Purpose: The Clinical Genome Resource (ClinGen) Gene Curation Expert Panels have historically focused on specific organ systems or phenotypes; thus, the ClinGen Syndromic Disorders Gene Curation Expert Panel (SD-GCEP) was formed to address an unmet need. Methods: The SD-GCEP applied ClinGen’s framework to evaluate the clinical validity of genes associated with rare syndromic disorders. A total of 111 gene-disease relationships (GDRs) associated with 100 genes spanning the clinical spectrum of syndromic disorders were curated. Results: From April 2020 through March 2024, 38 precurations were performed on genes with multiple disease relationships and were reviewed to determine if the disorders were part of a spectrum or distinct entities. A total of 14 genes were lumped into a single disease entity, and 24 were split into separate entities, of which 11 were curated by the SD-GCEP. A full review of 111 GDRs for 100 genes followed, with 78 classified as Definitive, 9 as Strong, 15 as Moderate, and 9 as Limited, highlighting cases in which further data are needed. All diseases involved 2 or more organ systems, whereas the majority (88/111 GDRs, 79.2%) had 5 or more organ systems affected. Conclusion: The SD-GCEP addresses a critical gap in gene curation efforts, enabling inclusion of genes for syndromic disorders in clinical testing and contributing to keeping pace with the rapid discovery of new genetic syndromes.

ImportanceThe feasibility of implementing genome sequencing as an adjunct to traditional newborn screening (NBS) in newborns of different racial and ethnic groups is not well understood.ObjectiveTo report interim results of acceptability, feasibility, and outcomes of an ongoing genomic NBS study in a diverse population in New York City within the context of the New York State Department of Health Newborn Screening Program.Design, Setting, and ParticipantsThe Genomic Uniform-screening Against Rare Disease in All Newborns (GUARDIAN) study was a multisite, single-group, prospective, observational investigation of supplemental newborn genome screening with a planned enrollment of 100 000 participants. Parent-reported race and ethnicity were recorded at the time of recruitment. Results of the first 4000 newborns enrolled in 6 New York City hospitals between September 2022 and July 2023 are reported here as part of a prespecified interim analysis.ExposureSequencing of 156 early-onset genetic conditions with established interventions selected by the investigators were screened in all participants and 99 neurodevelopmental disorders associated with seizures were optional.Main Outcomes and MeasuresThe primary outcome was screen-positive rate. Additional outcomes included enrollment rate and successful completion of sequencing.ResultsOver 11 months, 5555 families were approached and 4000 (72.0%) consented to participate. Enrolled participants reflected a diverse group by parent-reported race (American Indian or Alaska Native, 0.5%; Asian, 16.5%; Black, 25.1%; Native Hawaiian or Other Pacific Islander, 0.1%; White, 44.7%; 2 or more races, 13.0%) and ethnicity (Hispanic, 44.0%; not Hispanic, 56.0%). The majority of families consented to screening of both groups of conditions (both groups, 90.6%; disorders with established interventions only, 9.4%). Testing was successfully completed for 99.6% of cases. The screen-positive rate was 3.7%, including treatable conditions that are not currently included in NBS.Conclusions and RelevanceThese interim findings demonstrate the feasibility of targeted interpretation of a predefined set of genes from genome sequencing in a population of different racial and ethnic groups. DNA sequencing offers an additional method to improve screening for conditions already included in NBS and to add those that cannot be readily screened because there is no biomarker currently detectable in dried blood spots. Additional studies are required to understand if these findings are generalizable to populations of different racial and ethnic groups and whether introduction of sequencing leads to changes in management and improved health outcomes.Trial RegistrationClinicalTrials.gov Identifier: NCT05990179

Whole genome sequencing (WGS) comprehensively detects DNA sequence variation, enabling assessment of genetic disorders. The primary aim of this study was to investigate the diagnostic utility of WGS for pediatric musculoskeletal disorders by comparing it with whole exome sequencing (WES), which is more widely used but not as comprehensive in its coverage of the genome. This multi-center cohort study consists of WGS and WES analysis for 36 pediatric patients with musculoskeletal disorders of unknown etiology and, where available, their parents and siblings. WGS and WES were performed on DNA extracted from saliva samples. Secondary analysis of sequence data and tertiary analysis with interpretation of sequence variants were performed using the Illumina DRAGEN and Emedgene platforms, respectively. We evaluated 36 patients, and the median age was 11 years. The most common phenotypes included thoracolumbar scoliosis and gait disturbance. The median number of candidate variants per patient identified by WES and WGS were 57.5 and 90.5, respectively. 38 pathogenic or likely pathogenic variants were identified by WGS, providing a potentially diagnostic (tier-1) candidate for 22 of 36 (61.1%) patients. 12 of the 38 tier-1 variants (31.6%) were identified only by WGS. Of these 12 variants missed by WES, two candidates had variants that are likely to solve the respective case after undergoing manual curation. WGS resulted in a larger number of variants predicted as pathogenic/likely pathogenic in patients with musculoskeletal phenotypes, including variants potentially capable of solving their respective cases. WGS showed particular advantage in detecting CNVs. This study demonstrates that WGS is a promising method for improving our understanding of musculoskeletal disorders marked by genetic and phenotypic heterogeneity.

(Abstracted from JAMA 2025;333(3):232–240 Newborn screening (NBS) checks for congenital conditions that can be treated before symptoms start and lead to irreversible effects; these include conditions such as metabolic disease, cystic fibrosis, hearing impairment, endocrine disorders, hemoglobinopathies, severe combined immunodeficiency, and other conditions that are often genetic. Genetic sequencing is typically second-tier in terms of NBS, being used chiefly to identify genes involved in a condition such as cystic fibrosis.

Purpose: Despite monogenic and polygenic contributions to cardiovascular disease (CVD), genetic testing is not widely adopted, and current tests are limited by the breadth of surveyed conditions and variant interpretation burden. To address these limitations, a comprehensive clinical genome CVD test with semiautomated interpretation was developed. Methods: Monogenic conditions and risk alleles were selected based on the strength of disease association and evidence for increased disease risk, respectively. Non-CVD secondary findings genes, pharmacogenomic (PGx) variants, and CVD-associated polygenic risk scores (PRS) were also assessed for inclusion. Test performance was modeled using 2594 genomes from the 1000 Genomes Project and further investigated in 20 previously tested individuals. Results: The CVD genome test comprises a panel of 215 high-confidence CVD gene-disease pairs, 35 non-CVD secondary findings genes, 4 risk alleles or genotypes, 10 PGx genes, and a PRS for coronary artery disease. Modeling of test performance using samples from the 1000 Genomes Project revealed approximately 6% of individuals with a monogenic finding in a CVD-associated gene, 6% with a risk allele finding, 1% with a non-CVD secondary finding, and 93% with CVD-associated PGx variants. Assessment of blinded clinical samples showed concordance with prior testing. An average of 4 variants were reviewed per case, with interpretation and reporting time ranging from 9 to 96 minutes. Conclusion: A genome-sequencing-based CVD genetic risk assessment test can provide comprehensive genetic disease and genetic risk information to patients with CVD. The semiautomated and limited interpretation burden suggest that this testing approach can be scaled to support population-level initiatives in phenotypically enriched populations.

Elevated blood Lp(a) levels are an established cardiovascular disease (CVD) risk factor, but routine assessment of the LPA locus is confounded by a complex variable number tandem repeat (VNTR). A dedicated LPA variant caller for PCR-free genome sequencing data has been recently developed, enabling implementation of Lp(a) risk screening on a genome backbone. Here we describe the first clinical validation of Lp(a) risk factor screening as a component of a comprehensive CVD genomic risk assessment.

Disease-causing variants in the ZIC3 gene are associated with X-linked heterotaxy, congenital heart disease, and other malformations reminiscent of the VACTERL-H (vertebral abnormalities, anal atresia, cardiac defects, tracheoesophageal anomalies, renal or radial anomalies, limb anomalies and hydrocephalus), mainly affecting males. Disease-causing variants in ZIC3 are rare and known to have variable penetrance and expressivity. We present a Peruvian family with a novel variant in ZIC3. A 2-year-old boy (proband) was diagnosed at birth with anal stenosis with fistula, double outlet right ventricle (DORV) with transposition of the great arteries (TGA) and severe pulmonary valvar stenosis (PVS), operated on at 4-months with placement of a graft in the aorta and great vessels and Blalock-Taussig shunt without cardiopulmonary bypass. On physical examination, he presented with a broad forehead, long palpebral fissures, very sparse and short hair, and normal neurological examination. Abdominal and renal ultrasound were unremarkable. His 13-year-old maternal uncle presented at birth with an imperforate anus without fistula, DORV, TGA, PVS, common atrioventricular canal, dextrocardia and lipomyelomeningocele. He had a history of sensorineural deafness at the age of 4-years requiring hearing aids. On physical examination, colostomy carrier post-surgery for anorectal malformation, epicanthus, hypoplasia of antihelix, marked cyanosis, spastic paraparesis, absent osteotendinous reflexes and a soft lumbo-sacral tumor. He has used a wheelchair since He was 12-years-old and his intelligence quotient (IQ) was below average. Spinal X-ray with sacrococcygeal dysgenesis. Brain MRI visualized mild dilatation of the supratentorial ventricular system. Abdominal ultrasound revealed a midline-positioned liver, absent spleen in splenic space (left flank is not explored due to colostomy bag) and normal kidneys. Clinical genome (cGS) provided through the iHope program detected a hemizygous variant in the ZIC3 gene, c.973G>A p.(Glu325Lys), present in the proband and maternal uncle in a hemizygous state and in in the proband's 27 year-old asymptomatic mother in a heterozygous state. The ZIC3 c.973G>A p.(Glu325Lys) variant is not observed in version 2.1.1 or version 3.1.2 of the Genome Aggregation Database, is located in the nuclear export signal domain, which is important for appropriate cellular localization and trafficking of ZIC3, and multiple lines of computational evidence suggest the variant may impact the gene or gene product, resulting in a variant classification of likely pathogenic. We report the first Peruvian family with a disease-causing variant ZIC3, we show evidence of a novel mutation in the gene associated with variable phenotypic expression between two individuals within the same family. We highlight the importance of performing genetic tests in patients with congenital malformations, particularly those with affected relatives, to perform a personalized follow-up and genetic counseling for the family.

Variants in seven genes (LRRK2, GBA1, PRKN, SNCA, PINK1, PARK7 and VPS35) have been formally adjudicated as causal contributors to Parkinson's disease; however, individuals with Parkinson's disease are often unaware of their genetic status since clinical testing is infrequently offered. As a result, genetic information is not incorporated into clinical care, and variant-targeted precision medicine trials struggle to enrol people with Parkinson's disease. Understanding the yield of genetic testing using an established gene panel in a large, geographically diverse North American population would help patients, clinicians, clinical researchers, laboratories and insurers better understand the importance of genetics in approaching Parkinson's disease. PD GENEration is an ongoing multi-centre, observational study (NCT04057794, NCT04994015) offering genetic testing with results disclosure and genetic counselling to those in the US (including Puerto Rico), Canada and the Dominican Republic, through local clinical sites or remotely through self-enrolment. DNA samples are analysed by next-generation sequencing including deletion/duplication analysis (Fulgent Genetics) with targeted testing of seven major Parkinson's disease-related genes. Variants classified as pathogenic/likely pathogenic/risk variants are disclosed to all tested participants by either neurologists or genetic counsellors. Demographic and clinical features are collected at baseline visits. Between September 2019 and June 2023, the study enrolled 10 510 participants across >85 centres, with 8301 having received results. Participants were: 59% male; 86% White, 2% Asian, 4% Black/African American, 9% Hispanic/Latino; mean age 67.4 ± 10.8 years. Reportable genetic variants were observed in 13% of all participants, including 18% of participants with one or more 'high risk factors' for a genetic aetiology: early onset (<50 years), high-risk ancestry (Ashkenazi Jewish/Basque/North African Berber), an affected first-degree relative; and, importantly, in 9.1% of people with none of these risk factors. Reportable variants in GBA1 were identified in 7.7% of all participants; 2.4% in LRRK2; 2.1% in PRKN; 0.1% in SNCA; and 0.2% in PINK1, PARK7 or VPS35 combined. Variants in more than one of the seven genes were identified in 0.4% of participants. Approximately 13% of study participants had a reportable genetic variant, with a 9% yield in people with no high-risk factors. This supports the promotion of universal access to genetic testing for Parkinson's disease, as well as therapeutic trials for GBA1 and LRRK2-related Parkinson's disease.

Heritable connective tissue disorders (HCTDs) consist of a wide array of genetic disorders such as Ehlers-Danlos syndrome, Marfan syndrome, and osteogenesis imperfecta. The diagnosis relies on clinical presentation and family history to guide genetic testing with next-generation sequencing (NGS) for identification of gene variants in HCTDs. NGS was performed on a cohort of 100 consecutive, unrelated patients referred for a connective tissue disorder at Fulgent Genetics, an accredited commercial laboratory. One hundred seventeen gene variants were found in 76 patients with 10 recognized pathogenic or likely pathogenic variants seen in nine patients. The remaining variants were grouped as unknown clinical significance with 36 meeting three out of four pathogenicity criteria, or potentially pathogenic, as defined in our study in 33 patients. They were judged as potentially pathogenic for clinical care and management with disease surveillance based on the specific gene and phenotypic presentation. Gene variants in collagen-related proteins were the most frequent with ZNF469 and ADAMTSL2 variants most often identified. Joint hypermobility was the most frequent clinical finding. Variants were found in 76% of patients who had distinct clinical features of a HCTD. The data were stratified to provide insight into frequency and types of variants, their classification, and clinical manifestations.

Purpose: Genetic variants causing aberrant premessenger RNA splicing are increasingly being recognized as causal variants in genetic disorders. In this study, we devise standardized practices for polymerase chain reaction (PCR)-based RNA diagnostics using clinically accessible specimens (blood, fibroblasts, urothelia, biopsy). Methods: A total of 74 families with diverse monogenic conditions (31% prenatal-congenital onset, 47% early childhood, and 22% teenage-adult onset) were triaged into PCR-based RNA testing, with comparative RNA sequencing for 19 cases. Results: Informative RNA assay data were obtained for 96% of cases, enabling variant reclassification for 75% variants that can be used for genetic counseling (71%), to inform clinical care (32%) and prenatal counseling (41%). Variant-associated mis-splicing was highly reproducible for 28 cases with samples from >= 2 affected individuals or heterozygotes and 10 cases with >= 2 biospecimens. PCR amplicons encompassing another segregated heterozygous variant was vital for clinical interpretation of 22 of 79 variants to phase RNA splicing events and discern complete from partial mis-splicing. Conclusion: RNA diagnostics enabled provision of a genetic diagnosis for 64% of recruited cases. PCR-based RNA diagnostics has capacity to analyze 81.3% of clinically significant genes, with long amplicons providing an advantage over RNA sequencing to phase RNA splicing events. The Australasian Consortium for RNA Diagnostics (SpliceACORD) provide clinically-endorsed, standardized protocols and recommendations for interpreting RNA assay data. (C) 2021 Published by Elsevier Inc. on behalf of American College of Medical Genetics and Genomics.

Purpose Modern molecular genetics has revolutionized gene discovery, genetic diagnoses, and precision medicine yet many patients remain unable to benefit from these advances as disease-causing variants remain elusive for up to half of Mendelian genetic disorders. Patient-derived induced pluripotent stem (iPS) cells and transcriptomics were used to identify the fate of unsolved ABCA4 alleles in patients with Stargardt disease. Methods Multiple independent iPS lines were generated from skin biopsies of three patients with Stargardt disease harboring a single identified pathogenic ABCA4 variant. Derived retinal pigment epithelial cells (dRPE) from a normal control and patient cells were subjected to RNA-Seq on the Novaseq6000 platform, analyzed using DESeq2 with calculation of allele specific imbalance from the pathogenic or a known linked variant. Protein analysis was performed using the automated Simple Western system. Results Nine dRPE samples were generated, with transcriptome analysis on eight. Allele-specific expression indicated normal transcripts expressed from splice variants albeit at low levels, and missense transcripts expressed at near-normal levels. Corresponding protein was not easily detected. Patient phenotype correlation indicated missense variants expressed at high levels have more deleterious outcomes. Transcriptome analysis suggests mitochondrial membrane biodynamics and the unfolded protein response pathway may be relevant in Stargardt disease. Conclusions Patient-specific iPS-derived RPE cells set the stage to assess non-expressing variants in difficult-to-detect genomic regions using easily biopsied tissue. Translational Relevance This “Disease in a Dish” approach is likely to enhance the ability of patients to participate in and benefit from clinical trials while providing insights into perturbations in RPE biology.

Establishing or ruling out a molecular diagnosis of Prader–Willi or Angelman syndrome (PWS/AS) presents unique challenges due to the variety of different genetic alterations that can lead to these conditions. Point mutations, copy number changes, uniparental isodisomy (i-UPD) 15 of two subclasses (segmental or total isodisomy), uniparental heterodisomy (h-UPD), and defects in the chromosome 15 imprinting center can all cause PWS/AS. Here, we outline a combined approach using whole-exome sequencing (WES) and DNA methylation data with methylation-sensitive multiplex ligation-dependent probe amplification (MLPA) to establish both the disease diagnosis and the mechanism of disease with high sensitivity using current standard of care technology and improved efficiency compared to serial methods. The authors encourage the use of this approach in the clinical setting to confirm and establish the diagnosis and genetic defect which may account for the secondary genetic conditions that may be seen in those with isodisomy 15, impacting surveillance and counseling with more accurate recurrence risks. Other similarly affected individuals due to other gene disorders or cytogenetic anomalies such as Rett syndrome or microdeletions would also be identified with this streamlined approach.

When a potential disease-causing variant is detected in a proband, parental testing is used to determine the mode of inheritance. This study demonstrates that next-generation sequencing (NGS) is uniquely well suited for parental testing, in particular because of its ability to detect clinically relevant germline mosaicism. Parental variant testing by NGS was performed in a clinical laboratory for 1 year. The detection of mosaicism by NGS was compared with its detection by Sanger sequencing. Eight cases of previously unrevealed mosaicism were detected by NGS across eight different genes. Mosaic variants were differentiated from sequencing noise using custom bioinformatics analyses in combination with familial inheritance data and complementary Sanger sequencing. Sanger sequencing detected mosaic variants with allele fractions ≥8% by NGS, but could not detect mosaic variants below that level. Detection of germline mosaicism by NGS is invaluable to parents, providing a more accurate recurrence risk that can alter decisions on family planning and pregnancy management. Because NGS can also confirm parentage and increase scalability, it simultaneously streamlines and strengthens the variant curation process. These features make NGS the ideal method for parental testing, superior even to Sanger sequencing for most genomic loci.

Attention deficit hyperactivity disorder (ADHD) is a common and highly heritable neurodevelopmental disorder with poorly understood pathophysiology and genetic mechanisms. A balanced chromosomal translocation interrupts CTNND2 in several members of a family with profound attentional deficit and myopia, and disruption of the gene was found in a separate unrelated individual with ADHD and myopia. CTNND2 encodes a brain-specific member of the adherens junction complex essential for postsynaptic and dendritic development, a site of potential pathophysiology in attentional disorders. Therefore, we propose that the severe and highly penetrant nature of the ADHD phenotype in affected individuals identifies CTNND2 as a potential gateway to ADHD pathophysiology similar to the DISC1 translocation in psychosis or AUTS2 in autism.

OBJECTIVE:Description of a new variant of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) gene causing congenital myasthenic syndrome (CMS) in 3 children from 2 unrelated families.METHODS:Muscle biopsies, EMG, and whole-exome sequencing were performed.RESULTS:All 3 patients presented with congenital hypotonia, muscle weakness, respiratory insufficiency, head lag, areflexia, and gastrointestinal dysfunction. Genetic analysis identified a homozygous frameshift insertion in the GFPT1 gene (NM_001244710.1: c.686dupC; p.Arg230Ter) that was shared by all 3 patients. In one of the patients, inheritance of the variant was through uniparental disomy (UPD) with maternal origin. Repetitive nerve stimulation and single-fiber EMG was consistent with the clinical diagnosis of CMS with a postjunctional defect. Ultrastructural evaluation of the muscle biopsy from one of the patients showed extremely attenuated postsynaptic folds at neuromuscular junctions and extensive autophagic vacuolar pathology.CONCLUSIONS:These results expand on the spectrum of known loss-of-function GFPT1 mutations in CMS12 and in one family demonstrate a novel mode of inheritance due to UPD.

Baraitser-Winter cerebrofrontofacial syndrome (BWCFF) is a rare autosomal dominant developmental disorder associated with missense mutations in the genesACTBorACTG1. The classic presentation of BWCFF is discerned by the combination of unique craniofacial characteristics including ocular coloboma, intellectual disability, and hypertelorism. Congenital contractures and organ malformations are often present, including structural defects in the brain, heart, renal, and musculoskeletal system. However, there is limited documentation regarding its prenatal presentation that may encourage healthcare providers to be aware of this disorder when presented throughout pregnancy. Herein we describe a case of a pregnancy with large cystic hygroma and omphalocele. Whole exome sequencing (WES) was performed and a de novo, heterozygous, likely pathogenic mutation inACTBwas detected, c.1004G>A (p.Arg335His), conferring a diagnosis of BWCFF.

Compared to DNA, analysis of RNA is one step closer on the central dogma of biology to assessing cellular function. This makes it an extremely valuable target for research and clinical testing in nearly all areas of molecular biology. Most RNA molecules are ephemeral by nature. They exist as temporary intermediates, ostensibly enabling data transfer between the genome and the organism. Their ribose backbone renders them sensitive to simple degradation over time and they are the target molecule for numerous and abundant ribonucleases which have evolved to chop them to pieces with extreme efficiency. At the biochemical level, this means that they degrade rapidly in most physiological and laboratory conditions and are thus challenging to study. When considering specimen banking, it is critical to keep this reality in mind, as some commonly used banking modalities will not adequately preserve the relevant RNA molecules in a measureable state.In this chapter, we explore the broad range of RNA testing methodologies in current use, with particular focus on how specimen preparation impacts analysis. Following an overview in the introduction, Subheading 2 covers the major specimen types amenable to RNA analysis in the context of biobanking. Subheading 3 discusses the applications of various RNA analysis modalities to research and clinical testing.