Heterozygous variants in PLCG1 affect hearing, vision, cardiac, and immune function
- Department of Molecular and Human Genetics, Baylor College of Medicine, United States
- Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, United States
- Department of Neurology, Baylor College of Medicine, United States
- The Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, United States
- Division of Medical Genetics, Department of Medicine, University of Washington School of Medicine, United States
- Laboratory of Regulatory & Functional Genomics, Fondazione IRCCS Casa Sollievo della Sofferenza, Italy
- Department of Molecular Medicine & Medical Biotechnology, University of Naples Federico II, Italy
- Department of Interdisciplinary Medicine, University of Bari "Aldo Moro", Italy
- Department of Neurology, University of Washington and Seattle Children’s Hospital, United States
- SeqOIA Genomics Platform, Assistance Publique–Hôpitaux de Paris (AP-HP), France
- Division of Genetic Medicine, Department of Pediatrics, University of Washington School of Medicine, United States
- Brotman Baty Institute, United States
- Institute for Public Health Genetics, University of Washington, United States
- Medical Genetics and Rare Diseases Unit, Hospital of Perugia, Italy
- Laboratory of Human Genetics of Infectious Diseases, Imagine Institute, Necker Hospital for Sick Children, France
- Center for the Study of Immune Deficiencies, Necker-Enfants Malades Hospital, AP-HP Centre, University of Paris, France
- Genetics of Rare Ophthalmological, Auditory and Mitochondrial Disorders, Inserm UMR_S1163, Imagine Institute, France
- Reference Center for Genetic Deafness, Department of Genomic Medicine for Rare Diseases, Necker-Enfants Malades Hospital, AP-HP Centre, University of Paris, France
Figures
Figure 1 with 2 supplements
The PLCG1 ortholog is small wing (sl) in Drosophila.
(A) Schematic of human PLCG1 and fly Sl protein domains and positions of the variants identified in the affected individuals. Domain prediction is based on annotation from NCBI. (B) Alignment of protein domains near variants of PLCG1 and PLCG2 with PLCG1 from other species. The variants are marked with boxes. All the variants affect conserved amino acids (labeled in red). Isoforms for alignment: Human PLCG1 NP_877963.1; Human PLCG2 NP_002652.2; Mouse Plcg1 NP_067255.2; Zebrafish plcg1 NP_919388.1; Fly sl NP_476726.2. (C) Schematic of fly sl genomic span, transcript, alleles, and the 92 kb genomic rescue (GR) construct. Loss-of-function alleles of sl including sl2 (13 bp deletion, Thackeray et al., 1998), slKO (CRISPR-mediated deletion of the gene span; Trivedi et al., 2020), and slT2A (T2A cassette inserted in the first intron; Lee et al., 2018) are indicated. The T2A cassette in slT2A is flanked by FRT sites and can be excised by Flippase to revert loss-of-function phenotypes. GAL4 expression in slT2A is driven by the endogenous sl promoter, allowing assessment of sl gene expression pattern with a UAS-mCherry.nls reporter line. This system also allows in vivo modeling of proband-associated variants by crossing with human PLCG1 cDNAs or corresponding fly sl cDNAs. The primer pair used for real-time PCR is indicated.
Figure 1—figure supplement 1
Human PLC genes and orthologs in Drosophila.
In humans, at least 13 genes encoding Phospholipase C isozymes have been identified and classified into 6 protein families, whereas in Drosophila, 2 PLCβ genes and 1 PLCγ gene have been identified. The small wing (sl) gene is the fly ortholog of human PLCG1 and PLCG2.
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Figure 1—figure supplement 1—source data 1
Source data for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/95887/elife-95887-fig1-figsupp1-data1-v1.xls
Figure 1—figure supplement 2
Real-time PCR reveals that slT2A is a loss-of-function allele causing severely reduced mRNA expression of sl.
Relative sl mRNA expression levels are <5% and<1% in slT2A and slKO mutant larvae when compared to controls (w1118). The primers used for real-time PCR are shown in Figure 1C. Each dot represents a replicate per genotype. Unpaired t test, ***p<0.001, mean ± SEM.
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Figure 1—figure supplement 2—source data 1
Source data for Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/95887/elife-95887-fig1-figsupp2-data1-v1.xlsx
Figure 2 with 1 supplement
slT2A is a loss-of-function allele that affects fly wing and eye development.
(A) sl expression in wing and eye discs. Expression of UAS-mcherry.nls (red) was driven by slT2A to label the nuclei of the cells that expressed sl. sl is expressed in the 3rd instar larval wing disc (left) and eye disc (right). A higher magnification image of the wing disc pouch region indicated the by dashed rectangle is shown. The posterior/anterior and dorsal/ventral compartment boundaries are indicated by dashed lines in yellow. Scale bars, 100 μm. (B) slT2A cause a wing size reduction and ectopic veins (arrow) in hemizygous mutant male flies. The wing phenotypes can be rescued by introduction of a genomic rescue (GR) construct or the expression of Flippase. Scale bars, 0.5 mm. The quantification of adult wing size is shown in the right panel. Each dot represents the measurement of one adult wing sample. Unpaired t test, ∗∗∗∗p<0.0001, mean ± SEM. (C) slT2A causes extra photoreceptors (arrows) in the hemizygous mutant flies. The eye phenotype can be rescued by introduction of a genomic rescue (GR) construct. The photoreceptor rhabdomeres stain positive for phalloidin labeling F-actin. Scale bars, 10 μm. The quantification is shown in the right panel. Each dot represents the measurement of one retina sample. Unpaired t test, ****p<0.0001, mean ± SEM.
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Figure 2—source data 1
Source data for Figure 2B and C.
- https://cdn.elifesciences.org/articles/95887/elife-95887-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Trans-heterozygous sl mutant flies exhibit defects in wing and eye development.
(A) Representative images showing that slT2A/sl2 trans-heterozygous mutant flies have smaller wing and ectopic veins (indicated by arrow). Scale bars, 0.5 mm. (B) Representative images showing that slT2A/slKO trans-heterozygous mutant flies have extra photoreceptors (indicated by arrows). The schematic of the section of a normal ommatidia presenting seven photoreceptors (the R8 photoreceptor is not visible in such section) is shown. The photoreceptor rhabdomeres stain positive for phalloidin labeling F-actin. Scale bar, 10 μm.
Figure 3
sl is expressed in a subset of neurons and glia in the CNS, and loss of sl causes behavioral defects.
(A) Expression pattern of sl in the central nervous system observed by slT2A-driven expression of UAS-mCherry.nls reporter (red). In either larval or adult brain, sl is expressed in a subset of fly neurons and glia, which were labeled by pan-neuronal marker Elav (green, upper panel) and pan-glia marker Repo (green, lower panel). Higher magnification images of the regions indicated by dashed rectangles are shown. Scale bars, 20 μm in the magnified images, 50 μm in other images. (B) Loss of sl causes defects in longevity and locomotion. slT2A hemizygous flies have a shorter lifespan than w1118 control flies. The median lifespan of slT2A and w1118 flies is 40 days and 62 days, respectively. The shorter lifespan of slT2A flies can be rescued by a UAS transgene that expresses the wild-type sl cDNA (slWT). Fly locomotion was assessed by climbing assay (see Materials and methods). slT2A flies at the age of 7 days show reduced locomotion and become almost immotile at the age of 35 days. The reduced locomotion ability in slT2A flies can be fully rescued by slWT. For lifespan assay, Longrank test, ****p<0.0001; sample size n=114, 115, and 125 for w1118, slT2A, and slT2A>slWT flies, respectively. For climbing assay, each dot represents a measurement of one vial containing 17–22 flies for test. Unpaired t test, ****p<0.0001, mean ± SEM.
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Figure 3—source data 1
Source data for Figure 3B.
- https://cdn.elifesciences.org/articles/95887/elife-95887-fig3-data1-v1.xlsx
Figure 4 with 3 supplements
The human and corresponding fly variants are toxic when expressed in flies.
(A) Summary of the viability associated with expression of sl cDNAs in slT2A mutant or heterozygous flies. Cross strategy: heterozygous slT2A female flies were crossed to male flies carrying UAS-cDNAs or control (UAS-Empty) constructs, or crossed to the y w males as an extra control. The percentages of hemizygous slT2A/Y male progeny (red) or slT2A/yw heterozygous female progeny (blue) that express different UAS-cDNA constructs were calculated. The expected Mendelian ratio is 0.25 (indicated by the green line in the graph). The fly analogue variants of the proband-associated variants were tested. Each dot represents one independent replicate. Unpaired t test, ****p<0.0001, **p<0.01, *p<0.05, ns: not significant, mean ± SEM. (B) Summary of the viability associated with the expression of PLCG1 cDNAs in slT2A mutant (red, males) or heterozygous (blue, females) flies. The same cross strategy and progeny ratio measurement described in (A) was applied. The proband-associated variants, as well as three previously reported PLCG1 variants were assessed. We also included the PLCG2 reference cDNA. Each dot represents one independent replicate. Unpaired t test, **p<0.01, *p<0.05, ns: not significant, mean ± SEM.
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Figure 4—source data 1
Source data for Figure 4A and B.
- https://cdn.elifesciences.org/articles/95887/elife-95887-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Proband-associated variants exhibit deleterious impacts in adults.
(A) Cross strategy of the temperature shifting assay to express UAS-sl cDNAs only in the adult stages. GAL80ts is a temperature-sensitive inhibitor of GAL4, which allows temporal control of UAS- transgene expression by temperature manipulation. GAL80ts represses GAL4 activity at the permissive temperature (18°C) but becomes inactive at restrictive temperature (29°C), thereby allowing GAL4 to activate expression of UAS-transgenes. In this assay, Tub-GAL4, Tub-GAL80ts driver was used to express wild-type or variant sl cDNAs specifically in adult flies by shifting the temperature after eclosion. (B) Adult-stage expression of sl variants induces lifespan reduction. The adult flies with expression of slD1184G mostly die within 1 week after eclosion. The flies expressing slD1041G or slH384R have median lifespans of approximately 21 days and 26 days, respectively, both shorter than those expressing slWT, which have a median lifespan of 31 days. The sample size for each genotype is indicated (n). Longrank test, ****p<0.0001. (C) Adult-stage expression of slD1041G induces some locomotion defect. Flies aged to 20 days were assessed for locomotor ability using a climbing assay. The expression of slH384R did not induce significant difference compared to slWT. We did not test slD1184G as the flies are short-lived. Each dot represents the measurement of one fly. Unpaired t test, *p<0.05, ns: not significant, mean ± SEM. These results suggest that the two strong variants (slD1041G and slD1184G) contribute to both developmental and acute effects while the slH384R variant mainly contributes to developmental stages.
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1B and C.
- https://cdn.elifesciences.org/articles/95887/elife-95887-fig4-figsupp1-data1-v1.xlsx
Figure 4—figure supplement 2
The toxicity of human PLCG1 cDNAs in flies correlates with expression levels.
(A) Summary of the eclosion rate of animals expressing sl cDNAs in slT2A mutant (left panel) or heterozygous (right panel) flies at 22°C. The phenotypes are similar to, but slightly milder compared to the same assays performed at 25°C (Figure 4B). Expression of PLCG1D1019G or PLCG1D1165G caused lethality in both conditions, whereas expression of PLCG1Reference, PLCG1H380R, or PLCG1L597F led to reduced eclosion rate in mutant hemizygous but not in heterozygous flies. Each dot represents one independent replicate. Unpaired t test, ****p<0.0001, ***p<0.001, *p<0.05, ns: not significant, mean ± SEM. (B) Summary of the viability of expressing PLCG1Reference ubiquitously using Tub-GAL4. Expression levels of the UAS-PLCG1Reference transgenes can be manipulated at different temperatures. (C) Summary of the viability of expressing PLCG1 reference and variant cDNAs using a weak ubiquitous driver da-GAL4. Expression levels of the UAS- PLCG1Reference transgenes can be manipulated at different temperatures. (D) Representative images of the pupae of da-GAL4>UAS-PLCG1 cDNAs flies at 25°C. Expression of PLCG1D1019G or PLCG1D1165G caused reduced pupal size. Scale bars, 0.5mm.
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Figure 4—figure supplement 2—source data 1
Source data for Figure 4—figure supplement 2A–C.
- https://cdn.elifesciences.org/articles/95887/elife-95887-fig4-figsupp2-data1-v1.xlsx
Figure 4—figure supplement 3
Expressing human PLCG1 does not rescue wing or eye phenotypes associated with slT2A.
Representative images showing the adult wings (upper panel) and the photoreceptors (lower panel) expressing PLCG1Reference or slWT. Expression of slWT rescues the loss-of-function phenotypes including wing size reduction, ectopic veins (indicated by red arrows) and extra photoreceptors (indicated by yellow arrows), whereas expression of the PLCG1Reference or UAS-Empty shows no rescue. Scale bars, 0.5 mm for the wing images and 10 μm for the photoreceptor images. The photoreceptor rhabdomeres stain positive for phalloidin labeling F-actin. Quantification of the wing size (upper right panel) and the photoreceptors (lower right panel) is shown. Each dot represents a measurement of one wing or retina sample, respectively. Unpaired t test, ***p<0.001, *p<0.05. ns: not significant.
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Figure 4—figure supplement 3—source data 1
Source data for Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/95887/elife-95887-fig4-figsupp3-data1-v1.xlsx
Figure 5 with 2 supplements
Ectopic expression of PLCG1 variants causes variable phenotypes.
(A) The Ca2+ reporter CaLexA.GFP was expressed in the wing disc pouch, simultaneously with the PLCG1 cDNAs. Expression of PLCG1D1019G or PLCG1D1165G caused elevated CaLexA.GFP signal (green), indicating increased intracellular Ca2+ levels, indicating that these variants are hyperactive. Nuclei were labeled with DAPI (blue). Scale bars, 100 μm. (B) Representative images of the adult wing blades showing the morphological phenotypes caused by wing-specific expression of PLCG1 cDNAs. Expression of PLCG1D1019G or PLCG1D1165G caused severe wing morphology defects including notched margin (arrows) and fused/thickened veins (arrowheads). Expression of PLCG1L597F exhibited partial penetrance. Expression of PLCG1H380R exhibited very mild phenotypes, comparable to PLCG1Reference. Scale bars, 0.5 mm. (C) Representative images showing that eye-specific expression of PLCG1Reference or PLCG1H380R causes an ~15% eye size reduction compared to the UAS-Empty control construct, and expression of PLCG1L597F further reduced eye size. Expression of PLCG1D1019G or PLCG1D1165G causes a severe size reduction by ~30%. Scale bars, 100 μm.
Figure 5—figure supplement 1
Intracellular Ca2+ reporter assay suggests that the p.(Asp1019Gly) and p.(Asp1165Gly) are hyperactive variants.
(A) Three previously reported PLCG1 variants (PLCG1H380A, PLCG1D1165H, and PLCG1S1021F) were tested as controls. The Ca2+ reporter CaLexA.GFP was co-expressed with PLCG1 cDNAs in the wing disc pouch region. Expression of PLCG1D1165H or PLCG1S1021F caused elevated CaLexA.GFP signal (green), whereas expression of PLCG1H380A did not. Nuclei were labeled with DAPI (blue). Insets show merged images of DAPI and GFP channels. Scale bars, 100 μm. (B) The fly variants were analyzed using the CaLexA.GFP reporter. Expression of slD1041G or slD1184G caused elevated CaLexA.GFP signal (green), similar to the corresponding PLCG1 variants. Note that the wing discs expressing slD1184G are morphologically aberrant and have a diminished GFP signal. Nuclei were labeled with DAPI (blue). Insets show merged images of DAPI and GFP channels. Scale bars, 100 μm.
Figure 5—figure supplement 2
Wing and eye phenotypes associated with ectopic expression of PLCG1 variants.
(A) Quantification of the wing blade size in non-notched wings from the samples overexpressing PLCG1Reference or slWT. Wing-specific expression of PLCG1Reference or slWT caused an approximate 15% wing size reduction compared to the UAS-Empty control construct. Each dot represents one measured adult wing. Unpaired t test, ****p<0.0001, mean ± SEM. (B) Quantification of the percentage of adult wings with margin notching phenotype. Wing-specific expression of PLCG1Reference caused ~10% wings to display a notched margin. Expression of PLCG1H380R or PLCG1L597F caused approximately 18% and 23% of the wings to display notched margins, respectively. Expression of the enzymatic-dead PLCG1H380A caused less than 10% wings with notched margin, whereas expression of the hyperactive PLCG1S1021F is not significantly different from PLCG1Reference. Each dot represents one independent replicate. The total number of flies (N) was counted per genotype. Unpaired t test, ***p<0.001, **p<0.01, ns: not significant, mean ± SEM. (C) Representative images of adult wing blades showing morphological phenotypes caused by wing-specific expression of sl cDNAs. Expression of slD1041G caused severe wing defects including notched margins (arrows). Expression of slH384R or slL630F exhibited partial penetrance. Expression of slD1184G caused lethality before eclosion. Scale bars, 0.5 mm. (D) Quantification of eye size with expression of PLCG1 cDNAs. Eye size were normalized to the size of the ey-GAL4>UAS-Empty control. Each dot represents the measurement of one adult eye. Unpaired t test, ****p<0.0001, *p<0.05, ns: not significant, mean ± SEM. (E) Representative images showing that wing-specific expression of PLCG1D1165H caused similar but more severe morphological phenotypes compared to PLCG1D1019G or PLCG1D1165G. Expression levels of UAS-cDNA can be manipulated by raising the animals at different temperatures. Scale bars, 0.5 mm. (F) Representative images showing the phenotypes associated with ectopic expression of the control PLCG1 variants. Expression of PLCG1H380A or PLCG1S1021F did not induce obvious morphological phenotypes in adult wings or eyes compared to PLCG1Reference, whereas no eclosed adults were collected when overexpressing PLCG1D1165H at 29°C in these contexts. Scale bars, 0.5 mm for wing images, 100 μm for eye images.
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Figure 5—figure supplement 2—source data 1
Source data for Figure 5—figure supplement 2A–C.
- https://cdn.elifesciences.org/articles/95887/elife-95887-fig5-figsupp2-data1-v1.xlsx
Figure 6 with 1 supplement
PLCG1 variants affect important residues.
(A) 3D structure of full-length rat Plcg1 (rat Plcg1 shares 97% amino acid identity with human PLCG1). The conserved protein domains are labeled with different colors. Two major intracellular interfaces are circled by dashed lines: 1-The hydrophobic ridge between the sPH domain and the catalytic core (X-box and Y-box); and 2-The interface between the cSH2 domain and the C2 domain. The four amino acids affected by the variants are shown as bolded black and indicated by yellow balls. (B) Enlarged views of the Asp1019 residue within the autoinhibition interface between sPH domain and the Y box. The potential interactions with nearby residues are indicated. (C) Enlarged view of the Asp1165 residue within the autoinhibition interface between the cSH2 domain and the C2 domain. The potential interactions with nearby residues are indicated. (D) Enlarged view of the His380 residue within the X-box catalytic domain, in proximity to the Ca2+ cofactor. (E) Enlarged view of the Leu597 and nearby residues in the nSH2 domain. Structural analysis was performed via UCSF Chimera (Pettersen et al., 2004).
Figure 6—figure supplement 1
In silico analyses of PLCG1 variants.
(A) Molecular dynamics simulations for the p.(Asp1019Gly) and p.(Asp1165Gly) variants. These variants exhibit increased disorganization with a higher root mean square deviations (RMSD) compared to the reference PLCG, similar to the hyperactive p.(Asp1165His) variant. Color code: PLCG1 reference - red; p.(Asp1019Gly) - green; p.(Asp1165Gly) - cyan; p.(Asp1165His) - purple. (B) Prediction of the effects of PLCG1 variants on protein stability and folding using DDMut. DDMut is a platform designed to predict protein stability based on the Gibbs Free Energy change (ΔΔG), and larger ΔΔG indicates a more pronounced impact on protein stability. The protein structure of rat Plcg1 [Protein Data Bank (PDB) ID 6PBC] was used as a reference. The p.(Asp1019Gly), p.(Asp1165Gly), and p.(Asp1165His) variants are predicted to induce destabilizing, with ΔΔGStability wt->mt values of –0.91 kcal/mol, –1.54 kcal/mol, and –1.59 kcal/mol, respectively, suggesting impaired protein folding or altered conformational dynamics. These results are consistent with their proposed roles in disrupting autoinhibitory interactions (Hajicek et al., 2013; Siraliev-Perez et al., 2022). The ΔΔGStability wt->mt value for p.(His380Arg) and the enzymatically inactive p.(His380Arg) are predicted to be –0.22 kcal/mol and –0.13 kcal/mol, respectively. Given that the His380 residue’s influence on the phospholipase activity might not be through the autoinhibition mechanism, the impact of His380 residue variants on the protein folding and stability may not elucidate their effects on protein function. The ΔΔGStability wt->mt value for p.(Leu597Phe) is predicted to be –1.26 kcal/mol, which is not inconsistent with the potential impact of this variant on interaction between nSH2 domain and RTK activators. However, the p.(Ser1021Phe) variant has a predicted ΔΔGStability wt->mt value of 0.14 kcal/mol, indicating a slight stabilizing effect. These analyses indicate that the variants may act through various pathogenic mechanisms, but further investigations are needed.
Tables
Table 1
Clinical features of the affected individuals.
| Individual 1 | Individual 2 | Individual 3 | Individual 4 | Individual 5 | Individual 6 | Individual 7 | |
|---|---|---|---|---|---|---|---|
| PLCG1 variants | c.3056A>G | c.1139A>G | c.3494A>G | c.1798C>T | c.1798C>T | c.1798C>T | c.1798C>T |
| p.(Asp1019Gly) | p.(His380Arg) | p.(Asp1165Gly) | p.(Leu597Phe) | p.(Leu597Phe) | p.(Leu597Phe) | p.(Leu597Phe) | |
| Inheritance pattern | de novo, Sanger confirmed | de novo, Sanger confirmed | de novo, Sanger confirmed | Inherited | Inherited | Inherited | Unknown |
| Gender | Male | Female | Male | Female | Female | Male | Male |
| Age at evaluation | 18 years | 14 years | 9 years | 11 years | 42 years | 13 years | 66 years |
| Age of onset | Hearing loss since birth; other congenital anomalies recognized in infancy | Congenital microphthalmia/optic atrophy; episodic steroid-responsive inflammatory encephalomyelitis/optic neuritis from 11 years | Congenital hearing loss and heart defects | Congenital | 6 years | Childhood | Adulthood |
| Developmental milestones | Motor delays due to joint contractures; speech delay due to hearing loss | Developmental history limited; started walking at ~2 yo, articulation from 4 years | Normal | Motor delays | Normal | Normal | NA |
| Hearing | Mild hearing loss | Normal | Mild-moderate sensorineural hearing loss | Sensorineural bilateral, congenital progressive, profound hearing loss | Moderate bilateral sudden and progressive sensorineural hearing loss | Normal | Mild-moderate bilateral sensorineural hearing loss |
| Vision | Axenfeld anomaly bilaterally; posterior embryotoxon | Bilateral but variable congenital eye malformation | Normal | Unilateral posterior embryotoxon | High myopia | Normal | Normal |
| Heart | Cardiac septal defects (closed spontaneously) | Normal | Ventricular septal defect; atrial septal defect | Normal | Ventricular septal defect | Normal | NA |
| Brain MRI abnormality | Stable mild diffuse cerebral and cerebellar vermian volume loss, stable multifocal gliosis within the supratentorial white matter | Relapsing steroid-responsive inflammatory encephalomyelitis and progressive symmetrical white matter changes with swelling, and persistent diffuse T2 hyperintensities (deep and periventricular white matter), and bilateral frontoparietal lobe confluent hyperintensities | Normal | NA | NA | NA | NA |
| Immunological issues | No concerns reported | Symmetric steroid-responsive neuroinflammation | Lymphocytopenia (T lymphocytes), frequent infections during the first year of life | Episodes of joint inflammation, tarsal synovitis, recurrent upper respiratory and lung infections, and inflammatory lymphadenopathy Routine immunological evaluations revealed no biological abnormalities | Mild B lymphopenia, IgG2 severe deficit, splenectomy, post-vaccination sepsis septicemia, several autoimmune clinical manifestations, ITP (immune thrombocytopenic purpura) | Normal lymphocytes, IgA and IgG2 mild deficits; frequent oropharynx and lung infections | No concerns reported |
| Skin disorders | Multiple lentigines, keratosis pilaris | Striae seen over lower abdomen and bilateral inner thighs, possibly secondary to steroid use | Thin skin, prominent veins of the scalp | Absent | Absent | Absent | Absent |
| Joint | Joint stiffness/contractures; bilateral coxa profunda; trigger finger, cubitus valgus | Normal | Normal | Articular inflammations, tarsus synovitis episodes | Normal | Normal | NA |
| Dysmorphisms | Relative macrocephaly | Absent | Relative macrocephaly, facial dysmorphism | Absent | Absent | Absent | Absent |
| Short stature | Absent | Absent | Yes | Absent | Absent | Absent | Absent |
| Other potential variants | Intragenic PSD3 duplication, paternally inherited | ERAP2 and SEMA3G (compound heterozygous variants for both) | de novo heterozygous missense variant in PKP2 | No | No | No | No |
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Individual 1 carries an intragenic duplication in PSD3. PSD3 has not been associated with a Mendelian disorder but is potentially associated with an autosomal dominant arthrogryposis (Bayram et al., 2016). Hence, it may underlie the joint defects observed in individual 1.
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Individual 2 has compound heterozygous missense variants in ERAP2 and SEMA3G. ERAP2 [MIM: 609497] has not been associated with a Mendelian disorder. It encodes an ER-residential metalloaminopeptidase that functions in the major histocompatibility class I antigen presentation pathway. Some variants in ERAP2 are associated with a susceptibility to autoimmune diseases such as ankylosing spondylitis and Crohn’s disease (Franke et al., 2010; Cortes et al., 2013; Ebrazeh et al., 2021; Venema et al., 2024). Given that individual 2 exhibits neuroinflammation and encephalitis, these phenotypes may be associated with the ERAP2 variants. SEMA3G (Semaphorin 3G) has not been associated with a Mendelian disorder. However, a homozygous missense variant in SEMAG3 was observed in two affected siblings from a consanguineous family. The siblings exhibited dysmorphic features as well as developmental delay (Oleari et al., 2021).
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Individual 3 carries a de novo missense variant in PKP2 [MIM: 602861]. PKP2 encodes Pakophilin-2 and has been associated with dominant arrhythmogenic right ventricular dysplasia 9 [MIM: 609040] (Gerull et al., 2004; Dalal et al., 2006; Hakui et al., 2022). However, this individual was born with septal defects.
Table 2
Pathogenicity prediction of the proband variants.
| Individual 1 | Individual 2 | Individual 3 | Individual 4–7 | |
|---|---|---|---|---|
| PLCG1 variants (NM_002660.2) | c.3056A>G (p.Asp1019Gly) | c.1139A>G (p.His380Arg) | c.3494A>G (p.Asp1165Gly) | c.1789C>T (p.Leu597Phe) |
| CADD | 34 | 26.3 | 34 | 25.7 |
| M-CAP | Damaging, 0.7070 | Damaging, 0.8303 | Damaging, 0.7607 | Damaging, 0.5872 |
| PolyPhen2 hDiv (rare allele) | Probably Damaging, 0.9120 | Probably Damaging, 0.7456 | Probably Damaging, 0.9120 | Probably Damaging, 0.9058 |
| PolyPhen2 hVar (Mendelian Disease) | Probably Damaging, 0.8948 | Probably Damaging, 0.6982 | Probably Damaging, 0.9756 | Probably Damaging, 0.9737 |
| Mutation Taster | Disease Causing | Disease Causing | Disease Causing | Disease Causing |
| Count in gnomAD | Absent | Absent | Absent | Absent |
Table 3
Summary of the phenotypes observed in fly assays.
| 25°C | 29°C | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Lethality when expressed in slT2A/Y mutant | Lethality when expressed in slT2A/y w heterozygous | Ca2+ activity | Wing morphology when overexpressed | Eye morphology when overexpressed | |||||
| PLCG1 variants | Human variants | Fly variants | Human variants | Fly variants | Human variants | Fly variants | Human variants | Fly variants | Human variants |
| Reference | ++ | - | - | - | - | - | + | + | + |
| H380R | +++ | +++ | - | ++ | - | - | ++ | ++ | + |
| D1019G | 100% lethal | 100% lethal | 100% lethal | 100% lethal | + | + | ++++ | ++++ | +++ |
| D1165G | 100% lethal | 100% lethal | 100% lethal | 100% lethal | + | + | ++++ | lethal | +++ |
| L597F | +++ | ++ | - | + | - | - | +++ | +++ | ++ |
| H380A | +++ | NA | - | NA | - | NA | (+) | NA | - |
| D1165H | 100% lethal | NA | 100% lethal | NA | + | NA | lethal | NA | lethal |
| S1021F | ++ | NA | - | NA | + | NA | + | NA | - |
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‘-’: no obvious phenotypes observed.
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‘+’: phenotypes observed, the number of ‘+’ corresponds to the severity of the observed phenotype.
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NA: Not Available.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/95887/elife-95887-mdarchecklist1-v1.docx
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Supplementary file 1
Primers used in this study.
- https://cdn.elifesciences.org/articles/95887/elife-95887-supp1-v1.xlsx
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Heterozygous variants in PLCG1 affect hearing, vision, cardiac, and immune function
eLife 13:RP95887.
https://doi.org/10.7554/eLife.95887.3
