The BabySeq Project: A clinical trial of genome sequencing in a diverse cohort of infants.

Efforts to implement and evaluate genome sequencing (GS) as a screening tool for newborns and infants are expanding worldwide. The first iteration of the BabySeq Project (2015-2019), a randomized controlled trial of newborn sequencing, produced novel evidence on medical, behavioral, and economic outcomes. The second iteration of BabySeq, which began participant recruitment in January 2023, examines GS outcomes in a larger, more diverse cohort of more than 500 infants up to one year of age recruited from pediatric clinics at several sites across the United States. The trial aims for families who self-identify as Black/African American or Hispanic/Latino to make up more than 50% of final enrollment, and key aspects of the trial design were co-developed with a community advisory board. All enrolled families receive genetic counseling and a family history report. Half of enrolled infants are randomized to receive GS with comprehensive interpretation of pathogenic and likely pathogenic variants in more than 4,300 genes associated with childhood-onset and actionable adult-onset conditions, as well as larger-scale chromosomal copy number variants classified as pathogenic or likely pathogenic. GS result reports include variants associated with disease (Mendelian disease risks) and carrier status of autosomal-recessive and X-linked disorders. Investigators evaluate the utility and impacts of implementing a GS screening program in a diverse cohort of infants using medical record review and longitudinal parent surveys. In this perspective, we describe the rationale for the second iteration of the BabySeq Project, the outcomes being assessed, and the key decisions collaboratively made by the study team and community advisory board.

Long-read genome sequencing and variant reanalysis increase diagnostic yield in neurodevelopmental disorders.

Variant detection from long-read genome sequencing (lrGS) has proven to be more accurate and comprehensive than variant detection from short-read genome sequencing (srGS). However, the rate at which lrGS can increase molecular diagnostic yield for rare disease is not yet precisely characterized. We performed lrGS using Pacific Biosciences HiFi technology on 96 short-read-negative probands with rare diseases that were suspected to be genetic. We generated hg38-aligned variants and de novo phased genome assemblies, and subsequently annotated, filtered, and curated variants using clinical standards. New disease-relevant or potentially relevant genetic findings were identified in 16/96 (16.7%) probands, nine of which (8/96, ~9.4%) harbored pathogenic or likely pathogenic variants. Nine probands (~9.4%) had variants that were accurately called in both srGS and lrGS and represent changes to clinical interpretation, mostly from recently published gene-disease associations. Seven cases included variants that were only correctly interpreted in lrGS, including copy-number variants, an inversion, a mobile element insertion, two low-complexity repeat expansions, and a 1 bp deletion. While evidence for each of these variants is, in retrospect, visible in srGS, they were either not called within srGS data, were represented by calls with incorrect sizes or structures, or failed quality-control and filtration. Thus, while reanalysis of older srGS data clearly increases diagnostic yield, we find that lrGS allows for substantial additional yield (7/96, 7.3%) beyond srGS. We anticipate that as lrGS analysis improves, and as lrGS datasets grow allowing for better variant frequency annotation, the additional lrGS-only rare disease yield will grow over time.

Mosaicism in Tumor Suppressor Gene Syndromes: Prevalence, Diagnostic Strategies, and Transmission Risk.

A mosaic state arises when pathogenic variants are acquired in certain cell lineages during postzygotic development, and mosaic individuals may present with a generalized or localized phenotype. Here, we review the current state of knowledge regarding mosaicism for eight common tumor suppressor genes-NF1, NF2, TSC1, TSC2, PTEN, VHL, RB1, and TP53-and their related genetic syndromes/entities. We compare and discuss approaches for comprehensive diagnostic genetic testing, the spectrum of variant allele frequency, and disease severity. We also review affected individuals who have no mutation identified after conventional genetic analysis, as well as genotype-phenotype correlations and transmission risk for each tumor suppressor gene in full heterozygous and mosaic patients. This review provides new insight into similarities as well as marked differences regarding the appreciation of mosaicism in these tumor suppressor syndromes.

Errors in genome sequencing result disclosures: A randomized controlled trial comparing neonatology non-genetics healthcare professionals and genetic counselors.

PURPOSE: We compared the rate of errors in genome sequencing (GS) result disclosures by genetic counselors (GC) and trained non-genetics healthcare professionals (NGHPs) in SouthSeq, a randomized trial utilizing GS in critically ill infants. METHODS: Over 400 recorded GS result disclosures were analyzed for major and minor errors. We used Fisher's exact test to compare error rates between GCs and NGHPs and performed a qualitative content analysis to characterize error themes. RESULTS: Major errors were identified in 7.5% of disclosures by NGHPs and in no disclosures by GCs. Minor errors were identified in 32.1% of disclosures by NGHPs and in 11.4% of disclosures by GCs. Although most disclosures lacked errors, NGHPs were significantly more likely to make any error than GCs for all result types (positive, negative, or uncertain). Common major error themes include omission of critical information, overstating a negative result, and overinterpreting an uncertain result. The most common minor error was failing to disclose negative secondary findings. CONCLUSION: Trained NGHPs made clinically significant errors in GS result disclosures. Characterizing common errors in result disclosure can illuminate gaps in education to inform the development of future genomics training and alternative service delivery models.

Medical and psychosocial outcomes of state-funded population genomic screening.

As the uptake of population screening expands, assessment of medical and psychosocial outcomes is needed. Through the Alabama Genomic Health Initiative (AGHI), a state-funded genomic research program, individuals received screening for pathogenic or likely pathogenic variants in 59 actionable genes via genotyping. Of the 3874 eligible participants that received screening results, 858 (22%) responded to an outcomes survey. The most commonly reported motivation for seeking testing through AGHI was contribution to genetic research (64%). Participants with positive results reported a higher median number of planned actions (median = 5) due to AGHI results as compared to negative results (median = 3). Interviews were conducted with survey participants with positive screening results. As determined by certified genetic counselors, 50% of interviewees took appropriate medical action based on their result. There were no negative or harmful actions taken. These findings indicate population genomic screening of an unselected adult population is feasible, is not harmful, and may have positive outcomes on participants now and in the future; however, further research is needed in order to assess clinical utility.

The Genomic Medicine Integrative Research Framework: A Conceptual Framework for Conducting Genomic Medicine Research.

Conceptual frameworks are useful in research because they can highlight priority research domains, inform decisions about interventions, identify outcomes and factors to measure, and display how factors might relate to each other to generate and test hypotheses. Discovery, translational, and implementation research are all critical to the overall mission of genomic medicine and prevention, but they have yet to be organized into a unified conceptual framework. To fill this gap, our diverse team collaborated to develop the Genomic Medicine Integrative Research (GMIR) Framework, a simple but comprehensive tool to aid the genomics community in developing research questions, strategies, and measures and in integrating genomic medicine and prevention into clinical practice. Here we present the GMIR Framework and its development, along with examples of its use for research development, demonstrating how we applied it to select and harmonize measures for use across diverse genomic medicine implementation projects. Researchers can utilize the GMIR Framework for their own research, collaborative investigations, and clinical implementation efforts; clinicians can use it to establish and evaluate programs; and all stakeholders can use it to help allocate resources and make sure that the full complexity of etiology is included in research and program design, development, and evaluation.

Updated diagnostic criteria and nomenclature for neurofibromatosis type 2 and schwannomatosis: An international consensus recommendation.

PURPOSE: Neurofibromatosis type 2 (NF2) and schwannomatosis (SWN) are genetically distinct tumor predisposition syndromes with overlapping phenotypes. We sought to update the diagnostic criteria for NF2 and SWN by incorporating recent advances in genetics, ophthalmology, neuropathology, and neuroimaging. METHODS: We used a multistep process, beginning with a Delphi method involving global disease experts and subsequently involving non-neurofibromatosis clinical experts, patients, and foundations/patient advocacy groups. RESULTS: We reached consensus on the minimal clinical and genetic criteria for diagnosing NF2 and SWN. These criteria incorporate mosaic forms of these conditions. In addition, we recommend updated nomenclature for these disorders to emphasize their phenotypic overlap and to minimize misdiagnosis with neurofibromatosis type 1. CONCLUSION: The updated criteria for NF2 and SWN incorporate clinical features and genetic testing, with a focus on using molecular data to differentiate the 2 conditions. It is likely that continued refinement of these new criteria will be necessary as investigators study the diagnostic properties of the revised criteria and identify new genes associated with SWN. In the revised nomenclature, the term "neurofibromatosis 2" has been retired to improve diagnostic specificity.

Genome sequencing as a first-line diagnostic test for hospitalized infants.

PURPOSE: SouthSeq is a translational research study that undertook genome sequencing (GS) for infants with symptoms suggestive of a genetic disorder. Recruitment targeted racial/ethnic minorities and rural, medically underserved areas in the Southeastern United States, which are historically underrepresented in genomic medicine research. METHODS: GS and analysis were performed for 367 infants to detect disease-causal variation concurrent with standard of care evaluation and testing. RESULTS: Definitive diagnostic (DD) or likely diagnostic (LD) genetic findings were identified in 30% of infants, and 14% of infants harbored an uncertain result. Only 43% of DD/LD findings were identified via concurrent clinical genetic testing, suggesting that GS testing is better for obtaining early genetic diagnosis. We also identified phenotypes that correlate with the likelihood of receiving a DD/LD finding, such as craniofacial, ophthalmologic, auditory, skin, and hair abnormalities. We did not observe any differences in diagnostic rates between racial/ethnic groups. CONCLUSION: We describe one of the largest-to-date GS cohorts of ill infants, enriched for African American and rural patients. Our results show the utility of GS because it provides early-in-life detection of clinically relevant genetic variations not detected by current clinical genetic testing, particularly for infants exhibiting certain phenotypic features.

The seventh international RASopathies symposium: Pathways to a cure-expanding knowledge, enhancing research, and therapeutic discovery.

RASopathies are a group of genetic disorders that are caused by genes that affect the canonical Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Despite tremendous progress in understanding the molecular consequences of these genetic anomalies, little movement has been made in translating these findings to the clinic. This year, the seventh International RASopathies Symposium focused on expanding the research knowledge that we have gained over the years to enhance new discoveries in the field, ones that we hope can lead to effective therapeutic treatments. Indeed, for the first time, research efforts are finally being translated to the clinic, with compassionate use of Ras/MAPK pathway inhibitors for the treatment of RASopathies. This biannual meeting, organized by the RASopathies Network, brought together basic scientists, clinicians, clinician scientists, patients, advocates, and their families, as well as representatives from pharmaceutical companies and the National Institutes of Health. A history of RASopathy gene discovery, identification of new disease genes, and the latest research, both at the bench and in the clinic, were discussed.

Identifying rare, medically relevant variation via population-based genomic screening in Alabama: opportunities and pitfalls.

PURPOSE: To evaluate the effectiveness and specificity of population-based genomic screening in Alabama. METHODS: The Alabama Genomic Health Initiative (AGHI) has enrolled and evaluated 5369 participants for the presence of pathogenic/likely pathogenic (P/LP) variants using the Illumina Global Screening Array (GSA), with validation of all P/LP variants via Sanger sequencing in a CLIA-certified laboratory before return of results. RESULTS: Among 131 variants identified by the GSA that were evaluated by Sanger sequencing, 67 (51%) were false positives (FP). For 39 of the 67 FP variants, a benign/likely benign variant was present at or near the targeted P/LP variant. Variants detected within African American individuals were significantly enriched for FPs, likely due to a higher rate of nontargeted alternative alleles close to array-targeted P/LP variants. CONCLUSION: In AGHI, we have implemented an array-based process to screen for highly penetrant genetic variants in actionable disease genes. We demonstrate the need for clinical validation of array-identified variants in direct-to-consumer or population testing, especially for diverse populations.

A state-based approach to genomics for rare disease and population screening.

PURPOSE: The Alabama Genomic Health Initiative (AGHI) is a state-funded effort to provide genomic testing. AGHI engages two distinct cohorts across the state of Alabama. One cohort includes children and adults with undiagnosed rare disease; a second includes an unselected adult population. Here we describe findings from the first 176 rare disease and 5369 population cohort AGHI participants. METHODS: AGHI participants enroll in one of two arms of a research protocol that provides access to genomic testing results and biobank participation. Rare disease cohort participants receive genome sequencing to identify primary and secondary findings. Population cohort participants receive genotyping to identify pathogenic and likely pathogenic variants for actionable conditions. RESULTS: Within the rare disease cohort, genome sequencing identified likely pathogenic or pathogenic variation in 20% of affected individuals. Within the population cohort, 1.5% of individuals received a positive genotyping result. The rate of genotyping results corroborated by reported personal or family history varied by gene. CONCLUSIONS: AGHI demonstrates the ability to provide useful health information in two contexts: rare undiagnosed disease and population screening. This utility should motivate continued exploration of ways in which emerging genomic technologies might benefit broad populations.

Revised diagnostic criteria for neurofibromatosis type 1 and Legius syndrome: an international consensus recommendation.

PURPOSE: By incorporating major developments in genetics, ophthalmology, dermatology, and neuroimaging, to revise the diagnostic criteria for neurofibromatosis type 1 (NF1) and to establish diagnostic criteria for Legius syndrome (LGSS). METHODS: We used a multistep process, beginning with a Delphi method involving global experts and subsequently involving non-NF experts, patients, and foundations/patient advocacy groups. RESULTS: We reached consensus on the minimal clinical and genetic criteria for diagnosing and differentiating NF1 and LGSS, which have phenotypic overlap in young patients with pigmentary findings. Criteria for the mosaic forms of these conditions are also recommended. CONCLUSION: The revised criteria for NF1 incorporate new clinical features and genetic testing, whereas the criteria for LGSS were created to differentiate the two conditions. It is likely that continued refinement of these new criteria will be necessary as investigators (1) study the diagnostic properties of the revised criteria, (2) reconsider criteria not included in this process, and (3) identify new clinical and other features of these conditions. For this reason, we propose an initiative to update periodically the diagnostic criteria for NF1 and LGSS.

Pitfalls and challenges in genetic test interpretation: An exploration of genetic professionals experience with interpretation of results.

The interpretation of genetic testing results is subject to error. This observational study illustrates examples of pitfalls and challenges in interpretation of genetic testing results as reported by genetics professionals. We surveyed genetics professionals to describe interpretation challenges, the types of variants that were involved, and the reported clinical impact of misconception of a test result. Case studies were then collected from a select group to further explore potential causes of misunderstanding. A total of 83% of survey respondents were aware of at least one instance of genetic test misinterpretation. Both professionals with and without formal training in genetics were challenged by test reports, and variants of unknown significance were most frequently involved. Case submissions revealed that interpretation pitfalls extend beyond variant classification analyses. Inferred challenges in case submissions include lack of genetic counseling, unclear wording of reports, and suboptimal communication among providers. Respondents and case submitters noted that incorrect interpretation can trigger unnecessary follow-up tests and improperly alter clinical management. Further research is needed to validate and quantify large-scale data regarding challenges of genetic results interpretation.

Visual outcomes following everolimus targeted therapy for neurofibromatosis type 1-associated optic pathway gliomas in children.

Data for visual acuity (VA) after treatment of neurofibromatosis type 1-associated optic pathway gliomas (NF1-OPGs) are limited. We retrospectively collected VA, converted to logMAR, before and after targeted therapy with everolimus for NF1-OPG, and compared to radiologic outcomes (14/18 with NF1-OPG, 25 eyes [three without quantifiable vision]). Upon completion of treatment, VA was stable in 19 eyes, improved in four eyes, and worsened in two eyes; visual and radiologic outcomes were discordant. In summary, the majority of children with NF1-OPG exhibited stabilization of their VA after everolimus treatment. A larger, prospective study will help delineate visual outcomes after targeted therapy.

Clinical spectrum of individuals with pathogenic NF1 missense variants affecting p.Met1149, p.Arg1276, and p.Lys1423: genotype-phenotype study in neurofibromatosis type 1.

We report 281 individuals carrying a pathogenic recurrent NF1 missense variant at p.Met1149, p.Arg1276, or p.Lys1423, representing three nontruncating NF1 hotspots in the University of Alabama at Birmingham (UAB) cohort, together identified in 1.8% of unrelated NF1 individuals. About 25% (95% confidence interval: 20.5-31.2%) of individuals heterozygous for a pathogenic NF1 p.Met1149, p.Arg1276, or p.Lys1423 missense variant had a Noonan-like phenotype, which is significantly more compared with the "classic" NF1-affected cohorts (all p < .0001). Furthermore, p.Arg1276 and p.Lys1423 pathogenic missense variants were associated with a high prevalence of cardiovascular abnormalities, including pulmonic stenosis (all p < .0001), while p.Arg1276 variants had a high prevalence of symptomatic spinal neurofibromas (p < .0001) compared with "classic" NF1-affected cohorts. However, p.Met1149-positive individuals had a mild phenotype, characterized mainly by pigmentary manifestations without externally visible plexiform neurofibromas, symptomatic spinal neurofibromas or symptomatic optic pathway gliomas. As up to 0.4% of unrelated individuals in the UAB cohort carries a p.Met1149 missense variant, this finding will contribute to more accurate stratification of a significant number of NF1 individuals. Although clinically relevant genotype-phenotype correlations are rare in NF1, each affecting only a small percentage of individuals, together they impact counseling and management of a significant number of the NF1 population.

Two novel cases further expand the phenotype of TOR1AIP1-associated nuclear envelopathies.

Biallelic variants in TOR1AIP1, encoding the integral nuclear membrane protein LAP1 (lamina-associated polypeptide 1) with two functional isoforms LAP1B and LAP1C, have initially been linked to muscular dystrophies with variable cardiac and neurological impairment. Furthermore, a recurrent homozygous nonsense alteration, resulting in loss of both LAP1 isoforms, was identified in seven likely related individuals affected by multisystem anomalies with progeroid-like appearance and lethality within the 1st decade of life. Here, we have identified compound heterozygosity in TOR1AIP1 affecting both LAP1 isoforms in two unrelated individuals affected by congenital bilateral hearing loss, ventricular septal defect, bilateral cataracts, mild to moderate developmental delay, microcephaly, mandibular hypoplasia, short stature, progressive muscular atrophy, joint contractures and severe chronic heart failure, with much longer survival. Cellular characterization of primary fibroblasts of one affected individual revealed absence of both LAP1B and LAP1C, constitutively low lamin A/C levels, aberrant nuclear morphology including nuclear cytoplasmic channels, and premature senescence, comparable to findings in other progeroid forms of nuclear envelopathies. We additionally observed an abnormal activation of the extracellular signal-regulated kinase 1/2 (ERK 1/2). Ectopic expression of wild-type TOR1AIP1 mitigated these cellular phenotypes, providing further evidence for the causal role of identified genetic variants. Altogether, we thus further expand the TOR1AIP1-associated phenotype by identifying individuals with biallelic loss-of-function variants who survived beyond the 1st decade of life and reveal novel molecular consequences underlying the TOR1AIP1-associated disorders.