Engelhardt, Nicole M

Diagnosing rare genetic diseases remains a major challenge despite widespread clinical testing. Long-read RNA sequencing (RNA-seq) offers a powerful approach to capturing the effects of genetic variants on the transcriptome, yet challenges with sequencing coverage, cost, tissue selection, and scalability have limited its clinical adoption. To address this, we developed STRIPE (Sequencing Targeted RNAs Identifies Pathogenic Events), a targeted long-read RNA-seq–based strategy for rare disease diagnosis and variant interpretation. STRIPE enables deep sequencing of full-length transcripts for any customized disease-specific gene panel such that a wide range of clinically informative readouts, including transcript aberrations and sequence variants, can be detected at haplotype-level resolution. Applying STRIPE to 88 individuals spanning two major rare disease groups, we accurately reidentified known pathogenic variants and revealed their transcript consequences, including many unexpected ones. For 8 of 15 splice site region variants, we observed more complex RNA processing defects beyond single exon skipping or cryptic splice site activation. Notably, we find that donor splice site variants frequently activate cryptic intronic polyadenylation sites, leading to premature transcript termination. Leveraging unique strengths of long-read RNA-seq, STRIPE also resolved variants of uncertain significance and uncovered disease-causing variants in five previously undiagnosed individuals. Overall, STRIPE is a powerful, adaptable, and scalable strategy with broad potential to improve clinical variant interpretation and advance genetic diagnosis of rare diseases.

AbstractCongenital disorders of glycosylation are a continuously expanding group of monogenic disorders of glycoprotein and glycolipid glycan biosynthesis. These disorders mostly manifest with multisystem involvement. Individuals with ALG8‐CDG commonly present with hypotonia, protein‐losing enteropathy, and hepatic involvement. Here, we describe seven unreported individuals diagnosed with ALG8‐CDG based on biochemical and molecular testing and we identify nine novel variants in ALG8, bringing the total to 26 individuals with ALG8‐CDG in the medical literature. In addition to the typical multisystem involvement documented in ALG8‐CDG, our cohort includes the two oldest patients reported and further expands the phenotype of ALG8‐CDG to include stable intellectual disability, autism spectrum disorder and other neuropsychiatric symptoms. We further expand the clinical features in a variety of organ systems including ocular, musculoskeletal, dermatologic, endocrine, and cardiac abnormalities and suggest a comprehensive evaluation and monitoring strategy to improve clinical management.

AbstractCongenital disorders of glycosylation (CDGs) are a continuously expanding group of monogenic disorders of glycoprotein and glycolipid biosynthesis that cause multisystem diseases. Individuals with ALG3‐CDG frequently exhibit severe neurological involvement (epilepsy, microcephaly, and hypotonia), ocular anomalies, dysmorphic features, skeletal anomalies, and feeding difficulties. We present 10 unreported individuals diagnosed with ALG3‐CDG based on molecular and biochemical testing with 11 novel variants in ALG3, bringing the total to 40 reported individuals. In addition to the typical multisystem disease seen in ALG3‐CDG, we expand the symptomatology of ALG3‐CDG to now include endocrine abnormalities, neural tube defects, mild aortic root dilatation, immunodeficiency, and renal anomalies. N‐glycan analyses of these individuals showed combined deficiencies of hybrid glycans and glycan extension beyond Man5GlcNAc2 consistent with their truncated lipid‐linked precursor oligosaccharides. This spectrum of N‐glycan changes is unique to ALG3‐CDG. These expanded features of ALG3‐CDG facilitate diagnosis and suggest that optimal management should include baseline endocrine, renal, cardiac, and immunological evaluation at the time of diagnosis and with ongoing monitoring.