The NM_000330.4(RS1):c.216G>C (p.Glu72Asp) variant is a missense variant encoding the substitution of Glutamic acid and Aspartic acid at amino acid 72. This variant is absent from hemizygous individuals in gnomAD v4.1.0 (PM2_Supporting). The computational predictor REVEL gives a score of 0.930, which is within the ClinGen X-linked IRD VCEP range between 0.931 and 0.773 and predicts a damaging effect on RS1 function (PP3_moderate). The computational splicing predictor SpliceAI gives a delta score of 0.03 for acceptor gain, which is below the ClinGen X-linked IRD VCEP threshold of >0.2 and does not predict that the variant disrupts RS1 splicing. Another missense variant in the same codon, NM_000330.4(RS1):c.214G>C (p.Glu72Gln), (PMIDs: 19390641, 11295123, 28348004, 36460718, 39462066, 35456481) have been classified as pathogenic for X-linked retinoschisis by the ClinGen X-linked IRD VCEP, while no benign missense variants have been identified in this codon. The present variant has a higher Grantham’s distance (45) than the comparison variants (29), and SpliceAI has been used to confirm that neither variant has a predicted impact on RS1 splicing (PM5). This variant p.Glu72, of the RS1 gene, is a missense substitution affecting an established residue involved in cross-linking the adjacent RS1 subunit defined by the ClinGen X-linked IRD VCEP (PMID: 27114531, PM1). This variant has been reported in at least 3 apparently unrelated probands meeting one of the PS4 requirements of a male diagnosed with XLRS (PMIDs: 9618178, 34822951, 35456481, 38219857, PS4_Moderate). In summary, this variant is classified as likely pathogenic for X-linked retinoschisis based on the ClinGen X-linked Inherited Retinal Disease Expert Panel Specifications to the ACMG/AMP Variant Interpretation Guidelines for RS1 Version 1.0.0: PM2_Supporting, PP3_Moderate, PM5, PM1, and PS4_Moderate.