R-spondin 1 gene variant associated with 46,xx difference of sex development

R-spondin 1, encoded by RSPO1, is important for the differentiation of the bipotential gonad into the ovary. Rare homozygous RSPO1 frameshift variants, deletions, and intronic variants with exon skipping have been reported in four families with 46,XX, SRY negative sex reversal. We sought to characterize a novel RSPO1 homozygous exonic missense variant in a man with 46,XX difference of sex development (DSD), hypospadias, hypergonadotropic hypogonadism, azoospermia, and palmoplantar hyperkeratosis. A pedigree was obtained. The proband had a karyotype, FISH and PCR for SRY analysis. Familial segregation analysis was performed using Sanger sequencing to identify RSPO1 variants. In silico variant analyses were performed to assess pathogenicity with multiple sequence alignment, gnomAD allele frequency, CADD score, VarSome variant classification, and VARITY rare missense variant classification. The proband had a 46,XX karyotype and was SRY negative. Sanger sequencing identified that the proband had a homozygous RSPO1 missense variant (p.Cys105Arg). Both parents and one male sibling were unaffected and were heterozygous carriers for the RSPO1 p.Cys105Arg variant. Multiple sequence alignment identified cysteine amino acid conservation at position 105 in 99/100 species. This variant had a gnomAD carrier allele frequency 1.2 x 10-5 and zero homozygotes reported. The CADD score was 29.5 which predicts deleteriousness. VarSome American College of Medical Genetics & Genomics classification identified it as a variant of uncertain significance; however, 12/19 individual predictions in VarSome characterize it as pathogenic. VARITY Variant_R score of 0.982, which characterizes rare missense variants, predicted pathogenicity. In silico analyses support the pathogenicity of a rare RSPO1 c.Cys105Arg homozygous missense variant associated with 46,XX, SRY negative DSD.In vitro and in vivo studies can further characterize its deleteriousness and provide stronger evidence for pathogenicity.