Missense mutations in CRELD1 are associated with cardiac atrioventricular septal defects

Abstract

Atrioventricular septal defects (AVSD) are common cardiovascular malformations, occurring in 3.5/10,000 births. Although frequently associated with trisomy 21, autosomal dominant AVSD has also been described. Recently we identified and characterized the cell adhesion molecule CRELD1 (previously known as "cirrin") as a candidate gene for the AVSD2 locus mapping to chromosome 3p25. Analysis of the CRELD1 gene from individuals with non-trisomy 21-associated AVSD identified heterozygous missense mutations in nearly 6% of this population, including mutations in isolated AVSD and AVSD associated with heterotaxy syndrome. CRELD1 is the first human gene to be implicated in the pathogenesis of isolated AVSD and AVSD in the context of heterotaxy, which provides an important step in unraveling the pathogenesis of AVSD.

Figure 1

a, Sequence analyses identifying missense mutations in CRELD1. The arrows on each electropherogram indicate the variant nucleotides, with the wild-type sequence shown below. The altered nucleotides are shown in green. The single-letter–amino acid translation is under the first base of each codon. The altered amino acid residues are in red. Sequences from the complementary strands showed the same heterozygous changes. b, Diagrammatic representation of CRELD1 protein. The approximate positions of the amino acid changes are indicated above the diagram of CRELD1, with arrowheads pointing to the position of the substituted amino acid. c, Alignment of the sequence for the cb-EGF domain from the human, mouse, and bovine CRELD1 genes. d, Alignment of partial sequence of the WE domain from the human, mouse, and bovine CRELD1 genes. Amino acids that are identical among the three species are highlighted in green. The amino acid residues changed by the missense mutations in humans are boxed.

Figure 2

Diagrammatic representation of the cb-EGF domain harboring the T311I and R329C mutations. Conserved amino acid residues defining cb-EGF domains are indicated by the single-letter–amino acid code. Black lines show the disulfide-bonding pattern for the conserved cysteine residues. The positions of mutated amino acid residues are indicated by black circles; the β-hydroxylated asparagine residue is indicated by an asterisk.

Figure 3

a, Western blot showing the mobility shift of the C329 mutant protein, compared with wild-type (wt) CRELD1 without PNGase F digestion. Duplicate samples are loaded in alternating lanes. Digestion of the proteins with PNGase F results in a slight increase in mobility, compared with undigested protein, but does not resolve the difference in mobility between wt and C329 CRELD1. b, Western blot showing digestion of wt and 329C CRELD1 with PNGase F alone (+) or a mixture of glycohydrolases (PNGase F, sialidase A, endo-O-glycosidase, β(1,4)galactosidase, and glucosaminidase) that cleave all glycosidase linkages (++), compared with undigested protein (−). Note that digestion with additional glycohydrolases does not further shift the protein band, compared with digestion with PNGase F, indicating that CRELD1 is N-glycosylated but does not have other glycosylation sites to account for the decreased mobility of C329, compared with wt protein.