De novo variants in PLCG1 are associated with hearing impairment, ocular pathology, and cardiac defects

Reviewer #1 (Public Review):

Summary:

This manuscript provides an initial characterization of three new missense variants of the PLCG1 gene associated with diverse disease phenotypes, utilizing a Drosophila model to investigate their molecular effects in vivo. Through the meticulous creation of genetic tools, the study assesses the small wing (sl) phenotype - the fly's ortholog of PLCG1 - across an array of phenotypes from longevity to behavior in both sl null mutants and variants. The findings indicate that the Drosophila PLCG1 ortholog displays aberrant functions. Notably, it is demonstrated that overexpression of both human and Drosophila PLCG1 variants in fly tissue leads to toxicity, underscoring their pathogenic potential in vivo.

Strengths:

The research effectively highlights the physiological significance of sl in Drosophila. In addition, the study establishes the in vivo toxicity of disease-associated variants of both human PLCG1 and Drosophila sl.

Weaknesses:

The study's limitations include the human PLCG1 transgene's inability to compensate for the Drosophila sl null mutant phenotype, suggesting potential functional divergence between the species. This discrepancy signals the need for additional exploration into the mechanistic nuances of PLCG1 variant pathogenesis, especially regarding their gain-of-function effects in vivo.

Overall:

The study offers compelling evidence for the pathogenicity of newly discovered disease-related PLCG1 variants, manifesting as toxicity in a Drosophila in vivo model, which substantiates the main claim by the authors. Nevertheless, a deeper inquiry into the specific in vivo mechanisms driving the toxicity caused by these variants in Drosophila could significantly enhance the study's impact.

Reviewer #2 (Public Review):

The manuscript by Ma et al. reports the identification of three unrelated people who are heterozygous for de novo missense variants in PLCG1, which encodes phospholipase C-gamma 1, a key signaling protein. These individuals present with partially overlapping phenotypes including hearing loss, ocular pathology, cardiac defects, abnormal brain imaging results, and immune defects. None of the patients present with all of the above phenotypes. PLCG1 has also been implicated as a possible driver for cell proliferation in cancer.

The three missense variants found in the patients result in the following amino acid substitutions: His380Arg, Asp1019Gly, and Asp1165Gly. PLCG1 (and the closely related PLCG2) have a single Drosophila ortholog called small wing (sl). sl-null flies are viable but have small wings with ectopic wing veins and supernumerary photoreceptors in the eye. As all three amino acids affected in the patients are conserved in the fly protein, in this work Ma et al. tested whether they are pathogenic by expressing either reference or patient variant fly or human genes in Drosophila and determining the phenotypes produced by doing so.

Expression in Drosophila of the variant forms of PLCG1 found in these three patients is toxic; highly so for Asp1019Gly and Asp1165Gly, much more modestly for His380Arg. Another variant, Asp1165His which was identified in lymphoma samples and shown by others to be hyperactive, was also found to be toxic in the Drosophila assays. However, a final variant, Ser1021Phe, identified by others in an individual with severe immune dysregulation, produced no phenotype upon expression in flies.

Based on these results, the authors conclude that the PLCG1 variants found in patients are pathogenic, producing gain-of-function phenotypes through hyperactivity. In my view, the data supporting this conclusion are robust, despite the lack of a detectable phenotype with Ser1021Phe, and I have no concerns about the core experiments that comprise the paper.

Figure 6, the last in the paper, provides information about PLCG1 structure and how the different variants would affect it. It shows that the His380, Asp1019, and Asp1165 all lie within catalytic domains or intramolecular interfaces and that variants in the latter two affect residues essential for autoinhibition. It also shows that Ser1021 falls outside the key interface occupied by Asp1019, but more could have been said about the potential effects of Ser1021Phe.

Overall, I believe the authors fully achieved the aims of their study. The work will have a substantial impact because it reports the identification of novel disease-linked genes, and because it further demonstrates the high value of the Drosophila model for finding and understanding gene-disease linkages.

Reviewer #3 (Public Review):

Summary:

The paper attempts to model the functional significance of variants of PLCG2 in a set of patients with variable clinical manifestations.

Strengths:

A study attempting to use the Drosophila system to test the function of variants reported from human patients.

Weaknesses:

Additional experiments are needed to shore up the claims in the paper. These are listed below.

Major Comments:

(1) Does the pLI/ missense constraint Z score prediction algorithm take into consideration whether the gene exhibits monoallelic or biallelic expression?

(2) Figure 1B: Include human PLCG2 in the alignment that displays the species-wide conserved variant residues.

(3) Figure 4A:
Given that
(i) sl is predicted to be the fly ortholog for both mammalian PLCγ isozymes: PLCG1 and PLCG2 [Line 62]
(ii) they are shown to have non-redundant roles in mammals [Line 71] and
(iii) reconstituting PLCG1 is highly toxic in flies, leading to increased lethality.
This raises questions about whether sl mutant phenotypes are specifically caused by the absence of PLG1 or PLCG2 functions in flies. Can hPLCG2 reconstitution in sl mutants be used as a negative control to rule out the possibility of the same?

(4) Do slT2A/Y; UAS-PLCG1Reference flies survive when grown at 22{degree sign}C? Since transgenic fly expressing PLCG1 cDNA when driven under ubiquitous gal4s, Tubulin and Da, can result in viable progeny at 22{degree sign}C, the survival of slT2A/Y; UAS-PLCG1Reference should be possible.
and similarly
Does slT2A flies exhibit the phenotypes of (i) reduced eclosion rate (ii) reduced wing size and ectopic wing veins and (iii) extra R7 photoreceptor in the fly eye at 22{degree sign}C?
If so, will it be possible to get a complete rescue of the slT2A mutant phenotypes with the hPLCG1 cDNA at 22{degree sign}C? This dataset is essential to establish Drosophila as an ideal model to study the PLCG1 de novo variants.

(5) Localisation and western blot assays to check if the introduction of the de novo mutations can have an impact on the sub-cellular targeting of the protein or protein stability respectively.

(6) Analysing the nature of the reported gain of function (experimental proof for the same is missing in the manuscript) variants:
Instead of directly showing the effect of introducing the de novo variant transgenes in the Drosophila model especially when the full-length PLCG1 is not able to completely rescue the slT2A phenotype;
(i) Show that the gain-of-function variants can have an impact on the protein function or signalling via one of the three signalling outputs in the mammalian cell culture system: (i) inositol-1,4,5-trisphosphate production, (ii) intracellular Ca2+ release or (iii) increased phosphorylation of extracellular signal-related kinase, p65, and p38.
OR
(ii) Run a molecular simulation to demonstrate how the protein's auto-inhibited state can be disrupted and basal lipase activity increased by introducing D1019G and D1165G, which destabilise the association between the C2 and cSH2 domains. The H380R variant may also exhibit characteristics similar to the previously documented H335A mutation which leaves the protein catalytically inactive as the residue is important to coordinate the incoming water molecule required for PIP2 hydrolysis.

(7) Clarify the reason for carrying out the wing-specific and eye-specific experiments using nub-gal4 and eyless-gal4 at 29˚C despite the high gal4 toxicity at this temperature.

(8) For the sake of completeness the authors should also report other variants identified in the genomes of these patients that could also contribute to the clinical features.