After the pathologic examination, a gene sequencing panel targeting genes involved in tubulinopathies was performed. In the TUBB3 gene, we found a heterozygotic variant of unknown significance (c.156C>G (p.Asn52Lys) in the exon 2) but most likely pathogenic due to the abnormal brain findings. This variant was not found in the parents' genome, and was therefore de novo.  Our final diagnosis was therefore a TUBB3-related Tubulinopathy (OMIM #614039).

Discussion

Tubulins are proteins composed of α and β-tubulin monomers, resulting in blocks of microtubules. Microtubules are part of the cytoskeleton and are involved in brain embryogenesis through their role in mitosis, intracellular transport, cell migration and neuron morphology. Mutations in the genes of tubulins TUBA1A, TUBB2A, TUBB2B, TUBB3, TUBB (TUBB5), or TUBG1 can affect microtubules stability, thus disrupt neuronal division and migration, leading to brain malformations. Most pathological mutations are de novo [2]. The spectrum of tubulinopathies consists of non-syndromic cerebral malformations characterized by the association of cortical dysplasia and pontocerebellar hypoplasia, manifesting with global developmental delay, mild to severe intellectual disability, axial hypotonia, strabismus, nystagmus and occasionally, seizures and optic nerve hypoplasia [2].

Postnatal MRI of the brain reveals various malformations, including frontally predominant microgyria, gyral disorganization and simplification, dysmorphic and hypertrophic basal ganglia, cerebellar vermis dysplasia, brainstem/corpus callosum hypoplasia, and/or olfactory bulbs agenesis. Some authors try to delineate genotype-phenotype correlation depending on the tubulin gene involved [2,3], although there are overlapping features and variability in severity of the spectrum. Among this wide range of possible brain anomalies, fused basal ganglia is considered by many as the most specific imaging sign suggestive of tubulinopathies [3], a "hallmark" found in roughly 75% of tubulinopathy cases. However, this sign is not visible on prenatal MRI due to its lack of resolution, and not visible on prenatal ultrasound due to its inability to differentiate basal ganglia from white matter. Currently, it is only assessed on postnatal MRI. More recently Arrigoni et al. described another finding that is highly suggestive of tubulinopathy: asymmetric dysgenesis of the brainstem [4]. Prenatal ultrasound alone cannot reliably assess brainstem anomalies due to its anatomic location, while MRI can. Asymmetric appearance of the brainstem can also be found in isolated clastic events, which may sometimes be confusing. In the setting of prenatal diagnosis, the first step in raising the possibility of a tubulinopathy is to perform a fetal brain MRI. Cabet et al reported prenatal imaging features of the brain associated with tubulinopathies [1]. In five affected individuals from unrelated families with de novo heterozygous variants in tubulin genes (TUBA1A, TUBB2B, or TUBB3), they identified two distinct prenatal imaging patterns:

1. The severe prenatal form is characterized by voluminous germinal matrices, microlissencephaly, and a kinked brainstem.
2. A mild prenatal form is associated with non-specific features, including an asymmetric brainstem, distortion of the anterior part of the interhemispheric fissure with subsequent impacted medial borders of the frontal lobes, callosal dysgenesis, and a lack of Sylvian fissure operculation. In the absence of additional extra-cerebral anomalies, the combination is highly suggestive of tubulinopathy.

Our case of TUBB3-related Tubulinopathy (OMIM #614039) [5,6] is a mild form, though it did not feature a corpus callosum anomaly or a lack of Sylvian operculation.

The differential diagnosis for brainstem anomalies visible on prenatal MRI include:

1. Infectious foetopathies in the appropriate setting (for example microcephaly with pontocerebellar brainstem hypoplasia induced by Zika virus)
2. 5p-deletion (cri-du-chat or cat's cry syndrome)
3. True primitive pontocerebellar dysplasia described and classified by Barth PG (type 2 and type 4 linked to TSEN54 gene mutations are the most commonly identified prenatally)
4. Kinked brainstem in Walker-Warburg syndrome (for example with type 2 lissencephaly also known as "overmigration")
5. Brainstem dysplasia in KIAA1109 gene mutation which is often associated with arthrogryposis, heart and ocular defects
6. The recently described entity "pontine tegmental cap dysplasia"
7. Some congenital muscular dystrophies
8. Some metabolic conditions such as Congenital disorders of glycosylation

To add complexity to the physiopathology, TUBB3 mutations can also lead to a completely distinct phenotype called Congenital Fibrosis of Extraocular muscles 3A [6].

There is no curative treatment for tubulinopathies. Management is supportive and tailored to the severity of symptoms and disabilities. Individuals with severe symptoms may die at a young age as a result of complications, such as seizures or pneumonia [2].

References

[1] Cabet S, Karl K, Garel C, et al. Two different prenatal imaging cerebral patterns of tubulinopathy. Ultrasound Obstet Gynecol. 2021 Mar;57(3):493-497.
[2] Bahi-Buisson N, Maillard C. Tubulinopathies Overview. 2016 Mar 24 [Updated 2021 Sep 16]. In: Adam MP, Ardinger HH, Pagon RA, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2021. Available from: https://www.ncbi.nlm.nih.gov/books/NBK350554/
[3] Bahi-Buisson N, Poirier K, Fourniol F, et al. The wide spectrum of tubulinopathies: what are the key features for the diagnosis? Brain. 2014 Jun;137(Pt 6):1676-700. 
[4] Arrigoni F, Romaniello R, Peruzzo D, et al. The spectrum of brainstem malformations associated to mutations of the tubulin genes family: MRI and DTI analysis. Eur Radiol. 2019 Feb;29(2):770-782.
[5] Kniffin CL. "Cortical dysplasia, complex, with other brain malformations 1; CDCBM1 #614039" OMIM. https://www.omim.org/entry/614039, Publish date 6/2011.
[6] Rasooly RS. "Tubulin, BETA-3; TUBB3 #602661." OMIM. https://omim.org/entry/602661, Publish date 5/1998.

I especially want to thank Dr Catherine Garel and Pr Laurent Guibaud for their speech on fetal brainstem pathologies at the fetal medicine congress in Montpellier in November 2019. Without their presentation, I honestly would not have thought about a tubulinopathy at this early prenatal stage. They particularly reported cases of tubulinopathies with prenatal MRI that had striking similar findings. I also thank them for their kind advice to our team on this peculiar case.

Thanks also to Dr Khung Savatovsky for keeping in touch with great pathology images.