DHH pathogenic variants involved in 46,XY disorders of sex development differentially impact protein self-cleavage and structural conformation

Case Report: Long-term follow-up of desert hedgehog variant caused 46, XY gonadal dysgenesis with multiple complications in a Chinese child

“…The phenotype of patients with compound heterozygous variants is relatively mild, and in addition to the absence of peripheral neuropathy, the difference is also manifested in gonadal function; these patients may still have a certain degree of virilization and their gonadal pathology is PGD ( Tajouri et al, 2018 ; Ayers et al, 2019 ; Buonocore et al, 2019 ). Ayers and Elzaiat ( Ayers et al, 2019 ; Elzaiat et al, 2020 ) suggested that the DHH homozygous variant affects the overall conformation of the protein, thereby perturbing its self-cleavage, resulting in a significant loss (1%) of functional activity compared to the wild type, and they reported that compound heterozygotes show a significant decrease in activity (2% and 46%) and that single heterozygotes have nearly wild-type activity (90% and 98%). Similar to this case, patients with homozygous variants of DHH are more severe than those with compound heterozygous variants.…”

The Desert Hedgehog Signalling Pathway in Human Gonadal Development and Differences of Sex Development

“…Cellular localisation of the 11 DHH variants tested did not differ to the wild type, although the p.Arg-164Pro variant had a slightly reduced staining [Ayers et al, 2019]. In contrast, another study found that DHH is cleaved when expressed in HeLa cells, and that a number of patient variants had reduced or absent self-cleavage (continued) DOI: 10.1159/000518308 [Elzaiat et al, 2020] (Table 2). In addition, HeLa cells transfected with DHH-GFP variants had 2 patterns of intracellular localisation, termed by the authors "granular" (cytoplasmic expression) and "ring-type" (homogenous expression surrounding the nucleus).…”

“…In addition, HeLa cells transfected with DHH-GFP variants had 2 patterns of intracellular localisation, termed by the authors "granular" (cytoplasmic expression) and "ring-type" (homogenous expression surrounding the nucleus). A significantly lower proportion of the granular-type staining was described in some of the variants with impaired protein processing [Elzaiat et al, 2020] (Table 2). A cell-free in vitro cleavage assay has also shown that a fusion human DHH protein can undergo cleavage (with the addition of DTT or β-mercaptoethanol) [Tajouri et al, 2018] and 2 C-terminus patient variants led to reduced [p.( Glu212Lys)] or absent [p.(Asn337Lysfs*24)] cleavage efficiency in this system [Tajouri et al, 2018].…”

“…Based on this, it has been proposed that DHH is not processed or secreted in the Sertoli cells, instead eliciting a cellular response in the LCs in a juxtacrine fashion. Although based on the position and pro-posed effects of human DHH genetic variants, others have proposed that DHH auto-processing is necessary in the human gonads [Elzaiat et al, 2020].…”

“…It has not yet been established whether these phenotypes are due to primary failure in testis differentiation or later from a disrupted adrenal-pituitary-gonadal axis [Soliman et al, 1996], and therefore, further work is required to understand the exact role of primary cilia in DHH signalling in the testis. Reduced cleavage in HeLa cells [Elzaiat et al, 2020] Loss of activity in HEK/TM3 coculture assay [Ayers et al, 2019] p.Arg124Gln Slightly reduced cleavage in HeLa cells [Elzaiat et al, 2020] Partial loss of activity in HEK/TM3 co-culture assay [Ayers et al, 2019] p.Leu162Pro Abolished cleavage in HeLa cells [Elzaiat et al, 2020] Altered subcellular localisation in HeLa cells [Elzaiat et al, 2020] Loss of activity in HEK/TM3 coculture assay [Ayers et al, 2019] p.Arg164Pro Abolished cleavage in HeLa cells [Elzaiat et al, 2020] Slight reduction in expression levels [Ayers et al, 2019] Loss of activity in HEK/TM3 coculture assay [Ayers et al, 2019] p.Gly170Ser Reduced cleavage in HeLa cells [Elzaiat et al, 2020] Partial loss of activity in HEK/TM3 co-culture assay [Ayers et al, 2019] p.Trp173Cys Abolished cleavage in HeLa cells [Elzaiat et al, 2020] Altered subcellular localisation in HeLa cells [Elzaiat et al, 2020] p.Glu212Lys Reduced cleavage efficiency in in vitro time course [Tajouri et al, 2018] p.Ala227Val Abolished cleavage in HeLa cells [Elzaiat et al, 2020] Altered subcellular localisation in HeLa cells [Elzaiat et al, 2020] Loss of activity in HEK/TM3 coculture assay [Ayers et al, 2019] p.Arg249His Reduced cleavage in HeLa cells [Elzaiat et al, 2020] Partial loss of activity in HEK/TM3 co-culture assay [Ayers et al, 2019] p.Pro261Leu Abolished cleavage in HeLa cells [Elzaiat et al, 2020] Altered subcellular localisation in HeLa cells [Elzaiat et al, 2020] Loss of activity in HEK/TM3 coculture assay [Ayers et al, 2019] p.Asn337Lysfs*24 Abolished cleavage in in vitro assay [Tajouri et al, 2018] p.Arg353Gly Abolished cleavage in ...…”