Identifying genetic mutations in patients with Rasopathies using a Next Generation Sequencing Diagnostic Pipeline in Hong Kong

Abstract

Background and aims: RASopathies are a group of developmental syndromes, namely Noonan, Costello, Cardio-facio-cutaneous, Legius, and LEOPARD syndromes, collectively affecting 1 in 2000 livebirths. They were caused by mutations in genes involved in the RAS/ MAPK signaling pathway. Since they are characterised by clinical overlap and genetic heterogeneity, diagnoses are often challenging. We aim to develop a NGS-based strategy for molecular diagnosis of RASopathies. Method: Targeted NGS of 13 genes (A2ML1, BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NRAS, PTPN11, RAF1, SHOC2, SOS1, and SPRED1) in the RAS/MAPK pathway, where the targeted enrichment panel covered 98% of the gene coding regions, was performed on 57 RASopathies which were previously tested negative for mutation in PTPN11 and HRAS. Positive controls were run in parallel. Results: The average read-depth in the regions of interest was >500X, with 99% of target bases reaching minimal coverage of 30X. Eighteen known pathogenic mutations (SOS1, n=6; RAF1, n=2; KRAS, n=3; BRAF, n=2; SHOC2, n=2; or MAP2K1, n=3) were detected in 18/57 (32%) patients. Three novel mutations (1 nonsense and 2 missense) were found in four patients. All detected mutations were confirmed by Sanger sequencing. The novel missense mutations are in-silico demonstrated to be deleterious and are absent in unaffected control populations. Detailed genotype-phenotype correlation analysis is in progress. Functional analysis using Elk-1 reporter system and zebrafish modeling is underway to examine the pathogenicity of these novel mutations. Conclusion: To our knowledge, this study has the largest sample size of PTPN11 and HRAS negative patients from Hong Kong who received diagnosis of RASopathies from clinical geneticists. Our study has demonstrated that the strategy involving targeted NGS analysis can achieve an addition detection rate of 32%, showing an improvement over the conventional Sanger sequencing analysis merely of PTPN11 and HRAS mutations for RASopathies. Clinical correlations, customised bioinformatics pipelines and follow-up molecular characterisation in cell cultures or animal models are important to delineate the pathogenic role of novel mutations identified by NGS. Acknowledgement: We would like to thank the families for their participation and the SK Medical Foundation and SK Yee Medical Research Fund for financial support