Performance Evaluation of SpliceAI for the Prediction of Splicing of NF1 Variants

Abstract

Neurofibromatosis type 1, characterized by neurofibromas and café-au-lait macules, is one of the most common genetic disorders caused by pathogenic NF1 variants. Because of the high proportion of splicing mutations in NF1, identifying variants that alter splicing may be an essential issue for laboratories. Here, we investigated the sensitivity and specificity of SpliceAI, a recently introduced in silico splicing prediction algorithm in conjunction with other in silico tools. We evaluated 285 NF1 variants identified from 653 patients. The effect on variants on splicing alteration was confirmed by complementary DNA sequencing followed by genomic DNA sequencing. For in silico prediction of splicing effects, we used SpliceAI, MaxEntScan (MES), and Splice Site Finder-like (SSF). The sensitivity and specificity of SpliceAI were 94.5% and 94.3%, respectively, with a cut-off value of Δ Score > 0.22. The area under the curve of SpliceAI was 0.975 (p < 0.0001). Combined analysis of MES/SSF showed a sensitivity of 83.6% and specificity of 82.5%. The concordance rate between SpliceAI and MES/SSF was 84.2%. SpliceAI showed better performance for the prediction of splicing alteration for NF1 variants compared with MES/SSF. As a convenient web-based tool, SpliceAI may be helpful in clinical laboratories conducting DNA-based NF1 sequencing.

Keywords: NF1; SpliceAI; in silico prediction; neurofibromatosis type 1; splice variants.

Figure 1

Electrophoretogram of the (A) complementary DNA (cDNA) and (B) genomic DNA (gDNA) from novel splice variant c.7458-8T>G, resulting in creation of a new 3′ splice site that leads to a 7-nt insertion of c.7458-7_7458-1. Below, cDNA and gDNA sequences of wild type (wt) and splice variant are presented. In the cDNA sequences, the inserted nucleotides are italicized. In the gDNA sequences, the nucleotide substitution is underlined. Small letters indicate the intronic sequence.

Figure 2

Receiver operating characteristic curves and an area under the receiver operating characteristic curve comparing SpliceAI and MES/SSF for predicting the splice effect of 285 NF1 variants. Abbreviations: MES, MaxEntScan; SSF, splice site finder-like; AUC, area under the receiver operating characteristic curve.

Figure 3

Study workflow. ¹ Genomic DNA analysis showed no relevant variants. ² Variants other than single nucleotide polymorphisms and simple insertions and/or deletions of bases. N denotes different types of NF1 variants. Abbreviations: cDNA, complementary DNA; gDNA, genomic DNA; ACMG, American College of Medical Genetics and Genomics; AMP, Association for Molecular Pathology; PV, pathogenic variant; LPV, likely pathogenic variant; VUS, variant of uncertain significance; BV, benign variant; LBV, likely benign variant.