A syndromic neurodevelopmental disorder caused by rare variants in PPFIA3

Abstract

PPFIA3 encodes the protein-tyrosine phosphatase, receptor-type, F-polypeptide-interacting-protein-alpha-3 (PPFIA3), which is a member of the LAR-protein-tyrosine phosphatase-interacting-protein (liprin) family involved in synapse formation and function, synaptic vesicle transport, and presynaptic active zone assembly. The protein structure and function are evolutionarily well conserved, but human diseases related to PPFIA3 dysfunction are not yet reported in OMIM. Here, we report 20 individuals with rare PPFIA3 variants (19 heterozygous and 1 compound heterozygous) presenting with developmental delay, intellectual disability, hypotonia, dysmorphisms, microcephaly or macrocephaly, autistic features, and epilepsy with reduced penetrance. Seventeen unique PPFIA3 variants were detected in 18 families. To determine the pathogenicity of PPFIA3 variants in vivo, we generated transgenic fruit flies producing either human wild-type (WT) PPFIA3 or five missense variants using GAL4-UAS targeted gene expression systems. In the fly overexpression assays, we found that the PPFIA3 variants in the region encoding the N-terminal coiled-coil domain exhibited stronger phenotypes compared to those affecting the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin-α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 function is partially conserved in the fly. However, two of the tested variants failed to rescue the lethality at the larval stage and one variant failed to rescue lethality at the adult stage. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant-negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

Keywords: Mendelian phenotypes; active zone protein; fruit flies; neurodevelopmental disorder; synaptic protein.

Figure 1

Variant location and images of individuals with PPFIA3 variants (A) Location of PPFIA3 variants in the genomic locus corresponding to the exon-intron structure. Number of individuals with the rare PPFIA3 variant shown in the y axis. (B) Location of PPFIA3 variants in the corresponding protein domains. Number of individuals with the variant shown in the y axis. The fruit fly ortholog Liprin-α shows 48% identity and 62% similarity with the human PPFIA3. Sterile alpha motif, SAM. (C) Images of individuals with heterozygous or compound heterozygous PPFIA3 variants. The three individuals shown have dysmorphic features such as wide mouth, widely spaced teeth, prominent forehead, and hypotonic facies.

Figure 2

Molecular modeling of PPFIA3 missense variants and protein levels associated with PPFIA3 variants (A and B) PPFIA3 missense variants are modeled in PyMol (version 2.5.2) with GenBank: NP_003651.1. Human PPFIA3 WT residues are modeled in gray with coiled coils displayed in black and affected residues highlighted in orange. Local polar contacts (orange dashed lines) and residue interactions (highlighted in pink) are displayed before and after mutagenesis for (Bi) p.Arg415Trp, (Bii) p.Arg429Trp, (Ci) p.Arg784Trp, and (Cii) p.Ser906Leu. (C) (i) Western blot from PPFIA3 WT and variants show higher levels of HA in PPFIA3 p.Arg39Cys compared to WT. (ii) Quantification of relative HA in 4–6 sets of biological replicates show higher level of HA in PPFIA3 p.Arg39Cys flies. Statistical analysis conducted with one-way ANOVA and Tukey’s post-hoc analysis. Data shown as mean ± SEM. Significance shown as ^∗∗∗p < 0.001. Non-significance shown as ns.

Figure 3

Actin-GAL4-mediated ubiquitous expression of PPFIA3 variants cause developmental and anatomical defects in fruit flies (A) Pupal lethality and eclosion defect associated with Actin-GAL4-mediated overexpression of PPFIA3 variants. (i) Images showing overexpression of PPFIA3 p.Arg39Cys cause pupal lethality and eclosion defect, and p.Ala315Ser and p.Arg415Trp cause eclosion defect compared to the PPFIA3 WT and UAS-empty control. Uneclosed flies from p.Arg39Cys, p.Ala315Ser, and p.Arg415Trp remain in the pupal case. PPFIA3 p.Trp546Cys and p.Arg784Trp overexpression does not cause a difference in pupal lethality and eclosion defect compared to PPFIA3 WT and UAS-empty control flies. Scale bar = 100 μm. (ii) Bar graphs showing the percentage of eclosed pupae (overexpression), eclosion defect, and pupal lethal. Statistical analysis conducted with one-way ANOVA and Tukey’s post-hoc analysis. Data shown as mean ± SEM with the sample size of total number of pupae in three sets. Significance shown as ^∗∗p < 0.01 and ^∗∗∗p < 0.001. Non-significance shown as ns. (B) Images of leg morphology associated with Actin-GAL4-mediated overexpression of PPFIA3 variants. (i) Empty control and PPFIA3 WT flies have typical legs with three segments. PPFIA3 p.Arg39Cys, p.Ala315Ser, and p.Arg415Trp result in pronounced leg segment developmental defects compared to PPFIA3 WT. Mild leg segmental developmental defects found with PPFIA3 p.Arg784Trp but not significant compared to PPFIA3 WT. No leg defects were found in PPFIA3 p.Trp546Cys flies. Scale bar = 100 μm. (ii) Bar graph showing the percentage of flies with abnormal leg morphology. Statistical analysis conducted with one-way ANOVA and Tukey’s post-hoc analysis. Data shown as mean ± SEM with the sample size of total number of adult flies in three sets. Significance shown as ^∗∗∗p < 0.001. Non-significance shown as ns.

Figure 4

elav-GAL4-mediated neuronal overexpression of PPFIA3 variants result in climbing defect, bang sensitivity, and neuromuscular junction (NMJ) bouton loss (A) elav-GAL4 mediated neuronal expression of PPFIA3 p.Arg39Cys, PPFIA3 p.Ala315Ser, PPFIA3 p.Arg415Trp, and PPFIA3 p.Arg784Trp result in impaired motor coordination on the climbing assay compared to PPFIA3 WT and empty control flies. Crosses were set and maintained at 25°C. Behavioral testing was conducted at 20°C–21°C with both sexes. (B) elav-GAL4-mediated neuronal expression of PPFIA3 p.Arg39Cys, PPFIA3 p.Ala315Ser, and PPFIA3 p.Arg415Trp have bang sensitivity with delayed recovery from vortexing compared to PPFIA3 WT and UAS-empty control flies. Crosses were set and maintained at 25°C. Behavioral test was conducted at 20°C–21°C with both sexes. (C) elav-GAL4-mediated neuronal overexpression of PPFIA3 variants result in NMJ bouton loss without a significant change in NMJ length. (i) Model depicting the method for visualizing the NMJ in fruit fly third-instar larva. (ii) Representative images of third-instar larval NMJs of each genotype including elav GAL4>UAS empty, elav GAL4>PPFIA3 WT, p.Arg39Cys, p.Arg415Trp, p.Trp546Cys, and p.Arg784Trp are shown. Horseradish peroxidase (HRP) is a pan-neuronal marker (green) and Brp (Bruchpilot) is an active zone marker (magenta). Scale bar is 24 μm. (iii) Quantification of total number of boutons in the muscle 6/7 (abdominal segment 3) NMJ show that PPFIA3 p.Arg39Cys and p.Arg415Trp result in bouton loss compared to PPFIA3 WT and empty control. In contrast, PPFIA3 p.Trp546Cys and p.Arg784Trp show no alteration in bouton numbers. (iv) Quantification of total NMJ length in each genotype is shown, and there is no significant difference between PPFIA3 WT, variants, and UAS-empty control. Crosses were set and maintained at 25°C. Statistical analysis conducted with one-way ANOVA and Tukey’s post-hoc analysis. Data shown as mean ± SEM with the sample size of total number of quantified NMJs shown above the bars. Significance shown as ^∗∗p < 0.01, ^∗∗∗p < 0.001. Non-significance shown as ns.

Figure 5

PPFIA3 WT partially rescues the fly Liprin-α LOF lethality (A) Human PPFIA3 WT in the background of fly Liprin-α LOF results in a partial rescue of embryonic lethality. (i) Crossing scheme to delete fly Liprin-α and express human PPFIA3 WT and variants. The scheme describes the rescue larvae selection strategy. Crosses were set and maintained at 20°C. (ii) Quantification of n = 3 sets per genotype showing % GFP-negative larvae (rescue larvae) that survive to the larval stage. PPFIA3 WT expression can partially rescue larval viability compared to empty control. PPFIA3 p.Arg39Cys and p.Arg415Trp show impaired ability to rescue larval viability. (B) Human PPFIA3 WT expression partially rescues the lethality in adult stage. (i) Representative illustration of the different stages of fruit fly development. (ii) Quantification of 3 sets of rescued larvae per genotype that survive to the adult stage. PPFIA3 WT expression can partially rescue adult viability compared to empty control. PPFIA3 p.Arg39Cys and p.Arg415Trp show impaired ability to rescue adult viability. (iii) Quantification of 1–3 sets of rescued larvae per genotype that survived after 48 h post-eclosion. For the empty control larvae, only one escaper rescue larvae survived to adult stage but died within 2 days post-eclosion. None of the PPFIA3 p.Arg39Cys rescue larvae survived to adult stage. Due to the lack of any PPFIA3 p.Arg39Cys rescue larvae surviving to the adult stage, this variant was not quantifiable for the adult survival phenotype. PPFIA3 p.Arg415Trp and PPFIA3 p.Arg784Trp show impaired ability to rescue adult viability compared to the PPFIA3 WT. Sample size is shown in Table S2. Statistical analysis with one-way ANOVA and Tukey’s post-hoc analysis. Data shown as mean ± SEM with the sample size of flies scored shown in (Table S2). Significance shown as ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Non-significance shown as ns.