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 synaptic vesicle transport and presynaptic active zone assembly. The protein structure and function are well conserved in both invertebrates and vertebrates, but human diseases related to PPFIA3 dysfunction are not yet known. Here, we report 14 individuals with rare mono-allelic PPFIA3 variants presenting with features including developmental delay, intellectual disability, hypotonia, autism, and epilepsy. To determine the pathogenicity of PPFIA3 variants in vivo, we generated transgenic fruit flies expressing either human PPFIA3 wildtype (WT) or variant protein using GAL4-UAS targeted gene expression systems. Ubiquitous expression with Actin-GAL4 showed that the PPFIA3 variants had variable penetrance of pupal lethality, eclosion defects, and anatomical leg defects. Neuronal expression with elav-GAL4 showed that the PPFIA3 variants had seizure-like behaviors, motor defects, and bouton loss at the 3rd instar larval neuromuscular junction (NMJ). Altogether, in the fly overexpression assays, we found that the PPFIA3 variants in the N-terminal coiled coil domain exhibited stronger phenotypes compared to those in 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 protein function is partially conserved in the fly. However, the PPFIA3 variants failed to rescue lethality. 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.

The Department of Molecular and Human Genetics at Baylor College of Medicine derives revenue from the clinical exome sequencing services offered at Baylor Genetics. JLM, MJGS, and RP are employees of GeneDx, LLC.

This study was funded by The Robert and Janice McNair Foundation Burroughs Wellcome Fund Child Neurology Foundation and Society The Gordan and Mary Cain Foundation Annie and Bob Graham The Elkins Foundation Mark A. Wallace Endowment Award Texas Childrens Hospital Jan and Dan Duncan Neurological Research Institute Eunice Kennedy Shriver NICHD of the NIH under Award Number P50HD103555 NIH Common Fund under Award Number U01HG007709

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The Institutional Review Board of Baylor College of Medicine gave ethical approval for this work.

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I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

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I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

All data produced in the present study are available upon reasonable request to the authors. The accession numbers for the variants reported to ClinVar are (1) I:1, ClinVar: SUB12878798; GenBank:NM_003660.4 (PPFIA3); c.115 C>T (p.Arg39Cys); (2) I:2, ClinVar: SUB12879592; GenBank:NM_003660.4 (PPFIA3); c.239 A>C (p.Gln80Pro); (3) I:3, ClinVar: SUB12866061; GenBank:NM_003660.4 (PPFIA3); c.240+1G>A; (4) I:4, ClinVar: SUB12866110; GenBank:NM_003660.4 (PPFIA3); c.240+1G>A; (5) I:6, ClinVar: SUB12879585; GenBank:NM_003660.4 (PPFIA3); c.1243 C>T (p.Arg415Trp); (6) I:7, ClinVar: SUB12690823; GenBank: NM_003660.4 (PPFIA3); c.1285C>T (p.Arg429Trp); (7) I:8, ClinVar: SUB12947536; GenBank:NM_003660.4 (PPFIA3); c.1492 C>T (p.Arg498Trp); (8) I:9, ClinVar: SUB12879586; GenBank:NM_003660.4 (PPFIA3); c.1638 G>T (p.Trp546Cys); (9) I:10, ClinVar: SUB12689969; GenBank: NM_003660.4 (PPFIA3); c.2350 C>T (p.Arg784Trp); (10) I:11 and I:12, ClinVar: SUB12879588; GenBank:NM_003660.4 (PPFIA3); c.2609T>A (p.Ile870Asn); (11) I:13, ClinVar: SCV003035511; GenBank: NM_003660.4 (PPFIA3); c.2717 C>T (p.Ser906Leu); (12) I:14, ClinVar: SCV003035512; GenBank: NM_003660.4 (PPFIA3); c.3307del (p.Glu1103Asnfs*8); (13) I:15, ClinVar: SUB12693099; GenBank: NM_003660.4 (PPFIA3); c.1675C>T (p.Arg559Trp)