O36: Optical genome mapping for cytogenetic analysis in multiple myeloma: Real-world evidence

Results: Case 1: A 20-year-old male with developmental delay, seizures, tall stature, and hypermobility harbored an intronic SLC6A8 (NM_005629.4):c.912+3_912+8delVUS.RNA-seq showed exon 5 skipping in 8% of reads (54/661).This created an in-frame transcript unlikely to undergo nonsense-mediated decay (NMD), precluding the application of PVS1(RNA) despite evidence of altered splicing.Case 2: A 6-year-old male with elevated creatine kinase and MRI abnormalities carried a hemizygous, maternally inherited BCAP31(NM_001256447.2):c.193+3G>CVUS.RNA-seq revealed reduced exon 3 coverage (max 83 reads vs 545 in controls), confirmed as an outlier Z-score of -5.29 and Log2FC of -1.24.Although this supports of a deleterious impact, the PVS1(RNA) criterion could not be applied due to quantitative thresholds.Case 3: A 45-year-old female with progressive gait disturbance and brain calcifications carried bialleic VARS2 (NM_020442.6)variants: c.1087C>T, p.Arg363Ter (maternal) and c.1556G>C, p.Gly519Ala (paternal).RNA-seq showed absence of the c.1556G>C allele (0/11 reads), suggesting allele-specific expression loss, possibly due to NMD, but low read coverage precluded reclassification.Case 4: A 39-year-old female with basal ganglia calcifications had a heterozygous SLC20A2(NM_001257180.2):c.517-3C>GVUS.RNA-seq yielded only 5 reads spanning the splice junction (controls: 31 and 15 reads), demonstrating insufficient to read depth to meet technical requirements for variant reclassification.Conclusion: These cases underscore how RNA-seq can yield intriguing splicing findings that remain difficult to interpret within current clinical frameworks.Even when aberrant transcripts are detected, limitations in read depth, transcript completeness, and uncertain biological consequence often preclude confident variant reclassification, highlighting the need for clearer standards and interpretive criteria in diagnostic RNA-seq analysis.