Integrating Functional Analysis in the Next-Generation Sequencing Diagnostic Pipeline of RASopathies

Abstract

RASopathies are a group of heterogeneous conditions caused by germline mutations in RAS/MAPK signalling pathway genes. With next-generation sequencing (NGS), sequencing capacity is no longer a limitation to molecular diagnosis. Instead, the rising number of variants of unknown significance (VUSs) poses challenges to clinical interpretation and genetic counselling. We investigated the potential of an integrated pipeline combining NGS and the functional assessment of variants for the diagnosis of RASopathies. We included 63 Chinese patients with RASopathies that had previously tested negative for PTPN11 and HRAS mutations. In these patients, we performed a genetic analysis of genes associated with RASopathies using a multigene NGS panel and Sanger sequencing. For the VUSs, we evaluated evidence from genetic, bioinformatic and functional data. Twenty disease-causing mutations were identified in the 63 patients, providing a primary diagnostic yield of 31.7%. Four VUSs were identified in five patients. The functional assessment supported the pathogenicity of the RAF1 and RIT1 VUSs, while the significance of two VUSs in A2ML1 remained unclear. In summary, functional analysis improved the diagnostic yield from 31.7% to 36.5%. Although technically demanding and time-consuming, a functional genetic diagnostic analysis can ease the clinical translation of these findings to aid bedside interpretation.

Figure 1

Clinical photographs of patients with variants in genes of the RAS/MAPK signalling pathway. Patients with (a) pathogenic or likely pathogenic variants and (b) VUSs from our study.

Figure 2

Dual luciferase assay of the phosphorylation activity changes to ELK1 from 293 T cells transfected with the corresponding expression and reporter plasmids. Statistical significance was derived using an unpaired t-test comparing the mutants with the corresponding WT. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: not significant. Mutated human transcript with VUSs from (a) RAF1 (b) RIT1 and (c) A2ML1 from this study were compared with the wild-type transcript. The data are the mean ± SD from 3 determinations.

Figure 3

Transient expression of RNA transcripts in zebrafish embryos. The injection dosage of each RNA transcript was optimised for comparison (RAF1: 50 pg/embryo; RIT1: 400 pg/embryo; A2ML1: 200 pg/embryo). A morphometric analysis was performed to compare the effect of the VUSs compared to the wild-type transcript at three dpf. Statistical significance was derived using a two-sided Fisher’s exact test to compare mutants with the corresponding WT. **p < 0.01; ***p < 0.001; ns: not significant. NIC: No injection control; (a) Proportion of zebrafish embryos with normal or diseased phenotypes. (b) Representative zebrafish embryos with a normal phenotype (c) craniofacial dysmorphism (d) gross malformations and (e) cardiac oedema.

Figure 4

Craniofacial assessment of zebrafish embryos. The zebrafish embryos were treated with Alcian blue and washed with acidic alcohol 3 dpf. The ratio of the width of the ceratohyal (x) to the tip of Meckel’s cartilage (y) was used as a measure of craniofacial defects. (a) Comparison of the x-to-y ratio between mutant and wild-type zebrafish embryos. The data are the mean ± SD. Statistical significance was derived using an unpaired t-test comparing the mutants with the corresponding WT. **p < 0.01; ****p < 0.0001; ns: not significant. NIC: No injection control. The number in bold represents the total count of zebrafish embryos in the experimental group. (b) Alcian-blue stained cartilage in the zebrafish. The x-axis shows the width of the ceratohyal, and the y-axis shows the length of the tip of Meckel’s cartilage. (c) Representative embryos with different x-to-y ratios.

Figure 5

Structural assessment of cardiac tubes by in situ hybridization of cmlc1. Statistical significance was derived using a two-sided Fisher’s exact test to compare mutants with the corresponding WT. **p < 0.01; ns: not significant. (a) The proportion of zebrafish embryos with a normal or deformed heart tube structure. (b) Representative embryos with a normal and (c) deformed heart tube.