LRP6 mutation in a family with early coronary disease and metabolic risk factors

Abstract

Coronary artery disease (CAD) is the leading cause of death worldwide and is commonly caused by a constellation of risk factors called the metabolic syndrome. We characterized a family with autosomal dominant early CAD, features of the metabolic syndrome (hyperlipidemia, hypertension, and diabetes), and osteoporosis. These traits showed genetic linkage to a short segment of chromosome 12p, in which we identified a missense mutation in LRP6, which encodes a co-receptor in the Wnt signaling pathway. The mutation, which substitutes cysteine for arginine at a highly conserved residue of an epidermal growth factor-like domain, impairs Wnt signaling in vitro. These results link a single gene defect in Wnt signaling to CAD and multiple cardiovascular risk factors.

Fig. 1

Relationships of members of kindred CAD-100 are shown. The index case is indicated by the arrow. Numbered individuals correspond to those in table S1. Individuals with early CAD are indicated by black symbols; individuals without CAD who are beyond age 50 (men) or 55 (women) are shown as unfilled symbols; and individuals who are without symptomatic CAD, are below these ages, and have high LDL levels (range 157 to 192) are shown as half-black, half-gray symbols. Individuals who were not studied are indicated by symbols with dots. Circles represent females; squares represent males. Symbols with a slash through them indicate deceased subjects. Genotypes of informative microsatellite and SNP markers are shown in their chromosomal order below the symbol for each individual and their distance in centimorgans and megabases from 12pter is indicated. Segments of the haplotype seen in the index case which segregate with CAD and/or high LDL levels are indicated by shaded boxes. The presence of the wild-type LRP6 or LRP6_R611C mutation is indicated by a plus sign or a red asterisk, respectively. Subjects III-6, III-7, and III-8 are offspring of a homozygous mutation carrier and hence obligate mutation carriers; subjects III-3, III-4, and III-5 are also statistically likely to be offspring of a homozygous carrier. This explains the high proportion of mutation carriers in generation III.

Fig. 2

Mutation in LRP6 cosegregates with early CAD and high LDL. (A) Multipoint lod scores for linkage of early CAD and high LDL cholesterol to 12p. Multipoint lod scores for linkage of early CAD (solid line) or high LDL cholesterol (dashed line) to proximal 12p are shown under the stringent model of the trait locus (27). Lod scores were calculated with the use of all chromosome 12 SNP data from Affymetrix 10K chips (27). SNPs tightly linked to the location of the maximum lod score are indicated and the location of LRP6 is shown. The lod score peak occurs at zero recombination with marker rs958812 and the lod-1 interval spans 2.7 cM for both traits. (B) The DNA sequence of a segment of LRP6 exon 9 is shown from an unaffected kindred member (left), a heterozygous mutation carrier (middle), and the homozygous index case (right). A single base substitution (asterisk) changes the wild-type cytidine to thymidine leading to substitution of cysteine for arginine at codon 611. (C) A portion of the amino acid sequence of the second EGF-like domain of LRP6 is shown from diverse vertebrate species. This segment is highly conserved and arginine is completely conserved from Xenopus to human.

Fig. 3

LRP6_R611C impairs Wnt signaling. NIH3T3 cells were transfected with plasmids encoding wild-type (WT) or mutant hemagglutinin-tagged LRP6 and Wnt reporter genes and incubated with indicated concentrations of purified Wnt 3a protein followed by an assay of Wnt signaling (LEF-1–dependent expression of luciferase) (27). The results are shown as mean and standard error of the mean of quadruplicate experiments. RLU, relative light units.