In vitrofunctional characterization of the novelDHHmutations p.(Asn337Lysfs*24) and p.(Glu212Lys) associated with gonadal dysgenesis

Abstract

In humans, mutations of Desert Hedgehog gene (DHH) have been described in patients with 46,XY gonadal dysgenesis (GD), associated or not with polyneuropathy. In this study, we describe two patients diagnosed with GD, both harboring novel DHH compound heterozygous mutations p.[Tyr176*];[Asn337Lysfs*24] and p.[Tyr176*];[Glu212Lys]. To investigate the functional consequences of p.(Asn337Lysfs*24) and p.(Glu212Lys) mutations, located within the C‐terminal part of DHh on auto‐processing, we performed in vitro cleavage assays of these proteins in comparison with Drosophila melanogaster Hedgehog (Hh). We found that p.(Glu212Lys) mutation retained 50% of its activity and led to a partially abolished DHh auto‐processing. In contrast, p.(Asn337Lysfs*24) mutation resulted in a complete absence of auto‐proteolysis. Furthermore, we found a different auto‐processing profile between Drosophila Hh and human DHh, which suggests differences in the processing mechanism between the two species. Review of the literature shows that proven polyneuropathy and GD is associated with complete disruption of DHh‐N, whereas disruption of the DHh auto‐processing is only described with GD. We propose a model that may explain the differences between Schwann and Leydig cell development by autocrine versus paracrine DHh signaling. To our knowledge, this is the first study investigating the effect of DHH mutations on DHh in vitro auto‐processing.

Functional analysis of novel desert hedgehog gene variants improves the clinical interpretation of genomic data and provides a more accurate diagnosis for patients with 46,XY differences of sex development

“…Indeed, like us, Pettigrew et al found that DHH expressed in HEK 293 t cells was mostly uncleaved and yet it was still able to function in juxtacrine signalling. Tajouri et al suggest that DHH autoprocessing is necessary to mediate signalling from Sertoli cells to promote differentiation of the Leydig cells 16. While this may be the case in vivo, our cell culture experiments show that while DHH expressed in this system is mostly uncleaved, variants that lie within the C-terminal domain can still have a significant effect on signalling activation in TM3 mouse Leydig cells.…”

“…While none of our patients reported any signs of peripheral neuropathy, in several cases the genetic diagnosis prompted clinicians to test for this, and continued monitoring will now be carried out. Tajouri et al hypothesised that C-terminal variants affecting autoprocessing are more likely to lead to gonadal dysgenesis without neuropathies, as even unprocessed DHH protein may induce peripheral nerve development 16. Interestingly though, our patient with a homozygous variant in the N-terminal (patient 1; p.Arg164Pro) which showed a complete LOF has not reported neuropathies.…”

“…While it is well established that the N-terminal of DHH is the active molecule, the exact function of the C terminus is unclear. A recent study from the Werner Lab has found that, given the right conditions (addition of Dithiothreitol or β-mercaptoethanol), a cell-free in vitro transcribed and translated human fusion DHH protein does undergo cleavage 16. In addition, they have shown that two variants found in patients within the C terminus lead to reduced or absent cleavage efficiency.…”

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

“…Our searches were based on combinations of the following index terms: DHH , 46, XY gonadal dysplasia, 46, XY gonadal dysgenesis, 46, XY disorder of sex development, neuropathy, Hedgehog signaling pathway and the corresponding terms in Chinese. A total of 25 cases of DHH variants causing 46, XY GD in detail have been reported worldwide so far ( Umehara et al, 2000 ; Canto et al, 2004 ; Das et al, 2011 ; Werner et al, 2015 ; Paris et al, 2017 ; Sato et al, 2017 ; Baldinotti et al, 2018 ; Rothacker et al, 2018 ; Tajouri et al, 2018 ; Ayers et al, 2019 ; Buonocore et al, 2019 ; Neocleous et al, 2019 ; Akter et al, 2021 ; Kalinchenko et al, 2021 ; Mehta et al, 2021 ) (see Table 2 ), including 24 different variant types of missense, deletion, duplication, or transversion, and both homozygous and compound heterozygous variants have been reported. The reported ages ranged from 2.8 to 55 years.…”

DLC3/Cv-c function in testis development in humans and Drosophila: implication for variants of sex development

“…In the fly, somatic gonadal cells can coalesce into a gonad in the absence of PGCs, but the PGCs are unable to coalesce in the absence of somatic cells (Brookman et al , 1992). Similarly, subsets of somatic gonadal cells produce steroid hormones (testosterone in mammals (Zirkin & Papadopoulos, 2018) and ecdysone in insects (Tajouri et al , 2018)), which have a global influence on the organism.…”