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. 2019 Jul 20;142(9):2617–2630. doi: 10.1093/brain/awz198

Pathogenic WDFY3 variants cause neurodevelopmental disorders and opposing effects on brain size

Diana Le Duc 1, Cecilia Giulivi 2,3, Susan M Hiatt 4, Eleonora Napoli 2, Alexios Panoutsopoulos 5,6, Angelo Harlan De Crescenzo 5,6, Urania Kotzaeridou 7, Steffen Syrbe 7, Evdokia Anagnostou 8, Meron Azage 9, Renee Bend 10, Amber Begtrup 11, Natasha J Brown 12,13,14, Benjamin Büttner 1, Megan T Cho 11, Gregory M Cooper 4, Jan H Doering 7, Christèle Dubourg 15,16, David B Everman 10, Michael S Hildebrand 12,17, Francis Jeshira Reynoso Santos 18, Barbara Kellam 19, Jennifer Keller-Ramey 11, Johannes R Lemke 1, Shuxi Liu 11, Dmitriy Niyazov 9, Katelyn Payne 20, Richard Person 11, Chloé Quélin 21, Rhonda E Schnur 11, Brooke T Smith 10, Jonathan Strober 22, Susan Walker 19, Mathew Wallis 23,24, Laurence Walsh 20, Sandra Yang 11, Ryan K C Yuen 19,25, Andreas Ziegler 7, Heinrich Sticht 26, Michael C Pride 3,27, Lori Orosco 5,6, Verónica Martínez-Cerdeño 3,5,6, Jill L Silverman 3,27, Jacqueline N Crawley 3,27, Stephen W Scherer 19,28, Konstantinos S Zarbalis 3,5,6,✉, Rami Jamra 1,✉
PMCID: PMC6736092  PMID: 31327001

The autophagy scaffolding protein Wdfy3 helps regulate cortical size in mice. Le Duc et al. show that in humans, haploinsufficiency of WDFY3 leads to mild to moderate neurodevelopmental delay, psychiatric disorders, and macrocephaly, while a potential gain of function caused by variants in the PH domain of WDFY3 leads to microcephaly.

Keywords: WDFY3, brain size, neurodevelopmental delay, intellectual disability

Abstract

The underpinnings of mild to moderate neurodevelopmental delay remain elusive, often leading to late diagnosis and interventions. Here, we present data on exome and genome sequencing as well as array analysis of 13 individuals that point to pathogenic, heterozygous, mostly de novo variants in WDFY3 (significant de novo enrichment P = 0.003) as a monogenic cause of mild and non-specific neurodevelopmental delay. Nine variants were protein-truncating and four missense. Overlapping symptoms included neurodevelopmental delay, intellectual disability, macrocephaly, and psychiatric disorders (autism spectrum disorders/attention deficit hyperactivity disorder). One proband presented with an opposing phenotype of microcephaly and the only missense-variant located in the PH-domain of WDFY3. Findings of this case are supported by previously published data, demonstrating that pathogenic PH-domain variants can lead to microcephaly via canonical Wnt-pathway upregulation. In a separate study, we reported that the autophagy scaffolding protein WDFY3 is required for cerebral cortical size regulation in mice, by controlling proper division of neural progenitors. Here, we show that proliferating cortical neural progenitors of human embryonic brains highly express WDFY3, further supporting a role for this molecule in the regulation of prenatal neurogenesis. We present data on Wnt-pathway dysregulation in Wdfy3-haploinsufficient mice, which display macrocephaly and deficits in motor coordination and associative learning, recapitulating the human phenotype. Consequently, we propose that in humans WDFY3 loss-of-function variants lead to macrocephaly via downregulation of the Wnt pathway. In summary, we present WDFY3 as a novel gene linked to mild to moderate neurodevelopmental delay and intellectual disability and conclude that variants putatively causing haploinsufficiency lead to macrocephaly, while an opposing pathomechanism due to variants in the PH-domain of WDFY3 leads to microcephaly.

Introduction

Neurodevelopmental delay is a heterogeneous disorder that can be classified as mild if the functional age is above 67% of the development corresponding to the chronological age, or moderate if functional age is 34–66% of chronological age (McDonald et al., 2006). While next-generation sequencing has proven successful in understanding the genetic causes of severe forms of neurodevelopmental delay and associated intellectual disability, the genetic basis of mild to moderate neurodevelopmental delay has yet to be satisfactorily explained (Vissers et al., 2016).

Here we describe how the advent of next-generation sequencing corroborated with international collaboration (Sobreira et al., 2015) can facilitate the discovery of monogenic causes for mild to moderate neurodevelopmental delay and end a diagnostic odyssey. We report on 13 probands with mild to moderate neurodevelopmental delay and intellectual disability caused by heterozygous variants in WDFY3.

WDFY3 has been already reported as relevant for higher cognitive functions through its association with autism spectrum disorders (Iossifov et al., 2012, 2014; Wang et al., 2016; Stessman et al., 2017; Yuen et al., 2017) (SFARI gene category 2.1). Also, a missense variant, located in the PH-domain of WDFY3, has been linked to moderate intellectual disability and microcephaly in a large kindred, through modification of Dvl3 autophagy-mediated control of the canonical Wnt signalling pathway (Kadir et al., 2016).

WDFY3 encodes for autophagy-linked FYVE protein (also named ALFY), a large multidomain scaffolding protein implicated in the selective degradation of ubiquitinated protein aggregates by autophagy (Filimonenko et al., 2010) and clearance of mitochondria via mitophagy (Napoli et al., 2018). In mice, Wdfy3 is expressed in the neocortex of the developing embryo, where we have previously shown that it regulates the proliferation of neural progenitors with loss-of-function leading to an expansion of the radial glial cell population (Orosco et al., 2014).

In this study, we describe the impact of deleterious WDFY3 heterozygous variants on human neurodevelopment and provide a detailed clinical description of identified cases. We also show that the human macrocephaly phenotype is recapitulated in a Wdfy3-haploinsufficiency mouse model. Using proteomics analysis we investigated the effect of Wdfy3-haploinsufficiency on Wnt-pathway signalling and show different outputs based on different putative pathomechanisms.

Material and methods

Ethics approval

This study was approved and monitored by the ethics committee of the University of Leipzig, Germany (224/16-ek and 402/16-ek), the review boards at Western (20130675), and the University of Alabama at Birmingham (X130201001). Institutional ethics approval was not required if testing was part of routine clinical care. All families provided informed consent for clinical testing and publication.

Sequencing

Probands were tested mainly in trio-exome or -genome sequencing setup (Supplementary material), with the exception of Proband 13, whose deletion was identified by single nucleotide polymorphism microarray. Bioinformatic processing and evaluation of identified and annotated variants were performed at different centres using either in-house pipelines or commercial software. The evaluation of the cases was first performed in a diagnostic setting, which revealed no causative variant. Downstream analysis of genes not yet related to neurodevelopmental disorders revealed WDFY3 as the sole convincing candidate gene. All variants in WDFY3 that were not detected by sequencing a parent-child trio or where the quality of the massive parallel sequencing was insufficient were confirmed by Sanger sequencing. Further details on sequencing methods can be obtained from previous publications of the different centres (Bowling et al., 2017; Martin et al., 2017; Retterer et al., 2016; Yuen et al., 2017). A detailed description of the sequencing setup is provided in the Supplementary material.

Molecular modelling

The PH-BEACH domain pair of WDFY3 was modelled using the crystal structure of the homologous domain pair from human LRBA/BGL (PDB: 1T77; Gebauer et al., 2004) as a template. HHpred (Zimmermann et al., 2018) was used for sequence alignment and Modeller (Webb and Sali, 2017) for modelling. The model comprises residues 2534–2976 of WDFY3, with the exception of the sequence stretch 2582–2599 that exhibits no homology to the template structure. Mutations were modelled with SwissModel (Guex and Peitsch, 1997) and RasMol (Sayle and Milner-White, 1995) was used for structure analysis and visualization.

WDFY3 embryonic expression analysis

Immunostaining was carried out on slide-mounted 6-μm thick fixed sections of human forebrain. Human foetal tissue was donated by the next of kin to the Program of Body Donation for Teaching and Research at the Universidad Autónoma de Madrid School of Medicine. In brief, tissue was washed prior to antigen retrieval and after antigen retrieval the tissue was also repeatedly rinsed. α-Wdfy3 (Abnova) and secondary antibody (Biotin-SP-APure F(ab’)2 Frag Dnk Anti-Mse IgG (H + L), Jackson ImmunoResearch) were applied consequently. Then, we incubated the tissue with Avidin-Biotin (AB) Complex (Vector Laboratories) and visualized the signal using 3,3′-diaminobenzidine tetrahydrochloride (Sigma). A detailed description of antibody incubation is provided in the Supplementary material. After labelling procedures, tissue sections were post-fixed, dehydrated, mounted, and photomicrographs were taken on an Olympus BX61 microscope at ×10 and ×100 magnification.

Animal husbandry and behavioural tests

Generation and genotyping of Wdfy3+/lacZ mice was described elsewhere (Orosco et al., 2014). Animals tested were of either sex.

We tested the motor skill performance of 2–3-month-old mice on a rotarod apparatus (Rota-rod/RS, PanLab Harvard Apparatus). Trials were recorded using the SeDaCom software system (version 1.4.02, PanLab Harvard Apparatus).

Contextual and cued fear conditioning was used to evaluate memory of an aversive experience associated with the stimuli present during the aversive event. On Day 1, we placed 3–4-month-old mice in a metal chamber with a grid floor and allowed them to explore for 2 min. An auditory stimulus was presented for 30 s during the last second of which a 2-s 0.5 mA AC current foot-shock was delivered. On Day 2, mice were returned to the same testing chamber without foot-shock administration and we tested whether they remembered the association of foot-shock with contextual features of the chamber by measuring freezing time. On Day 3, we placed the mice in a different testing chamber (curved shape, Plexiglas floor surfaces, white light, and vanilla extract odour). Freezing in the new context was minimal since no aversive experience had occurred in the new context. Then we played the auditory stimulus for 3 min with no foot-shock and we scored freezing as the measure of cued fear conditioning. A detailed description of the behavioural tests setup is provided in the Supplementary material.

Wnt proteomics of cortical lysates of Wdfy3 haploinsufficient mice

Sample preparation

We used cortices from seven wild-type and seven Wdfy3+/lacZ 3-month-old female mice to obtain protein-enriched fractions as described previously (Napoli et al., 2018). Proteins were evaluated using the Pierce BCA protein assay (Thermo Scientific).

Mass spectrometry

Liquid chromatography (LC) with tandem mass spectrometry (MS) was performed at the University of California Davis Genome Center Core Proteomics Facility. Protein pellets were digested overnight with a trypsin to protein ratio of 1:30. For each sample, the equivalent of 2–5 µg of protein was loaded into the LC-MS/MS.

Database searching

All LC/MS samples were analysed using X! Tandem [The GPM, thegpm.org; version TORNADO (2010.01.01.4)]. X! Tandem was set up to search the uniprot__20120523_ gTmkm3 database (89 576 entries) assuming trypsin digestion. X! Tandem was searched with a fragment ion mass tolerance of 20 ppm and a parent ion tolerance of 1.8 Da. Deamidation of Asn and Gln, oxidation of Met and Trp, sulphonation of Met, Trp oxidation to formylkynurenin, and acetylation of the N-terminus were specified in X! Tandem as variable modifications.

Criteria for protein identification

Scaffold (v. 3.00.07, Proteome Software Inc., Portland, OR) was used to validate LC/MS based peptide and protein identification. Peptide identification was accepted if it could be established at >80% probability by the Peptide Prophet algorithm (Keller et al., 2002). Protein identifications were accepted if they could be established at >80% probability and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Proteins that contained similar peptides and could not be differentiated based on LC/MS analysis alone were grouped to satisfy the principles of parsimony.

Proteomics analysis

The proteome profiles from all 14 animals were normalized by their spectral counting sum. We used partial least squares discriminant analysis to identify the features that separated the two genotypes the most. The proteins that best described the differences between the two groups were selected by setting a variable in importance projection score of >0.8.

Statistical analysis

To test whether there is a significant enrichment of WDFY3 de novo variants in a neurodevelopmental delay cohort compared to the occurrence by chance, we used a binomial test in R (Core, 2017). Further, we used a Bonferroni correction to adjust the P-value for multiple testing considering 18 894 coding sequences according to the latest release of the consensus coding sequence (CCDS) project (Pruitt et al., 2009) (v.20 interrogated in May 2018).

For the proteomics analyses, proteins associated with the gene ontology term ‘Wnt’ (under biological process) and a P-value corrected by the FDR (false discovery rate) of ≤0.10 were considered to have a significant differential expression.

To test the enrichment of probands with occipital-frontal circumference (OFC) over the 87th percentile compared to the OFC distribution in normal population we used a binomial test function (Core, 2017). All other statistics related to Wdfy3+/lacZ were performed with unpaired t-tests and bars represent mean ± standard error of the mean (SEM). The difference between groups was considered statistically significant at P-value < 0.05.

Data availability

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

Results

Phenotypic characterization of probands

Thirteen probands (2–18 years old) were clinically examined at several centres in North America, Europe, and Australia. All individuals were reported to have cognitive and/or developmental deficits. Mild to moderate intellectual disability (defined as mild intellectual disability if the functional age was above 67% of the corresponding chronological age development or moderate if the functional age corresponded to 34–66% of chronological age) was observed in 8/10 (80%) cases for whom intellectual assessment was performed (Fig. 1A and Table 1). Information on quantitative diagnostic standardization was not obtainable, because testing was performed in multiple clinical centres that are, however, specialized in developmental delay. Delayed speech was common in examined individuals (11/13, 85%), as was gross motor development delay or muscular hypotonia (7/13, 54%; Fig. 1A and Table 1). Behavioural concerns were noted in 9/12 assessed individuals (75%) including autism spectrum disorder (7/12) and attention deficit hyperactivity disorder (4/12) (Fig. 1A and Table 1). There was no apparent shared facial gestalt. Macrocephaly or large-normal OFC was observed in 9/11 evaluated cases (5/9 >97th percentile and 4/9 between 87th and 95th percentiles) (Fig. 1A and Table 1). Statistical analysis revealed an enrichment of probands with OFC over the 87th percentile in our cohort compared to the distribution in general population (P = 4.548 × 10−7). We did not observe any differences in the phenotypical spectrum of probands bearing missense variants located outside organized protein domains (Probands 1 and 2; Fig. 1 and Table 1) or in the BEACH-Domain (Proband 4; Fig. 1 and Table 1) compared to probands bearing truncating variants (Probands 5–12; Fig. 1 and Table 1). However, the proband with a de novo variant located in the PH-domain (Proband 3; Fig. 1 and Table 1) displayed overt microcephaly [OFC = 42 cm, under the 3rd centile; −8.78 standard deviation (SD)]. This patient was also diagnosed with cystic fibrosis and showed typical signs of dystrophy related to the condition [short stature: 92 cm, under the 3rd centile, −3.95 SD; low weight 10.86 kg, under the 3rd centile, −4.72 SD; body mass index (BMI) 12.8 kg/m2, under the 3rd centile, −2.02 SD]. Nonetheless, microcephaly is not associated with cystic fibrosis. Brain MRI was available on nine individuals, of whom six were reported as normal or unremarkable, and abnormal in the remaining three without specific or overlapping features. Detailed clinical information is described in the Supplementary material, Case reports are summarized in Fig. 1 and Table 1.

Figure 1.

Figure 1

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Summary of genotypes and phenotypes of individuals with WDFY3-related neurodevelopmental delay. (A) Outer circle: proband number (Table 1). Second to seventh circles: clinical symptoms, phenotype. Inner circles: genotype. (B) De novo missense, nonsense, and frameshift variants (NM_014991.4) from patients with neurodevelopmental delay and macrocephaly described in the present study (red, proband number in square brackets; Table 1). De novo variants described in previous autism spectrum diorder cohorts [bottom: black; Autism Speaks MSSNG, Autism Sequencing Consortium (ASC) and Simons Simplex Collection (SSC)] (Wang et al., 2016; Yuen et al., 2017). Variants in yellow occurred in probands with neurodevelopmental delay and microcephaly (upper panel variant occurred de novo, lower panel variant previously described in a kindred (Kadir et al., 2016)]. Variants depicted in purple are inherited from an affected father, otherwise all presented variants occurred de novo.

Table 1.

Main clinical features of the probands

Patient identifier 1 2 3 4 5 6 7
Genotype
Genomic position chr4 85752586T>C 85658484A>G 85639657C>T 85623635G>A 85750272C>A 85731395del 85678261G>A
cDNA (NM_014991.4) c.749A>G c.6610T>C c.7672G>A c.8467C>T c.841G>T c.1990delT c.5242C>T
Protein alteration p.(Asn250Ser) p.(Trp2204Arg) p.(Gly2558Ser) p.(Arg2823Trp) p.(Glu281*) p.(Ser664Leufs*2) p.(Arg1748*)
Zygosity Het Het Het Het Het Het Het
Inheritance De novo De novo De novo De novo De novo De novo De novo
Patient details
Age of patient, sex 14 y, M 15y, F 5 y, F 2 y, M 4 y, M 5 y, M 4 y, M
OFC 57.7 cm (P87th) Not available 42 cm (<P3th; −8.78 SD) 52 cm (>P97th; +2.03 SD) 54.8 cm (>P97th; +2.88 SD) 55.5 cm (>P97th; +3.01 SD) 52 cm (P90th)
Height 168 cm (P67th) 163.5 cm (P41th) 92 cm (<P3rd; −3.95 SD) 84 cm (P12th) 88. 3 cm (<P3rd; −3,61 SD) 114.8 cm (P89th) 95 cm (<P3rd; −2.08 SD)
Weight 59.8 kg (P74th) 65.4 kg (P83th) 10.86 kg (<P3th; −4.72 SD) 9.5 kg (P2th; −2.12 SD) 14.3 kg (P25th) 20.1 kg (P74th) 14.4 kg (P10th)
Neurodevelopment
Neurodevelopmental delay Yes Yes Yes Yes Yes Yes Yes
Intellectual disability Mild ID Mild ID Moderate ID Not available Normal Not available Normal
Speech and language Speech delay, stutter Normal Speech delay (a few words, no word association) Speech delay Normal Speech delay; first words after 2y Speech delay
Motor development Gross motor delay, mild truncal hypotonia, abnormal gait Normal Gross motor delay (walk at 2 y) Gross motor delay at 2 y Gross motor delay, truncal hypotonia, improved, intoeing Gross motor delay Motor delay, muscular hypotonia
Behavioural issues ADHD ADHD, anxiety, depression None Not available None None Suspected ASD
Patient identifier 8 9 10 11a 12a 13
Genotype
Genomic position chr4 85645644_85645645dup 85617157_85617160dup 85614060_85614070del 85605108_85605111del 85605095del 4q21.23del: 85,665, 448−86,686,764del
cDNA (NM_014991.4) c.7371_7372insGA c.8867_8868insTTGG c.9017_9027del c.9711_9714del c.9726+1del c.?
Protein alteration p.(Ser2459Argfs*30) p.(Phe2957Trpfs*4) p.(Asp3006Alafs*21) p.(Asp3238Glufs*25) p.? p.?
Zygosity Het Het Het Het Het Het
Inheritance De novo De novo Paternally inherited Paternally inherited De novo De novo
Patient details
Age of patient, sex 2 y 9 m, M 7 y, F 17 y, M ca. 12 y, M 10 y 2 m, F 11 y 9 m, M
OFC 53.8 cm (>P97th; +2.67 SD) Not available 59 cm (P94th) 57.5 cm (>P97th; +2.18 SD) 50.8 cm (P6th) 56.5 cm (P95th)
Height 95 cm (P50–75th) 132 cm (P93th) 181.5 cm (P75–90th) 153.7 cm (P59th) 127 cm (<P3rd, −2.22 SD) 153 cm (P76th)
Weight 17.5 kg (P97th) 21.32 kg (P20th) 97.7 kg (>P97th, +3 SD) 50.8 kg (P82nd) 25.54 kg (P4th) 43.2 kg (P67th)
Neurodevelopment
Neurodevelopmental delay Yes Yes Yes Yes Yes Yes
Intellectual disability Mild ID Moderate ID Mild ID Not available Moderate ID Mild ID
Speech and language Speech delay; single words at age of 2 y Speech delay Speech delay Speech delay; below average based on Vineland scores Speech delay Speech delay
Motor development Normal Normal Motor delay, muscular hypotonia in early childhood Normal Normal Normal
Behavioural issues ASD, easily frustrated ASD ASD ASD, ADHD ASD ASD, ADHD
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ADHD = attention deficit hyperactivity disorder; ASD = autism spectrum disorder; F = female; Het = heterozygous; ID = intellectual disability; m = months; M = male; OFC = occipitofrontal circumference; P = percentile; y = years.

aPatients from the Autism Speaks MSSNG cohort (Yuen et al., 2017).

Identified variants and their characteristics

We identified heterozygous de novo missense-variants in WDFY3 (MIM: 617485, NM_014991.4) in four probands: c.749A > G: p.(Asn250Ser), c.6610T > C: p.(Trp2204 Arg), c.7672G > A: p.(Gly2558Ser), and c.8467C > T: p.(Arg2823Trp). Five probands had de novo truncating-variants: c.841G > T: p.(Glu281*), c.1990delT: p.(Ser664 Leufs*2), c.5242C > T: p.(Arg1748*), c.7372_7373insGA: p.(Ser2459Argfs*30), and c.8867_8868insTTGG: p.(Phe 2957Trpfs*4). In one proband we identified a de novo splicing variant involving the conserved consensus splicing site c.9726+1del and affecting the donor site located on intron 64 and another proband had a de novo heterozygous 1.0 Mb deletion within 4q21.23 (chr4: g.85,665,448–86,686,764) including the first 36 exons of WDFY3 and the first three exons of ARHGAP24 (tolerates haploinsufficiency with a pLi score of 0). We also identified a familial variant of a 11-bp deletion, c.9017_9027del: p.(Asp3006Alafs*21), present in a proband and inherited from his similarly affected father, who presented with a moderate intellectual deficit (Supplementary material, Case reports, Proband 10). An additional paternally inherited variant, c.9711_9714del: p.(Asp3238Glufs*25, was identified in a child with mild intellectual deficit and macrocephaly (cognitive performance has not been assessed for the parents). None of these variants was present in public databases [gnomAD; accessed in September 2018 (Lek et al., 2016) or 1000 Genomes (Genomes Project et al., 2015)]. For the missense variants, most of the in silico tools predicted pathogenicity (Kumar et al., 2009; Adzhubei et al., 2010; Pollard et al., 2010; Schwarz et al., 2014). The scaled CADD scores of the missense variants (Supplementary Table 1) ranged between 21 (corresponding to the top 1% deleterious variants in the human genome) and 32 (top 0.1% deleterious variants in the human genome) (Rentzsch et al., 2019). An overview of the genetic variants together with previously published variants of association studies in probands with autism spectrum disorder/neurodevelopmental delay is presented in Fig. 1B.

This patient series and the identified variants were collected through international collaboration with colleagues and data-sharing resources such as GeneMatcher (Sobreira et al., 2015). The size of the tested cohorts at all contributing centres sums up to 11 743 probands with neurodevelopmental delay. We further added the MSSNG cohort [5205 whole genomes (Yuen et al., 2017)] and the Chinese autism cohort from Wang et al. (2016) (4998 whole exomes, including Autism Sequencing Consortium and Simons Simplex Collection) that have previously reported de novo variants in WDFY3 (Fig. 1B). These sum to 20 de novo variants in 21 946 individuals. The occurrence of de novo variants in these neurodevelopmental delay cohorts is significantly higher (P = 1.745 × 10−7; corrected P-value = 0.003) than expected under the mutation rate of 1.08997 × 10−4 (missense, nonsense, and frameshift variants) estimated for WDFY3 by Samocha et al. (2014).

Truncating variants may activate the mRNA surveillance nonsense-mediated decay, or if translated, may result in a non-functional protein or a dominant negative effect. Such a pathogenic effect is not always clear for missense variants. All four missense variants in this study were conserved and predicted as pathogenic by most interrogated in silico evaluation tools. However, of the four missense variants, we were not able to model the structural effect of the variants p.(Asn250Ser) and p.(Trp2204Arg) and infer the impact on protein functionality since these were not located in a domain with a described function. Since WDFY3 binds phospholipids (Simonsen et al., 2004; Cullinane et al., 2013), we also checked whether the loss of a positive charge as consequence of the p.(Arg2823Trp) or the p.(Arg2637Trp) exchange might additionally affect phospholipid binding of the PH-BEACH domain pair. p.(Arg2637Trp) is located in the PH segment close to the canonical phospholipid binding pocket found in homologous PH domains. However, because this pocket is partially blocked by the protein chain in WDFY3, phospholipid binding would require some conformational rearrangement. p.(Arg2823Trp) is located in the BEACH segment of the domain pair, for which no structural information about ligand binding sites is known. Thus, based on the structural data available, no final conclusion can be drawn, whether the variants observed in the PH-BEACH domain pair affect phospholipid binding in addition to disturbing the protein structure.

The variant p.(Gly2558Ser) located in a β-turn of the PH-domain could not be reliably modelled due to low local sequence similarity to the template structure. The modelling template (PDB:1T77) has a bulky threonine instead of glycine at the corresponding sequence position thus favouring a different loop conformation. Still, in the PH-domain we could model the effect of the previously reported variant p.(Arg2637Trp) (Kadir et al., 2016) (Fig. 2C). The results suggest that Arg2637 forms electrostatic interactions with Asp2635, which are lost in the p.(Arg2637Trp) variant leading to steric clashes and domain destabilization. Further, we also modelled the effect of the variant p.(Arg2823Trp), located in the BEACH-domain. The residue Arg2823 forms a salt-bridge with Asp2819 that stabilizes a turn in the BEACH-domain (Fig. 2). This bridge is lost in the p.(Arg2823Trp) variant (Fig. 2B), which may result in BEACH domain destabilization.

Figure 2.

Figure 2

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Structural effects of WDFY3 sequence variants located in the BEACH- and PH-domains. (A) Modelled ribbon diagram of the wildtype domain pair of PH-domain (green) and BEACH-domain (cyan). The connecting linker is shown in yellow and the sites of the variants investigated are marked as black spheres. (B) Effect of the p.(Arg2823Trp) exchange in the BEACH-domain, associated in our study with neurodevelopmental delay and macrocephaly. Arg2823 forms a salt-bridge with Asp2819 (green dotted lines) that is lost in the Arg2823Trp variant. This likely leads to BEACH-domain destabilization. (C) Effect of the Arg2637Trp exchange, which has been associated with neurodevelopmental delay and microcephaly (Kadir et al., 2016). Arg2637 forms electrostatic interactions with Asp2635, which are lost in the p.(Arg2637Trp) variant and steric clashes are observed instead. The magenta arrow marks the site of these opposing interactions. This likely leads to PH-domain destabilization.

WDFY3 expression in human prenatal brain

To examine WDFY3 expression during developmental neurogenesis in humans, we proceeded to analyse WDFY3 expression in human forebrain sections of gestational Weeks 15 and 17. WDFY3 immunostaining revealed widespread and overlapping expression in the pallium of both brains encompassing all layers, yet with distinct differences between layers (Fig. 3A–C). Importantly, as in the murine Wdfy3 KO model (Orosco et al., 2014), radial glial cell-containing ventricular zone showed the strongest expression with high levels in radial units of the cortical plate. Progressively lower WDFY3 expression was observed in the outer subventricular zone, inner subventricular zone, and subplate/intermediate zone. WDFY3-expressing cells do not show uniform expression levels, as they differ in signal intensity. Notably, dividing progenitors in the ventricular zone exhibit some of the strongest WDFY3 immunolabelling replicating aspects of WDFY3 expression previously observed in mice (Orosco et al., 2014) (Fig. 3A–C).

Figure 3.

Figure 3

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WDFY3 expression in human embryonic pallium. Sections of human ventral pallium reveal WDFY3 immunostaining at gestational Weeks (GW) 15 (A) and 17 (B) Pallial expression can be seen in all layers at varying degrees of intensity, with ventricular zone (VZ) and cortical plate (CP) containing the greatest proportion of WDFY3+ cells. Lower WDFY3 expression is present in outer subventricular zone (oSVZ), inner subventricular zone (iSVZ), and subplate/intermediate zone (SP/IZ). Respective positions of high-magnification images in C are indicated as numbers in B. Some dividing progenitors in the ventricular zone exhibit strong WDFY3 immunolabeLling (arrowheads) as some radial units of the cortical plate do (dotted lines). Scale bars = 500 μm in A and B; 10 μm in C.

Macrocephaly in WDFY3 variant carriers and Wdfy3+/lacZ mice

As previously described in Wdfy3 mouse models (Orosco et al., 2014), probands with truncating variants had large head circumference (Fig. 1 and Table 1). While strongest cortical size increases were recorded in homozygous mouse mutants that are subject to perinatal lethality, heterozygous Wdfy3+/lacZ mice, which we analysed here further, survived into adulthood, were fertile, and presented at birth with structural abnormalities including cortical lengthening. Comparing whole-mount Wdfy3+/lacZ brains to wild-type controls at postnatal Days 7 and 10 revealed overt changes including significant cortical enlargement that apparently affected the anteroposterior axis to a greater extent than the medio-lateral axis (Fig. 4A). Cortical area enlargement was ∼9% at postnatal Day 7 (wild-type, 0.753 ± 0.01 cm2, Wdfy3+/lacZ, 0.824 ± 0.01 cm2; Fig. 4B) and ∼12% at postnatal Day 10 (wild-type, 1.095 ± 0.03 cm2, Wdfy3+/lacZ, 1.229 ± 0.02 cm2; Fig. 4C) without overt sex differences, mirroring the human large head circumference upon heterozygous WDFY3 loss.

Figure 4.

Figure 4

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Wdfy3+/lacZ mice characteristics. Developmental megalencephaly in Wdfy3+/lacZ mice. (A) Dorsal views of whole-mount PND7 brains show cerebral enlargement in Wdfy3+/lacZ mice (indicated by the arrow; Cx = cortex, Cb = cerebellum, MB = midbrain). (B) For two postnatal stages [postnatal Day 7 (P7): wild-type n = 4, Wdfy3+/lacZ n = 8 and postnatal Day 10 (P10): wild-type n = 6, Wdfy3+/lacZ n = 6] both total cortical area (postnatal Day 7, P = 0.0273; postnatal Day 10, P = 0.0096) and cortical length (postnatal Day 7, P = 0.0012; postnatal Day 10, P = 0.008) were analysed and found to be significantly increased. Motor coordination and learning and memory deficits in Wdfy3+/lacZ mice. (C) Wdfy3+/lacZ mice show diminished motor coordination in the rotarod test [wild-type (WT) n = 8, Wdfy3+/lacZ n = 6; Day 1, t(1,12) = 2.747, P = 0.0177; Day 2, t(1,12) = 4.258, P = 0.0011; Day 3, t(1,12) = 2.945, P = 0.0123]. (D) After footshock conditioning in the cued fear conditioning assay (Day 1), Wdfy3+/lacZ mice exhibit learning and memory deficits in context freezing (Day 2), [t(1,37) = 2.353, P = 0.024], but not in cued freezing (Day 3) [t(1,37) = 1.158, P = 0.254]. All statistics were performed with unpaired t-tests and bars represent mean ± SEM.

Motor coordination and associative learning deficits in Wdfy3+/lacZ mice

Multiple probands show gross motor abnormalities (Fig 1A and Table 1). Consequently, we tested motor coordination in Wdfy3+/lacZ mice by using the rotarod test. On three consecutive days, we compared the performance of Wdfy3+/lacZ to wild-type. Wdfy3+/lacZ mice (n = 6) showed decreased endurance, learning, and motor coordination compared to wild-type mice (n = 8), as the latency to fall off the rod was shorter for the Wdfy3+/lacZ mice in the acceleration test (4–40 rpm in 60 s; Fig. 4D).

Subsequently, we examined cognitive performance in Wdfy3+/lacZ mice, by testing associative learning with the contextual and cued fear conditioning test (Fanselow, 2000; LeDoux, 2003) in young males and nulliparous females. As shown in Fig. 4E, freezing to context on test Day 2 was significantly lower in the Wdfy3+/lacZ mice as compared to wild-type [Student’s t(1,37) = 2.353, P = 0.024]. No differences between genotype groups were seen during the training Day 1, indicating normal perception and response to the foot-shock. No differences between Wdfy3+/lacZ and wild-type mice were detected on auditory cue test Day 3, either during the pre-cue period or during the presentation of the auditory cue, indicating similar sensory, auditory skills (Fig. 4E).

Altogether, these results indicated that Wdfy3+/lacZ mice ranging from 2 to 4 months of age showed impairment in motor coordination and the associative learning component of fear conditioned learning and memory.

Wnt pathway analysis

Considering that Wnt signalling plays critical roles in normal brain development and neuropsychiatric disorders (Okerlund and Cheyette, 2011; Krumm et al., 2014; Hormozdiari et al., 2015; Kwan et al., 2016), and that Drosophila with a missense heterozygous variant p.(Arg2637Trp) (Kadir et al., 2016) in the WDFY3 homologue Bchs showed a lack of canonical Wnt signalling inhibition with its consequent uncontrolled upregulation, we performed an untargeted proteomic study on cortical neurons from 3-month-old wild-type and Wdfy3+/lacZ females and focused our analysis on the entire Wnt pathway. We observed that Wdfy3 haploinsufficiency was associated with a downregulation of the degradation of beta-catenin by the destruction complex (FDR = 1.45 × 10−10) and by a relatively milder downregulation of both TCF-dependent signalling in response to Wnt (FDR = 1.84 × 10−14) and beta-catenin-independent Wnt-Ca2+ signalling (FDR = 1.84× 10−14; Supplementary Table 1 and Supplementary Fig. 1). The downregulation of the canonical Wnt pathway was supported mainly by the decreased levels of cytoplasmic b-catenin (Ctnnb1; Tacchelly-Benites et al., 2013) as well as DVL1, CK2, GSK-3 b, Pontin52. The lower levels of DVL1 seem to drive, albeit at a lower degree, the downregulation of the Wnt-dependent planar cell polarity (decreased levels of DVL1, Rac, and increased of RhoA) and Wnt/Ca2+ pathways (decreased levels of CaN and increased of PLC and PKC; Supplementary Table 1 and Supplementary Fig. 2) based on the fact that Dvl proteins act at the node of divergence between the canonical and the non-canonical Wnt pathways (Gao and Chen, 2010). These results were extended by the upregulation of the proteasome pathway emphasizing the ubiquitin-mediated proteolytic catabolism of several key targets within the Wnt pathway, likely including Dvl (Supplementary Table 1 and Supplementary Fig. 2).

Discussion

Our results show that pathogenic variants in WDFY3 cause a monogenic, autosomal dominant neurodevelopmental disorder with mild to moderate neurodevelopmental delay and intellectual disability. Also, affected individuals showed deficits in motor coordination as well as behavioural disorders, with autism spectrum disorder as leading diagnosis. While putative haploinsufficiency of the gene leads to large head circumference, missense variants in the PH domain can cause the opposite phenotype—overt microcephaly.

Although data show that WDFY3 is under constraint for missense variants (Z-score 5.72) (Lek et al., 2016) and intolerant to loss of function [pLI = 1.00 (Lek et al., 2016), RVIS = 0.1564% (Petrovski et al., 2013)], there are 25 individuals with loss of function variants in the Genome Aggregation Database (gnomAD). Since WDFY3 loss of function variants are mainly associated with mild developmental disorders, it is possible that such individuals are included in control cohorts. This is also supported by a recent study, which showed that 2.8% of the ExAC population (consequently also the overlapping gnomAD cohort) bears possible disease-associated genotypes, mainly for phenotypes with variable clinical outcomes or those that occur as mild forms of the disease (Tarailo-Graovac et al., 2017). The number of identified de novo WDFY3 variants in our combined cohort of probands with neurodevelopmental delay is significantly higher than the expected occurrence by chance, even after stringent multiple-testing correction (P = 0.003).

Intolerance to loss of function is also supported by homozygous Wdfy3 knock-out mouse models, which show perinatal lethality (Orosco et al., 2014) We now show that heterozygous Wdfy3+/lacZ mice survive into adulthood and are megalencephalic at young age (Fig. 3D–F), recapitulating the observation in the probands (Fig. 1A and Table 1). The molecular underpinnings of WDFY3’s essential role in brain size regulation were previously tested in Drosophila for a heterozygous missense variant p.(Arg2637Trp), which segregated in a large kindred with an autosomal dominant phenotype of microcephaly and moderate intellectual disability (Kadir et al., 2016). This study concluded that the underlying molecular mechanism was based on a lack of canonical Wnt signalling inhibition with its consequent uncontrolled upregulation. Conversely, Wdfy3+/lacZ mice show megalencephaly with a downregulation of the canonical Wnt pathway supported by our proteomic data (Supplementary Table 1 and Supplementary Fig. 1). Wnt proteins are required for basic developmental processes (e.g. cell-fate specification, progenitor-cell proliferation, and control of asymmetric cell division), and Wnt signalling plays critical roles in brain development and neuropsychiatric disorders (Okerlund and Cheyette, 2011), including autism spectrum disorders (Krumm et al., 2014; Hormozdiari et al., 2015; Kwan et al., 2016). Our proteomic studies on cortical lysates of haploinsufficient mice showed a downregulation of the canonical Wnt pathway and a relatively milder downregulation of the Wnt-dependent planar cell polarity pathway and Wnt/Ca2+ pathways (Supplementary Table 1, Supplementary Figs 1 and 2). While the exact sequence of events leading to macrocephaly in humans and mice upon WDFY3 loss remain uncertain, the downregulation of the canonical Wnt pathway will likely lead to cell cycle acceleration increasing proliferative and neurogenic rates of neural progenitors, a phenomenon that we had earlier described in Wdfy3 knock-out mice with macrocephaly (Orosco et al., 2014). Our results seem to contrast the WDFY3-dependent autophagic attenuation of Wnt signalling through removal of DVL3 aggregates (Gao and Chen, 2010; Kadir et al., 2016). Dvl turnover is accelerated by autophagy under stress conditions (Gao and Chen, 2010), but Dvl degradation takes place through the proteasome pathway (Angers et al., 2006; Chan et al., 2006; Madrzak et al., 2015) even in cells in which autophagy is impaired (Gao and Chen, 2010). We observed no differences in DVL2 or DVL3 in our proteomic analysis of brain cortices, thus the haploinsufficiency in Wdfy3 and impaired autophagy (Napoli et al., 2018) could be compensated by the upregulation of ubiquitination and proteasome-dependent degradation of Dvl, thereby attenuating Wnt pathway. In accordance with this we see a down-regulation of DVL1 in our proteomics data (Supplementary Table 1) and canonical Wnt signalling was shown to be sensitive to changes in the abundance of either DVL3 or DVL1.

In regard to human macro- and microcephaly, molecular modelling of variant p.(Arg2823Trp) found in one of our macrocephalic probands together with the known microcephaly-associated variant p.(Arg2637Trp) (Kadir et al., 2016) revealed that each variant might cause a destabilization of different WDFY3 domains, namely the BEACH- (Fig. 2B) and the PH-domain respectively (Fig. 2C). Thus, destabilization of different domains may lead to distinct effects on the canonical Wnt pathway and opposite phenotypes with respect to brain size resulting from predicted loss or gain of protein function. In support of this we report on an additional proband with a de novo missense-variant also located in the PH-domain, who also presented with intellectual disability and microcephaly (Proband 3; Fig. 1 and Table 1). The function of the PH-domain is unknown, but the close interaction of PH and BEACH domains may lead to functional autoinhibition. Namely, an operative PH-domain would constitutively inhibit the function of the BEACH-domain. While this hypothesis remains purely speculative, we propose a model in which variants that disrupt the PH domain would result in a loss of the autoinhibition and a consequent gain of function. This would have an opposite effect to truncating variants and variants that are located in the BEACH-domain, which would lead to loss of function.

Further we show that basic parameters of mouse Wdfy3 neocortical expression during embryonic development are preserved in humans. In human, during the second trimester, WDFY3 is highly expressed in pallium; however, not only in a subset of dividing radial glial cells of the ventricular zone as in the mouse, but throughout all layers with ventricular zone and cortical plate showing strongest expression (Fig. 3A–C). This likely points at additional roles that WDFY3 may exert during human neocortical histogenesis, which requires its continued activity in post-mitotic cells. We noted that in the cortical plate WDFY3 is retained in distinct radially aligned columns that likely present radial clones originating from the same proliferative units and precursors to the later minicolumnar organization of the neocortex (Fig. 3C). WDFY3 loss of function may thus promote minicolumn disorganization. Minicolumn structure has been implicated in ASD pathogenesis (McKavanagh et al., 2015) and cognitive performance (Opris and Casanova, 2014).

Additional support for an association between WDFY3 and cognitive performance comes from the knockdown of the Drosophila WDFY3 orthologue Bchs. Bchs-deficient flies show defects in habituation, reflecting alterations in non-associative learning and memory (Stessman et al., 2017). Indeed, the majority of our probands show mild to moderate intellectual disability, sometimes accompanied by behavioural concerns including attention deficit hyperactivity disorder and autism spectrum disorder. In addition, 7 of 13 cases (54%) showed gross motor development delay or muscular hypotonia. This appears to be paralleled by the deficits we observed in associative learning and motor coordination in haploinsufficient mice (Fig. 4).

Based on the concordant human and murine phenotype-genotype correlation and the direct role of WDFY3 in neuronal development, we conclude that WDFY3 haploinsufficiency causes mild-to-moderate neurodevelopmental delay, intellectual disability, macrocephaly, and behavioural disorders. WDFY3 variants produce different phenotypic outcomes with respect to brain size depending on how Wnt pathway is regulated. While, as previously demonstrated, PH domain mutations cause microcephaly, variants putatively resulting in haploinsufficiency lead to macrocephaly.

Web resources

ClinVar, https://www.ncbi.nlm.nih.gov/clinvar/

GenBank, https://www.ncbi.nlm.nih.gov/genbank/

Genome Aggregation Database (gnomAD), http://gnomad.broadinstitute.org/

HGMD, http://www.hgmd.cf.ac.uk/ac/index.php

MutationTaster, http://www.mutationtaster.org/

OMIM, http://omim.org/

PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/

Protter, http://wlab.ethz.ch/protter/

PubMed, https://www.ncbi.nlm.nih.gov/pubmed/

REVEL: Rare Exome Variant Ensemble Learner, https://sites.google.com/site/revelgenomics/

RSCB Protein Data Bank, https://www.rcsb.org/pdb/home/home.do

UCSC Genome Browser, https://genome.ucsc.edu/

Varvis, https://www.limbus-medtec.com/

wANNOVAR, http://wannovar.wglab.org/

Supplementary Material

awz198_Supplementary_Data
Click here for additional data file. (738.2KB, zip)

Acknowledgements

The authors are thankful to Dr Jimenez-Amaya (Universidad Autónoma de Madrid, Spain) for providing the human embryo brains. We also wish to acknowledge the resources of MSSNG (www.mss.ng), Autism Speaks and The Centre for Applied Genomics at The Hospital for Sick Children, Toronto, Canada. We thank the participants in the Autism Speaks MSSNG program and all families for their time and contributions.

Funding

D.L.D. is funded through “Clinician Scientist Programm, Medizinische Fakultät der Universität Leipzig”. This study was in part funded by Shriners Hospitals for Children, NICHD R21HD67855 and NIMH R21MH115347 to K.S.Z., the Simons Foundation with SFARI 286567 to K.S.Z., and the Nancy Lurie Marks Family Foundation to K.S.Z., as well as a Shriners Hospitals for Children Postdoctoral Fellowship Grant to L.A.O. Additional support was provided by NICHD U54 HD079125 to J.L.S., M.C.P., J.N.C. NHGRI UM1HG007301 applies to S.M.H and G.M.C. The MIND Institute IDDRC is funded by the National Institute of Child Health and Human Development (U54 HD079125).

Competing interests

All authors declare no conflict of interest. Authors affiliated to GeneDx10 are employees of GeneDx, Inc., a wholly owned subsidiary of OPKO Health, Inc. S.W.S. holds the GlaxoSmithKline-CIHR Endowed Chair in Genome Sciences at the Hospital for Sick Children and University of Toronto.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

awz198_Supplementary_Data
Click here for additional data file. (738.2KB, zip)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

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