R-Spondins regulate the WNT/β-catenin signaling pathway by interacting with leucine rich-repeat containing G-protein coupled receptors (LGR4-6). These receptors share unique sequence and structural similarities with each other. Here, we report comparative binding analysis of R-Spondin-4 (RSPO4) with LGRs through structural characterization of a missense variant (GLY67ARG) identified in two consanguineous families of Pakistani origin. The modeled structure of RSPO4 comprises two contiguous Furin-like cysteine-rich domains that are involved in binding with LGRs. We observed an overall conservation of overlapping interacting residues among LGRs which recognized RSPO4 at two specific parallel positions (sites 'a' and 'b'). The residual contributions of RSPO4 reconciled previously defined interactions of RSPO1 with LGRs. To check the comparative expression pattern of β-catenin, we quantified β-catenin levels in normal and anonychia patients. β-catenin level was significantly reduced in the patients exhibiting mutated RSPO4 as compared to control individuals. These findings confirm that RSPO4 modulates the LGR-dependent WNT/β-catenin signaling pathway and may have therapeutic potential in anonychia patients.