Enhancer Variants Synergistically Drive Dysfunction of a Gene Regulatory Network In Hirschsprung Disease.

Common sequence variants in cis-regulatory elements (CREs) are suspected etiological causes of complex disorders. We previously identified an intronic enhancer variant in the RET gene disrupting SOX10 binding and increasing Hirschsprung disease (HSCR) risk 4-fold. We now show that two other functionally independent CRE variants, one binding Gata2 and the other binding Rarb, also reduce Ret expression and increase risk 2- and 1.7-fold. By studying human and mouse fetal gut tissues and cell lines, we demonstrate that reduced RET expression propagates throughout its gene regulatory network, exerting effects on both its positive and negative feedback components. We also provide evidence that the presence of a combination of CRE variants synergistically reduces RET expression and its effects throughout the GRN. These studies show how the effects of functionally independent non-coding variants in a coordinated gene regulatory network amplify their individually small effects, providing a model for complex disorders.

Genomic Answers for Kids: Toward more equitable access to genomic testing for rare diseases in rural populations.

Next-generation sequencing has revolutionized the speed of rare disease (RD) diagnoses. While clinical exome and genome sequencing represent an effective tool for many RD diagnoses, there is room to further improve the diagnostic odyssey of many RD patients. One recognizable intervention lies in increasing equitable access to genomic testing. Rural communities represent a significant portion of underserved and underrepresented individuals facing additional barriers to diagnosis and treatment. Primary care providers (PCPs) at local clinics, though sometimes suspicious of a potential benefit of genetic testing for their patients, have significant constraints in pursuing it themselves and rely on referrals to specialists. Yet, these referrals are typically followed by long waitlists and significant delays in clinical assessment, insurance clearance, testing, and initiation of diagnosis-informed care management. Not only is this process time intensive, but it also often requires multiple visits to urban medical centers for which distance may be a significant barrier to rural families. Therefore, providing early, "direct-to-provider" (DTP) local access to unrestrictive genomic testing is likely to help speed up diagnostic times and access to care for RD patients in rural communities. In a pilot study with a PCP clinic in rural Kansas, we observed a minimum 5.5 months shortening of time to diagnosis through the DTP exome sequencing program as compared to rural patients receiving genetic testing through the "traditional" PCP-referral-to-specialist scheme. We share our experience to encourage future partnerships beyond our center. Our efforts represent just one step in fostering greater diversity and equity in genomic studies.

Committing to genomic answers for all kids: Evaluating inequity in genomic research enrollment.

PURPOSE: Persistent inequities in genomic medicine and research contribute to health disparities. This analysis uses a context-specific and equity-focused strategy to evaluate enrollment patterns for Genomic Answers for Kids (GA4K), a large, metropolitan-wide genomic study on children. METHODS: Electronic health records for 2247 GA4K study participants were used to evaluate the distribution of individuals by demographics (race, ethnicity, and payor type) and location (residential address). Addresses were geocoded to produce point density and 3-digit zip code maps showing local and regional enrollment patterns. Health system reports and census data were used to compare participant characteristics with reference populations at different spatial scales. RESULTS: Racial and ethnic minoritized and populations with low-income were underrepresented in the GA4K study cohort. Geographic variation demonstrates inequity in enrollment and participation among children from historically segregated and socially disadvantaged communities. CONCLUSION: Our findings illustrate inequity in enrollment related to both GA4K study design and structural inequalities, which we suspect may exist for similar US-based studies. Our methods provide a scalable framework for continually evaluating and improving study design to ensure equitable participation in and benefits from genomic research and medicine. The use of high-resolution, place-based data represents a novel and practical means of identifying and characterizing inequities and targeting community engagement.

Insurance denials and diagnostic rates in a pediatric genomic research cohort.

PURPOSE: This study aimed to assess the amount and types of clinical genetic testing denied by insurance and the rate of diagnostic and candidate genetic findings identified through research in patients who faced insurance denials. METHODS: Analysis consisted of review of insurance denials in 801 patients enrolled in a pediatric genomic research repository with either no previous genetic testing or previous negative genetic testing result identified through cross-referencing with insurance prior-authorizations in patient medical records. Patients and denials were also categorized by type of insurance coverage. Diagnostic findings and candidate genetic findings in these groups were determined through review of our internal variant database and patient charts. RESULTS: Of the 801 patients analyzed, 147 had insurance prior-authorization denials on record (18.3%). Exome sequencing and microarray were the most frequently denied genetic tests. Private insurance was significantly more likely to deny testing than public insurance (odds ratio = 2.03 [95% CI = 1.38-2.99] P = .0003). Of the 147 patients with insurance denials, 53.7% had at least 1 diagnostic or candidate finding and 10.9% specifically had a clinically diagnostic finding. Fifty percent of patients with clinically diagnostic results had immediate medical management changes (5.4% of all patients experiencing denials). CONCLUSION: Many patients face a major barrier to genetic testing in the form of lack of insurance coverage. A number of these patients have clinically diagnostic findings with medical management implications that would not have been identified without access to research testing. These findings support re-evaluation of insurance carriers' coverage policies.

Consideration and Disclosure of Group Risks in Genomics and Other Data-Centric Research: Does the Common Rule Need Revision?

Harms and risks to groups and third-parties can be significant in the context of research, particularly in data-centric studies involving genomic, artificial intelligence, and/or machine learning technologies. This article explores whether and how United States federal regulations should be adapted to better align with current ethical thinking and protect group interests. Three aspects of the Common Rule deserve attention and reconsideration with respect to group interests: institutional review board (IRB) assessment of the risks/benefits of research; disclosure requirements in the informed consent process; and criteria for waivers of informed consent. In accordance with respect for persons and communities, investigators and IRBs should systematically consider potential group harm when designing and reviewing protocols, respectively. Research participants should be informed about any potential group harm in the consent process. We call for additional public discussion, empirical research, and normative analysis on these issues to determine the right regulatory and policy path forward.

Molecular Genetic Anatomy and Risk Profile of Hirschsprung's Disease.

BACKGROUND: Hirschsprung's disease, or congenital aganglionosis, is a developmental disorder of the enteric nervous system and is the most common cause of intestinal obstruction in neonates and infants. The disease has more than 80% heritability, including significant associations with rare and common sequence variants in genes related to the enteric nervous system, as well as with monogenic and chromosomal syndromes. METHODS: We genotyped and exome-sequenced samples from 190 patients with Hirschsprung's disease to quantify the genetic burden in patients with this condition. DNA sequence variants, large copy-number variants, and karyotype variants in probands were considered to be pathogenic when they were significantly associated with Hirschsprung's disease or another neurodevelopmental disorder. Novel genes were confirmed by functional studies in the mouse and human embryonic gut and in zebrafish embryos. RESULTS: The presence of five or more variants in four noncoding elements defined a widespread risk of Hirschsprung's disease (48.4% of patients and 17.1% of controls; odds ratio, 4.54; 95% confidence interval [CI], 3.19 to 6.46). Rare coding variants in 24 genes that play roles in enteric neural-crest cell fate, 7 of which were novel, were also common (34.7% of patients and 5.0% of controls) and conferred a much greater risk than noncoding variants (odds ratio, 10.02; 95% CI, 6.45 to 15.58). Large copy-number variants, which were present in fewer patients (11.4%, as compared with 0.2% of controls), conferred the highest risk (odds ratio, 63.07; 95% CI, 36.75 to 108.25). At least one identifiable genetic risk factor was found in 72.1% of the patients, and at least 48.4% of patients had a structural or regulatory deficiency in the gene encoding receptor tyrosine kinase (RET). For individual patients, the estimated risk of Hirschsprung's disease ranged from 5.33 cases per 100,000 live births (approximately 1 per 18,800) to 8.38 per 1000 live births (approximately 1 per 120). CONCLUSIONS: Among the patients in our study, Hirschsprung's disease arose from common noncoding variants, rare coding variants, and copy-number variants affecting genes involved in enteric neural-crest cell fate that exacerbate the widespread genetic susceptibility associated with RET. For individual patients, the genotype-specific odds ratios varied by a factor of approximately 67, which provides a basis for risk stratification and genetic counseling. (Funded by the National Institutes of Health.).

Genomic answers for children: Dynamic analyses of >1000 pediatric rare disease genomes.

PURPOSE: This study aimed to provide comprehensive diagnostic and candidate analyses in a pediatric rare disease cohort through the Genomic Answers for Kids program. METHODS: Extensive analyses of 960 families with suspected genetic disorders included short-read exome sequencing and short-read genome sequencing (srGS); PacBio HiFi long-read genome sequencing (HiFi-GS); variant calling for single nucleotide variants (SNV), structural variant (SV), and repeat variants; and machine-learning variant prioritization. Structured phenotypes, prioritized variants, and pedigrees were stored in PhenoTips database, with data sharing through controlled access the database of Genotypes and Phenotypes. RESULTS: Diagnostic rates ranged from 11% in patients with prior negative genetic testing to 34.5% in naive patients. Incorporating SVs from genome sequencing added up to 13% of new diagnoses in previously unsolved cases. HiFi-GS yielded increased discovery rate with >4-fold more rare coding SVs compared with srGS. Variants and genes of unknown significance remain the most common finding (58% of nondiagnostic cases). CONCLUSION: Computational prioritization is efficient for diagnostic SNVs. Thorough identification of non-SNVs remains challenging and is partly mitigated using HiFi-GS sequencing. Importantly, community research is supported by sharing real-time data to accelerate gene validation and by providing HiFi variant (SNV/SV) resources from >1000 human alleles to facilitate implementation of new sequencing platforms for rare disease diagnoses.

Identifying Needs, Challenges, and Benefits Among Adults and Parents of Children With Hirschsprung Disease.

ABSTRACT: Adults with Hirschsprung disease (HD), unaffected parents of children with HD, and affected adults with an affected child completed a cross-sectional survey with open-ended questions about greatest needs at diagnosis and at current time, greatest challenges encountered, and any benefits of having HD or having a child with HD. In the 297 respondents, information and good medical care were common needs at diagnosis and at the time of survey, but the information needed evolved with time. Managing ongoing symptoms was a frequently cited need and challenge, along with managing medical care and the social and emotional impact of HD. Perceived benefits included empathy for others and new perspectives on life. The needs and challenges identified in this study can guide healthcare providers in discussions with families. Provision of information, recommendations, and referrals based on each individual family's needs can support families with HD throughout the lifecycle and facilitate adaptation.

Challenges in genetic testing: clinician variant interpretation processes and the impact on clinical care.

PURPOSE: Efforts have been made to standardize laboratory variant interpretation, but clinicians are ultimately tasked with clinical correlation and application of genetic test results in patient care. This study aimed to explore processes clinicians utilize when reviewing and returning genetic test results, and how they impact patient care. METHODS: Medical geneticists, genetic counselors, and nongenetics clinicians from two Midwestern states completed surveys (n = 98) and in-depth interviews (n = 29) on practices of reviewing and returning genetic test results. Retrospective chart review (n = 130) examined discordant interpretations and the impact on care. RESULTS: Participants reported variable behaviors in both reviewing and returning results based on factors such as confidence, view of role, practice setting, and relationship with the lab. Providers did not report requesting changes to variant classifications from laboratories, but indicated relaying conflicting classifications to patients in some cases. Chart reviews revealed medically impactful differences in interpretation between laboratories and clinicians in 18 (13.8%) records. CONCLUSION: Clinician practices for reviewing and integrating genetic test results into patient care vary within and between specialties and impact patient care. Strategies to better incorporate both laboratory and clinician expertise into interpretation of genetic results could result in improved care across providers and settings.

Parents of newborns in the NICU enrolled in genome sequencing research: hopeful, but not naïve.

PURPOSE: In 2014, our institution launched a randomized controlled trial (RCT) comparing rapid genome sequencing (GS) to standard clinical evaluations of infants with suspected genetic disorders. This study aimed to understand parental response to the use of GS for their newborn babies. METHODS: Twenty-three of 128 parents whose infant had enrolled in the RCT completed a retrospective survey and interview addressing attitudes about GS and responses to receiving diagnostic information. We also collected information about participants' genetic literacy, genetic knowledge, numeracy, and symptoms of anxiety and depression. RESULTS: The majority reported positive (13; 56.5%) or neutral 4 (4; 17.4%) feelings when approached about GS for their infant and 100% felt that GS was generally beneficial. The 12 participants who had received a unifying diagnosis for their child's symptoms described personal utility of the information. Some reported the diagnosis led to changes in medical care. Participants showed understanding of some of the psychological risks of GS. For example, 21 (91.3%) agreed or strongly agreed that genetic testing could reveal disturbing results. CONCLUSIONS: Parents who enrolled their newborn in a RCT of GS demonstrated awareness of a psychological risk, but generally held positive beliefs about GS and perceived the benefits outweighed the risk.

Clinical genome sequencing in an unbiased pediatric cohort.

PURPOSE: We report for the first time, the use of clinical genome sequencing (GS) in an unbiased pediatric cohort. We describe the clinical validation, patient metrics, ordering patterns, results, reimbursement, and physician retrieval of results for the first consecutive 80 cases. METHODS: Clinical GS was performed for both inpatients and outpatients undergoing etiologic evaluations. Results were reported in the electronic medical record. Evidence of report retrieval by clinicians and whether interpretation was concordant with laboratory report was obtained through retrospective chart review. RESULTS: Twenty definitive diagnoses were made in 19 patients (24%; n = 80). Except for two partial gene deletions, all diagnostic variants would have been detectable by our exome methods. Surprisingly, there was no documentation of communication of results to the family in the medical record for 17.5% of patients, and in 7.5%, physician and laboratory interpretations were discordant. Average insurance reimbursement was 30.2%, with yield for commercial payers significantly higher, at 54.1%. CONCLUSIONS: The detection rate of GS is equivalent and potentially superior to exome sequencing (ES). Reimbursement rates were variable but overall satisfactory for commercial insurers, and poor for government entities. In addition, we identify opportunities for improvement in the communication of results to families, likely translatable to other tests and other institutions.

High Levels of Interest in Reproductive Genetic Information in Parents of Children and Adults With Hirschsprung Disease.

OBJECTIVES: Families affected by Hirschsprung disease (HSCR) have opportunities to learn recurrence risks to their children from statistical genetic and empiric studies and, in some cases, prenatal genetic testing or preimplantation genetic diagnosis (PGD). This study aimed to assess interest in reproductive genetic information for HSCR and factors that predict this interest in 2 groups with elevated risk of having a child with HSCR. METHODS: Adult patients with HSCR and parents of children with HSCR were surveyed about their interest in learning reproductive genetic information regarding HSCR through genetic counseling, prenatal testing, and PGD. Covariates assessed in this cross-sectional study included quality of life, illness perceptions, depressive symptoms, and adaptation to the condition. Bivariate analyses assessed differences between affected adults and parents. Logistic modeling was used to identify predictors of interest in reproductive genetic information. RESULTS: Study participants (n = 368) reported high interest in reproductive genetic information through genetic counseling (yes = 60%/unsure = 16%), prenatal testing (yes = 59%/unsure = 16%), and PGD (yes = 43%/unsure = 18%). Illness perceptions differed between affected adults and parents, but perceived severity of HSCR was high among all participants (µâ€Š= 3.42, SD = 0.67, 4-point scale). Interest in reproductive information was associated with being an affected adult, not having a family history of HSCR, negative emotional representations, and adaptation to the condition. CONCLUSIONS: Findings from this study support a desire among the surveyed HSCR patient groups to attend genetic counseling and be offered testing when available. Exploration of perceptions and experiences with the condition should be incorporated into the counseling to insure informed preference-based decision making.

Using the diffusion of innovations model to guide participant engagement in the genomics era.

Exome and genome sequencing (EGS) are increasingly the genetic testing modalities of choice among researchers owing to their ready availability, low cost, and large data output. Recruitment of larger, more diverse cohorts into long-term studies with extensive data collection is fundamental to the success of EGS research and to the widespread benefit of genomic medicine to various populations. Effective engagement will be critical to meeting this demand. The Diffusion of Innovation (DOI) model provides a framework for how new technologies are adopted in communities, including antecedents of an individual's decision to adopt the technology, how the technology's attributes influence its acceptability, the predictors of sustained use of that technology, and its diffusion through society. We apply the DOI model to frame participant engagement in EGS research and to guide the proposal of potential strategies that aim to overcome forecasted challenges. Finally, we suggest a variety of ways genetic counselors can apply their skills and training to the development and implementation of these strategies.

Trans-ethnic meta-analysis of genome-wide association studies for Hirschsprung disease.

Hirschsprung disease (HSCR) is the most common cause of neonatal intestinal obstruction. It is characterized by the absence of ganglia in the nerve plexuses of the lower gastrointestinal tract. So far, three common disease-susceptibility variants at the RET, SEMA3 and NRG1 loci have been detected through genome-wide association studies (GWAS) in Europeans and Asians to understand its genetic etiologies. Here we present a trans-ethnic meta-analysis of 507 HSCR cases and 1191 controls, combining all published GWAS results on HSCR to fine-map these loci and narrow down the putatively causal variants to 99% credible sets. We also demonstrate that the effects of RET and NRG1 are universal across European and Asian ancestries. In contrast, we detected a European-specific association of a low-frequency variant, rs80227144, in SEMA3 [odds ratio (OR) = 5.2, P = 4.7 × 10-10]. Conditional analyses on the lead SNPs revealed a secondary association signal, corresponding to an Asian-specific, low-frequency missense variant encoding RET p.Asp489Asn (rs9282834, conditional OR = 20.3, conditional P = 4.1 × 10-14). When in trans with the RET intron 1 enhancer risk allele, rs9282834 increases the risk of HSCR from 1.1 to 26.7. Overall, our study provides further insights into the genetic architecture of HSCR and has profound implications for future study designs.

Enrolling Genomics Research Participants through a Clinical Setting: the Impact of Existing Clinical Relationships on Informed Consent and Expectations for Return of Research Results.

Genetic counselors working in a clinical setting may find themselves recruiting, enrolling, and returning results for genomic research, and existing clinical relationships with study participants may impact these research interactions. We present a qualitative study using semi-structured interviews of participants enrolled in a genome sequencing/exome sequencing (GS/ES) study at the same institution where they receive clinical care. Interviews were coded for motivations to participate and expectations of this research. The interviews revealed common motivations for participation, including altruism and hope for benefit for themselves, family members, and/or others with their condition. Additionally, themes emerged related to unintentional influence based on trust of the clinical provider that recruited them to the study. Participant trust in the enrolling provider at times appeared to extend to the study team to decide which research results to return and to do so in an appropriate format. Participants also based expectations for research results return on previous clinical genetic testing experiences, which may or may not be realistic depending on study design. It is imperative that genetic counselors enrolling patients into research studies be aware of the potential influence of their clinical relationship on potential subjects, be transparent about their role on the study team, and help set expectations about the study process, including results return.

Population variation in total genetic risk of Hirschsprung disease from common RET, SEMA3 and NRG1 susceptibility polymorphisms.

The risk of Hirschsprung disease (HSCR) is ∼15/100 000 live births per newborn but has been reported to show significant inter-individual variation from the effects of seven common susceptibility alleles at the RET, SEMA3 and NRG1 loci. We show, by analyses of these variants in 997 samples from 376 HSCR families of European ancestry, that significant genetic risk can only be detected at RET (rs2435357 and rs2506030) and at SEMA3 (rs11766001), but not at NRG1. RET rs2435357 also showed significant frequency differences by gender, segment length of aganglionosis and familiality. Further, in combination, disease risk varied >30-fold between individuals with none and up to 6 susceptibility alleles. Thus, these polymorphisms can be used to stratify the newborn population into distinct phenotypic classes with defined risks to understand HSCR etiology.

Comparing Attitudes About Genomic Privacy and Data Sharing in Adolescents and Parents of Children Enrolled in a Genomic Research Repository.

BACKGROUND: Sharing of genomic data aims to make efficient use of limited resources, which may be particularly valuable in rare disease research. Adult research participants and parents of pediatric research participants have shown support for data sharing with protections, but little is known about adolescent attitudes on genomic privacy and data sharing. METHODS: In-depth interviews were conducted with 10 adolescents and 18 parents of children enrolled in a pediatric genomic research repository. Interview transcripts were analyzed for themes on attitudes toward genomic privacy, restricted-access data sharing, and open-access data sharing. Findings in adolescent and parent participants were compared and contrasted. RESULTS: No adolescents endorsed privacy concerns for restricted-access data sharing. Both adolescents and parents saw value in data sharing for reaching the goals of research and discussed trust in institutions and researchers to protect their data and use it as intended. Adolescents were more likely than parents to accept open-access data sharing, including after risks were discussed. CONCLUSIONS: In this exploratory study, adolescents and parents enrolled in a genomic research repository shared many attitudes about genomic data sharing, but adolescents were less concerned about privacy and more agreeable toward open-access data sharing. Future research is needed to investigate this hypothesis in expanded populations and settings, and to clarify whether adolescent attitudes change with age and experiences.

Developing a community-led rare disease ELSI research agenda.

BACKGROUND: Research priorities are best defined through engagement with communities who will be impacted by the research and have lived experience of the topics to be studied. We aimed to establish a pediatric rare disease community stakeholder group and empower them in (1) eliciting perspectives from affected families in the wider region and (2) synthesizing collective ideas into a research agenda focused on shared ethical, legal, and social implications (ELSI) across rare disease. METHODS: This two-year project utilized a community-centered approach to engage rare disease community members as equal partners in developing a research agenda for ELSI in rare disease. We established "Rare Voices" (RV), a 22-member stakeholder group of patients, parents, clinicians and researchers. Following capacity-building trainings, RV designed and conducted listening sessions with teen patients and parents of children with rare diseases to explore challenges, positive experiences, and ethical concerns. Listening session findings were synthesized and contextualized into research topics, which RV members further refined and prioritized. We used established measures to assess RV member engagement and satisfaction. RESULTS: From 14 listening sessions with parents (n = 52) and teen patients (n = 13), RV identified eight core research topics as most important for future rare disease research: coordinating care, communication, accessing resources and care, impact on family unit, community and support in society, mental health and identity, ethical aspects of care, and uncertainty. RV members were highly engaged throughout the two-year project and reported high levels of satisfaction with the experience and research agenda. CONCLUSIONS: Through capacity-building and authentic engagement, this project resulted in a community-led rare disease research agenda to guide future rare disease ELSI research that aligns with patients' and families' priorities. An environment of equal partnership and respect created a space for mutual learning where community members were empowered to shape the research agenda based on their collective experiences. The agenda recognizes the shared psychosocial and healthcare experiences of rare disease and offers practical areas of research to address patient and family needs.