In Vitro and Molecular Modeling Analysis of Two Mutant Desert Hedgehog Proteins Associated with 46,XY Gonadal Dysgenesis

Abstract

Mutations of Desert hedgehog (DHH) have been associated to 46,XY pure gonadal dysgenesis (PGD) and to mixed gonadal dysgenesis (MGD); however, there have been no functional studies of mutations described in DHH. To determine if mutations p.L162P and D1086delG yield functional impairment, we performed in vitro and in silico analysis of both DHH mutants. In complementary DNA of DHH, we performed site-directed mutagenesis, which was confirmed by DNA sequencing. Protein extracts were obtained from HEK293cells transfected with different constructs and analyzed by Western blot; besides, densitometric analysis of chemiluminescent signals was performed. In addition, the structure of the wt-DHH and its two mutant proteins was inferred using in silico protein molecular modeling. In the Western blot analysis, we observed the absence of signal for p.L162P in DHH-N and a diminished signal for D1086delG in DHH-C, when compared to wt-DHH. Protein modeling showed notable conformational changes for the side chains of p.L162P, while the secondary structure was drastically modified in D1086delG, when compared to wt-DHH. To our knowledge, this is the first study focused to determine by in vitro studies, the effect of two specific mutations in DHH associated with 46,XY PGD and MGD. Our results suggest that both mutations have a deleterious effect on the expression of the DHH mutant proteins.

46,XY Gonadal Dysgenesis due to a Homozygous Mutation in Desert Hedgehog (DHH) Identified by Exome Sequencing

“…and are predicted to abolish DHhN protein ( Table 1). The missense mutation P162L and small deletion p.E91del detected in the two further patients have been analyzed by protein modeling and are predicted to alter the binding interface to CDO and BOC (22,38). With respect to the gonadal phenotype of Dhh Ϫ/Ϫ mice and unaffected fathers carrying heterozygous mutations in DHH, we conclude that loss of function in DHh signaling is responsible for the phenotype of 46,XY patients with gonadal dysgenesis.…”

Partial duplication of DHH causes minifascicular neuropathy

“…(Table S2). Although nerve conduction studies, nerve biopsy, and/or functional study of the mutations were performed in a limited number of patients, mutations presumably leading to complete loss of function of DHH (c.304–572dup, c.2T>C, c.485T>C,17 and c.57–60dupAGCC) does not always cause both neuropathy and GD. It is still uncertain whether there is a phenotype correlation with complete loss versus partial loss of function of DHH gene.…”

Genetic testing of XY newborns with a suspected disorder of sex development

“…SOX9 variants were detected in patients with gonadal dysgenesis and concomitant bone abnormalities due to the lack of chondrocyte-specific enhancer activity [78]. Although a small number of individuals were found to be carriers of variants in DHH, gonadal cancer was evident in almost 30% of them [60] and it was commonly associated with peripheral minifascicular neuropathy [61,79,80]. 46, XY PGD and CGD due to missense variants in WT1 were recognised in Denys Drash syndrome [81] and concurrent renal abnormalities [82].…”