Radial Microbrain (Micrencephaly) Is Caused by a Recurrent Variant in the RTTN Gene

Abstract

Background and objectives: Genetic primary microcephaly (PM) is a defect in early brain development leading to congenital microcephaly, mostly recessively inherited, and mild-to-moderate intellectual disability. PM has been largely elucidated, thanks to exome and genome sequencing. However, radial microbrain, the most severe form of genetic PM or micrencephaly described in the 1980s, which leads to early lethality or very severe intellectual handicap, remains without a molecular diagnosis. We sought to identify the cause of radial microbrain by analyzing the genotype of children/adults and fetuses with an extremely small brain.

Methods: We searched for individuals with the smallest head circumference among patients with a confirmed diagnosis of PM included in 2 French and European observational studies coordinated at the Robert Debré Children's Hospital in Paris. Their neurodevelopment and brain imaging were analyzed, as well as next-generation sequencing for a panel of microcephaly genes or exome sequencing. Neuropathologic and immunohistologic analyses of extremely severe microcephalic fetal brains and stage-matched controls were performed. A nonparametric test and Mann-Whitney post-test were used to compare the cortical thickness between groups.

Results: We identified 5 individuals (4 female patients, 7 years 10 months-19 years) with a particularly small brain among a series of 50, all suffering from a severe neurodevelopmental disorder with no ability to communicate verbally and, in 3 of them, no ability to walk. Genetic analysis revealed in all individuals the presence of the same homozygous variant c.2953A>G (p.R985G) in the RTTN gene (ROTATIN). The same variant was found in 2 fetuses whose neuropathologic evaluation showed a major reduction in the thickness of the ventricular zone and neuronal heterotopias. The cortical plate was reduced by 70% compared with controls, irrespective of the region considered. Immunostaining with vimentin showed a 50% loss of radial glial columns, characteristic of radial microbrain.

Discussion: Our data show that the homozygous c.2953A>G substitution in RTTN is a recurrent variant responsible for radial microbrain, the most severe form of primary microcephaly. Our combined neurologic, imaging, and histopathologic approaches provide a better understanding of the severity of this condition and its prognosis.

Trial registration information: ClinicalTrials.gov number: NCT01565005.

Figure 1. Clinical and Imaging Data From Patients Carrying the c.2953A>G Variant in the RTTN Gene Showing the Severity of Their Phenotype Compared With the Reported Patients

(A) Head circumference at follow-up of the 50 patients with primary microcephaly enrolled in the Microfanc and Euromicro research projects. Red dots indicate head circumference <−10 SD in the 5 patients included in this study. (B and C) HC (B) and height (C) of reported patients with RTTN-related phenotypes. The empty round symbol represents individuals carrying the c.2953A>G variant in the RTTN gene: previously reported patients in black and those in our series in red. (D) Brain MRI with axial, coronal, and sagittal T2-weighted images performed in the third trimester of pregnancy showing the lissencephalic appearance of the cortex at WG 32 (D1-3), the hypoplasia of the corpus callosum, and the interhemispheric cyst (D4). (E) Brain MRI with axial and coronal T1-weighted (E1-2) and coronal and sagittal T2-weighted images (E3-4) performed at the age of 6 showing a simplified gyral pattern of the cortex (E1-3), periventricular neuronal heterotopia, hypoplasia of the corpus callosum, and an interhemispheric cyst (E4). (F) Brain MRI (F) with axial and coronal FLAIR (F1-2) and coronal and sagittal T2-weighted images (F3-4) performed at the age of 14 showing a simplified gyral pattern of the cortex and periventricular neuronal heterotopia (F1-3).

Figure 2. Neuropathologic Analysis of Fetuses Carrying the c.2953A>G Variants in the RTTN Gene Revealing Micrencephaly

Images of whole-mount histologic preparation (stained with Cresyl violet, imaged using a Leica microscope) of coronal sections. (A–L) Fetus carrying the homozygous c.2953A>G RTTN variant (G–L) at WG 16 and stage-matched control (A–F). Control: Staining of a coronal section of both hemispheres in the frontal lobe (A), frontoparietal area (B), parietal lobe (D), and occipital lobe (F). Box area zoom (images 1 and 2) (H&E staining ×40) showing normal separation of both thalami (C) and normal CP (E). RTTN: Staining of a coronal section of both hemispheres in the frontal lobe (G). The intermediate mass between the 2 thalami (H) and in the frontoparietal area (I) can be observed. The posterior corpus callosum hypoplasia responsible for a single posterior ventricle (I) and irregular VZ (J) can be observed. Box area zoom (H&E staining ×40) showing the thalamic fusion on midline with an intermediate mass between the 2 hypoplastic thalamic nuclei (H) and irregular VZ with periventricular heterotopia (J and K). (M–AA) Fetus carrying the homozygous c.2953A>G RTTN variant (T–AA) at WG 21 and stage-matched control (M–S). Control: Staining of a coronal section of the left hemisphere in the frontal lobe (M), frontoparietal area (O), parietal lobe (Q), and occipital lobe (S). Box area zoom (H&E staining ×40) showing normal CP (P) and normal VZ (R). RTTN: Staining of a coronal section of both hemispheres in the frontal lobe (T), frontoparietal area (V), parietal lobe (Y), and occipital lobe (AA). Box area zoom (H&E staining ×40) showing a reduction in CP thickness (U), a thalamic fusion on midline with an intermediate mass between the 2 thalami (W and X), and irregular VZ with periventricular heterotopia (Z). Scale bars for A–B, D, F, G, I, L, M, O, Q, S-T, V, Y, AA: 5 mm; for C, E, H, J, N, P, R, U, W, X, Z: 500 µm; and for K: 100 µm.

Figure 3. Histopathologic Analysis Illustrating the Significant Thickness Reduction in the Ventricular Zone and Cortical Plate in the Fetus Carrying the c.2953A>G RTTN Variant

(A–D) Cresyl violet staining of coronal sections of the frontal and parietal cortex of the WG 16 fetus carrying the homozygous c.2953A>G variant in the RTTN gene showing the severe reduction of the cortical plate (A–B) and the VZ (C–D) thickness compared with the stage-matched control (p < 0.05, nonparametric test, Mann-Whitney post-test). (E–F) Cresyl violet staining of coronal sections of the parietal and occipital cortex of the WG 21 fetus carrying the homozygous c.2953A>G variant in the RTTN gene showing the strong reduction of the cortical plate compared with the stage-matched control (p < 0.0001, Welch t test). Scale bar: 100 µm.

Figure 4. The c.2953A>G Variants in the RTTN Gene Cause a Loss of Radial Glial Units Underlying the Radial Microbrain

Immunohistochemistry against vimentin of coronal sections of the frontoparietal cortex of the WG 16 fetus carrying the homozygous c.2953A>G variant in the RTTN gene (A–C) showing the significant loss of radial glial fibers compared with the stage-matched control (p = 0.0009, unpaired t test, Figure 3D). Scale bar: 500 µm. Quantification of the number of glial columns per surface unit (66-mm² rectangle, 400-µm length).