Sönke Behrends

The purpose of this study was to characterize the phenotype associated with a de novo gain-of-function variant in the GUCY1A2 gene. An individual carrying the de novo heterozygous variant c.1458G>T p.(E486D) in GUCY1A2 was identified by exome sequencing. The effect of the corresponding enzyme variant α₂E486D/β₁ was evaluated using concentration-response measurements with wild-type enzyme and the variant in cytosolic fractions of HEK293 cells, UV-vis absorbance spectra of the corresponding purified enzymes, and examination of overexpressed fluorescent protein-tagged constructs by confocal laser scanning microscopy. The patient presented with precocious peripheral puberty resembling the autonomous ovarian puberty seen in McCune-Albright syndrome. Additionally, the patient displayed severe intellectual disability. In vitro activity assays revealed an increased nitric oxide affinity for the mutant enzyme. The response to carbon monoxide was unchanged, while thermostability was decreased compared to wild type. Heme content, susceptibility to oxidation, and subcellular localization upon overexpression were unchanged. Our data define a syndromic autonomous ovarian puberty likely due to the activating allele p.(E486D) in GUCY1A2 leading to an increase in cGMP. The overlap with the ovarian symptoms of McCune-Albright syndrome suggests an impact of this cGMP increase on the cAMP pathway in the ovary. Additional cases will be needed to ensure a causal link.

The identification and characterization of a compound that selectively activates a particular isoform provides unique insight into the molecular differences and functional divergence between the isoforms. This not only allows for a more precise understanding of cellular signalling pathways but also potentially opens up new avenues for targeted therapy of diseases where dysregulated activity of a specific isoform is implicated. In humans, there are two distinct active isoforms of the soluble guanylyl cyclase (sGC): α1/β1 (GC-1) and α2/β1 (GC-2). In the present study we show that runcaciguat is the first isoform-specific sGC activator with a strong preference for GC-1. The activator exhibits antagonistic behaviour towards GC-2, leading to reduced efficacy of the nitric oxide donor DEA/NO. Similar results were generated with the related activator BAY-543. In addition, comparing the sGC activators BAY 60-2770 and BI 703704, a much stronger activation of GC-2 is measurable with BI 703704, whereas the potency of both activators in GC-1 is approximately the same. The classification of the investigated activators into mono- and dicarboxylic acids does not seem to be significant for the observed isoform specificity. It is tempting to hypothesize that the activation of GC-2 may rather increase with the lengthening of the lipophilic tail of the activators. Our laboratory is currently investigating this hypothesis.

An individual carrying the de novo heterozygous variant c.1458G>T p.(E486D) in GUCY1A2 was identified by exome sequencing. The effect of the corresponding enzyme variant a ₂E486D/b ₁ was evaluated using concentration-response measurements with wild-type enzyme and the variant in cytosolic fractions of HEK293 cells, UV-VIS absorbance spectra of the corresponding purified enzymes, and examination of overexpressed fluorescent protein-tagged constructs by confocal laser scanning microscopy. The patient presented with precocious peripheral puberty resembling the autonomous ovarian puberty seen in McCune-Albright syndrome. Additionally, the patient displayed severe intellectual disability. In vitro activity assays revealed an increased nitric oxide affinity for the mutant enzyme. The response to carbon monoxide was unchanged, while thermostability was decreased compared to wildtype. Heme content, susceptibility to oxidation and subcellular localisation upon overexpression were unchanged. Our data defines a syndromic autonomous ovarian puberty likely due to the activating allele p.(E486D) in GUCY1A2 leading to an increase in cGMP. The overlap with the ovarian symptoms of McCune-Albright syndrome suggests an impact of this cGMP increase on the cAMP pathway in the ovary. Additional cases will be needed to ensure a causal link. The data are currently being published in the European Journal of Endocrinology, but we expect additional results by the time of the meeting in Lübeck.