ANKLE2-related microcephaly: A variable microcephaly syndrome resembling Zika infection

Abstract

Objective: This study delineates the clinical and molecular spectrum of ANKLE2-related microcephaly (MIC), as well as highlights shared pathological mechanisms between ANKLE2 and the Zika virus.

Methods: We identified 12 individuals with MIC and variants in ANKLE2 with a broad range of features. Probands underwent thorough phenotypic evaluations, developmental assessments, and anthropometric measurements. Brain imaging studies were systematically reviewed for developmental abnormalities. We functionally interrogated a subset of identified ANKLE2 variants in Drosophila melanogaster.

Results: All individuals had MIC (z-score ≤ -3), including nine with congenital MIC. We identified a broad range of brain abnormalities including simplified cortical gyral pattern, full or partial callosal agenesis, increased extra-axial spaces, hypomyelination, cerebellar vermis hypoplasia, and enlarged cisterna magna. All probands had developmental delays in at least one domain, with speech and language delays being the most common. Six probands had skin findings characteristic of ANKLE2 including hyper- and hypopigmented macules. Only one individual had scalp rugae. Functional characterization in Drosophila recapitulated the human MIC phenotype. Of the four variants tested, p.Val229Gly, p.Arg236*, and p.Arg536Cys acted as partial-loss-of-function variants, whereas the c.1421-1G>C splicing variant demonstrated a strong loss-of-function effect.

Interpretation: Deleterious variants in the ANKLE2 gene cause a unique MIC syndrome characterized by congenital or postnatal MIC, a broad range of structural brain abnormalities, and skin pigmentary changes. Thorough functional characterization has identified shared pathogenic mechanisms between ANKLE2-related MIC and congenital Zika virus infection. This study further highlights the importance of a thorough diagnostic evaluation including molecular diagnostic testing in individuals with MIC.

Figure 1

Pedigrees of all families with ANKLE2 variants. Family pedigrees and segregation of identified ANKLE2 variants shown in respective probands including parents. Shaded icons represent affected individuals. Gene name, nucleotide changes, protein changes, and respective variants (underlined) are indicated. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

Graph plot of growth measurements of probands with ANKLE2 variants. Available OFC (A) and stature (B) measurements at birth and subsequent clinical evaluations are plotted with the age in months on the x‐axis and z‐scores on the y‐axis. Most affected probands had congenital microcephaly (OFC z‐scores ≤ −2) and all probands for whom postnatal OFC measurements were available had severe microcephaly (OFC z‐scores ≤ −3). Microcephaly was notably progressive in nature in all affected probands. OFC, occipitofrontal circumference. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Brain imaging in ANKLE2‐related microcephaly. Select brain MR images of individuals with ANKLE2 variants are shown. (A and B) (proband 1), diffuse and severe undersulcation of the cortical gyral pattern with thin‐normal cortical thickness (arrows), increased extra‐axial space (asterisks), severe white matter involvement, dysplastic ventricles with complete agenesis of the corpus callosum with open communication between the trigones and medial extra‐axial space; (C and D) (proband 5), diffuse simplification and undersulcation of the cortical gyral pattern, partial agenesis of the corpus callosum (arrowhead), cerebellar vermis hypoplasia (arrow) (limited images available); (E and F) (proband 6), diffuse simplification of the cortical gyral pattern with severely foreshortened frontal lobes, very short and thick corpus callosum with partial agenesis (arrowhead), relatively preserved brainstem and cerebellum; (G and H) (proband 7), similar appearance as the sibling (proband 6) with diffuse simplification of the cortical gyral pattern, partial agenesis of the corpus callosum (arrowhead), and relatively preserved brainstem and cerebellum; (I and J) (proband 8), mild simplification of the cortical gyral pattern with mild foreshortening of the frontal lobes and mildly thin corpus callosum; (K and L) (proband 9), diffuse and severe simplification of the cortical gyral pattern (arrows), diffuse white matter abnormalities, mildly increased extra‐axial spaces; (M and N) (proband 10), diffuse severe simplification of the cortical gyral pattern (arrows), severe foreshortening of the frontal lobes, partial agenesis of the corpus callosum (arrowhead), relatively preserved brainstem and cerebellum; (O and P) (proband 11), mildly thin corpus callosum, with mild simplification of the cortical gyral pattern; (Q and R) (proband 12), diffusely simplified gyral pattern with paucity of the sulci in various areas, mild reduction in supratentorial white matter and a cisterna magna; (S and T) (normal), normal mid‐sagittal and axial images.

Figure 4

Clinical photographs of individuals with ANKLE2 variants. Proband 9 at 4 months (A–D) and 23 months of age (E–H). (A and B) frontal and lateral profile of the head at 4 months demonstrating apparent microcephaly with a low‐sloping forehead. (C) anterior view of the chest demonstrating hypopigmented and hyperpigmented macules. (D and H) full length photographs at 4 and 23 months further demonstrating skin pigmentary abnormalities and apparent microcephaly. (E and F) frontal and lateral profile of the head at 23 months. (G) anterior view of the chest demonstrating hypopigmented and hyperpigmented macules. Proband 11 at 10 months (I and J) and 20 months of age (K–M). (I and J) frontal and lateral profile of the head at 10 months demonstrating a narrow skull shape with prominent supraorbital ridges. (K and L) frontal and oblique profile of the head at 24 months demonstrating a mildly sloping forehead. (M) mid‐length body photograph demonstrating apparent microcephaly. Proband 1 (N–Q). Multiple hypopigmented and hyperpigmented macules located on (N) anterior chest, (O) back, (P) dorsal hand, (Q) posterior thigh. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5

Diagram of the ANKLE2 protein and identified variants. Diagram of the ANKLE2 protein with the most important functional domains and the identified ANKLE2 variants in this manuscript. ANK, ankyrin repeats; Cauli‐VI, Caulimovirus viroplasmin VI domain; LEM, Lap2, Emerin, MAN1 domain; TMD, transmembrane domain. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6

Functional modeling of ANKLE2 variants recapitulates microcephaly phenotypes in Drosophila melanogaster. (A–F) Bright field images of third instar larval brains from (A) Ankle2 ^A , da‐hANKLE2 wt, (B) Ankle2 ^A , (C) Ankle2 ^A , da‐hANKLE2 p.Val229Gly, (D) Ankle2 ^A , da‐hANKLE2 p.Arg236*, (E) Ankle2 ^A , da‐hANKLE2 p.Arg536Cys, (F) Ankle2 ^A , da‐hANKLE2 c.1421‐1G>C. (G) Brain volume quantification of animals from (A–F). One‐way ANOVA with post hoc test. *p < 0.05, **p < 0.01, ****p < 0.0001.