Functional analysis of a novel FBN1 deep intronic variant causing Marfan syndrome in a Chinese patient

FIGURE 1. Photographs of our patient with MFS. Note bilateral temporal skull flattening, enophthalmos, retrognathia, arachnodactyly, pes planus, long, narrow feet, hammer toes and skin striae. (A–D) X-ray showed significant enlargement of the heart shadow with a cardiothoracic ratio of 0.67 (normal value < 0.6) (E). Echocardiography showed atrial septal defect, mitral regurgitation, tricuspid regurgitation and dilatation of the aortic root, 15 mm with Z-score of 4.48 (F–H).

FIGURE 2. Sanger sequencing results for the patient, and the patient’s father and mother. The analysis demonstrated the presence of a deep intronic variant in FBN1 (c.4943-8_4943-7insTATGTGATATTCATTCAC) in the patient and the absence of the variant in her parents. The red arrow indicates the variant site.

FIGURE 3. Minigene assay for FBN1 c.4943-8_4943-7insTATGTGATATTCATTCAC variant and schematic diagram of the splicing pattern. (A) The construction of FBN1-pcMINI minigene plasmid; (B) Gel electrophoresis of RT-PCR revealed a single band for wild-type (wt) and two bands for mutant-type (mut); (C) minigene product sequencing demonstrated that the wild-type minigene formed normal mRNA, but the c.4943-8_4943-7insTATGTGATATTCATTCAC variant in FBN1 caused a splicing abnormality, which abrogates the canonical splice site of intron 40, resulting in exon 41 skipping.

FIGURE 4. Schematic representation of FBN1 deep intronic variants identified to date. The structure of FBN1 contained 66 exons (blue rectangles), introns (black horizontal line); The localization of variants identified is depicted with dots. The dots above FBN1 indicate deep intronic variants determined by functional analysis, and those below FBN1 indicate deep intronic variants not determined by functional analysis. Red: Novel variants identified in this study.