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usc Refereed/Peer-reviewed 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.

Acute promyelocytic leukaemia with PML-RARA fusion is usually associated with the t(15;17)(q24.1;q21.1) translocation but may also arise from complex or cryptic rearrangements. The fusion usually resides on chromosome 15 but occasionally on others. We describe a cryptic PML-RARA fusion within a novel chromosome 17 rearrangement. We performed interphase fluorescence in situ hybridisation (FISH) using a dual-fusion PML-RARA probe, followed by reverse transcriptase-polymerase chain reaction (RT-PCR) for PML-RARA, karyotyping, and metaphase FISH using RARA break-apart, locus-specific, and subtelomere probes for chromosome 17. An 850K SNP microarray was also employed. Interphase and metaphase FISH showed atypical results involving a single PML-RARA fusion, no second fusion, but instead separate diminished PML and RARA signals. RT-PCR confirmed PML-RARA fusion; however, karyotyping detected only an altered chromosome 17. Metaphase FISH showed the single fusion and diminished 5' RARA signals located unexpectedly in the subtelomeric short-arm and long-arm regions of the rearranged chromosome 17, respectively. SNP microarray revealed no copy number abnormality. This paediatric patient with PML-RARA fusion reflects a cryptic insertion that resides within a complex and novel chromosome 17 rearrangement. This rearrangement likely arose via 7 chromosome breaks with the insertion occurring first followed by sequential paracentric and then pericentric inversions.

Complete hydatidiform moles (CHM) are abnormal pregnancies with no fetal development resulting from having two paternal genomes with no maternal contribution. It is important to distinguish CHM from partial hydatidiform moles, and non-molar abortuses, due to the increased risk of gestational trophoblastic neoplasia. We evaluated a series of products of conception (POC) (n=643) investigated by genome-wide microarray comparative genomic hybridisation (CGH) with the aim of refining our strategy for the identification of complete moles. Among 32 suspected molar pregnancies investigated by STR genotyping to supplement microarray CGH testing, we found 31.3% (10/32) CHM; all identified among 3.6% (10/272) early first trimester POC. We suggest that when using microarray CGH that genotyping using targeted STR analysis should be performed for all POC referrals to aid in the identification of CHM.