Bi-allelic loss-of-function variants in PPFIBP1 cause a neurodevelopmental disorder with microcephaly, epilepsy and periventricular calcifications

PPFIBP1 encodes for the liprin-{beta}1 protein which has been shown to play a role in neuronal outgrowth and synapse formation in Drosophila melanogaster. By exome sequencing, we detected nine ultra-rare homozygous loss-of-function variants in 14 individuals from 10 unrelated families. The individuals presented with moderate to profound developmental delay, often refractory early-onset epilepsy and progressive microcephaly. Further common clinical findings included muscular hypertonia, spasticity, failure to thrive and short stature, feeding difficulties, impaired hearing and vision, and congenital heart defects. Neuroimaging revealed abnormalities of brain morphology with leukoencephalopathy, cortical abnormalities, and intracranial periventricular calcifications as major features. In a fetus with intracranial calcifications, we identified a rare homozygous missense variant that by structural analysis was predicted to disturb the topology of the SAM-domain region that is essential for protein-protein interaction. For further insight in the effects of PPFIBP1 loss-of-function, we performed automated behavioural phenotyping of a Caenorhabditis elegans PPFIBP1/hlb-1 knockout model which revealed defects in spontaneous and light-induced behaviour and confirmed resistance to the acetylcholinesterase inhibitor aldicarb suggesting a defect in the neuronal presynaptic zone. In conclusion, we present bi-allelic loss-of-function variants in PPFIBP1 as a novel cause of an autosomal recessive neurodevelopmental disorder.

1, also known as liprin-β1. Liprin-β1 belongs to the liprin-protein family whose members are terminal sterile alpha motifs (SAM domains) that form multiple protein binding surfaces and 1 0 7 allow for protein-protein interaction. [1][2][3] In mammals, the liprin family comprises four liprin-α 1 0 8 proteins (liprin-α1-4) and two liprin-β proteins (liprin-β1 and -β2). Liprin-β1 has the ability to in synapse formation, synaptic signaling and axonal transport processes via the assembly of 1 1 3 large protein complexes. 5,6 Although yet to be confirmed, it has been suggested, that liprin-β1 1 1 4 could play a role in the regulation of liprin-α mediated protein assemblies. 1,6,7 In line with this 1 1 5 is the observation that liprin-β1 forms a ternary complex with liprin-α2 and CASK, 3 a 1 1 6 presynaptic scaffolding protein essential for neurodevelopment. 8,9 A previous knock-out 1 1 7 model of the PPFIBP1 homolog hlb-1 in C. elegans showed abnormal locomotion behavior. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 Here we describe a cohort of 14 individuals with a neurodevelopmental disorder from ten 1 2 8 unrelated families harboring homozygous loss-of-function (LoF) variants and a fetus with a 1 2 9 missense variant in PPFIBP1. The study was approved by the ethics committee of the University of Leipzig (402/16-ek). Written informed consent for molecular testing and permission for publication of the data was  All individuals were ascertained in the context of local diagnostic protocols followed by  , 4, 5, 6, 7, 8, 9, 10 and 11. Family 3 was recently published as part of a larger cohort of 1 4 2 individuals with congenital microcephaly. 12 In the context of our study we describe the 1 4 3 phenotype in detail. Phenotypic and genotypic information were obtained from the referring 1 4 4 collaborators using a standardized questionnaire. Trio exome sequencing (ES) and trio genome sequencing were performed for the affected 1 4 8 individuals and the parents in the families 1, 2, 5 and 11 and in family 7, respectively.

4 9
. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 6, 2022. Pharyngeal pumps per minute (ppm) of C. elegans strains were determined by counting 2 2 4 grinder movements over a 15 second period by eye using a stereomicroscope 28 , n = 120 2 2 5 worms per strain. Grinder movements of a single worm were counted three times and the 2 2 6 results recorded as an average of these values. Statistical differences in ppm between N2 2 2 7 reference strain and hlb-1(syb4896) were calculated using block permutation t-tests, using n = 2 2 8 10 3 permutations. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 6, 2022. All individuals share a core phenotype of global developmental delay / intellectual disability 2 3 3 (GDD/ID) and epilepsy. 13 were affected by profound or severe GDD/ID (13/14). They had 2 3 4 not acquired speech (13/14) and showed impaired motor development (13/14). Most of them 2 3 5 never achieved gross motor milestones such as sitting and walking except for individual 6-1 2 3 6 who was able to sit independently at the age of (removed due to medRxivpolicy) and 2 3 7 individual 7 who could stand and walk. Individual 1 presented with moderate ID, developed 2 3 8 expressive language skills and had a normal motor development. All individuals were affected 2 3 9 by epilepsy: most commonly with focal seizures (9/14) including cases of impaired awareness (1/14) and focal to bilateral tonic-clonic (1/14) seizures. Furthermore, generalized onset 2 4 1 seizures occurred in six of the individuals (6/14). Epileptic spasms were described in half of 2 4 2 the cohort (7/14). Other reported seizure types included tonic (3/14) and myoclonic seizures 2 4 3 (6/14). The median age of seizure onset was at two months with a range from the first day of and with VPA and Diazepam, respectively. Individual 9 was reported to have had a seizure-2 5 3 free period. Electroencephalography (EEG) was performed in 12 individuals (Table S4). EEG 2 5 4 findings included focal (3/12) or multifocal (5/12) interictal epileptiform discharges. In one 2 5 5 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. ;

9 3
Calcifications mostly appeared in a scattered pattern with periventricular localisation (8/8) but 2 9 4 also the basal ganglia (4/8), centra semiovale (2/8) and internal capsule (1/8) were affected 2 9 5 ( Figure 1C). Furthermore, CT scan also showed ventriculomegaly in individual 5-2 who did 2 9 6 not have MRI. The fetus (individual 11) showed severe intrauterine growth retardation and microcephaly 3 0 0 during pregnancy and the pregnancy was terminated in the 25 th gestational week. Autopsy 3 0 1 confirmed length and weight below -2 SD and an occipitofrontal circumference below -4 SD. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

2 1
In family 1, a homozygous splice-site variant c.1146+1G>A, p.? affecting the consensus 5'-3 2 2 splice site of exon 13 was identified. Multiple in silico tools consistently predict a loss of the 3 2 3 splice site (Table S2). This could lead to out-of-frame exon skipping or to intron retention. 30,31 3 2 4 Thus, the mRNA resulting from this allele is likely to encode a truncated protein as well. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.04.22273309 doi: medRxiv preprint tools consistently predicted a damaging effect of the variant (Table S3)  All of the identified variants are very rare in the general population, represented by the 3 3 5 gnomAD database. 14 The variants detected in the families 1, 2, 3, 4, 6, 8, 9, 10 and in the fetus  The hlb-1(syb4896) mutant showed a significant increase in body curvature ( Figure 3A). In existing C. elegans models of epilepsy "head bobbing" is a phenotype associated with convulsions and the onset of seizures. 32 We saw no statistically significant difference in the 3 5 0 head movement of hlb-1(syb4896) compared to N2 during baseline (pre-stimulus) tracking 3 5 1 ( Figure 3B). However, upon stimulation with pulses of blue light, a significant increase in the 3 5 2 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.04.22273309 doi: medRxiv preprint 1 7 acceleration of the head tip (indicative of increased head movement) was observed for mutant suggests some elements of a mild epileptic phenotype may be present in hlb-1(syb4896).

6 1
A previous study into the function of hlb-1 in C. elegans identified a defect in pharyngeal 3 6 2 pumping rate, 10 that we also confirm for hlb-1(syb4896) (Figure 3D), and enlarged pre-and epileptic seizures and the hypothesis that liprin-ß1 acts as a core scaffold to mediate protein 3 6 5 assembly in the presynaptic zone, 3 we investigated if our quantitative phenotyping approach 3 6 6 could detect a defect in the synaptic transmission apparatus of hlb-1(syb4896).

6 7
Aldicarb is an acetylcholinesterase inhibitor that induces paralysis of the body-wall muscles give rise to defects in presynaptic function, as ACh accumulates in the neuromuscular 3 7 1 junction at a slower rate. 33 Indeed, hlb-1(syb4896) showed a significant dose-dependent 3 7 2 decrease in the fraction of paused worms that were exposed to 1 -10 µM aldicarb for 1h mutations only affect the presynaptic site. 34 In contrast to previous hlb-1 studies 10 , we do not 3 7 7 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. increased sensitivity observed at 10 µM levamisole for 4h ( Figure 4B).

7 9
These findings provide evidence that a defect arises in the presynaptic, but not postsynaptic, supports presynaptic defects as a cause the pathologies arising from mutations in PPFIBP1. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. Here we describe 14 individuals from ten unrelated families with a core phenotype of 3 8 7 moderate to profound developmental delay, progressive microcephaly, epilepsy and in PPFIBP1. In addition, we describe a fetus with severe growth restriction, microcephaly and 3 9 0 intracranial calcifications with a homozygous missense variant that is in silico and structurally 3 9 1 predicted to be disrupting. gnomAD. Since all described variants are ultra-rare, (MAF < 0.01) it is highly unlikely to 3 9 6 assemble a cohort with this level of phenotypical overlap and homozygous LoF variants in 3 9 7 PPFIBP1 by coincidence which further strengthens disease causality. The 13 individuals harbouring homozygous frameshift or nonsense variants exhibit a . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. signs. This could be due to an incomplete splice defect, either leading to the expression of a   domain region and its function in protein-protein interactions. Given the hypothesis that 4 3 3 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.04.22273309 doi: medRxiv preprint 1 liprin-β1 acts as a core scaffold to mediate protein assembly in the presynaptic zone, 3 the 4 3 4 ability to precisely interact with other proteins would appear to be critical for protein function. screening for infections of the TORCH spectrum was performed with negative results and 4 5 0 also the fetus was tested negative for CMV.

5 1
The biological function of liprin-β1 and its molecular mechanisms are still largely unstudied. zone and at the postsynaptic density and also play a role in intracellular transport, cell motility 4 5 8 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.04.22273309 doi: medRxiv preprint 2 and protein assembly. 3,5,6,[45][46][47] Wei et al. found that liprin-α2 forms a ternary complex 4 5 9 simultaneously binding liprin-β1 and CASK, another presynaptic scaffold protein, supporting 4 6 0 the hypothesis that liprin-β1 could act as a core scaffold and mediate large protein assemblies hearing, ophthalmologic anomalies, muscular hypo-or hypertonia and spasticity, as well as with the clinical signs found in this cohort, it seems possible that PPFIBP1 and CASK are showing that null-allele mutants of the drosophila orthologues liprin-β and liprin-α CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10. 1101/2022 In summary, we establish bi-allelic loss-of-function variants in PPFIBP1 as a novel cause for 4 8 3 an autosomal recessive neurodevelopmental disorder with early-onset epilepsy, microcephaly 4 8 4 and periventricular calcifications.
CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. ; https://doi.org/10.1101/2022.04.04.22273309 doi: medRxiv preprint J O'Brien, Thomas. Pharyngeal Pumping Assay V1.    . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 Supplemental information 5 0 8 Supplemental data includes detailed case reports of the described individuals, three tables 5 0 9 (Table S1, Table S2, Table S3) and supplemental methods. Table S4 is provided separately as 5 1 0 an excel file. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 Acknowledgements 5 1 3 We thank all families that participated in this study. This project has received funding from . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

2 1
The authors declare no competing interests. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022  . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. ;https://doi.org/10.1101/2022 doi: medRxiv preprint alternative splicing and nonsense-mediated mRNA decay in humans. Proceedings of the National 5 9 9 Academy of Sciences 100, 189-192. genetic selection for Caenorhabditis elegans synaptic transmission mutants. Proceedings of the 6 0 9 National Academy of Sciences 93, 12593-12598. 6 1 0 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2022.