To date, only seven EIF2B mutated patients from four unrelated families affected by congenital form of VWMD with antenatal onset have been reported [3, 7]. Antenatal symptoms consisted in severe growth restriction, oligohydramnios and decreased foetal movements or arthrogryposis. Microcephaly was present at birth in one neonate whereas progressive cerebellar atrophy and delayed gyration appeared later on MRI in all cases. In some patients with early disease onset, involvement of other organs has been reported such as hepatosplenomegaly, pancreatic abnormalities, kidney hypoplasia, ovary dysgenesis resulting in premature ovarian failure and cataracts [8]. Intriguingly, no visceral lesions were identified in the two foetuses at autopsy. Furthermore, no cataracts were present and GFAP immunohistochemistry did not reveal any lesion of Muller glia. Neuropathological examination performed in two affected infants with antenatal onset aged 8 and 10 months disclosed severe white matter loss in the cerebral hemispheres, pons, cerebellum and spinal cord with a diffuse decrease in oligodendrocyte number and no reactive gliosis. As in our cases, periventricular pseudo-cysts were reported [8, 9]. In a neuropathological review of affected children by Bugiani et al. in 2018, white matter rarefaction usually appears after birth but cystic degeneration of the deep white matter appears later, contrary to our cases in which cavitated lesions were present from the second trimester [10]. Histologically, an increased number of oligodendrocytes in the hemispheric white matter despite myelin paucity has been reported in children’s or adult brains [11] conversely to infants’ brains in which mature oligodendrocyte numbers appear to be normal, a finding that could not be confirmed in both foetuses as mature, myelinating oligodendrocytes are mostly produced 30 WG onward. Nevertheless, immunohistochemical studies revealed that at 32 WG the cerebellar white matter mainly contained oligodendrocyte precursors instead of maturing oligodendrocytes, resulting rather from delayed than defective oligodendrocyte maturation, in so far as normal numbers of mature oligodendrocytes have been reported from birth to childhood [4, 9, 10]. Immunohistochemical analyses also revealed severe astrocytic lesions in the cerebellar white matter. Dysmorphic astrocytes presented severely altered morphologies consisting in smaller size, thin or coarse and short processes. Moreover, they still expressed nestin, indicating that they remained at an astroglial progenitor stage, as previously shown in a mouse model by Dooves et al. [12, 13]. Such alterations have also been recently described using patient-derived induced pluripotent stem cells (iPSCs). VWMD iPSCs-derived astrocytes expressed nestin as in the present cases, and the mean length of the longest processes was diminished by a factor of 2. Astrocytes also overexpressed alpha-B-crystallin contrary to foetal astrocytes in which no accumulation was visualized, an event that probably occurs after birth [4, 14]. Besides, it has been shown in several previous studies that astrocytes influence oligodendrocyte differentiation and maturation. In a recent study on human and mouse VWM iPSCs, Leferink et al. demonstrated that the white matter astrocytic subpopulation is selectively vulnerable to VWM mutations and that oligodendrocyte precursor cell maturation is inhibited by VWM astrocytes [15]. These findings very likely explain the over-representation of PDGFRα-positive oligodendrocyte precursors observed in our cases, resulting from impaired astrocyte differentiation and functioning. Moreover, it has been shown that in older VWMD affected patients, Bergmann glial somata are translocated to the molecular layer whereas they should be located close to Purkinje cells, this translocation representing a hallmark of VWMD for Dooves et al. [13, 16]. This disease marker has also been reported in a spontaneous mutant mouse in the Eif2b5 gene called the “toy mouse” [17]. In the two foetuses, virtually absent Bergmann glia somata and processes suggested a defect of Bergmann glia development and/or maintenance from early stages of gestation. Even if a specific granular neuron loss could not be evaluated in our cases as these cells are mainly generated from the 30th WG to 13 post-natal months, the transient external granular cell layer as well as Purkinje cell and internal granular cell layers were poorly developed with almost absent stellate and Golgi II cells, suggesting that EIF2B5 is not only required for astrocyte and oligodendrocyte generation and differentiation [18] but also for the production, migration and survival of cerebellar neurons resulting in cerebellar hypoplasia.
More than 80 different pathogenic amino acid changes of EIF2B5 on 67 different positions have been reported in patients with VWMD (Fig. 3a) according to the HGMD Pro [7] and Clinvar databases [19] (Supplementary information S2). The average normalized Consurf conservation score on 150 species of the amino acids affected by pathogenic variants is − 0,434, that means that mutated residues are more conserved than other EIF2B5 residues. However, as previously reported, the range of scores (min − 1421, max 1563) indicates that several mutated residues are not conserved [16, 20]. EIF2B5 variants are located across various domains of the protein with no clear hotspot. Consurf analysis shows that the p.Ile156 and p.Val389 amino acids which belong to different domains are conserved, with scores of − 0.918 and − 1.069 respectively (Fig. 3b). Concerning the p.Ile156 amino-acid, even if this position may be occupied depending on species by an Isoleucine or a Valine, two very similar hydrophobic amino acids (Grantham score: 29), the p.Ile156 may be considered as conserved. Besides, genotype-phenotype correlation studies have shown that in EIF2B related disorders, the severity of the disease directly depends on the nature of the EIF2B5 deleterious variants [21]. From these observations, it could be speculated that the disease severity observed in our foetal cases may be due to a specific effect of the two variants, especially the newly described p.Val389Met variant. But one cannot exclude that the extremely severe phenotype observed in our two siblings may be related to the impact of not yet identified modifier genes or of critical post-transcriptional regulators such as miRNAs. It could also be hypothesized that additional variants in other genes not detected using WES or random developmental processes involving other cellular pathways could be responsible for the phenotypic severity.
In conclusion, neuropathology findings together molecular data reported here further expand the phenotypic spectrum of VWMD which may occur early during gestation and may manifest from the second part of the pregnancy by defective Bergmann glia responsible for severe cerebellar hypoplasia.