P655: Improved representation of functional data in ClinVar

Methods: We recruited patients with suspected genetic disorders and conducted comprehensive evaluations, including medical histories, physical exams, and relevant diagnostic tests.Initial molecular and cytogenetic testing performed at clinical laboratories either yielded negative results or identified variants of uncertain significance.As a result, analysis of genome sequencing and RNA-seq data, including splicing evaluation, were performed at the University of Utah and ARUP Laboratories to aid in these patients' diagnostic journeys.Results: The study analyzed 20 patients (9 males, 11 females) with various clinical presentations.RNA-seq analysis provided functional data that helped confirm diagnoses or identify likely diagnoses in eight cases (3 males and 5 females).Specifically, RNA-seq played a pivotal role in upgrading variant pathogenicity in seven cases, including three with intronic splicing variants identified by genome sequencing and confirmed as pathogenic through RNA-seq.Case 1: A 16-month-old boy with suspected Diamond-Blackfan Anemia (DBA) presented with severe anemia, low reticulocytes, and elevated erythrocyte adenosine deaminase (eADA) levels, requiring frequent transfusions.Despite initial negative genetic testing, genome sequencing revealed a novel maternally inherited splicing variant in RPS7 (c.-19G>C), which was predicted to impair splicing.RNA-seq confirmed significant intron retention in RPS7 transcripts in both the proband and his mother, indicating a strong allelic association.The variant was classified as pathogenic and consistent with DBA8.The patient underwent a successful hematopoietic stem cell transplant (HSCT) from an HLA-matched sibling who did not carry the variant RPS7, achieving transfusion independence for over 8 months with 100% donor chimerism and currently normal hematologic indices.Case 2: A 20-month-old girl presented with severe anemia, red cell aplasia, and congenital heart defects.Initial genetic testing was negative, but genome sequencing identified a de novo intronic RPS19 variant (c.172+350C>T), predicted to create a novel splice donor site.RNA-seq confirmed the presence of an 81-bp novel exon downstream of RPS19 exon 3 in about half of the proband's transcripts, absent in controls.This variant was classified as pathogenic based on its de novo occurrence, phenotype match, absence from population databases, and functional evidence.This is consistent with a diagnosis of DBA1.The patient is preparing for HSCT with a 12/12 HLA-matched sibling donor, who does not carry the RPS19 variant.Case 3: A 10-year-old girl presented with short stature, metaphyseal irregularities around the knee, vertebral abnormalities, and rib widening, suggestive of a spondyloepiphyseal dysplasia.Initial genetic testing, including a 54-gene skeletal dysplasia panel and exome sequencing, was negative.Genome sequencing identified compound heterozygous splicing variants in EFL1: a maternally inherited likely pathogenic variant (c.932+2T>G) and a paternally inherited variant of uncertain significance (c.245-12A>G).RNA-seq confirmed splicing defects, including exon skipping for c.932+2T>G and intron retention for c.245-12A>G, supporting the pathogenicity of both variants and confirming a diagnosis of Schwachman-Diamond syndrome 2. Conclusion: This study underscores the value of RNA-seq in unraveling complex diagnostic cases, particularly for intronic variants of uncertain significance.By integrating RNA-seq with genome sequencing, we have enhanced our ability to identify elusive genetic causes of disease.This combined approach proves crucial for resolving diagnostic challenges, establishing RNA-seq and DNA sequencing as indispensable tools in clinical genetics for rare and undiagnosed diseases.