A framework for individualized splice-switching oligonucleotide therapy.

Splice-switching antisense oligonucleotides (ASOs) could be used to treat a subset of individuals with genetic diseases1, but the systematic identification of such individuals remains a challenge. Here we performed whole-genome sequencing analyses to characterize genetic variation in 235 individuals (from 209 families) with ataxia-telangiectasia, a severely debilitating and life-threatening recessive genetic disorder2,3, yielding a complete molecular diagnosis in almost all individuals. We developed a predictive taxonomy to assess the amenability of each individual to splice-switching ASO intervention; 9% and 6% of the individuals had variants that were 'probably' or 'possibly' amenable to ASO splice modulation, respectively. Most amenable variants were in deep intronic regions that are inaccessible to exon-targeted sequencing. We developed ASOs that successfully rescued mis-splicing and ATM cellular signalling in patient fibroblasts for two recurrent variants. In a pilot clinical study, one of these ASOs was used to treat a child who had been diagnosed with ataxia-telangiectasia soon after birth, and showed good tolerability without serious adverse events for three years. Our study provides a framework for the prospective identification of individuals with genetic diseases who might benefit from a therapeutic approach involving splice-switching ASOs.

Contribution and therapeutic implications of retroelement insertions in ataxia telangiectasia.

Certain classes of genetic variation still escape detection in clinical sequencing analysis. One such class is retroelement insertion, which has been reported as a cause of Mendelian diseases and may offer unique therapeutic implications. Here, we conducted retroelement profiling on whole-genome sequencing data from a cohort of 237 individuals with ataxia telangiectasia (A-T). We found 15 individuals carrying retroelement insertions in ATM, all but one of which integrated in noncoding regions. Systematic functional characterization via RNA sequencing, RT-PCR, and/or minigene splicing assays showed that 12 out of 14 intronic insertions led or contributed to ATM loss of function by exon skipping or activating cryptic splice sites. We also present proof-of-concept antisense oligonucleotides that suppress cryptic exonization caused by a deep intronic retroelement insertion. These results provide an initial systematic estimate of the contribution of retroelements to the genetic architecture of recessive Mendelian disorders as ∼2.1%-5.5%. Our study highlights the importance of retroelement insertions as causal variants and therapeutic targets in genetic diseases.

"It's hard to wait": Provider Perspectives on Current Genomic Care in Safety-Net NICUs.

PURPOSE: Critically ill infants from marginalized populations disproportionately receive care in neonatal intensive care units (NICUs) that lack access to state-of-the-art genomic care, leading to inequitable outcomes. We sought provider perspectives to inform our implementation study (VIGOR) providing rapid genomic sequencing within these settings. METHODS: We conducted semi-structured focus groups with neonatal and genetics providers at five NICUs at safety-net hospitals, informed by the Promoting Action on Research Implementation in Health Services framework, which incorporates evidence, context, and facilitation domains. We iteratively developed codes and themes until thematic saturation was reached. RESULTS: Regarding evidence, providers felt that genetic testing benefits infants and families. Regarding context, the major barriers identified to genomic care were genetic testing cost, lack of genetics expertise for disclosure and follow-up, and navigating the complexity of selecting and ordering genetic tests. Providers had negative feelings about the current status quo and inequity in genomic care across NICUs. Regarding facilitation, providers felt that a virtual support model like VIGOR would address major barriers and foster family-centered care and collaboration. CONCLUSION: NICU providers at safety-net hospitals believe that access to state-of-the-art genomic care is critical for optimizing infant outcomes, yet substantial barriers exist that the VIGOR study may address.

Data sharing to advance gene-targeted therapies in rare diseases.

Recent advancements in gene-targeted therapies have highlighted the critical role data sharing plays in successful translational drug development for people with rare diseases. To scale these efforts, we need to systematize these sharing principles, creating opportunities for more rapid, efficient, and scalable drug discovery/testing including long-term and transparent assessment of clinical safety and efficacy. A number of challenges will need to be addressed, including the logistical difficulties of studying rare diseases affecting individuals who may be scattered across the globe, scientific, technical, regulatory, and ethical complexities of data collection, and harmonization and integration across multiple platforms and contexts. The NCATS/NIH Gene-Targeted Therapies: Early Diagnosis and Equitable Delivery meeting series held during June 2021 included data sharing models that address these issues and framed discussions of areas that require improvement. This article describes these discussions and provides a series of considerations for future data sharing.

Whole-genome sequencing holds the key to the success of gene-targeted therapies.

Rare genetic disorders affect as many as 3%-5% of all babies born. Approximately 10,000 such disorders have been identified or hypothesized to exist. Treatment is supportive except in a limited number of instances where specific therapies exist. Development of new therapies has been hampered by at least two major factors: difficulty in diagnosing diseases early enough to enable treatment before irreversible damage occurs, and the high cost of developing new drugs and getting them approved by regulatory agencies. Whole-genome sequencing (WGS) techniques have become exponentially less expensive and more rapid since the beginning of the human genome project, such that return of clinical data can now be achieved in days rather than years and at a cost that is comparable to other less expansive genetic testing. Thus, it is likely that WGS will ultimately become a mainstream, first-tier NBS technique at least for those disorders without appropriate high-throughput functional tests. However, there are likely to be several steps in the evolution to this end. The clinical implications of these advances are profound but highlight the bottlenecks in drug development that still limit transition to treatments. This article summarizes discussions arising from a recent National Institute of Health conference on nucleic acid therapy, with a focus on the impact of WGS in the identification of diagnosis and treatment of rare genetic disorders.

Moving away from one disease at a time: Screening, trial design, and regulatory implications of novel platform technologies.

Most rare diseases are caused by single-gene mutations, and as such, lend themselves to a host of new gene-targeted therapies and technologies including antisense oligonucleotides, phosphomorpholinos, small interfering RNAs, and a variety of gene delivery and gene editing systems. Early successes are encouraging, however, given the substantial number of distinct rare diseases, the ability to scale these successes will be unsustainable without new development efficiencies. Herein, we discuss the need for genomic newborn screening to match pace with the growing development of targeted therapeutics and ability to rapidly develop individualized therapies for rare variants. We offer approaches to move beyond conventional "one disease at a time" preclinical and clinical drug development and discuss planned regulatory innovations that are necessary to speed therapy delivery to individuals in need. These proposals leverage the shared properties of platform classes of therapeutics and innovative trial designs including master and platform protocols to better serve patients and accelerate drug development. Ultimately, there are risks to these novel approaches; however, we believe that close partnership and transparency between health authorities, patients, researchers, and drug developers present the path forward to overcome these challenges and deliver on the promise of gene-targeted therapies for rare diseases.

Are we prepared to deliver gene-targeted therapies for rare diseases?

The cost and time needed to conduct whole-genome sequencing (WGS) have decreased significantly in the last 20 years. At the same time, the number of conditions with a known molecular basis has steadily increased, as has the number of investigational new drug applications for novel gene-based therapeutics. The prospect of precision gene-targeted therapy for all seems in reach… or is it? Here we consider practical and strategic considerations that need to be addressed to establish a foundation for the early, effective, and equitable delivery of these treatments.

Interpretation of Genomic Sequencing Results in Healthy and Ill Newborns: Results from the BabySeq Project.

Genomic sequencing provides many opportunities in newborn clinical care, but the challenges of interpreting and reporting newborn genomic sequencing (nGS) results need to be addressed for its broader and effective application. The BabySeq Project is a pilot randomized clinical trial that explores the medical, behavioral, and economic impacts of nGS in well newborns and those admitted to a neonatal intensive care unit (NICU). Here we present childhood-onset and actionable adult-onset disease risk, carrier status, and pharmacogenomics findings from nGS of 159 newborns in the BabySeq Project. nGS revealed a risk of childhood-onset disease in 15/159 (9.4%) newborns; none of the disease risks were anticipated based on the infants' known clinical or family histories. nGS also revealed actionable adult-onset disease risk in 3/85 (3.5%) newborns whose parents consented to receive this information. Carrier status for recessive diseases and pharmacogenomics variants were reported in 88% and 5% of newborns, respectively. Additional indication-based analyses were performed in 29/32 (91%) NICU newborns and 6/127 (5%) healthy newborns who later had presentations that prompted a diagnostic analysis. No variants that sufficiently explained the reason for the indications were identified; however, suspicious but uncertain results were reported in five newborns. Testing parental samples contributed to the interpretation and reporting of results in 13/159 (8%) newborns. Our results suggest that nGS can effectively detect risk and carrier status for a wide range of disorders that are not detectable by current newborn screening assays or predicted based on the infant's known clinical or family history, and the interpretation of results can substantially benefit from parental testing.

De Novo Variants in WDR37 Are Associated with Epilepsy, Colobomas, Dysmorphism, Developmental Delay, Intellectual Disability, and Cerebellar Hypoplasia.

WD40 repeat-containing proteins form a large family of proteins present in all eukaryotes. Here, we identified five pediatric probands with de novo variants in WDR37, which encodes a member of the WD40 repeat protein family. Two probands shared one variant and the others have variants in nearby amino acids outside the WD40 repeats. The probands exhibited shared phenotypes of epilepsy, colobomas, facial dysmorphology reminiscent of CHARGE syndrome, developmental delay and intellectual disability, and cerebellar hypoplasia. The WDR37 protein is highly conserved in vertebrate and invertebrate model organisms and is currently not associated with a human disease. We generated a null allele of the single Drosophila ortholog to gain functional insights and replaced the coding region of the fly gene CG12333/wdr37 with GAL4. These flies are homozygous viable but display severe bang sensitivity, a phenotype associated with seizures in flies. Additionally, the mutant flies fall when climbing the walls of the vials, suggesting a defect in grip strength, and repeat the cycle of climbing and falling. Similar to wall clinging defect, mutant males often lose grip of the female abdomen during copulation. These phenotypes are rescued by using the GAL4 in the CG12333/wdr37 locus to drive the UAS-human reference WDR37 cDNA. The two variants found in three human subjects failed to rescue these phenotypes, suggesting that these alleles severely affect the function of this protein. Taken together, our data suggest that variants in WDR37 underlie a novel syndromic neurological disorder.

Patient-Customized Oligonucleotide Therapy for a Rare Genetic Disease.

Genome sequencing is often pivotal in the diagnosis of rare diseases, but many of these conditions lack specific treatments. We describe how molecular diagnosis of a rare, fatal neurodegenerative condition led to the rational design, testing, and manufacture of milasen, a splice-modulating antisense oligonucleotide drug tailored to a particular patient. Proof-of-concept experiments in cell lines from the patient served as the basis for launching an "N-of-1" study of milasen within 1 year after first contact with the patient. There were no serious adverse events, and treatment was associated with objective reduction in seizures (determined by electroencephalography and parental reporting). This study offers a possible template for the rapid development of patient-customized treatments. (Funded by Mila's Miracle Foundation and others.).

Exome and genome sequencing for pediatric patients with congenital anomalies or intellectual disability: an evidence-based clinical guideline of the American College of Medical Genetics and Genomics (ACMG).

PURPOSE: To develop an evidence-based clinical practice guideline for the use of exome and genome sequencing (ES/GS) in the care of pediatric patients with one or more congenital anomalies (CA) with onset prior to age 1 year or developmental delay (DD) or intellectual disability (ID) with onset prior to age 18 years. METHODS: The Pediatric Exome/Genome Sequencing Evidence-Based Guideline Work Group (n = 10) used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) evidence to decision (EtD) framework based on the recent American College of Medical Genetics and Genomics (ACMG) systematic review, and an Ontario Health Technology Assessment to develop and present evidence summaries and health-care recommendations. The document underwent extensive internal and external peer review, and public comment, before approval by the ACMG Board of Directors. RESULTS: The literature supports the clinical utility and desirable effects of ES/GS on active and long-term clinical management of patients with CA/DD/ID, and on family-focused and reproductive outcomes with relatively few harms. Compared with standard genetic testing, ES/GS has a higher diagnostic yield and may be more cost-effective when ordered early in the diagnostic evaluation. CONCLUSION: We strongly recommend that ES/GS be considered as a first- or second-tier test for patients with CA/DD/ID.

Discordant results between conventional newborn screening and genomic sequencing in the BabySeq Project.

PURPOSE: Newborn screening (NBS) is performed to identify neonates at risk for actionable, severe, early-onset disorders, many of which are genetic. The BabySeq Project randomized neonates to receive conventional NBS or NBS plus exome sequencing (ES) capable of detecting sequence variants that may also diagnose monogenic disease or indicate genetic disease risk. We therefore evaluated how ES and conventional NBS results differ in this population. METHODS: We compared results of NBS (including hearing screens) and ES for 159 infants in the BabySeq Project. Infants were considered "NBS positive" if any abnormal result was found indicating disease risk and "ES positive" if ES identified a monogenic disease risk or a genetic diagnosis. RESULTS: Most infants (132/159, 84%) were NBS and ES negative. Only one infant was positive for the same disorder by both modalities. Nine infants were NBS positive/ES negative, though seven of these were subsequently determined to be false positives. Fifteen infants were ES positive/NBS negative, all of which represented risk of genetic conditions that are not included in NBS programs. No genetic explanation was identified for eight infants referred on the hearing screen. CONCLUSION: These differences highlight the complementarity of information that may be gleaned from NBS and ES in the newborn period.

Integrated genome and transcriptome sequencing identifies a noncoding mutation in the genome replication factor DONSON as the cause of microcephaly-micromelia syndrome.

While next-generation sequencing has accelerated the discovery of human disease genes, progress has been largely limited to the "low hanging fruit" of mutations with obvious exonic coding or canonical splice site impact. In contrast, the lack of high-throughput, unbiased approaches for functional assessment of most noncoding variants has bottlenecked gene discovery. We report the integration of transcriptome sequencing (RNA-seq), which surveys all mRNAs to reveal functional impacts of variants at the transcription level, into the gene discovery framework for a unique human disease, microcephaly-micromelia syndrome (MMS). MMS is an autosomal recessive condition described thus far in only a single First Nations population and causes intrauterine growth restriction, severe microcephaly, craniofacial anomalies, skeletal dysplasia, and neonatal lethality. Linkage analysis of affected families, including a very large pedigree, identified a single locus on Chromosome 21 linked to the disease (LOD > 9). Comprehensive genome sequencing did not reveal any pathogenic coding or canonical splicing mutations within the linkage region but identified several nonconserved noncoding variants. RNA-seq analysis detected aberrant splicing in DONSON due to one of these noncoding variants, showing a causative role for DONSON disruption in MMS. We show that DONSON is expressed in progenitor cells of embryonic human brain and other proliferating tissues, is co-expressed with components of the DNA replication machinery, and that Donson is essential for early embryonic development in mice as well, suggesting an essential conserved role for DONSON in the cell cycle. Our results demonstrate the utility of integrating transcriptomics into the study of human genetic disease when DNA sequencing alone is not sufficient to reveal the underlying pathogenic mutation.

Prospective, phenotype-driven selection of critically ill neonates for rapid exome sequencing is associated with high diagnostic yield.

PURPOSE: To investigate the impact of rapid-turnaround exome sequencing in critically ill neonates using phenotype-based subject selection criteria. METHODS: Intensive care unit babies aged <6 months with hypotonia, seizures, a complex metabolic phenotype, and/or multiple congenital malformations were prospectively enrolled for rapid (<7 day) trio-based exome sequencing. Genomic variants relevant to the presenting phenotype were returned to the medical team. RESULTS: A genetic diagnosis was attained in 29 of 50 (58%) sequenced cases. Twenty-seven (54%) patients received a molecular diagnosis involving known disease genes; two additional cases (4%) were solved with pathogenic variants found in novel disease genes. In 24 of the solved cases, diagnosis had impact on patient management and/or family members. Management changes included shift to palliative care, medication changes, involvement of additional specialties, and the consideration of new experimental therapies. CONCLUSION: Phenotype-based patient selection is effective at identifying critically ill neonates with a high likelihood of receiving a molecular diagnosis via rapid-turnaround exome sequencing, leading to faster and more accurate diagnoses, reducing unnecessary testing and procedures, and informing medical care.

A phenotypically severe, biochemically "silent" case of HIBCH deficiency in a newborn diagnosed by rapid whole exome sequencing and enzymatic testing.

3-Hydroxyisobutyryl-CoA dehydrogenase (HIBCH) deficiency is a rare error in valine catabolism associated with a Leigh syndrome-like phenotype, mitochondrial dysfunction, and increased C4-OH. We report the most severe case to date in a full-term female who presented with poor feeding and nystagmus on day of life (DOL) 1. Although initial neuroimaging findings were concerning for metabolic disease, further metabolic testing was nondiagnostic and she was discharged on DOL 18. She was readmitted on DOL 22 after severe apneic episodes requiring intubation, with EEG demonstrating multifocal seizures and MRI/MRS demonstrating worsening findings. Care was withdrawn DOL 27 and she expired. Rapid whole exome sequencing (WES) demonstrated compound heterozygous variants in HIBCH with a paternal pathogenic variant (c.852delA, p.L284FfsX10) and a maternal likely pathogenic variant (c.488G>T, p.C163F). Fibroblast enzymatic testing demonstrated marked reduction in HIBCH levels. This case demonstrates the importance of rapid WES and follow-up functional testing in establishing a diagnosis when metabolic disease is suspected but lacks an expected biochemical signature.

Rare variant association test in family-based sequencing studies.

The objective of this article is to introduce valid and robust methods for the analysis of rare variants for family-based exome chips, whole-exome sequencing or whole-genome sequencing data. Family-based designs provide unique opportunities to detect genetic variants that complement studies of unrelated individuals. Currently, limited methods and software tools have been developed to assist family-based association studies with rare variants, especially for analyzing binary traits. In this article, we address this gap by extending existing burden and kernel-based gene set association tests for population data to related samples, with a particular emphasis on binary phenotypes. The proposed approach blends the strengths of kernel machine methods and generalized estimating equations. Importantly, the efficient generalized kernel score test can be applied as a mega-analysis framework to combine studies with different designs. We illustrate the application of the proposed method using data from an exome sequencing study of autism. Methods discussed in this article are implemented in an R package 'gskat', which is available on CRAN and GitHub.

Parental interest in genomic sequencing of newborns: enrollment experience from the BabySeq Project.

PURPOSE: Newborn genomic sequencing (nGS) has great potential to improve pediatric care. Parental interest and concerns about genomics are relatively unexplored. Understanding why parents decline research consent for nGS may reveal implementation barriers. METHODS: We evaluated parental interest in a randomized trial of nGS in well-baby and intensive care unit nursery settings. Interested families attended an informational enrollment session (ES) with a genetic counselor prior to consenting. Reason(s) for declining participation and sociodemographic associations were analyzed. RESULTS: Of 3860 eligible approached families, 10% attended ES, 67% of whom enrolled. Of 1760 families queried for decline reasons, 58% were uninterested in research. Among 499 families considering research, principal reasons for decline prior to ES included burdensome study logistics (48%), feeling overwhelmed postpartum (17%), and lack of interest/discomfort with genetic testing (17%). Decliners after ES more often cited concerns about privacy/insurability (41%) and uncertain/unfavorable results (23%). CONCLUSION: Low interest in research and study logistics were major initial barriers to postpartum enrollment and are likely generic to many postpartum research efforts. Concerns over privacy and result implications were most commonly cited in decliners after ES. Understanding parental concerns around research nGS may inform future integration of nGS into newborn screening, predictive testing, and pediatric diagnostics.

DCC mutation update: Congenital mirror movements, isolated agenesis of the corpus callosum, and developmental split brain syndrome.

The deleted in colorectal cancer (DCC) gene encodes the netrin-1 (NTN1) receptor DCC, a transmembrane protein required for the guidance of commissural axons. Germline DCC mutations disrupt the development of predominantly commissural tracts in the central nervous system (CNS) and cause a spectrum of neurological disorders. Monoallelic, missense, and predicted loss-of-function DCC mutations cause congenital mirror movements, isolated agenesis of the corpus callosum (ACC), or both. Biallelic, predicted loss-of-function DCC mutations cause developmental split brain syndrome (DSBS). Although the underlying molecular mechanisms leading to disease remain poorly understood, they are thought to stem from reduced or perturbed NTN1 signaling. Here, we review the 26 reported DCC mutations associated with abnormal CNS development in humans, including 14 missense and 12 predicted loss-of-function mutations, and discuss their associated clinical characteristics and diagnostic features. We provide an update on the observed genotype-phenotype relationships of congenital mirror movements, isolated ACC and DSBS, and correlate this to our current understanding of the biological function of DCC in the development of the CNS. All mutations and their associated phenotypes were deposited into a locus-specific LOVD (https://databases.lovd.nl/shared/genes/DCC).

The BabySeq project: implementing genomic sequencing in newborns.

BACKGROUND: The greatest opportunity for lifelong impact of genomic sequencing is during the newborn period. The "BabySeq Project" is a randomized trial that explores the medical, behavioral, and economic impacts of integrating genomic sequencing into the care of healthy and sick newborns. METHODS: Families of newborns are enrolled from Boston Children's Hospital and Brigham and Women's Hospital nurseries, and half are randomized to receive genomic sequencing and a report that includes monogenic disease variants, recessive carrier variants for childhood onset or actionable disorders, and pharmacogenomic variants. All families participate in a disclosure session, which includes the return of results for those in the sequencing arm. Outcomes are collected through review of medical records and surveys of parents and health care providers and include the rationale for choice of genes and variants to report; what genomic data adds to the medical management of sick and healthy babies; and the medical, behavioral, and economic impacts of integrating genomic sequencing into the care of healthy and sick newborns. DISCUSSION: The BabySeq Project will provide empirical data about the risks, benefits and costs of newborn genomic sequencing and will inform policy decisions related to universal genomic screening of newborns. TRIAL REGISTRATION: The study is registered in ClinicalTrials.gov Identifier: NCT02422511 . Registration date: 10 April 2015.