Genetic Complexity of Mitral Valve Prolapse Revealed by Clinical and Genetic Evaluation of a Large Family

Abstract

Background: A genetic component to familial mitral valve prolapse (MVP) has been proposed for decades. Despite this, very few genes have been linked to MVP. Herein is described a four-generation pedigree with numerous individuals affected with severe MVP, some at strikingly young ages.

Methods: A detailed clinical evaluation performed on all affected family members demonstrated a spectrum of MVP morphologies and associated phenotypes.

Results: Linkage analysis failed to identify strong candidate loci, but revealed significant regions, which were investigated further using whole-exome sequencing of one of the severely affected family members. Whole-exome sequencing identified variants in this individual that fell within linkage analysis peak regions, but none was an obvious pathogenic candidate. Follow up segregation analysis of all exome-identified variants was performed to genotype other affected and unaffected individuals in the family, but no variants emerged as clear pathogenic candidates. Two notable variants of uncertain significance in candidate genes were identified: p.I1013S in PTPRJ at 11p11.2 and FLYWCH1 p.R540Q at 16p13.3. Neither gene has been previously linked to MVP in humans, although PTPRJ mutant mice display defects in endocardial cushions, which give rise to the cardiac valves. PTPRJ and FLYWCH1 expression was detected in adult human mitral valve cells, and in-silico analysis of these variants suggests they may be deleterious. However, neither variant segregated completely with all of the affected individuals in the family, particularly when 'affected' was broadly defined.

Conclusions: While a contributory role for PTPRJ and FLYWCH1 in this family cannot be excluded, the study results underscored the difficulties involved in uncovering the genomic contribution to MVP, even in apparently Mendelian families.

Figure 1:

Pedigree demonstrating apparently autosomal dominant segregation of MVP. A five-generation family history was obtained during a family research visit in which medical histories could be elicited from members of each of the major branches of the family. The initial self-reported diagnoses of mitral valve prolapse or other cardiovascular phenotypes were subsequently evaluated in more detail by study investigators to define the specific phenotypes (definitive MVP, equivocal MVP, dilated AR) that were present or absent.

Figure 2:

Genome-wide linkage analysis identifies suggestive multipoint LOD scores. Linkage analysis was performed in the MVP family using the Illumina Human CytoSNP-12 genotyping bead chip assay, with two different schemes for affected status based on the stringency of the echocardiographic findings. There was no definitive evidence for linkage to any particular locus. A.) In the broader scheme 1, suggestive LOD scores were identified at 2q, 6q, and 16p. In the stricter scheme 2, suggestive LOD scores were identified at additional chromosomal loci, including 3p, 8, proximal 11p, and 12.

Figure 3:

Segregation analysis of candidate variants in family members available for genotyping. Candidate variants in the WES data from family member V:2 were investigated for co-segregation in affected and unaffected family members using Sanger sequencing. Inferred genotypes are shown in parentheses. Each column corresponds to a single individual from the MVP family pedigree that was available for genotyping. The number at the top of the column refers to the individual as they are labeled in the full pedigree of Figure 1. D=definitive MVP, U=unaffected, E=equivocal MVP, R=MVP per external report. Columns that correspond to the proband’s affected mother, grandmother, maternal great-aunt, and great-grandmother are shaded grey. FLYWCH1 and PTPRJ genotypes for informative unaffected individuals are boxed in bold.

Figure 4:

Variants identified in FLYWCH1 and PTPRJ provide strong candidates for a molecular etiology of MVP in the family. The FLYWCH1 and PTPRJ variants identified in the MVP family are rare, predicted deleterious, and found in genes expressed in human mitral and aortic valve cells. A. Top: Graphic depiction of human FLYWCH1, with FLYWCH zinc-finger domains shown in black, and p.R540Q variant position as yellow vertical line. Graphic depiction of human PTPRJ showing p.I1013S residue (yellow line) within catalytic cytoplasmic domain (black), downstream of the extracellular region (grey) and transmembrane domain (TM), and adjacent to residue 1016 (red line) reported to result in decreased binding to ERK½. FLYWCH1 p.R540Q and PTPRJ p.I1013S variants are conserved among mammals, rare in the general population, and predicted to be damaging. B). RT-PCR results show relative levels of FLYWCH1, PTPRJ, and COL6A3 transcripts in cDNA from human mitral and aortic valve normalized to ACTB. Delta Ct = number of amplification cycles to reach the threshold when actin Ct is set as zero. Higher positive delta Ct indicates lower levels of expression compared to ACTB. Data are presented as mean of three experiments with standard deviation error bars. C). The fourth and fifth FLYWCH domains of FLYWCH1 are 98% similar. Residues flanking R540 (bold) are shown in the top sequence, and the cognate region of the fifth FLYWCH domain used in the model is shown the bottom sequence. Bottom left panel: PyMol model of protein data bank structure 2rprA, human flywch1 residues 595–674. Residues comprising a helical structure are colored red, beta strands as yellow, and looping regions as green. The arginine occupying the cognate position to residue R540 is depicted as white sticks with hydrogens colored blue, and can be seen in close association to the zinc molecule, shown as a magenta sphere. Inset: Modeled substitution of glutamine at the cognate position 540, normally occupied by arginine. Insets zoom in on the variant – zinc ion region.

Figure 5:

Clinical and genomic evaluation strategy to identify candidate MVP-associated variants.