ADDRESSING THE PROBLEM OF VARIANTS OF UNCERTAIN SIGNIFICANCE IN GENETIC DIAGNOSIS OF VASCULAR PULMONARY DISEASE: A ROLE FOR TRANSCRIPT EXPRESSION IN BLOOD MONOCYTES?

Introduction and objectives Variants of uncertain significance (VUS) represent an increasing issue for NHS diagnostics, as they are not clinically actionable for patients and their families. ACMG-AMP guidance1 emphasises that in-vitro evidence is often required to validate computational predictions. Ahead of the introduction of the National Genomic Test Directory, the objective was to use hereditary haemorrhagic telangiectasia (HHT) to quantify the unmet need and provide proof-of-concept for a functional assay deliverable within clinical diagnostic laboratories. Methods Clinically reported missense variants in HHT-causing genes were examined within the HHT Mutation Database,2 ClinVar3 and in reports for patients reviewed in our clinical HHT service. Blood monocytes isolated from HHT patients and controls were cultured overnight to upregulate endoglin, and experimentally treated with various stimuli prior to RNA extraction, cDNA synthesis and quantitative reverse transcriptase PCR. Results Of 389 missense variants currently listed on the HHT Mutation Database (254 ACVRL1, 110 ENG, 25 SMAD4), 285 (73.3%) are classified as a VUS or equivalent (‘pending classification’). Similarly in ClinVar, of 192 missense variants listed (80 ACVRL1, 93 ENG, 19 SMAD4), 113 (58.9%) are classified as a VUS or having ‘conflicting interpretations for pathogenicity’. Evaluating patient reports received from our institution, where since 1999 genetic tests have been reported by 4 different NHS laboratories, 24 missense variants were classified as pathogenic/likely pathogenic, suitable for predictive familial testing. However, following ACMG-AMP guidelines1, 8 (33.3%) have since been re-classified as being VUS or equivalent by the HHT Mutation Database2 and/or ClinVar3. Monocytes from 16 HHT patients and 4 controls have been isolated for transcript analyses. Using normalised ID1 expression as a readout, differential responses following BMP9 and TGFβI stimulation are being assessed for potential differences between controls and different HHT genotypes. Conclusion Identification of variants within disease-causing genes does not indicate pathogenicity. With an agglomeration of variants pending classification or assigned as a VUS, further functional assays which may reconcile unresolved classifications are crucial to provide ACMG-AMP supportive criterion for pathogenicity assignments. Preliminary results from monocyte readouts support further optimisation of this functional assay for potential use. References Genet Med 2015:17;405–424. www.arup.utah.edu/database/HHT/ www.clinvar.com/