Hereditary disorders often present remarkable genetic and clinical heterogeneities, but bridging the gap between pathogenic mutation and clinical consequences remains challenging. One plausible explanation is the presence of second-hit modifier mutations, which could create additional genetic complexity to contribute to clinical heterogeneities. We propose to trace modifier mutations by focusing on single-pedigree patients who present divergent clinical phenotypes; this scenario would ensure shared pathogenic mutations and highly related genetic makeups, thus simplifying the detection of second-hit modifiers. An appropriate window to test this idea lies in the case of low-density lipoprotein receptor-related protein 6 (LRP6), which encodes one essential co-receptor of Wnt signaling and is involved in multiple human diseases. In particular, LRP6 has been discovered as a frequent pathogenic gene for tooth agenesis (absence of one or more permanent teeth) or in rare cases for ectodermal dysplasia where affected members showed variable abnormalities of teeth, hair, and sweat glands.1 Yet the pathogenic mechanism of LRP6-related tooth agenesis and the genetic cause for its clinical heterogeneity remain unclear. Here, we report a novel LRP6 C1032F mutation in one ectodermal dysplasia family, highlighting YWTD-motif targeting as a generalized mechanism in LRP6-associated tooth agenesis and involvement of second-hit modifier mutations in LRP6-associated clinical heterogeneity.