Genetic analysis of a novel FBN1 mutation in a pediatric Marfan syndrome patient

Abstract

The aim of this study was to investigate a novel FBN1 gene mutation in a pediatric patient with Marfan syndrome (MFS) to provide a theoretical basis for genetic counseling. The subject was a 5-month-old male infant. With informed consent from the proband and his family, 2 mL of peripheral venous blood was collected from the patient, his father, mother, and sister. DNA was extracted using a DNA extraction kit with EDTA-K as an anticoagulant. The extracted DNA was subjected to minigene transcription and bioinformatics analysis. For minigene construction, wild-type and mutant minigenes were inserted into pcMINI and pcMINI-C vectors, respectively. Four recombinant vectors were transfected into the HeLa and 293T cell lines. After transfection for 48 hours, RNA was extracted from eight samples. DNA was also extracted from the family members' samples to construct a library. Target regions were captured using the SureSelect Human All Exon V6 (Agilent) kit and were sequenced with Illumina NovaSeq (sequencing read length 2×150 bp). Bioinformatic analysis identified the c.8226+5del mutation as a variant of uncertain clinical significance (VOUS). Literature and database reviews confirmed that this mutation had not been previously reported, identifying it as a novel mutation. The study identified a novel FBN1 mutation, c.8226+5del, that may be associated with clinical features such as low-set ears and distinctive facial characteristics in the proband. This mutation likely affects normal mRNA splicing, altering the structure and function of Exon 64 and potentially contributing to the development of autosomal dominant MFS.

Keywords: FBN1; Marfan syndrome (MFS); gene mutation; mutation; novel.

Figure 1.

Detection of the pcMINI vector. (A) Sequencing diagram of minigene construction, with wt (wild type) shown on top and mut (mutation) shown below; (B) Gel electrophoresis of the results of RT-PCR transcription analysis, with bands labeled as a, b, c in HeLa and 293T cells; (C) Schematic diagram illustrating the minigene construction strategy and splicing pattern; (D) Sequencing results corresponding to splicing bands. The red asterisk (*) indicates the mutation site.

Figure 2.

Detection of the pcMINI-C vector. (A) Sequencing diagram of minigene construction, with wt (wild type) shown on top and mut (mutation) shown below; (B) Gel electrophoresis of the results of RT-PCR transcription analysis, with bands labeled as a, b, c in HeLa and 293T cells; (C) Schematic diagram illustrating the minigene construction strategy and splicing pattern; (D) Sequencing results corresponding to splicing bands. The red asterisk (*) indicates the mutation site.

Figure 3.

IGV schematic diagram of sequencing results for FBN1 gene variant c.8226+5del. (A), Patient; (B), Mother of the patient; (C), Father of the patient.

Figure 4.

Schematic representation of Sanger sequencing validation results for FBN1 gene variant c.8226+5del in the patient and parents. (S), Sequencer; (A), Patient; (B), Mother of the patient; (C), Father of the patient.