Novel FBN1 intron variant causes isolated ectopia lentis via in-frame exon skipping

Abstract

Mutations in fibrillin-1 (FBN1) cause various clinical conditions, such as Marfan syndrome (MFS). However, the genotype-phenotype relationships underlying MFS and other conditions relevant to FBN1 mutations have not been fully elucidated. We performed whole-exome sequencing on three participants, including an affected mother-daughter pair, in a three-generation Japanese family with isolated ectopia lentis (IEL). The sequencing identified a novel single-nucleotide variant (c.1327+3A>C) in intron 11 of FBN1 that was shared between the two patients. We confirmed the co-segregation of the variant with IEL in two additional affected relatives in the family. The Combined Annotation-Dependent Depletion score of the variant was 26.1, which was indicated by SpliceAI to influence splicing, with a score of 0.93. Reverse transcription-polymerase chain reaction (RT-PCR) of mRNAs isolated from peripheral blood mononuclear cells revealed aberrant bands in all four affected individuals. Subsequent sequencing revealed that these bands originated from FBN1 transcripts lacking exon 11. The causality of the variant in the skipping of exon 11, which results in an in-frame deletion of 60 amino acids corresponding to the "hinge" region of FBN1 protein, was confirmed in a minigene experiment. Interestingly, the same result was observed for a minigene for c.1327+1G>A, a variant previously identified in two unrelated EL families without MFS manifestations. These results suggest that the c.1327+3A>C mutation in FBN1 likely leads to IEL. The findings expand our knowledge of FBN1 and provide insights into FBN1-related diseases.

Fig. 1

Pedigree of the studied family. The distribution of the affected individuals in the family suggests an autosomal dominant mode of inheritance. It is unknown whether patient I-1 had been diagnosed with lens dislocation, but it seems reasonable to assume that he was the founder of the causative mutation in this family and that the acute ocular hypertension that caused blindness was due to EL, as were several descendants. Patient II-1, who died at 47 years of age due to systemic infection, had no ophthalmological abnormalities

Fig. 2

Images of the eyes of the affected individuals. A Right anterior eye of III-3 with ectopia lentis and the lens dislocated downwards. B Optical coherence tomography of the anterior segment of the left eye of patient IV-1 upon referral to our hospital. The anterior lens deviated, and the anterior chamber depth was as short as 1.46 mm. C Sac shrinkage in the right eye of patient IV-1 4 months after cataract surgery. The patient subsequently underwent YAG laser capsulotomy. D Right eye of patient IV-1. Partial tearing of the ciliary zonule and deviation of the lens capsule was clearly observed soon after YAG laser capsulotomy

Fig. 3

Novel intronic variant of FBN1 identified in this study. Electropherograms from PCR direct sequencing of the FBN1 exon 11–intron 11 boundary for patient III-3 are shown

Fig. 4

Analysis of the splicing of FBN1 pre-RNAs from the patients’ peripheral blood mononuclear cells. A Image of the RT-PCR products resolved by gel electrophoresis. PC positive control (healthy adult), NC negative control (distilled water). In addition to the amplicon of the same size as that seen in PC (1), aberrant bands, including smaller (2) and larger smeary bands (*) were observed in patients IV-1, III-2, II-2, and III-3. Sanger sequencing result of amplicon (1) (B) and amplicon (2) (C)

Fig. 5

Analysis of splicing patterns using the FBN1 minigene model. A Structure of pcDNA3.1-FBN1 harboring partial exon 10, intron 10, exon 11, intron 11, and partial exon 12 of the FBN1 gene. B Results of RT-PCR for RNAs expressed from wild-type (WT) or mutant (MT) minigene constructs in HEK293 cells. NC negative control (distilled water)