome sequencing. Depending on the mode of inheritance and locus heterogeneity of the disease, a sample population of affected individuals (at least 1) and unaffected controls (if any) per unknown MD is selected on the basis of sample DNA quality and quantity, status of affectedness, and pedigree analysis.

7. Clinical knowledgebase

A critical component of our interpretation process is our database which contains all clinically interpreted variants as a well as a case repository of patient-level data. It has an automatic rule-based reporting engine that can create draft reports by pulling in stored interpretations for the variant(s) present in a patient.

8. Clinical Decision Support (CDS)

Barriers to implementing CDS extend beyond technology to other factors including organizational, environmental, process, and patient factors.68,69 Adding to the challenge is that physicians and other organizational decision-makers do not fully understand the complex sociotechnical factors affecting decision-making from genomic test results.70 In addition, health screening and intervention workflows are often markedly different in pediatric settings.71 A well-designed CDS development and validation plan is critical to addressing these barriers and creating a CDS model that can be implemented across phenotypes.

In collaboration with leading CHOP investigators, we have developed interactive CDS models based on ACMG recommendations for reporting incidental findings.72 The models included advanced EHR integrated CDS functionality feasible via our CDS programming framework, Care Assistant. Models were presented to 26 primary care pediatricians (via a cognitive walkthrough method).
The models include an EHR based result notification, access to the test report, an expert-curated set of clinician and patient focused education resources, and EHR actions such as problem list entry and template for note entry. PCP feedback indicates that this platform would have high utility, with a high likelihood of adoption.

9. Data Legacy

CHOP pursues clinical programs through the Epic© EHR vendor-supported Care Everywhere network, which is available for some healthcare systems within 49 out of the 50 United States including four of the largest adult healthcare providers in the Philadelphia area (Penn Health, Temple Health, Thomas Jefferson University Hospital, and Main Line Health). For family members whose primary care providers are members of Care Everywhere and who consent to review of their outside EHRs through this system, this arrangement allows healthcare professionals to exchange patient medical information electronically, including visits, tests, procedures, diagnoses, medications, and documents (e.g., Summary of Care Documents). Once the family member is registered at CHOP, we can return genetic risk assessments through Care Everywhere to participants using the CHOP EHR and monitor their follow-up and outcomes even if that care is provided at an outside institution. This strategy also enhances follow-up of adolescent patients who may have transitioned to adult care during this study.


PART C. Putting the Pieces Together – A Translational Blueprint

A critical component of CAG’s mission is moving beyond gene discovery toward building translational initiatives that can improve health outcomes, either by innovating diagnostic markers or novel therapies. We have built a mature translational team that is highly integrated in terms of ethos and personnel: data-mangers, analysts, clinicians and pharmacologists share a common workspace and expertise. Fundamental to this effort is a coordinated approach among stakeholders that includes weekly meetings of more than 25 doctoral-level researchers and clinicians who span a wide spectrum of the translational continuum. Individuals of disparate but complementary skillsets collaborate at close quarters; this approach has been particularly productive as a means of delineating and targeting the functional consequences of disease-associated variants. Our success with remediating rare lymphatic anomalies has become our blueprint for tracking different stages of our translational pipeline.

Step 1. Recruitment from an enriched cohort

In 2015, a twelve-year old boy presented to a local hospital in Virginia with buildup of lymphatic fluid around his heart and lungs. He did not respond to treatment and was transferred to CHOP for palliative care, including cauterization of lymphatic vessels and pharmacotherapy with sirolimus (rapamycin), an immune-suppressing drug that has been shown to remediate some forms of lymphatic disease.73 He was subsequently recruited to the rare disease sequencing program at CAG. With over 22,000 participants already in the database, the large scope of this program allowed us to match the boy’s case with 75 other patients with complicated lymphatic anomalies (matching was even before whole exome sequencing (WES) of his sample). We note that even though these anomalies have a frequency of ~1 in 100,000, CHOP’s standing as the leading referral site for rare pediatric disease means we were significantly enriched for this disease population.

Step 2. Deep phenotyping and Clinical Expertise

Complicated lymphatic anomalies include a variety of diagnoses: lymphangiectasia, Central Conducting Lymphatic Anomaly (CCLA), Generalized Lymphatic Anomaly (GLA), Kaposiform Lymphangiomatosis (KLA), and Gorham Stout Disease (GSD), all of which are chronically debilitating and often life-threatening. The absence of data on molecular etiology and mechanisms hampers further research into causes and treatments of these conditions.
Deep clinical experience and expertise is patently needed to identify and discriminate between sub-types. CHOP collaborates widely with the broader clinical community; in the case of lymphatic anomalies, with CHOP’s Center for Lymphatic Imaging and Interventions (CLII), who have been leaders in spearheading new imaging and interventional methods to better understand lymphatic anatomy, physiology, and pathophysiology. Leveraging these collaborations, we additionally sequenced several families with complex lymphatic anomalies, including a sample from a deceased female patient diagnosed with lymphangiomatosis in 2012 (although prior to publication of the International Society for the Study of Vascular Anomalies classification (2015)).

Step 3. Analysis Infrastructure and Patient Families

CAG is heavily invested in genomic analysis, and the Lab has published some of the most widely-utilized tools in the community, including ANNOVAR, PennCNV, and ParseCNV.66,67,74 Previous WES of the unrelated probands was negative for known lymphatic anomaly-associated genes, prompting a deeper dive into potential genomic drivers. Leveraging in-house expertise that has resolved over 200 novel and unique diseases, we subsequently used a gene prioritization approach to reveal a novel X chromosomal ARAF mutation, c.640T>C:p.S214P, in both patients. The mutation affects a conserved phosphorylation site, which is consistent with a gain-of-function (GoF) effect, given that the residue Ser 214 is a paralogous regulatory site in the homologous protein C-RAF (or RAF1) for inhibition by 14-3-3 proteins.75

As discussed above, one of the unique features of a pediatric cohort is that it offers streamlined access to parent(s), the large majority of whom live with and accompany probands to CHOP. Access to family members has allowed us to develop rich pedigrees with sequence data from a large number of families in our lymphatic cohort. For the male proband discussed above, Sanger sequencing of blood-derived DNA from both parents confirmed that the X-linked ARAF mutation occurred as a de novo event during early stage of meiosis (50% mutation load). In the second proband, Sanger sequencing established that an unaffected daughter and mother of the second proband did not carry the mutation. The father was unavailable for sequencing but had no history of respiratory symptoms, indicating the ARAF mutation was most likely a de novo or somatic mosaic mutation.

Step 4. The Trametinib Story: Generating Functional Data from Gene to Treatment

In this section, we provide a detailed outline of how the CAG team brought together its many strengths – genomic data, family history, laboratory science – to identify a possible therapeutic intervention, trametinib, after identifying a gene (ARAF) of interest.

The observation in Step 3 regarding the ARAF mutation led to the insight that the Ser214 residue, a 14-3-3 binding site in conserved region 2 (CR2) in ARAF, might provide a path forward to a treatment. Ser214 is highly conserved across vertebrate species and within the RAF proteins, indicating a critical role in these kinases. The binding of 14-3-3 proteins to phosphorylated Ser214 of ARAF would prevent ARAF protein from recruiting plasma membrane by activated RAS.76 Supporting this, we showed that HEK293T cells transfected with ARAF‑S214P had reduced co‑immunoprecipitation of 14-3-3 proteins, leading to significantly greater activation of ERK1/2 (per increased phosphorylation versus HEK293T cells expressing wild-type (WT) ARAF). Conspicuously, AKT, p70S6K, mTOR and p38 did not show changes in phosphorylation associated with ARAF-S214P, consistent with patients not responding to sirolimus.

Furthermore, human dermal lymphatic endothelial cells (HDLECs) expressing ARAF‑S214P have enhanced lymphangiogenic capacity versus wild-type, as measured by sprout quantity, length, and volume. Critically, we showed that the phenotype is rescued in these cells by the MEK inhibitor, trametinib, as are alterations in actin organization. HDLECs transduced with the mutant ARAF show elevated ERK1/2 activity, enhanced lymphangiogenic capacity, and disassembly of actin skeleton and VE-cadherin junctions, all of which are rescued with trametinib. In ARAF-S214P-expressing HDLECs, sprouting persists in the absence of VEGFC, a known and potent lymphangiogenic factor (77). Cells expressing wild type ARAF, however, do not show evidence of sprouting in the absence of VEGFC. This phenomenon strongly supports the hypothesis that the ARAF mutant is either mimicking the stimulatory behavior of VEGFC or inducing the expression of VEGFC.
We also reproduced the anomalous lymphatic phenotype in a zebrafish model. Zebrafish embryos were engineered to carry the mutation, and within five days, the fish had developed a lymphatic system, and confirmed that the mutation causes overgrowth of lymphatic vessels. Critically, the zebrafish model showed rescue of the phenotype following intervention with MEK inhibitor therapy. We trialed several drug regimens that were MEK inhibitors and observed maximum improvement with trametinib.

Step 5. Personalize and Precise Treatment – The Results!

Trametinib, which is approved to treat patients with BRAF V600E/K-mutant metastatic melanoma, is not approved for use in children. We received approval for compassionate use in the male proband. This therapy yielded a dramatic improvement in his symptoms that included remodeling of his dilated and torturous lymphatic vasculature, resolution of the lymphatic edema and resumption of regular daily activities within 12 months. Within two months, his breathing had improved significantly, and at three months of therapy, he no longer needed supplemental oxygen. The swelling in his legs disappeared, and the lymphatic vessels reshaped, essentially remodeling an entire organ system.

We have now sequenced over 100 patients with lymphatic anomaly, including cases with Noonan (or Noonan-related) syndrome, Gorham–Stout disease, kaposiform lymphangiomatosis (KLA), lymphangiectasia and CCLA. This has allowed us to discover and confirm multiple mutations in 9 different genes (both germline and somatic), including 6 different RASopathy genes (PTPN11, RASA1, RAF1, RIT1, and BRAF), with mosaicism levels for the somatic variants ranging from 5% to 12%. These results, suggesting that the RAS–MAPK signaling is a common pathway responsible for the various clinical lymphatic disease manifestations.

Step 6. Program Acceleration

Another patient in our cohort was recently found to harbor a germline mutation in EPHB4 in a four-generation family with CCLA. CRISPR knock-in of the EPHB4 mutation in 293T cells showed higher phosphorylation of p70S6K levels, and zebrafish studies demonstrated lymphatic vessel mis-branching and developmental deformities that were effectively rescued by mTOR and MEK inhibitor.78 Taken together with the studies above, we have strong evidence that RAS/MAPK signaling is a common pathway responsible for the various clinical lymphatic disease manifestations and emphasizes the importance of both germline and somatic mutations in the RAS/MAPK pathway in lymphatic anomalies.

PIK3CA is mainly involved in PI3K/mTOR pathway, which we hypothesize explains some of the heterogeneity in response to treatment; different underlying mutations will respond to different drugs, opening the possibility of precision therapy. Addressing this question requires a systematic investigation of an unbiased cohort. We have now recruited over 100 highly-phenotyped patients with undiagnosed lymphatic anomaly, despite a multidisciplinary evaluation at CHOP involving Interventional Radiology, Plastic Surgery, Cardiology, Oncology, and Dermatology. We envision high yield of somatic mosaic mutations in these patients that may unfold only by sequencing lymphatic tissue.

Step 7. The Translational Model

Eleven centers in the United States are forming a consortium to facilitate multi-center clinical trials for this group of lymphatic anomalies. There are now than 3,000 patients recruited with moderate to severe disease course, with ~300 new cases/year. Based on the current molecular diagnostic yield, we anticipate that about 20% will have defects in the RAS–MAPK pathway, suggesting that several thousand patients in the USA alone could benefit from MEK inhibitor therapy.

Thus, our work exemplifies how genetic discoveries can impact disease classification and uncover novel biological and life-saving treatments as represented here in a patient with lymphatic anomaly of a previously unknown etiology, a realization of a precision medicine approach.

PART D. Other Representative CAG Rare Disease Programs

Reflecting our high diagnostic yield, we have published over 60 manuscripts identifying the molecular etiology for novel syndromes, or expanding the genotype or phenotype associated with specific genes. We are working on multiple other papers at various stages of preparation and review. We cite below a sampling of our work that is available for further reading (or will be available online or in print shortly):

1. Rare Multiple Congenital Anomaly (MCA)
In a recent WES study of MCA with dysmorphic features (for example, heart defects and/or neurological manifestations), we sequenced 652 subjects from 281 families. We evaluated these data with our analytical platform; by using unique filtering techniques, we achieved an overall molecular diagnostic yield of 40% – an exceptional outcome in the context of undiagnosed disease. Furthermore, these data served to validate our analysis platform.

The following items represent significant results from our findings:

  • Successfully identified causal mutations in known Mendelian genes for 30% of families (n=84);
  • Identified likely pathogenic variants in ~14 genes not previously implicated in rare multiple congenital anomalies in 18 patients (6.4%);
  • Identified and cataloged 34 disease-causing variants responsible for a range of bone and mineral metabolism disorders, including hypoparathyroidism, pseudohypoparathyroidism type 1a, pseudohypoparathyroidism type 1b, metaphyseal anadysplasia, Kenney Caffey syndrome, and hypophosphatemic rickets, in 59 independent families (molecular diagnostic yield of 57.6%).

One of our discoveries involving the role of GRIN2D in epilepsy has already led to a clinical treatment. The epileptic encephalopathies are a spectrum of conditions manifesting with intractable seizures, neurodevelopmental disabilities, and have a diverse range of, including increasing numbers of monogenic disorders. Disovery and study on a novel N-methyl-D-asparate receptor (NMDAR) gene, we found that GRIN2D in two unrelated children with epileptic encephalopathies indicated that the FDA-approved NMDAR drugs Memantine and Ketamine could provide therapeutic control of symptoms. Two patients with epileptic encephalopathies have now been successfully treated, yielding significant improvements in symptoms and quality of life.79

2. Re-evaluation of ‘Negative’ Clinical Exome

The analytic pipeline at CAG is optimized not only for known genes, but also for novel gene discoveries. More importantly, our pipeline has also proved to be robust for reanalyzing ‘negative’ clinical exomes. Based on a variety of clinician-initiated referrals and prominent clinical labs in the US, we reanalyzed 49 cases after initial WES failed to identify causative variants (outlined in Table 1). Through in vitro assays, ex vivo and in vivo models, we resolved an additional 16.1% of these cases:

Ashkenazi Founder LS Mutation:

Leigh Syndrome (LS) is genetically heterogeneous neurodevelopmental disorder with recurrent metabolic strokes and intermittent regression and progressive neurological decline. Through reanalysis of a negative clinical exome from another program, we identified a homozygous, autosomal recessive splice site mutation, c.87+1G>C, in USMG5 (OMIM 615204). USMG5 encodes the DAPIT protein, associated with function and assembly of mitochondrial respiratory chain complex V (CV). We showed that the mutation causes aberrant USMG5 mRNA splicing with complete loss of DAPIT protein expression. Blue native gel analysis in fibroblast cells from an affected proband showed complete absence of holo-CV assembly. Integrated high-resolution respirometry in their intact fibroblasts showed an increase in maximal, uncoupled oxygen consumption rate, consistent with CV deficiency.

We subsequently used a usmg5 morpholino in a Zebrafish model to knock down USMG5 expression and then analyzed the results. Though not previously implicated in human disease, we confirmed that loss of usmg5 function results in severe survival, neurodevelopmental, and cardiac defects.80 Through collaboration, we found the exact same mutation in two additional unrelated Ashkenazi families with childhood onset LS.

Causal USP9X Variant Missed by Undiagnosed Disease Program (UDP):

This is an example of a misdiagnosis that was corrected by our analytical pipeline in a patient previously analyzed and subsequently submitted to the UDP for review of two missense UBE3B variants. Although the patient’s phenotype was not an exact match and all published cases had truncating indels rather than missense mutations, the UDP review board concluded that the patient had a UBE3B-associated disease. However, our pipeline identified a novel hemizygous variant, c.235A>G:p.I79V, in USP9X. Looking at the fibroblast cell line derived from the patient, USP9X substrates, SMURF1, and beta catenin, are down-regulated consistent with a loss of function effect.81

Intellectual Disability, Novel Cause & Translational Impact:

For the novel gene TBCK, identified as a cause of intellectual disability (ID) by our re-analysis pipeline (Table 1), the causative mutations suggest that leucine supplement may offer a targeted treatment for children with ID.82 Follow-up trials are in planning.

Causal Gene for Hoffman syndrome:

Hoffman syndrome is characterized by complete B cell immunodeficiency, facial dysmorphism, and limb and genital anomalies – all of unknown genetic etiology. Through reanalysis we identified a de novo mutation in TOP2B, encoding topoisomerase IIb that relaxes topological stress during DNA replication and gene transcription. A role for TOP2B in B cell development and differentiation has not previously been described. We used in vitro, ex vivo and in vivo models to show that the mutation inactivates topoisomerase II function and that loss of TOP2B negatively affects B cell development. More importantly, this discovery provides a unique window into the relationship between human B cell differentiation and bone pathophysiology.83

CAG’s vision and mission is focused on applying genomic strategies using large datasets to identify genetic underpinnings in complex medical disorders, both common and rare. In parallel, we also search for individuals who, based on their genetic make-up, are predicted to have a favorable response to drugs that modify specific pathways and gene networks involved in their disease, including variants in specific genes tagging a subset of patients who may benefit from genetically-tailored interventions.

To meet our vision and mission, CAG has built the largest pediatric biorepository in the world, containing samples and associated clinical data from >100,000 consented participants from CHOP and over 400,000 subjects from other worldwide-collaborative studies. The CHOP pediatric samples are linked to electronic health care records data, with parents and family members available for approximately 50% of subjects. There is rich longitudinal phenotypic data from the EHR with consent to update health records as well as re-contact permission for >85% of subjects. CAG updates clinical records on a quarterly basis, and the mean length of EHR follow-up is >6.5 years.
The CAG recruitment team enrolls patients from CHOP and over 50 satellite clinics within the Philadelphia metropolitan area. Taking advantage of the city- and world-wide diversity available to CHOP, the repository is minority-enriched with >38% African American, 6% Hispanic, 5% Asian and 1% other; the balance consists of 50% of subjects of European Ancestry. While the cohort is enriched for both rare and common diseases, CAG also has a high number of typically-developing children, due to recruitment in primary care and satellite clinics. Our recruitment pool renders the CAG repository with a very different composition from disease-based recruitment repositories.
Biospecimens from our patients are divided into DNA, PBMCs and plasma/serum, which affords us the opportunity to generate LCLs and iPSCs from any subject of interest. Nearly all samples are genotyped on GWAS arrays and imputed, with a growing number of sequenced exomes or genomes. CAG has made numerous discoveries and published over 800 manuscript since 2006.
Many examples were cited: progress in inflammatory bowel disease, as well as a novel precision medicine approach to lymphatic disorders. Several rare disease examples were discussed, where new discoveries have led to repurposing opportunities based on state-of-the-art genomic approaches with opportunities to expand the indication to broader disease populations.


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John J Connolly, Ph.D., is a researcher at the Center for Applied Genomics (CAG) at The Children’s Hospital of Philadelphia (CHOP). Since joining CAG in 2010, he has led a range of neuropsychiatric studies, with a focus on autism and ADHD. He also maintained a lead role in the Philadelphia Neurodevelopmental Cohort project, which integrates deeply-characterized data from 9,500 children and young adults that includes electronic health records (EHRs), neuroimaging, genotype, sequencing, and methylation profiles. He is currently a project leader for CHOP as part of our collaboration with the electronic Medical Records and Genomics (eMERGE) Consortium, a major NHGRI 9-Center initiative to integrate EHRs and genomics. Dr. Connolly trained as neuropsychologist at Trinity College Dublin, where he studied stroke rehabilitation and neural correlates of ADHD. He subsequently joined Cold Spring Harbor Laboratory to lead development of Genes to Cognition Online, an online resource that examines how the brain, biochemicals, and genes interact to produce neuropsychiatric. He also created 3D Brain, an award-winning app that has been downloaded more than 4 million times.

Joseph T. Glessner, PhD. Technical Director, Genetics Core Facility,
Center for Applied Genomics.

Dr.Glessner’s current research focuses on childhood neuropsychiatric and neurodevelopmental disorders along with the genetic architecture associated with them, including single nucleotide polymorphisms, single nucleotide variations, and copy number variations ascertained by genomic technologies.
Dr Li’s research interests are focused on novel gene discovery, and specifically how gene discovery can inform basic science research and targeted therapeutic development. He trained in molecular biology and bioinformatics with the goal of discovering and assessing the functional impact of genetics variants on human disease, as well as to ensure the success of highly translational research projects. He received his Ph.D. in Molecular Biology from the Southwest University at Chongqing, China in 2011, with a thesis detecting the signatures of artificial selection in the silkworm genome using NGS technology and large-scale bioinformatics processing. Since his arrival at Center for Applied Genomics (CAG), Children’s Hospital of Philadelphia in August 2011, his roles have included management of several collaborations focused on resolving the genetic causes of rare diseases of unknown etiology.

Dr Sleiman is associate professor of pediatrics at the University of Pennsylvania Medical School and the associate director and lead statistical geneticist at the Center for Applied Genomics of the Children’s Hospital of Philadelphia. He received his PhD in genetics from the University of London before completing postdoctoral training at the Institute of Neurology in London where he led a study that resulted in the identification of a novel early onset Parkinson’s disease gene, PINK1. His research interests lay in uncovering the genetic basis of human disease with a view towards providing more patient focused treatment through both mining of the CAG internal EMR-based patient collection and work with external collaborators across academia and the pharmaceutical industry. His work has resulted in the identification of numerous novel genes and disease loci across multiple phenotypes including eosinophilic esophagitis, schizophrenia, frontotemporal dementia, progressive supranuclear palsy, Parkinson’s disease and asthma that have been reported in over 150 peer reviewed publications, garnering over 23,000 citations.
HakonHakonarson, M.D., Ph.D., is Director of the Center for Applied Genomics, Endowed Chair in Genomics Research and Professor of Pediatrics at The University of Pennsylvania, Perelman School of Medicine. Dr.Hakonarson leads a major commitment from CHOP to genomically characterize approximately 100,000 children, an initiative that has gained nationwide attention in the Wall Street Journal, New York Times, Time Magazine, Nature and Science. Dr.Hakonarson is a Principal Investigator within the Kids First program and the TopMed genomics program funded by the NIH. Dr.Hakonarson published the first pediatric GWAS in T1D (Hakonarson, Nature, 2007) and he has over >25 T1D discovery and translational publications since then. Dr.Hakonarson has previously held several senior posts within the biopharmaceutical industry, directing a number of genomics and pharmacogenomics projects as vice president of Clinical Sciences and Development at deCODE genetics, Inc. Dr. Hakonarson has been the principal investigator (PI) on multiple National Institute of Health-sponsored grants, and he was a principal investigator on the Neurodevelopmental Genomics: Trajectories of Complex Phenotypes, the largest research project ever supported by the National Institute of Mental Health. Dr.Hakonarson recently completed a clinical biomarker study in ADHD demonstrating strong efficacy and safety of a neuromodulator compound (NFC-1) in children with specific mutations in the glutamate metabotropic (mGluR) receptor family of genes with ADHD and Autism (; Elia et al, Nat Comm, 2018). Dr.Hakonarson has published over 750 scientific papers, including numerous high-impact papers on genomic discoveries and their translations in some of the most prestigious scientific medical journals, including Nature, Nature Medicine, Nature Genetics, Cell and The New England Journal of Medicine. Time Magazine listed Dr.Hakonarson’s autism gene discovery project, reported in Nature in 2009, among the top 10 medical breakthroughs of that year. With over 20 years of experience in pioneering genomics research and genome-wide mapping and association studies, Dr.Hakonarson has intimate knowledge of the complexities of large-scale genomics and drug development projects, and he has put together the necessary infrastructure and workflow processes to unravel these complexities for optimized deliverables of precision medicine programs.

Major Landmarks In CAG’s Bench to Bedside Research Strategy. To date, our group has published more than 900 peer-reviewed studies. Ultimately, our objective is to generate new diagnostic tests and to guide physicians to the most appropriate therapies.