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

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.

Keywords: WDFY3; brain size; intellectual disability; neurodevelopmental delay.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.