Genetics and Genomics

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

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Houston, TX 77030, USA
Current affiliation: State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty of Medicine and Life Sciences, Xiamen University, Xiamen 361102, China
Division of Medical Genetics, Department of Medicine, University of Washington School of Medicine, Seattle, WA 98195, USA
Current affiliation: Children’s National Medical Center and George Washington University, Washington DC 20010, USA
Laboratory of Regulatory & Functional Genomics, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Foggia 71013, Italy
Department of Molecular Medicine & Medical Biotechnology, University of Naples Federico II, Naples 80131, Italy
Department of Interdisciplinary Medicine, University of Bari “Aldo Moro”, Bari 70121, Italy
Department of Neurology, Baylor College of Medicine, Houston, TX 77030, USA
Division of Genetic Medicine, Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98195, USA
Brotman Baty Institute, Seattle, WA 98195, USA
Institute for Public Health Genetics, University of Washington, Seattle, WA 98195, USA
Medical Genetics Unit, Hospital of Perugia, Perugia 06129, Italy

https://doi.org/10.7554/eLife.95887.1

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Figures and data

The PLCG1 homolog, sl is conserved in Drosophila
(A) Schematic of human PLCG1 and fly Sl protein domains and positions of the variants identified in the probands. Domain prediction is based on annotation from NCBI. Alignment of human PLCG1 and the homologous proteins. The variants are marked with boxes.
(B) All the variants affect conserved amino acids (labeled in red). Isoforms for alignment: Human PLCG1 NP_877963.1; 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 92kb genomic rescue (GR) construct.
Loss-of-function alleles of sl including sl² (13bp deletion ⁵²), sl^KO (CRISPR-mediated deletion of the gene span ⁵³), and sl^T2A (T2A cassette inserted in the first intron, ⁵⁴) are indicated. The T2A cassette in sl^T2Ais flanked by FRT sites and can be excised by Flippase to revert loss-of-function phenotypes. GAL4 expression in sl^T2A under the control by sl endogenous promoter can be used to assess sl gene expression pattern by crossing with a UAS-mCherry.nls reporter line, or be used to model patient variants in vivo by crossing with human PLCG1 cDNAs or corresponding fly sl cDNAs. The primer pair used for real-time PCR is indicated.

sl^T2A is a loss-of-function allele causing phenotypes in wing and eye
(A) sl expression in wing and eye. Expression of UAS-mcherry.nls (red) was driven by sl^T2Ato label the nuclei of the cells that expressed sl. sl is expressed in the 3rd instar larval wing disc (left) and eye disc (right). Higher magnification image of the wing disc pouch region indicated by dashed rectangle is shown. The posterior/anterior and dorsal/ventral compartment boundaries are indicated by dashed lines in yellow. Scale bars, 100μm
(B) sl^T2A causes wing size reduction and ectopic veins (arrowhead) 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, 0.5mm. 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) sl^T2A 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, 10μm. 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.

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 sl^T2A-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, 20μm in the magnified images, 50μm in other images.
(B) Loss of sl causes behavioral defects in longevity and locomotion. sl^T2A hemizygous flies have a shorter lifespan than w¹¹¹⁸ control flies. The median life span of sl^T2A and w¹¹¹⁸ flies is 40 days and 62 days respectively. The short lifespan in sl^T2A flies can be rescued by a UAS transgene that expresses wild-type sl cDNA (sl^WT). Fly locomotion ability was assessed by climbing assay (see methods). sl^T2A flies at the age of 7 days show reduced locomotion ability and become almost immotile at the age of 35 days. The reduced locomotion ability in sl^T2A flies can be fully rescued by sl^WT. For lifespan assay, Longrank test, ****p<0.0001. For climbing assay, each dots represents one vial containing 18-22 flies for test. Unpaired t test, ****p<0.0001.

The variant cDNAs are toxic
(A) Summary of the viability of expressing sl or PLCG1 cDNAs in sl^T2A mutant flies. Cross strategy: heterozygous sl^T2A 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 the hemizygous sl^T2A male progeny expressing different UAS-cDNA constructs were calculated. The expected Mendelian ratio is 0.25 (indicated by the green line in the graph). The dark gray columns represent human cDNAs, the light gray columns represent fly cDNAs, and the clear columns represent controls (y w or UAS-Empty). Each dot represents one independent replicate. Unpaired t test, *p<0.05, ****p < 0.0001, mean ± SEM.
(B) Summary of the viability of ubiquitous overexpression of sl or PLCG1 cDNAs driven by Tub-GAL4. Expression of wild-type fly sl cDNA shows no impact on viability whereas expression of the sl variants reduces viability. Expressing human PLCG1 cDNAs at 29°C is toxic in flies. Expression of PLCG1^Referenceor PLCG1^H380R causes semi-lethality, whereas expression of PLCG1^D1019G or PLCG1^D1165G is 100% lethal at early larval stage.
(C) Summary of the viability of ubiquitous overexpression PLCG1 cDNAs using a strong driver Tub-GAL4 (left panel) or a weaker driver da-GAL4 (right panel) at different temperatures. The expression level rises with higher temperatures and decreases with lower temperatures, and the toxic impact on viability correlates with the expression level. Expression of PLCG1^D1019Gor PLCG1^D1165G causes more severe toxicity than expression of PLCG1^Reference or PLCG1^H380R.

The PLCG1 p.Asp1019Gly and p.Asp1165Gly variants are likely to be gain-of-function alleles
(A) Wing-specific overexpression of PLCG1^D1019G or PLCG1^D1165G causes severe wing morphology defects including notched margin (arrows) and fused/thickened veins (arrowheads), fully penetrant (the penetration ratio is indicated). Note that overexpression of the fly sl^D1041G (corresponding to the human PLCG1^D1019G) shows similar phenotypes. Scale bars, 0.5mm.
(B) Wing-specific overexpression of an established hyperactive variant PLCG1^D1165H causes wing morphology phenotypes similar to, but more severe than that of PLCG1^D1019G or PLCG1^D1165G. Scale bars, 0.5mm.

PLCG1 variants affect important residues
(A) 3D structure of full-length rat Plcg1 (PDB code: 6PBC; rat Plcg1 shares 96.9% 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 three residues affected by the variants are 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. Notably, the Ser1021 residue is outside of the hydrophobic ridge and shows no predicted interaction with the residues in the sPH domain.
(C) Enlarged views 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. Structural analysis was performed via UCSF Chimera ⁸³.

sl^T2A is a loss-of-function allele causing wing and eye phenotypes
(A) Relative sl mRNA expression are <5% and <1% in sl^T2A and sl^KO mutant larvae when compared to controls (w¹¹¹⁸). The primers used for real-time PCR are shown in Figure 2A. Each dot represents a replicate per genotype. Unpaired t test, ***p<0.001.
(B) Representative images showing that sl^T2A/sl² trans-heterozygous mutant flies have smaller wing and ectopic veins (indicated by arrow). Scale bars, 0.5mm.
(C) Representative images showing that sl^T2A/sl^KO trans-heterozygous mutant flies have extra photoreceptors (indicated by arrows). The schematic of the section of an 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, 10μm.

Ubiquitous overexpressing of PLCG1^D1019G or PLCG1^D1165G driven by da-GAL4 causes pupal size reduction
Representative images showing the pupal size of animals overexpressing PLCG1 cDNAs by da-GAL4 at 25°C. Overexpression of PLCG1^D1019G or PLCG1^D1165G causes pupae size reductions when compared to PLCG1^Reference. Overexpression of PLCG1^H380Ris indistinguishable from PLCG1^Reference. Scale bars, 0.5mm.

Wing-specific overexpression of PLCG1^Reference or sl^WT causes wing size reduction, while overexpressing the PLCG1^H380R or sl^H384Rfurther reduces wing size
Representative images showing that wing-specific overexpression of PLCG1^Referenceor sl^WT causes ∼15% wing size reduction compared to the UAS-Empty control construct. Overexpression of PLCG1^H380R or sl^H384R causes a further ∼5% size reduction. Each dot represents one measured adult wing. Unpaired t test, ***p<0.001, ****p < 0.0001, mean ± SEM. Scale bars, 0.5mm

Eye-specific overexpression of human PLCG1 leads to size reduction in the eyes, and expression of the PLCG1^D1019G or PLCG1^D1165G causes more severe phenotype than expression of _PLCG1Reference _{or PLCG1}H380R
Representative images showing that eye-specific overexpression of PLCG1^Reference or PLCG1^H380R causes ∼15% eye size reduction compared to the UAS-Empty control construct, whereas overexpression of PLCG1^D1019G or PLCG1^D1165Gcauses a ∼30% size reduction. Overexpression of PLCG1^D1165H causes lethality. Each dot represents one measured adult eye. Unpaired t test, **p<0.01, ****p < 0.0001, ns: not significant, mean ± SEM. Scale bars, 100μm.

Ectopic expression of PLCG1^S1021F causes indistinguishable phenotypes in the wings or eyes compared to PLCG1^Reference
Representative images showing the adult wings and eyes with PLCG1^S1021F overexpression using nub-GAL4 or ey-GAL4, respectively. There is no obvious morphological difference compared to PLCG1^Reference. Scale bars, 0.5mm in the wing images, 100μm in the eye images.

Expressing human PLCG1 does not rescue wing and eye phenotypes associated with sl^T2A
Representative images showing the adult wings (upper panel) and the photoreceptors (lower panel) expressing PLCG1^Reference or sl^WT. Expression of sl^WT 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 PLCG1^Referenceor UAS-Empty shows no rescue. Scale bars, 0.5mm for the wing images and 10μm 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) are shown. Each dot represents one wing or one retina sample, respectively. Unpaired t test, ***p<0.001, *p<0.05. ns: not significant.

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