Deregulated expression of EZH2 in congenital brainstem disconnection

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Congenital brainstem disconnection (CBSD) is an enigmatic embryo‐fetal defect presenting as (sub)total absence of a segment between mesencephalon and lower brainstem. Rostro‐caudal limits of the defect vary while the basal pons is always involved and the cerebellum is globally hypoplastic. A recent update and review 1 lists 14 cases, including three brain autopsy studies 1. Necrosis and glial or inflammatory reactions were absent. Inferior olivary nuclei were small or absent, pontine nuclei depleted and the cerebellar dentate nuclei dysplastic. Supra‐tentorial parts were normal in size, shape and microscopic structure. Frequent associated findings were vertebral segmentation defects (4/14) and hydronephrosis (3/14). No intra‐familial recurrence or consanguinity was recorded.
We considered interference with the developing rhombencephalon by an epigenetic mechanism as possible cause of CBSD. We probed the role of PRC2 (Polycomb Repressive Complex 2), member of the polycomb group of chromatin modifying proteins (PcG) with a cell‐fate conserving function in organ development 4. PRC2 acts by promotor binding as well as by modification of histone H3 through its catalytic subunit EZH2 (Enhancer of Zeste2) that tri‐methylates the free ending tail of H3 at lysine 27 to H3K27me3 (with H3K27me2 as intermediate step). H3K27me3 blocks transcription of developmental genes to consolidate the mature stage of cell lineage on completed development 7. Advantageous use can be made of anti‐H3K27me3 antibody staining of nuclear chromatin on routinely prepared autopsy tissue sections to study EZH2 activity, an application more commonly used in neoplastic studies 8, but not routinely applied in human malformations. The absence of immunoreactivity in a developmental setting is due to immaturity, as in embryonic stem cells, or to an abnormal persistence (or even reversal) to that state with loss of control over tissue‐specific development. A large number of genes are known to undergo maturational silencing by histone modification. For example, EZH2 stabilizes the identity of individual vertebral body segments as well as brainstem development by silencing HOX genes 9. Another role for EZH2, sustaining normal pontine neuron migration from the embryonic rhombic lip to the mature site of function has been identified by rhombomere‐specific knockout of EZH2 in mouse embryos 11. This finding prompted this study.
Antibody staining to H3K27me3 on routinely prepared autopsy material was applied in this study to probe a possible role for EZH2. In situ hybridization of EZH2 mRNA was used to monitor EZH2 transcription at sites of negative H3K27me3 staining. Antibody staining to histone H3K14ac, which is independent of EZH2 was used as tissue quality control for histone staining.
We studied a new patient (case 1) and a previously published patient with CBSD 1 (case 2 in this report) for local involvement of EZH2. We included brain material from the first reported autopsy of pontine tegmental cap dysplasia (PTCD) 12 (case 3) because characteristic aspects of brainstem morphology in PTCD are similar to CBSD 12. Prominent features linking these three patients are summarized in Table 1.
Clinical and neuropathological findings of the three patients are as follows.
Patient 1 is the second male child of nonconsanguineous parents of European ancestry. Birth at 35 weeks was normal following an uncomplicated pregnancy. Birthweight was 2945 g, length 48.5 cm, head circumference 34 cm. Breathing was spontaneous. He had an absent gag reflex and hypertonia of jaw muscles. Radiographs showed multiple segmentation defects of the ribs and spinal column (Figure 1D). MRI of the brain on day 3 showed a defect separating the tegmentum from the ventral pons (Figure 1A,B) at the exit level of the trigeminal nerves. The cerebellum was normally shaped but hypoplastic (Figure 1C). He went home with tube feeding.
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