Introduction

The inositol lipid-specific phospholipase C (PLC) isozymes are key signaling proteins that play critical roles in transducing signals from hormones, growth factors, neurotransmitters, and many extracellular stimuli (Berridge and Irvine 1984; Exton 1996; Balla 2013). The PLCs selectively hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP₂) into inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) (Nishizuka 1984; Majerus et al. 1986). PIP2 functions as a membrane anchor for numerous proteins and affects membrane dynamics and ion transport (Hilgemann et al. 2001; Hilgemann 2007; Suh and Hille 2008). The two products, IP₃ and DAG, are important intracellular second messengers involved in Ca²⁺ signaling regulation and protein kinase C signaling activation, respectively (Nishizuka 1992; Berridge 1993). Hence, PLC orchestrates diverse cellular processes and behaviors, including cell growth, differentiation, migration, and cell death (Yang et al. 2012; Cocco et al. 2015; Gomes et al. 2021; Asano et al. 2022). There are at least thirteen PLC isozymes grouped in 6 classes (β, δ, ε, γ, η, ζ) in mammals with similar enzymatic function, but each PLC has its own spectrum of activators, expression pattern, and subcellular distribution (Suh et al. 2008; Kadamur and Ross 2013; Katan and Cockcroft 2020).

PLCG1 [MIM: 172420] encodes the PLCγ1 isozyme. PLCγ1 can be directly activated by receptor tyrosine kinases (RTKs) as well as cytosolic receptors coupled to tyrosine kinases (Gresset et al. 2012). Upon tyrosine phosphorylation, PLCγ1 undergoes conformational changes that release its autoinhibition upon which it associates with the plasma membrane to bind and hydrolyze its substrates (Gresset et al. 2010; Hajicek et al. 2019; Nosbisch et al. 2022). There is a second PLCγ isozyme, PLCγ2, encoded by PLCG2 [MIM: 600220]. Although these two isozymes have similar protein structure and activation mechanism, they are differentially expressed and regulated, and play non-redundant roles (Homma et al. 1989; Regunathan et al. 2006). PLCG2 is mostly expressed in cells of the hematopoietic system and mainly functions in immune response, causing human diseases associated with immune disorders (Yu et al. 2005; Ombrello et al. 2012; Zhou et al. 2012; Neves et al. 2018; Baysac et al. 2024). However, PLCG1 is ubiquitously expressed and is enriched in the central nervous system (CNS) (Consortium 2015). Plcg1 is essential in mice, and a null allele causes embryonic lethality with developmental defects in the vascular, neuronal, and immune system (Ji et al. 1997; Liao et al. 2002). PLCG1 has emerged as a possible driver for cell proliferation, and increased expression levels of PLCG1 have been observed in breast cancer, colon cancer, and squamous cell carcinoma (Arteaga et al. 1991; Noh et al. 1994; Park et al. 1994; Xie et al. 2010). Moreover, hyperactive somatic mutations of PLCG1 have been observed in angiosarcomas and T cell leukemia/lymphomas (Behjati et al. 2014; Kunze et al. 2014; Vaque et al. 2014; Kataoka et al. 2015). However, the genotype-phenotype association of germline PLCG1 variants has yet to be explored.

Here, we reported seven individuals carrying heterozygous variants in PLCG1 (GenBank: NM_002660.3) who exhibit partially overlapping clinical features including hearing impairment (5/7), ocular pathology (4/7), cardiac defects (3/6), abnormal brain MRI findings (2/3), and immunological issues with diverse manifestations (5/7). Utilizing Drosophila to model the variants in vivo, we provide evidence that the missense PLCG1 variants are toxic and affect protein function to varying degrees. We argue that these variants contribute to the clinical symptoms observed in the affected individuals.

Results

Individuals with heterozygous missense variants in PLCG1 exhibit hearing impairment, cardiac defects, ocular pathology, and immune dysregulation

Seven individuals with heterozygous missense variants in PLCG1 were recruited trough the Undiagnosed Diseases Network (UDN) (Splinter et al. 2018) (Individuals 1-2) and GeneMatcher (Sobreira et al. 2015) (Individuals 3-7). Individuals 1-3 are de novo cases from unrelated families: Individual 1 [c.3056A>G, p.(Asp1019Gly)], Individual 2 [c.1139A>G, p.(His380Arg)] and Individual 3 [c.3494A>G, p.(Asp1165Gly)]. Individuals 4-7 are from the same family, and all carry the PLCG1 variant [c.1789C>T p.(Leu597Phe)]. The phenotypes of the individuals partially overlap but show a spectrum of clinical manifestations. [As per medRxiv policy, the whole and detailed case history for the probands have been removed. To obtain more detailed information, please contact the authors]

The missense PLCG1 variants affect conserved protein domains and are predicted to be deleterious

PLCG1 is predicted to be tolerant to loss-of-function alleles with a pLI score (Lek et al. 2016) of 0.16, suggesting that loss of one copy of the gene is unlikely to cause haploinsufficiency in humans, consistent with the presence of many protein truncating variants in gnomAD (Karczewski et al. 2020). However, the missense constraint Z score (Lek et al. 2016) of PLCG1 is 3.69, suggesting that it is intolerant to missense variants. Consistently, all variants are located within regions or stretches depleted in missense variants according to scores such as regional missense constraint (RMC) (Chao et al. 2024) or missense tolerance ratio (MTR) (Sun et al. 2024). In addition, the prediction based on the DOMINO algorithm indicates that PLCG1 variants are likely to have a dominant effect (Quinodoz et al. 2017). Several other in-silico pathogenicity predictions also suggest that these variants are likely to be pathogenic (Table S1) based on MARRVEL (Wang et al. 2017).

The four variants identified from the affected individuals map to different conserved domains of PLCγ1, and each variant affects an amino acid residue that is conserved from flies to humans (Figure 1A and 1B). The p.(Asp1019Gly) and p.(His380Arg) variants map to the catalytic core domains (X and Y regions, respectively), the p.(Asp1165Gly) variant is in the C-terminal C2 domain and the p.(Leu597Phe) variant is in the nSH2 domain. The latter is part of the PLCγ-specific regulatory array composed of a split PH domain (sPH), two Src homology 2 (nSH2 and cSH2) domains and a Src homology 3 (SH3) domain. PLCγ1 also contains other conserved domains including an N-terminal pleckstrin homology (PH) domain and four EF hand motifs.

Figure 1.

The small wing (sl) is the fly ortholog of human PLCG1

Flies have three genes encoding PLC isozymes (Table S2). Among them, small wing (sl) is predicted to be the ortholog of PLCG1 with a DIOPT (DRSC Integrative Ortholog Prediction Tool) score of 17/18 (DIOPT version 9.0) (Hu et al. 2021). The encoded proteins share 39% identity and 57% similarity and are composed of similar conserved domains (Figure 1A). The sl gene is also predicted to be the ortholog of PLCG2 with a DIOPT score of 12/18. These data suggest that sl corresponds to two human genes encoding the PLCγ isozymes. To obtain information about the nature of the PLCG1 variants, we utilize Drosophila to model them in vivo using the binary GAL4 system (Brand and Perrimon 1993). We generated transgenic flies carrying the UAS-human PLCG1 cDNAs for both the reference (UAS-PLCG1^Reference) and the variants (UAS-PLCG1^D1019G, UAS-PLCG1^H380R, UAS-PLCG1^D1165G, and UAS-PLCG1^L597F). Given the high level of protein sequence homology and the conservation of the affected amino acids (Figure 1B), we also generated transgenic flies for the reference and analogous variants in the fly sl cDNA, namely UAS-sl^WT and UAS-sl^variants (UAS-sl^D1041G, UAS-sl^H384R, UAS-sl^D1184G, and UAS-sl^L630F).

In Drosophila, sl is on the X chromosome, and several alleles of sl have been isolated or previously generated, including sl², sl^KO and sl^T2A(Figure 1C). sl² carries a 13bp deletion in the third exon that leads to a frameshift and early stop gain (Thackeray et al. 1998). sl² is a strong loss-of-function allele that causes small wing size, ectopic wing veins and extra R7 photoreceptors (Thackeray et al. 1998). sl^KO was generated by CRISPR-mediated genomic editing that removes nearly the entire gene (Trivedi et al. 2020). sl^T2A allele was generated by inserting an FRT-SA-T2A-GAL4-polyA-FRT cassette as an artificial exon into the first coding intron of sl (Figure 1C) (Diao et al. 2015; Lee et al. 2018). The polyA arrests transcription, and sl^T2A is a strong loss-of-function allele (Figure S1A). The T2A viral sequence triggers ribosomal skipping and leads to the production of GAL4 proteins (Donnelly et al. 2001; Diao and White 2012) that are expressed in the proper spatial and temporal pattern of sl. This allows us to assess the expression pattern of sl by driving the expression of a UAS-fluorescent protein (Lee et al. 2018), or to assess the function of variants by expressing the human UAS-reference/variant cDNAs (Huang et al. 2022a; Huang et al. 2022b; Lu et al. 2022a; Lu et al. 2022b; Ma et al. 2023; Pan et al. 2023). In addition, the cassette is flanked by two FRT sites and can therefore be excised from the cells that express the gene in the presence of UAS-Flippase to revert the mutant phenotypes (Figure 1C) (Lee et al. 2018).

We first assessed the expression pattern of sl by driving UAS-mCherry.nls (an mCherry that localizes to nuclei) with sl^T2A. sl is expressed in the 3^rd larval wing discs and eye discs (Figure 2A), consistent with the loss-of-function phenotypes observed in the wings and eyes (Thackeray et al. 1998). The expression pattern of sl in the wing discs is not homogenous. Higher expression levels are observed in the anterior compartment and along both the anterior/posterior and dorsal/ventral compartment boundaries (Figure 2A). The hemizygous sl^T2A/Y male flies and the trans-heterozygous sl^T2A/sl² or sl^T2A/sl^KOfemale flies show reduced wing size and ectopic wing veins (Figure 2B and Figure S1B), as well as additional photoreceptors in the eye (Figure 2C and Figure S1C). These phenotypes can be rescued by UAS-Flippase or by introducing a genomic rescue construct (Dp(1;3)DC313 (Venken et al. 2010), Figure 1C) that covers the sl locus (Figure 2B and Figure 2C). These data show that all the observed phenotypes in sl^T2A mutants can be attributed to the loss of sl.

Figure 2.

The sl gene is expressed in the fly CNS and loss of sl causes longevity and locomotion defects

Given that human PLCG1 is highly expressed in the central nervous system (CNS) (Consortium 2015) and that the affected individuals present with neurologic phenotypes including hearing or vision deficits (Table 1), we investigated the expression pattern and the cell type specificity of sl in the CNS of flies. sl is expressed in the larval CNS as well as the adult brain, and co-staining with the pan-neuronal marker Elav (Robinow and White 1991) and glial marker Repo (Sepp et al. 2001) show that sl is expressed in many neurons and glia cells in the CNS (Figure 3A). We therefore assessed the longevity and climbing of sl^T2A flies. Compared to the wild-type w¹¹¹⁸ flies, sl^T2A/Y hemizygous mutant flies show a shortened lifespan and a progressively reduced climbing ability. These phenotypes can be rescued by expression of the wild-type sl cDNA(sl^T2A/Y; UAS-sl^WT) (Figure 3B).

Figure 3.

Functional assays in flies indicate that the PLCG1 variants are toxic

To assess the impact of the variants, we expressed the sl variant cDNAs in the sl^T2A/Y hemizygous mutant males(sl^T2A/Y; UAS-sl^variants) and compared their rescue ability with the wild-type sl(sl^T2A/Y; UAS-sl^WT). As shown in Figure 4A (middle panel), the sl^T2A/Y mutant flies (or the ones expressing a UAS-Empty control construct) have a slightly reduced eclosion rate, but expression of the sl^WT cDNA fully rescues the percentage of eclosing progeny as measured by the Mendelian ratio. In contrast, expression of sl^L630F (sl^T2A/Y; UAS-sl^L630F) reduced the percentage of hemizygous male progeny from the expected 25% to approximately 17%, while expression of sl^H384R causes a severe reduction in the number of eclosing flies, with only a few escapers (sl^T2A/Y; UAS-sl^H384R). Expression of the sl^D1041G or sl^D1184G leads to 100% lethality. These data clearly indicate that these variants are toxic but at different levels.

Figure 4.

Since the sl^T2A/Y; UAS-cDNA hemizygous males lack the endogenous sl⁺, we tested sl^T2A/yw; UAS-cDNA heterozygous female flies that carry a copy of wild-type sl⁺ while simultaneously expressing UAS-cDNAs driven by the sl^T2A driver in the cells that endogenously express sl (Figure 4A, right panel). The eclosion rates of heterozygous female progeny expressing sl variants were significantly reduced compared to those expressing sl^WT. Expression of sl^H384R or sl^L630F in the heterozygous progeny reduced the expected 25% proportion to approximately 10% and 20%, respectively, whereas expression of sl^D1041G or sl^D1184Gresulted in complete lethality in heterozygous flies. These results suggest that the missense variants exert a dominant toxic effect. Additionally, we observed that the toxicity may have both developmental and acute effects in adults, with varying severity among the different variants (Figure S2), indicating that sl function is required in adult flies, implying that PLCG1 variants may cause long-term deficits in affected individuals.

To compare the sl and PLCG1 associated phenotypes, we conducted similar assays using human PLCG1 cDNAs (Figure 4B). Expression of PLCG1^Reference in the sl^T2A/Y mutant flies (sl^T2A/Y; UAS-PLCG1^Reference) reduces viability by 80%, and expression of the other PLCγ coding gene, PLCG2, is also toxic and causes similar viability reduction compared to PLCG1^Reference. This suggests that expression of human PLCγ genes is toxic in flies. This toxicity appears to be associated with expression level (Figure S3), and the survivals of sl^T2A/Y; UAS-PLCG1^Reference did not show rescue of the loss-of-function phenotypes in the wings or eyes (Figure S4). Expression of PLCG1^H380Ror PLCG1^L597F in the sl^T2A/Y mutant flies (sl^T2A/Y; UAS-PLCG1^H380Ror sl^T2A/Y; UAS-PLCG1^L597F) leads to a significant but very modest increase in lethality when compared to PLCG1^Reference, whereas expression of PLCG1^D1019G or PLCG1^D1165Gresults in 100% lethality (Figure 4B, left panel). When the reference and variants are assayed in the presence of a wild type copy of sl⁺, the heterozygous female progeny expressing the reference cDNA of PLCG1 or PLCG2 exhibited normal eclosion rate, as did the ones expressing PLCG1^H380R or PLCG1^L597F, suggesting that the presence of a wild-type copy of sl⁺ combined with the reduced expression levels (typically 50% due to dosage compensation for the cells on X chromosome) mask some of the potential toxicity. However, expression of PLCG1^D1019G or PLCG1^D1165Gstill resulted in complete lethality in the females (Figure 4B, right panel). In summary, expression of the PLCG1 variants and the corresponding fly sl variants exhibit greater toxicity than the reference or wild-type proteins with varying degrees of severity, suggesting that the variants are likely to be gain-of-function or neomorphic alleles. Among them, the PLCG1^D1019G and sl^D1041G, as well as PLCG1^D1165G and sl^D1184G are very strong toxic alleles whereas PLCG1^H380R, PLCG1^L597F, and their fly analogues are mild variants.

The p.Asp1019Gly and p.Asp1165Gly variants are hyperactive

To assess whether the variants act as gain-of-function alleles that enhance the enzymatic activity of the PLCγ1 isozyme, we tested them using a Ca²D reporter assay. Since one of the products of the PLCγ1 isozyme, IPD, binds to receptors on the endoplasmic reticulum to trigger Ca²D release (Foskett et al. 2007), intracellular Ca²D levels can serve as a proxy of the PLCγ1 enzymatic activity. We expressed the CaLexA (calcium-dependent nuclear import of LexA) reporter (Masuyama et al. 2012) in the wing disc pouch region using a specific GAL4 driver (nub-GAL4 > UAS-CaLexA.GFP) while simultaneously expressing UAS-PLCG1 cDNAs. We first assessed three control variants: PLCG1^H380A, PLCG1^D1165H, and PLCG1^S1021F. Substitution of His380 with Ala (H380A) has been reported to suppress PIP₂ hydrolysis and IP₃ production (Smith et al. 1994; Wada et al. 2022), acting as an enzymatic-dead loss-of-function allele. On the other hand, the p.Asp1165His (D1165H) variant was previously identified as a strong gain-of-function somatic variant in adult T cell leukemia/lymphoma (Kataoka et al. 2015; Hajicek et al. 2019; Siraliev-Perez et al. 2022), and has been documented to cause a dramatic increase in phospholipase activity in vitro (Hajicek et al. 2019; Siraliev-Perez et al. 2022). The p.Ser1021Phe variant was reported recently in a de novo case and was characterized as a gain-of-function germline variant (Tao et al. 2023). As shown in Figure S5A, the GFP signal of the CaLexA.GFP reporter was low in wing discs expressing PLCG1^H380A, whereas the signal was significantly enhanced in those expressing PLCG1^D1165H or PLCG1^S1021F, showing that this is a robust assay for detecting increased enzymatic activity. We next tested the variants of the affected individuals. As shown in Figure 5A, expression of PLCG1^Reference did not induce obvious GFP signals, suggesting that the protein is not enzymatically active, possibly because of autoinhibition. Similarly, expression of PLCG1^H380R or PLCG1^L597F did not significantly alter the GFP signal, suggesting that they are not constitutively active. However, expression of PLCG1^D1019G or PLCG1^D1165G markedly increased the GFP signal, similar to the PLCG1^D1165Hand PLCG1^S1021F positive controls (Figure 5A and S4A). The same observations were made with the fly sl variants (Figure S5B). These results indicate that the PLCG1^D1019G and PLCG1^D1165G variants are hyperactive, whereas the PLCG1^H380R and PLCG1^L597F variants are not hyperactive based on this assay.

Figure 5.

The PLCG1 variants affect size and morphology of wings and eyes

To further assess the impact of the PLCG1 variants on normal development, we analyzed the morphology of the adult wings upon wing-specific expression of PLCG1 or sl cDNAs (nub-GAL4 > UAS-cDNAs). Interestingly, ectopic expression of either PLCG1^Reference or sl^WT in the wing disc leads to a ∼10% reduction in adult wing size when compared to the UAS-Empty control (Figure S6A). This observation, together with the reduced wing size seen in the loss-of-function context (Figures 2B), suggests that both reduced and elevated levels of PLCγ1 can impair wing growth. This implies a dosage-dependent regulation on wing growth by the PLCγ1 isozymes, while the underlying mechanism is unknown. Additionally, as shown in Figure 5B and S6B, approximately 10% of the wings expressing PLCG1^Reference exhibit notching along the wing margin, a phenotype not observed in wings expressing sl^WT. Expression of PLCG1^H380R or PLCG1^L597F caused notched wings in approximately 18% and 23% of the flies, respectively (Figure S6B), whereas expression of PLCG1^D1019G or PLCG1^D1165G results in severe wing phenotypes characterized by notched wing margins, fused/thickened veins and reduced wing sizes with >95% penetrance (Figure 5B). Notably, expression of fly sl^variantscould lead to similar morphological defects as their corresponding human variants, arguing that these wing phenotypes are due to alterations of PLCG1 or Sl protein function (Figure 5B and S6C).

We also assessed the effect of expression of human PLCG1 on eye development using the eyeless-GAL4 (ey-GAL4). Expression of PLCG1^Referenceor PLCG1^H380R in fly eyes leads to a mild reduction in eye size when compared to UAS-Empty control (Figure 5C and S6D). However, expression of PLCG1^L597F results in rough eyes that are reduced in size whereas overexpression of PLCG1^D1019G or PLCG1^D1165Gleads to a more severe eye phenotype (Figure 5C and S6D). In summary, the eye data are consistent with the wing data, showing that PLCG1^D1019G and PLCG1^D1165G are more toxic than PLCG1^Reference. On the other hand, the toxicity of PLCG1^H380R and PLCG1^L597Fare stronger than the PLCG1^Reference but not as severe as PLCG1^D1019G and PLCG1^D1165G. Interestingly, the morphological defects in wings or eyes caused by ectopic expression of PLCG1 cDNAs correlate with the expression level (Figure S6E), but do not directly correlate with the phospholipase enzymatic activity. For example, expression of PLCG1^S1021F does not cause obvious morphological defects when compared to PLCG1^Reference (Figure S6B and S6F), even though PLCG1^S1021Fis hyperactive and induces significantly elevated intracellular Ca²⁺ in the CaLexA reporter assay (Figure S5A).

Discussion

Here we report seven individuals who carry heterozygous missense variants in PLCG1 which encodes the phospholipase C γ1 isozyme. The individuals present with partially overlapping clinical features including hearing impairment, eye abnormality, heart defects and immune phenotypes. We show that the fly ortholog, small wing (sl), is widely expressed in wings and eyes, as well as in the central nervous system. Consistent with its expression pattern, we report that sl not only regulates wing and eye development, as previously documented, but also plays critical roles in the nervous system and affects locomotion and longevity. Furthermore, we assessed the function of the variants in the context of the human and fly cDNAs and show that their expression induces variable levels of toxicity when compared to the reference PLCG1 or wild-type sl. Two of the variants are clearly hyperactive, and all the variants exhibit neomorphic effects (discussed in Supplemental Figure Notes). These observations show that the variants impair the normal function in vivo and suggest that they contribute to the symptoms observed in the affected individuals. Similarly to inborn error caused by the paralogous PLCG2 (Baysac et al. 2024), germline variants in PLCG1 can be pathogenic and dominant by different mechanisms.

Structural analysis of the PLCG1 variants

Previously, studies based on biochemical assays and protein structures provided insights into how the variants studied here may affect the enzymatic activity of PLCγ1 (the protein structure of full-length rat Plcg1 is shown in Figure 6A). In its basal state, the PLCγ-specific regulatory array (sPH-nSH2-cSH2-SH3) forms autoinhibitory interfaces with the catalytic domains. Upon activation by the RTKs through binding with nSH2, PLCγ1 is phosphorylated, which induces the dissociation of the inhibitory cSH2 domain from the C2 domain. This triggers conformational rearrangements, allowing the enzyme to associate with the membrane and to expose the catalytic domains to allow hydrolysis of PIP2 (Gresset et al. 2010; Hajicek et al. 2019; Liu et al. 2020; Le Huray et al. 2022; Nosbisch et al. 2022). As shown in Figure 6A, the proband-associated variants map to conserved domains of the protein, either within the catalytic domains or at intramolecular and intermolecular interfaces. The p.Asp1019Gly and p.Asp1165Gly variants impact key residues involved in autoinhibition, leading to increased enzymatic activity. Specifically, the p.Asp1019Gly variant affects a conserved residue within the hydrophobic ridge of the Y box (Figure 6B), which is important for interaction with the sPH domain. This interaction is critical for the autoinhibition by blocking the membrane engagement of the catalytic core domain prior to enzymatic activation (Ellis et al. 1998; Hajicek et al. 2019). Notably, a substitution at the same position (Asp1019Lys, D1019K) has been demonstrated to enhance basal phospholipase activity in vitro (Hajicek et al. 2013), supporting its regulatory importance. Similarly, another hotspot somatic variant, p.Ser345Phe, located in the corresponding hydrophobic ridge within the X box, is also hyperactive (Vaque et al. 2014; Manso et al. 2015). On the other hand, the p.Asp1165Gly variant affects a residue situated within a loop of the C2 domain (Figure 6C). The Asp1165 residue plays a key role in stabilizing the interaction between the cSH2 domain and the C2 domain to maintain the autoinhibited state (DeBell et al. 2007). As mentioned above, the somatic variant p.Asp1165His leads to significantly elevated phospholipase activity in vitro (Liu et al. 2020; Siraliev-Perez et al. 2022), and results in severe phenotypes in vivo (Figure 4B, S5E and S5F). Molecular dynamics simulation data consistently indicate that autoinhibition is likely disrupted by the p.Asp1019Gly and p.Asp1165Gly variants (Figure S7A). In contrast, the p.His380Arg variant impacts the His380 residue within the X box, situated near a Ca²⁺ ion in the catalytic core (Figure 6D). His380 plays a role in coordination of the phosphate group at the 1-position of IP₃ (Le Huray et al. 2022). While this residue may not be key to autoinhibition, it is important for the phospholipase activity. Substitution of His380 with Phe or Ala (H380F, H380A) have been reported to suppress PIP₂ hydrolysis and IP₃ production (Smith et al. 1994; Wada et al. 2022). Hence, substitution of the His380 with Arg in p.His380Arg variant may create a more basic environment, impacting the lipase activity. On the other hand, the p.Leu597Phe variant affects a residue within the nSH2 domain, which is part of the PLCγ-specific regulatory array (Figure 6E). The nSH2 domain mediates interactions with phosphorylated tyrosine residues on RTKs to initiate activation (Bae et al. 2009). Leu597 is located near the phosphotyrosine-binding pocket and this variant may therefore alter receptor specificity or induce novel protein interactions. Additionally, we utilized the DDMut platform (Zhou et al. 2023) to predict protein stability and folding of the variants, which are discussed in Figure S7B. In summary, our in vivo data are consistent with previous reports and in silico analyses, showing that the affected amino acids map to critical residues and strengthening the conclusion that the variants are pathogenic and likely impact the protein function through distinct mechanisms.

Figure 6.

The PLCG1 variants affect protein function to varying degrees and are associated with variable clinical manifestations

To better assess the genotype-phenotype relationship of the variants, we summarize the clinical features of affected individuals in Table 1, and the phenotypic effects observed in fly assays in Table S3. The p.(Asp1019Gly) (carried by Individual 1) and p.(Asp1165Gly) (carried by Individual 3) variants and their corresponding fly variants induce severe phenotypes across all assays performed. Individuals 1 and 3 share several obvious clinical features including hearing loss and heart septal defect. In contrast, the p.(His380Arg) and p.(Leu597Phe) variants cause mild or partially penetrant phenotypes across the different fly assays. Individual 2 who carries the p.(His380Arg) variant does not exhibit hearing impairment or heart defects observed in Individuals 1 and 3, but has eye malformations and neuroinflammation features that are shared with individuals 1 and 3, although the ocular and immunological defects manifest differently among individuals. Interestingly, individuals 4-7 are from the same family and all carry the p.(Leu597Phe) variant but also differ in their phenotypes, yet all share some clinical features with Individuals 1-3 (Table 1).

The heterogeneity in clinical manifestations may be influenced by additional genetic variants (see Table 1 legend) and environmental factors. Additionally, the variable expressivity observed in carriers of the same variant may be explained by allelic expression bias through autosomal random monoallelic expression (aRME) (Reinius and Sandberg 2015), a phenomenon that is thought to be common among carriers of genetic defects associated with inborn errors of immunity (IEIs). Indeed, these conditions often exhibit non-Mendelian segregation patterns and variable clinical features (Stewart et al., 2025). Moreover, the PLCγ1 isozyme is an integral component of multiple signaling pathways, and the consequences of its dysregulation are likely to be context dependent. It is likely that different PLCG1 variants impact distinct cellular processes across various tissues and cell types, resulting in a spectrum of pathological changes. In summary, the symptoms observed in affected individuals appear to correlate, to some extent, with the severity of the variants as indicated by fly assays. However, the penetrance and expressivity of these phenotypes will require further investigation to better understand the genotype-phenotype associations of PLCG1 variants.

Acknowledgements

We thank the individuals and families for their participation in this study. We thank Ms. Hongling Pan for helping in the generation of transgenic fly lines. We thank Dr. Meisheng Ma for suggestions about protein structure interpretation. We thank Dr. Zhandong Liu for providing computational resources for performing the molecular dynamics simulations. We thank the Bloomington Drosophila Stock Center (BDSC) for providing stocks.

Additional information

Data and code availability

This study did not generate datasets. All reagents developed in this study are available upon request.

Funding Statement

This work was supported by the Huffington Foundation; the Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, and the Undiagnosed Diseases Network funded by grants from the National Institutes of Health (U01 HG010233, U01 NS134355, U01 HG007709, U01 HG007942). Sequence data analysis was supported by the University of Washington Center for Rare Disease Research (UW-CRDR; U01 HG011744, UM1 HG006493, U24 HG011746). The content of this paper is the sole responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. H.J.B. receives support from the NIH Common Fund through the Office of Strategic Coordination/Office of the NIH Director and the NINDS (U54 NS093793) as well as ORIP (R24 OD022005 and R24 OD031447). Confocal microscopy was performed in the BCM IDDRC Neurovisualization Core, supported by the NICHD (U54 HD083092).

Author contributions

Conceptualization: M.M., Y.Z., S.R.L., I.A.G., P.P., J.R., S.M., H.J.B.; Data curation: M.M., Y.Z., S.R.L., I.A.G., E.B., P.P., S.M., H.J.B.; Formal analysis: M.M., Y.Z., S.L., J.A.R., A.A., M.D., X.L., P.B.; Funding acquisition: UDN, H.J.B.; Investigation: M.M., Y.Z., S.L., M.D., D.L., M.E.; Resources: M.M., Y.Z., D.L., K.W.C., B.L.C., B.L.S., M.G., M.B., S.Y., M.F.W., S.R.L., I.A.G., P.P., J.R., S.M., H.J.B.; Supervision: H.J.B.; Visualization: M.M., Y.Z., M.D., S.L., X.P. L.D.; Writing-original draft: M.M., Y.Z.; Writing-review & editing: M.M., Y.Z., M.D., X.L., X.P., S.L., D.L., D.D., J.A.R., J.C., W-L.C., E.B., S.R.L., I.A.G., P.P., S.M., H.J.B..

The Undiagnosed Diseases Network Consortia (Version 3.31.25)

Alyssa A. Tran, Arjun Tarakad, Ashok Balasubramanyam, Brendan H. Lee, Carlos A. Bacino, Daryl A. Scott, Elaine Seto, Gary D. Clark, Hongzheng Dai, Hsiao-Tuan Chao, Ivan Chinn, James P. Orengo, Jennifer E. Posey, Jill A. Rosenfeld, Kim Worley, Lindsay C. Burrage, Lisa T. Emrick, Lorraine Potocki, Monika Weisz Hubshman, Richard A. Lewis, Ronit Marom, Seema R. Lalani, Shamika Ketkar, Tiphanie P. Vogel, William J. Craigen, Jared Sninsky, Lauren Blieden, Sandesh Nagamani, Hugo J. Bellen, Michael F. Wangler, Oguz Kanca, Shinya Yamamoto, Christine M. Eng, Patricia A. Ward, Pengfei Liu, Adeline Vanderver, Cara Skraban, Edward Behrens, Gonench Kilich, Kathleen Sullivan, Kelly Hassey, Ramakrishnan Rajagopalan, Rebecca Ganetzky, Vishnu Cuddapah, Anna Raper, Daniel J. Rader, Giorgio Sirugo, Vaidehi Jobanputra, Allyn McConkie-Rosell, Kelly Schoch, Mohamad Mikati, Nicole M. Walley, Rebecca C. Spillmann, Vandana Shashi, Alan H. Beggs, Calum A. MacRae, David A. Sweetser, Deepak A. Rao, Edwin K. Silverman, Elizabeth L. Fieg, Frances High, Gerard T. Berry, Ingrid A. Holm, J. Carl Pallais, Joan M. Stoler, Joseph Loscalzo, Lance H. Rodan, Laurel A. Cobban, Lauren C. Briere, Matthew Coggins, Melissa Walker, Richard L. Maas, Susan Korrick, Jessica Douglas, Cecilia Esteves, Emily Glanton, Isaac S. Kohane, Kimberly LeBlanc, Rachel Mahoney, Shamil R. Sunyaev, Shilpa N. Kobren, Brett H. Graham, Erin Conboy, Francesco Vetrini, Kayla M. Treat, Khurram Liaqat, Lili Mantcheva, Stephanie M. Ware, Breanna Mitchell, Brendan C. Lanpher, Devin Oglesbee, Eric Klee, Filippo Pinto e Vairo, Ian R. Lanza, Kahlen Darr, Lindsay Mulvihill, Lisa Schimmenti, Queenie Tan, Surendra Dasari, Abdul Elkadri, Brett Bordini, Donald Basel, James Verbsky, Julie McCarrier, Michael Muriello, Michael Zimmermann, Adriana Rebelo, Carson A. Smith, Deborah Barbouth, Guney Bademci, Joanna M. Gonzalez, Kumarie Latchman, LéShon Peart, Mustafa Tekin, Nicholas Borja, Stephan Zuchner, Stephanie Bivona, Willa Thorson, Herman Taylor, Rakale C. Quarells, Ayuko Iverson, Bruce Gelb, Charlotte Cunningham-Rundles, Eric Gayle, Joanna Jen, Louise Bier, Mafalda Barbosa, Manisha Balwani, Mariya Shadrina, Rachel Evard, Saskia Shuman, Susan Shin, Andrea Gropman, Barbara N. Pusey Swerdzewski, Camilo Toro, Colleen E. Wahl, Donna Novacic, Ellen F. Macnamara, John J. Mulvihill, Maria T. Acosta, Precilla D’Souza, Valerie V. Maduro, Ben Afzali, Ben Solomon, Cynthia J. Tifft, David R. Adams, Elizabeth A. Burke, Francis Rossignol, Heidi Wood, Jiayu Fu, Joie Davis, Leoyklang Petcharet, Lynne A. Wolfe, Margaret Delgado, Marie Morimoto, Marla Sabaii, MayChristine V. Malicdan, Neil Hanchard, Orpa Jean-Marie, Wendy Introne, William A. Gahl, Yan Huang, Andrew Stergachis, Danny Miller, Elisabeth Rosenthal, Elizabeth Blue, Elsa Balton, Emily Shelkowitz, Eric Allenspach, Fuki M. Hisama, Gail P. Jarvik, Ghayda Mirzaa, Ian Glass, Kathleen A. Leppig, Katrina Dipple, Mark Wener, Martha Horike-Pyne, Michael Bamshad, Peter Byers, Runjun Kumar, Seth Perlman, Sirisak Chanprasert, Virginia Sybert, Wendy Raskind, Nitsuh K. Dargie, Chun-Hung Chan, Dr. Francisco Bustos velasq, Isum Ward, Jason Schend, Jennifer Morgan, Megan Bell, Miranda Leitheiser, Mohamad Saifeddine, Paul Berger, Rachel Li, Taylor Beagle, Alexander Miller, Beatriz Anguiano, Beth A. Martin, Brianna Tucker, Chloe M. Reuter, Devon Bonner, Elijah Kravets, Hector Rodrigo Mendez, Holly K. Tabor, Jacinda B. Sampson, Jason Hom, Jennefer N. Kohler, Jennifer Schymick, John E. Gorzynski, Jonathan A. Bernstein, Kevin S. Smith, Laura Keehan, Laurens Wiel, Matthew T. Wheeler, Meghan C. Halley, Mia Levanto, Page C. Goddard, Paul G. Fisher, Rachel A. Ungar, Raquel L. Alvarez, Sara Emami, Shruti Marwaha, Stephen B Montgomery, Suha Bachir, Tanner D Jensen, Taylor Maurer, Terra R. Coakley, Euan A. Ashley, Ali Al-Beshri, Anna Hurst, Brandon M Wilk, Bruce Korf, Elizabeth A Worthey, Kaitlin Callaway, Martin Rodriguez, Tammi Skelton, Tarun KK Mamidi, Andrew B. Crouse, Jordan Whitlock, Mariko Nakano-Okuno, Matthew Might, William E. Byrd, Albert R. La Spada, Changrui Xiao, Elizabeth C. Chao, Eric Vilain, Jose Abdenur, Kirsten Blanco, Maija-Rikka Steenari, Rebekah Barrick, Richard Chang, Sanaz Attaripour, Suzanne Sandmeyer, Tahseen Mozaffar, Alden Huang, Andres Vargas, Bianca E. Russell, Brent L. Fogel, Esteban C. Dell’Angelica, George Carvalho, Julian A. Martínez-Agosto, Layal F. Abi Farraj, Manish J. Butte, Martin G. Martin, Naghmeh Dorrani, Neil H. Parker, Rosario I. Corona, Stanley F. Nelson, Yigit Karasozen, Aaron Quinlan, Alistair Ward, Ashley Andrews, Corrine K. Welt, Dave Viskochil, Erin E. Baldwin, John Carey, Justin Alvey, Laura Pace, Lorenzo Botto, Nicola Longo, Paolo Moretti, Rebecca Overbury, Russell Butterfield, Steven Boyden, Thomas J. Nicholas, Matt Velinder, Gabor Marth, Pinar Bayrak-Toydemir, Rong Mao, Monte Westerfield, Brian Corner, John A. Phillips III, Kimberly Ezell, Lynette Rives, Rizwan Hamid, Serena Neumann, Ashley McMinn, Joy D. Cogan, Thomas Cassini, Alex Paul, Dana Kiley, Daniel Wegner, Erin McRoy, Jennifer Wambach, Kathy Sisco, Patricia Dickson, F. Sessions Cole, Dustin Baldridge, Jimann Shin, Lilianna Solnica-Krezel, Stephen C. Pak, Timothy Schedl, Allen Bale, Carol Oladele, Caroline Hendry, Emily Wang, Hua Xu, Hui Zhang, Lauren Jeffries, María José Ortuño Romero, Mark Gerstein, Michele Spencer-Manzon, Monkol Lek, Nada Derar, Odelya Kaufman, Shrikant Mane, Teodoro Jerves Serrano, Vasilis Vasiliou, Winston Halstead, Yong-Hui Jiang

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