We have introduced new several sentences in this paragraph of Materials and Methods, which define these ad-hoc rules. Lines 154-155: “mgR51C read-outs have been introduced into the classification system as PVS1_O or BP7_O codes of variable evidence strength depending on the splicing outcome [P, Sup-porting (±1 point); M, Moderate (±2); Strong (±4); Very Strong (±8)].” Lines 163-179: “we have developed some ad-hoc rules that take into consideration the coding potential of each individual transcript and its relative contribution to the overall expression to reach the appropriate PVS1_O or BP/_O evidence strength. In brief, for each complex read-out we have applied the following algorithm: (i) De-convolute mgR51C read-outs in-to individual transcripts; (ii) apply ACMG/AMP evidences to each individual transcript; (iii) produce an overall PVS1_O (or BP7_O) code strength based on the relative contribu-tion of individual transcripts/evidences to the overall expression. Thus, if pathogenic supporting transcripts contribute ≥90% to the overall expression, PVS1_O_ code is applied (if different transcripts support different pathogenic evidence strengths, the lowest strength contributing >10% to the overall expression is selected for overall evidence strength). Similarly, BP7_O_ code is applied if benign supporting transcripts contribute ≥90% to the overall expression (if different transcripts support different pathogenic evi-dence strengths, the lowest strength contributing >10% to the overall expression is selected for overall evidence strength). If neither pathogenic nor benign supporting transcripts contribute ≥90% to the overall expression, the splicing assay is considered not providing any evidence in favor, or against, pathogenicity. Recently, we have used a similar ap-proach to deal with complex PALB2/ATM minigene read-outs [20,30].” 2 -
Line 156 – the authors refer to “ad-hoc rules” that they have developed for consideration of the different coding transcripts associated with the same spliceogenic variant in variant interpretation and classification. Although a reference is provided so that the reader can look up what these ad-hoc rules are, it would also be helpful to briefly describe these in the current manuscript.