Two Novel NF1 Pathogenic Variants Causing the Creation of a New Splice Site in Patients With Neurofibromatosis Type I

Abstract

Neurofibromatosis type I (NF1) is one of the most common autosomal dominant disorders, since the estimated incidence is one in 3,500 births. In this study, we present bioinformatical and functional characterization of two novel splicing NF1 variants, detected in NF1 patients. Patient 1, carrying NF1:c.122A>T, which introduces a new exonic 5' donor splice site, was diagnosed with hormone-positive, Her-2-negative breast cancer at the age of 47. She had an atypical presentation of NF1, with few café-au-lait spots and no Lisch nodules. Patient developed a hemothorax due to subclavian artery rupture, which has previously been described as an extremely rare complication of NF1. Patient 2, carrying NF1:c.7395-17T>G that creates a new intronic 3' acceptor splice site, had quite a typical clinical presentation of NF1: formations on her tongue in the region of her left metacarpal bones and on her left foot, plexiform neurofibroma in her pelvis, several café-au-lait spots, and axillary freckling. She was also diagnosed with cognitive impairment. In the report, we are presenting two novel variants which were successfully classified based on NGS and mRNA analysis. Based on results of mRNA analysis, both variants were classified as likely pathogenic according to ACMG guidelines applying evidence categories PS3, PM2, PP3, and PP1 supporting. By characterizing those two novel NF1 splicing variants, we have confirmed the neurofibromatosis type I phenotype in the two probands.

Keywords: NF1; NGS; functional analysis; mRNA; neurofibromatosis type I; splicing; splicing alteration; variant of uncertain significance.

Figure 1

(A) Pedigree of a family carrying variant NF1:c.122A > T; (B) Pedigree of a family harboring NF1:c.7395-17T > G.

Figure 2

Two novel NF1 mutations and their effect on mRNA splicing. (A) Sanger sequencing electropherogram and in silico splicing prediction of variants using Alamut Visual software for variant c.122A > T and (B) for variant c.7395-17A > G.

Figure 3

Fragment analysis profiles of (A) patient 1, carrier of NF1: c.122A > T and two controls (廼primers spanning 5’UTR and exon–exon junction of exons 4 and 5); (B) patient 2, carrier of NF1:c.7395-17T > G and two controls (primers flanking exons 48/49 and 53/54), showing the amount of abberant transcripts,alternative isoform lacking exon 52 (Δ52) and full-length transcript (FL). (C, D) Ratio between abberant and wild-type transcript for patients 1 and 2 measured by capillary electrophoresis analysis of FAM-labeled fragments.