Characterization of LTBP2 mutation causing mitral valve prolapse

Abstract

Aims: Mitral valve prolapse (MVP) is a common valvular disorder associated with significant morbidity and mortality, with a strong genetic basis. This study aimed to identify a mutation in a family with MVP and to characterize the valve phenotype in LTBP2 knockout (KO) mice.

Methods and results: Exome sequencing and segregation analysis were performed on a large family with MVP. Two mouse strains were generated: a complete KO of the LTBP2 gene and a knockin (KI) of the human mutation. At 6 months, phenotyping was conducted using echocardiography, histology, eye optical coherence tomography, and quantitative polymerase chain reaction analysis for TGF-β signalling targets (periostin/POSTN, RUNX2, and CTGF) in valve tissues. LTBP2 rs117800773 V1506M mutation exhibited segregation with MVP. LTBP2 KO mice had a higher incidence of myxomatous changes by histology (7 of 9 of KO vs. 0 of 7 control animals, P = 0.00186) and echocardiography (7 of 9 vs. 0 of 8, P = 0.0011). LTBP2 KI mice for the human mutation showed a significantly elevated myxomatous histological phenotype (8 of 8 vs. 0 of 9, P = 0.00004) as well as by echocardiography (6 of 8 vs. 0 of 9, P = 0.00123). Knockout mice demonstrated an increase in the depth of the anterior chamber as well as reduced visual acuity. LTBP2 KO mice demonstrated overexpression of both TGF-β signalling targets RUNX2 and periostin (P = 0.0144 and P = 0.001826, respectively).

Conclusion: We report a KO mouse strain with an LTBP2 mutation, demonstrating a valve phenotype, alongside a family with a novel mutation linked to MVP.

Keywords: LTBP2; Mitral valve prolapse; Myxomatous valve.

Graphical abstract

Figure 1

(A) Pedigree demonstrating LTBP2 V1506M mutation. Segregation with mitral valve prolapse in the pedigree. Dark shapes are mitral valve prolapse, and empty shapes are normal phenotype. A question mark denotes either equivocal phenotype or that an echocardiogram was not available. LTBP2 genotype is at the bottom: CC is the wild-type allele and CT heterozygote for the V1506M mutation. The left top number is a serial number for the individual in the family. The right top number when available is the age of phenotyping. (B) The sequence variation with a blue arrow pointing to the mutation. (C) A demonstrative parasternal long axis with and without colour Doppler of individual 004, demonstrating mitral valve prolapse and significant regurgitation.

Figure 2

Valve phenotype of LTBP2 deficiency. Echocardiograms of 6-month-old knockout (A) and wild type (B). The line delineates annular line. The arrow is pointing to the posterior leaflet. Mitral valve LTBP2 immunofluorescence in a knockout mouse (C) and wild type (D). Notice the Cy5 colouring (red) of the wild-type valve that is lacking in the knockout. (E–G) Movat pentachrome straining of knockout, knockin, and wild-type mice, respectively. Black arrows are pointing at the leaflets. Notice the marked thickening and fibrosis with myxomatous changes of the valve leaflets in the knockout (black arrows) (E) and the widespread blue staining of mucinous substance (arrow) (F). The leaflets of the wild-type mice are thin and are not stained blue (G).

Figure 3

Ocular structure and visual function. Ocular structure and visual function in 12–14-month-old LTBP2 knockout mice. Left bars represent wild-type mice, and right bars represent LTBP2 knockout mice. Results are presented as mean ± SEM. Number of animals in each group is written within the corresponding bar. *P < 0.05; **P < 0.0001. (A) Comparison between wild-type and LTBP2 knockout mouse eyes revealed a significant increase in the anterior chamber depth in mutant mice as measured using in vivo optical coherence tomography imaging of the anterior segment. Representative images from 12-month-old mice are shown to the right of the bar graph. Anterior chamber depth reflects the distance between the corneal endothelium and the anterior capsule of the lens and is marked by red lines. Scale bars, 240 μm. (B) Knockout of the LTBP2 gene also led to significant enlargement of the iridocorneal angle. Representative images of iridocorneal angle (marked by yellow dotted lines) in 12-month wild-type and LTBP2 knockout mice. (C) From 12 months of age and on, LTBP2 knockout mice exhibited very poor dilation of the pupils in response to topical application of mydriatic drops. Measurements revealed a statistically significant reduction in pharmacologically dilated pupil diameter in mutant mice compared with age-matched wild-type mice. Representative images of eyes of 12-month-old mice captured in infra-red mode are presented to the right of the graph. (D) Representative images of eye sections from 12-month-old mice, taken through the central cornea and optic nerve and stained with haematoxylin and eosin, align with our in vivo optical coherence tomography findings. Mutant eyes show a deeper anterior chamber caused by posterior subluxation of the lens accompanied by an increased iridocorneal angle, reduced depth of the vitreous cavity, and a narrow pupil. Scale bar, 500 μm. (E) Visual acuity as measured using the optomotor response revealed a significant reduction in LTBP2 knockout mice compared with wild-type mice.