Determining the characteristics of genetic disorders that predict inclusion in newborn genomic sequencing programs.

Over 30 international research studies and commercial laboratories are exploring the use of genomic sequencing to screen apparently healthy newborns for genetic disorders. These programs have individualized processes for determining which genes and genetic disorders are queried and reported in newborns. We compared lists of genes from 26 research and commercial newborn screening programs and found substantial heterogeneity among the genes included. A total of 1,750 genes were included in at least one newborn genome sequencing program, but only 74 genes were included on >80% of gene lists, 16 of which are not associated with conditions on the Recommended Uniform Screening Panel. We used a linear regression model to explore factors related to the inclusion of individual genes across programs, finding that a high evidence base as well as treatment efficacy were two of the most important factors for inclusion. We applied a machine learning model to predict how suitable a gene is for newborn sequencing. As knowledge about and treatments for genetic disorders expand, this model provides a dynamic tool to reassess genes for newborn screening implementation. This study highlights the complex landscape of gene list curation among genomic newborn screening programs and proposes an empirical path forward for determining the genes and disorders of highest priority for newborn screening programs.

Heterozygous MAP3K20 variants cause ectodermal dysplasia, craniosynostosis, sensorineural hearing loss, and limb anomalies.

Biallelic pathogenic variants in MAP3K20, which encodes a mitogen-activated protein kinase, are a rare cause of split-hand foot malformation (SHFM), hearing loss, and nail abnormalities or congenital myopathy. However, heterozygous variants in this gene have not been definitively associated with a phenotype. Here, we describe the phenotypic spectrum associated with heterozygous de novo variants in the linker region between the kinase domain and leucine zipper domain of MAP3K20. We report five individuals with diverse clinical features, including craniosynostosis, limb anomalies, sensorineural hearing loss, and ectodermal dysplasia-like phenotypes who have heterozygous de novo variants in this specific region of the gene. These individuals exhibit both shared and unique clinical manifestations, highlighting the complexity and variability of the disorder. We propose that the involvement of MAP3K20 in endothelial-mesenchymal transition provides a plausible etiology of these features. Together, these findings characterize a disorder that both expands the phenotypic spectrum associated with MAP3K20 and highlights the need for further studies on its role in early human development.

Evidence review and considerations for use of first line genome sequencing to diagnose rare genetic disorders.

Early use of genome sequencing (GS) in the diagnostic odyssey can reduce suffering and improve care, but questions remain about which patient populations are most amenable to GS as a first-line diagnostic test. To address this, the Medical Genome Initiative conducted a literature review to identify appropriate clinical indications for GS. Studies published from January 2011 to August 2022 that reported on the diagnostic yield (DY) or clinical utility of GS were included. An exploratory meta-analysis using a random effects model evaluated DY based on cohort size and diagnosed cases per cohort. Seventy-one studies met inclusion criteria, comprising over 13,000 patients who received GS in one of the following settings: hospitalized pediatric patients, pediatric outpatients, adult outpatients, or mixed. GS was the first-line test in 38% (27/71). The unweighted mean DY of first-line GS was 45% (12-73%), 33% (6-86%) in cohorts with prior genetic testing, and 33% (9-60%) in exome-negative cohorts. Clinical utility was reported in 81% of first-line GS studies in hospitalized pediatric patients. Changes in management varied by cohort and underlying molecular diagnosis (24-100%). To develop evidence-informed points to consider, the quality of all 71 studies was assessed using modified American College of Radiology (ACR) criteria, with five core points to consider developed, including recommendations for use of GS in the N/PICU, in lieu of sequential testing and when disorders with substantial allelic heterogeneity are suspected. Future large and controlled studies in the pediatric and adult populations may support further refinement of these recommendations.

Genomic newborn screening: Are we entering a new era of screening?

Population newborn screening (NBS) for phenylketonuria began in the United States in 1963. In the 1990s electrospray ionization mass spectrometry permitted an array of pathognomonic metabolites to be identified simultaneously, enabling up to 60 disorders to be recognized with a single test. In response, differing approaches to the assessment of the harms and benefits of screening have resulted in variable screening panels worldwide. Thirty years on and another screening revolution has emerged with the potential for first line genomic testing extending the range of screening conditions recognized after birth to many hundreds. At the annual SSIEM conference in 2022 in Freiburg, Germany, an interactive plenary discussion on genomic screening strategies and their challenges and opportunities was conducted. The Genomics England Research project proposes the use of Whole Genome Sequencing to offer extended NBS to 100 000 babies for defined conditions with a clear benefit for the child. The European Organization for Rare Diseases seeks to include "actionable" conditions considering also other types of benefits. Hopkins Van Mil, a private UK research institute, determined the views of citizens and revealed as a precondition that families are provided with adequate information, qualified support, and that autonomy and data are protected. From an ethical standpoint, the benefits ascribed to screening and early treatment need to be considered in relation to asymptomatic, phenotypically mild or late-onset presentations, where presymptomatic treatment may not be required. The different perspectives and arguments demonstrate the unique burden of responsibility on those proposing new and far-reaching developments in NBS programs and the need to carefully consider both harms and benefits.

Elective genomic testing: Practice resource of the National Society of Genetic Counselors.

Genetic counseling for patients who are pursuing genetic testing in the absence of a medical indication, referred to as elective genomic testing (EGT), is becoming more common. This type of testing has the potential to detect genetic conditions before there is a significant health impact permitting earlier management and/or treatment. Pre- and post-test counseling for EGT is similar to indication-based genetic testing. Both require a complete family and medical history when ordering a test or interpreting a result. However, EGT counseling has some special considerations including greater uncertainties around penetrance and clinical utility and a lack of published guidelines. While certain considerations in the selection of a high-quality genetic testing laboratory are universal, there are some considerations that are unique to the selection of a laboratory performing EGT. This practice resource intends to provide guidance for genetic counselors and other healthcare providers caring for adults seeking pre- or post-test counseling for EGT. Genetic counselors and other genetics trained healthcare providers are the ideal medical professionals to supply accurate information to individuals seeking counseling about EGT enabling them to make informed decisions about testing and follow-up.

Applying the Clinician-reported Genetic testing Utility InDEx (C-GUIDE) to genome sequencing: further evidence of validity.

Genome sequencing (GS) outperforms other rare disease diagnostics, but standardized approaches to assessing its clinical utility are limited. This study assessed the validity of the Clinician-reported Genetic testing Utility InDEx (C-GUIDE), a novel tool for assessing the utility of genetic testing from a clinician's perspective, for GS. C-GUIDE ratings were completed for patients who received GS results. For each patient, total C-GUIDE and single item global scores were calculated. Construct validity was assessed using linear regression to determine the association between C-GUIDE total and global item scores and measure the effects of potential explanatory variables. Ratings were completed for 67 pediatric and 36 adult patients. GS indications were neurological for 70.9% and results were diagnostic for 28.2%. When the C-GUIDE assessed primary (PV), secondary (SV), and pharmacogenomic (PGx) variants, on average, a one unit increase in the global item score was associated with an increase of 7.3 in the C-GUIDE score (p < 0.05). Diagnostic results were associated with an increase in C-GUIDE score of 5.0 compared to non-diagnostic results (p < 0.05) and an increase of one SV was associated with an increase of 2.5 (p < 0.05). For children, decreased age of one year was associated with an increase in C-GUIDE score of 0.3 (p < 0.05). Findings provide evidence that C-GUIDE measures the construct of clinical utility in pediatric and adult rare disease populations and is sensitive to changes in utility related to variant type. Quantifying the clinical utility of GS using C-GUIDE can inform efforts to optimize its use in patient care.

Newborn Screening by Genomic Sequencing: Opportunities and Challenges.

Newborn screening for treatable disorders is one of the great public health success stories of the twentieth century worldwide. This commentary examines the potential use of a new technology, next generation sequencing, in newborn screening through the lens of the Wilson and Jungner criteria. Each of the ten criteria are examined to show how they might be applied by programmes using genomic sequencing as a screening tool. While there are obvious advantages to a method that can examine all disease-causing genes in a single assay at an ever-diminishing cost, implementation of genomic sequencing at scale presents numerous challenges, some which are intrinsic to screening for rare disease and some specifically linked to genomics-led screening. In addition to questions specific to routine screening considerations, the ethical, communication, data management, legal, and social implications of genomic screening programmes require consideration.

Developing a National Newborn Genomes Program: An Approach Driven by Ethics, Engagement and Co-design.

The transformative potential of whole genome sequencing (WGS) as a diagnostic tool in healthcare has been demonstrated by initiatives including the 100,000 Genomes Project and is now offered to certain patients in the National Health Service (NHS) in England. Building on these foundations, the utility of WGS in the newborn period can now be explored. Genomics England is working in partnership with NHS England and NHS Improvement and other healthcare, patient and public interest groups to design a research program embedded in the NHS to explore the potential challenges and implications of offering WGS in all newborns. The program will aim to: 1) evaluate the feasibility, utility and impact on the NHS of screening for childhood-onset rare actionable genetic conditions; 2) understand how, with consent, genomic and healthcare data could be used to enable research to develop new diagnostics and treatments; and 3) explore the implications of storing an individual's genome for use over their lifetime. Recognizing the important practical, scientific and ethical questions that we must explore in dialogue with the public and experts, we are taking a collaborative, evidence-based and ethically deliberate approach to designing the program. An iterative co-design process including a nationwide public dialogue has identified emergent themes and ethical considerations which are the focus of the program's design. These themes will be further developed through continued engagement with healthcare professionals, researchers, ethics experts, patient groups and the public, with an ongoing commitment to embedding ongoing ethics research and co-design into the delivery of the program.

Best practices for the interpretation and reporting of clinical whole genome sequencing.

Whole genome sequencing (WGS) shows promise as a first-tier diagnostic test for patients with rare genetic disorders. However, standards addressing the definition and deployment practice of a best-in-class test are lacking. To address these gaps, the Medical Genome Initiative, a consortium of leading health care and research organizations in the US and Canada, was formed to expand access to high quality clinical WGS by convening experts and publishing best practices. Here, we present best practice recommendations for the interpretation and reporting of clinical diagnostic WGS, including discussion of challenges and emerging approaches that will be critical to harness the full potential of this comprehensive test.

A study of elective genome sequencing and pharmacogenetic testing in an unselected population.

BACKGROUND: Genome sequencing (GS) of individuals without a medical indication, known as elective GS, is now available at a number of centers around the United States. Here we report the results of elective GS and pharmacogenetic panel testing in 52 individuals at a private genomics clinic in Alabama. METHODS: Individuals seeking elective genomic testing and pharmacogenetic testing were recruited through a private genomics clinic in Huntsville, AL. Individuals underwent clinical genome sequencing with a separate pharmacogenetic testing panel. RESULTS: Six participants (11.5%) had pathogenic or likely pathogenic variants that may explain one or more aspects of their medical history. Ten participants (19%) had variants that altered the risk of disease in the future, including two individuals with clonal hematopoiesis of indeterminate potential. Forty-four participants (85%) were carriers of a recessive or X-linked disorder. All individuals with pharmacogenetic testing had variants that affected current and/or future medications. CONCLUSION: Our study highlights the importance of collecting detailed phenotype information to interpret results in elective GS.

Next-generation sequencing for constitutional variants in the clinical laboratory, 2021 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG).

Next-generation sequencing (NGS) technologies are now established in clinical laboratories as a primary testing modality in genomic medicine. These technologies have reduced the cost of large-scale sequencing by several orders of magnitude. It is now cost-effective to analyze an individual with disease-targeted gene panels, exome sequencing, or genome sequencing to assist in the diagnosis of a wide array of clinical scenarios. While clinical validation and use of NGS in many settings is established, there are continuing challenges as technologies and the associated informatics evolve. To assist clinical laboratories with the validation of NGS methods and platforms, the ongoing monitoring of NGS testing to ensure quality results, and the interpretation and reporting of variants found using these technologies, the American College of Medical Genetics and Genomics (ACMG) has developed the following technical standards.

Functional and structural analysis of cytokine-selective IL6ST defects that cause recessive hyper-IgE syndrome.

BACKGROUND: Biallelic variants in IL6ST, encoding GP130, cause a recessive form of hyper-IgE syndrome (HIES) characterized by high IgE level, eosinophilia, defective acute phase response, susceptibility to bacterial infections, and skeletal abnormalities due to cytokine-selective loss of function in GP130, with defective IL-6 and IL-11 and variable oncostatin M (OSM) and IL-27 levels but sparing leukemia inhibitory factor (LIF) signaling. OBJECTIVE: Our aim was to understand the functional and structural impact of recessive HIES-associated IL6ST variants. METHODS: We investigated a patient with HIES by using exome, genome, and RNA sequencing. Functional assays assessed IL-6, IL-11, IL-27, OSM, LIF, CT-1, CLC, and CNTF signaling. Molecular dynamics simulations and structural modeling of GP130 cytokine receptor complexes were performed. RESULTS: We identified a patient with compound heterozygous novel missense variants in IL6ST (p.Ala517Pro and the exon-skipping null variant p.Gly484_Pro518delinsArg). The p.Ala517Pro variant resulted in a more profound IL-6- and IL-11-dominated signaling defect than did the previously identified recessive HIES IL6ST variants p.Asn404Tyr and p.Pro498Leu. Molecular dynamics simulations suggested that the p.Ala517Pro and p.Asn404Tyr variants result in increased flexibility of the extracellular membrane-proximal domains of GP130. We propose a structural model that explains the cytokine selectivity of pathogenic IL6ST variants that result in recessive HIES. The variants destabilized the conformation of the hexameric cytokine receptor complexes, whereas the trimeric LIF-GP130-LIFR complex remained stable through an additional membrane-proximal interaction. Deletion of this membrane-proximal interaction site in GP130 consequently caused additional defective LIF signaling and Stüve-Wiedemann syndrome. CONCLUSION: Our data provide a structural basis to understand clinical phenotypes in patients with IL6ST variants.

Reducing Sanger confirmation testing through false positive prediction algorithms.

PURPOSE: Clinical genome sequencing (cGS) followed by orthogonal confirmatory testing is standard practice. While orthogonal testing significantly improves specificity, it also results in increased turnaround time and cost of testing. The purpose of this study is to evaluate machine learning models trained to identify false positive variants in cGS data to reduce the need for orthogonal testing. METHODS: We sequenced five reference human genome samples characterized by the Genome in a Bottle Consortium (GIAB) and compared the results with an established set of variants for each genome referred to as a truth set. We then trained machine learning models to identify variants that were labeled as false positives. RESULTS: After training, the models identified 99.5% of the false positive heterozygous single-nucleotide variants (SNVs) and heterozygous insertions/deletions variants (indels) while reducing confirmatory testing of nonactionable, nonprimary SNVs by 85% and indels by 75%. Employing the algorithm in clinical practice reduced overall orthogonal testing using dideoxynucleotide (Sanger) sequencing by 71%. CONCLUSION: Our results indicate that a low false positive call rate can be maintained while significantly reducing the need for confirmatory testing. The framework that generated our models and results is publicly available at https://github.com/HudsonAlpha/STEVE .

An online compendium of treatable genetic disorders.

More than 4,000 genes have been associated with recognizable Mendelian/monogenic diseases. When faced with a new diagnosis of a rare genetic disorder, health care providers increasingly turn to internet resources for information to understand the disease and direct care. Unfortunately, it can be challenging to find information concerning treatment for rare diseases as key details are scattered across a number of authoritative websites and numerous journal articles. The website and associated mobile device application described in this article begin to address this challenge by providing a convenient, readily available starting point to find treatment information. The site, Rx-genes.com (https://www.rx-genes.com/), is focused on those conditions where the treatment is directed against the mechanism of the disease and thereby alters the natural history of the disease. The website currently contains 633 disease entries that include references to disease information and treatment guidance, a brief summary of treatments, the inheritance pattern, a disease frequency (if known), nonmolecular confirmatory testing (if available), and a link to experimental treatments. Existing entries are continuously updated, and new entries are added as novel treatments appear in the literature.

Clinical utility of genomic sequencing: a measurement toolkit.

Whole-genome sequencing (WGS) is positioned to become one of the most robust strategies for achieving timely diagnosis of rare genomic diseases. Despite its favorable diagnostic performance compared to conventional testing strategies, routine use and reimbursement of WGS are hampered by inconsistencies in the definition and measurement of clinical utility. For example, what constitutes clinical utility for WGS varies by stakeholder's perspective (physicians, patients, families, insurance companies, health-care organizations, and society), clinical context (prenatal, pediatric, critical care, adult medicine), and test purpose (diagnosis, screening, treatment selection). A rapidly evolving technology landscape and challenges associated with robust comparative study design in the context of rare disease further impede progress in this area of empiric research. To address this challenge, an expert working group of the Medical Genome Initiative was formed. Following a consensus-based process, we align with a broad definition of clinical utility and propose a conceptually-grounded and empirically-guided measurement toolkit focused on four domains of utility: diagnostic thinking efficacy, therapeutic efficacy, patient outcome efficacy, and societal efficacy. For each domain of utility, we offer specific indicators and measurement strategies. While we focus on diagnostic applications of WGS for rare germline diseases, this toolkit offers a flexible framework for best practices around measuring clinical utility for a range of WGS applications. While we expect this toolkit to evolve over time, it provides a resource for laboratories, clinicians, and researchers looking to characterize the value of WGS beyond the laboratory.

Best practices for the analytical validation of clinical whole-genome sequencing intended for the diagnosis of germline disease.

Whole-genome sequencing (WGS) has shown promise in becoming a first-tier diagnostic test for patients with rare genetic disorders; however, standards addressing the definition and deployment practice of a best-in-class test are lacking. To address these gaps, the Medical Genome Initiative, a consortium of leading healthcare and research organizations in the US and Canada, was formed to expand access to high-quality clinical WGS by publishing best practices. Here, we present consensus recommendations on clinical WGS analytical validation for the diagnosis of individuals with suspected germline disease with a focus on test development, upfront considerations for test design, test validation practices, and metrics to monitor test performance. This work also provides insight into the current state of WGS testing at each member institution, including the utilization of reference and other standards across sites. Importantly, members of this initiative strongly believe that clinical WGS is an appropriate first-tier test for patients with rare genetic disorders, and at minimum is ready to replace chromosomal microarray analysis and whole-exome sequencing. The recommendations presented here should reduce the burden on laboratories introducing WGS into clinical practice, and support safe and effective WGS testing for diagnosis of germline disease.

The Medical Genome Initiative: moving whole-genome sequencing for rare disease diagnosis to the clinic.

Clinical whole-genome sequencing (WGS) offers clear diagnostic benefits for patients with rare disease. However, there are barriers to its widespread adoption, including a lack of standards for clinical practice. The Medical Genome Initiative consortium was formed to provide practical guidance and support the development of standards for the use of clinical WGS.

Design and Reporting Considerations for Genetic Screening Tests.

Testing asymptomatic individuals for unsuspected conditions is not new to the medical and public health communities. Protocols to develop screening tests are well established. However, the application of screening principles to inherited diseases presents unique challenges. Unlike most screening tests, the natural history and disease prevalence of most rare inherited diseases in an unselected population are unknown. It is difficult or impossible to obtain a truth set cohort for clinical validation studies. As a result, it is not possible to accurately calculate clinical positive and negative predictive values for likely pathogenic variants, which are commonly returned in genetic screening assays. In addition, many of the genetic conditions included in screening panels do not have clinical confirmatory tests. All these elements are typically required to justify the development of a screening test, according to the World Health Organization screening principles. Nevertheless, as the cost of DNA sequencing continues to fall, more individuals are opting to undergo genomic testing in the absence of a clinical indication. Despite the challenges, reasonable estimates can be deduced and used to inform test design strategies. Herein, we review basic test design principles and apply them to genetic screening.

Expanding the phenotype of the CDKL5 deficiency disorder: Are seizures mandatory?

Pathogenic variants in the cyclin-dependent kinase-like 5 (CDKL5) gene cause the neurodevelopmental disorder, the CDKL5 deficiency disorder. Reports of individuals with pathogenic variants in CDKL5 without seizures are exceedingly rare, and in-depth analyses of their variants have been lacking. Whole-genome sequencing was performed on a 29-year-old female with mild intellectual disability who, in the absence of overt seizures, presented with multiple episodes of altered mental status over a 24-year period. Clinical history was supplemented by a parent completed questionnaire from the International CDKL5 Disorder Database. We identified a de novo heterozygous variant in CDKL5 (NM_003159.2:c.645T>A;p.Ser215Arg). In-depth computational analysis performed to predict the impact of the variant on protein structure and function demonstrated that the variant was likely pathogenic. In this light, cell-based studies showed that the S215R substitution causes a marked reduction in CDKL5 kinase activity. Similarities between our case and one previously reported case are striking. These cases, both without seizures but with apparent behavioral symptomatology, together question whether seizures are mandatory in this neurodevelopmental disorder.