Neuroradiologic, Clinical, and Genetic Characterization of Cerebellar Heterotopia: A Pediatric Multicentric Study

DISCUSSION

CH has received poor attention in previous hindbrain malformation classifications.^8,16 Lack of awareness of this easily overlooked imaging finding may be the primary driver behind the lack of identification.

This retrospective multicenter study describes CH neuroradiologic patterns in a cohort of pediatric patients, clinically and genetically characterized. Such comprehensive evaluation of CH imaging features and associated clinical characteristics and genotypes represents a first attempt to highlight the importance of this finding in the diagnostic pathway. In fact, the neurodevelopmental and functional outcomes of several cerebellar malformations are far from being defined, and the phenotypic spectrum is often broad, ranging from normal or near-normal functioning to profound disability for a given malformation.¹⁷ Like in other brain regions, cerebellar neuronal migration relies on appropriate spatiotemporal patterns.¹⁸ The migration process takes place both prenatally and postnatally and is controlled by several molecules, leading to the establishment of elaborate compartments and circuitry.

Wright and colleagues⁹ in 2019 described CH as a recurrent finding in a cohort of 35 patients with CHARGE syndrome, with a prevalence of 77%. Moreover, CH has been previously reported in patients with trisomy 21 or trisomy 18 and is rarely associated with other genetic conditions such as Turner syndrome,^19,20 ornithine carbamoyltransferase deficiency,²¹ MKS3-related Meckel syndrome,²² occipital horn syndrome,²³ OPHN1-related syndrome,²⁴ and Fryns syndrome²⁵ in single patients. Importantly, cerebellar hemorrhages should be excluded since these could be mistaken for CH, especially in premature infants; in the presented cohort, there was no history of cerebellar hemorrhage, no imaging signs of blood products, and only 1 patient was born preterm.

In our cohort, clinical findings of patients with CHARGE syndrome are in line with the literature, showing a pattern of CH characterized by recurrent appearance and location. More precisely, the distribution pattern mainly resulted in a bilateral, symmetric, and peripheral subcortical disposition, typically located in the inferior portion of the cerebellar hemispheres.⁹ CHARGE syndrome is part of the CHD7-related disorder spectrum, caused by point mutations or deletions of the CHD7 gene (MIM *608892),²⁶ which is known to be involved in embryonic development. Indeed, Reddy and colleagues²⁷ demonstrated a critical role for CHD7 in the formation, differentiation, and migration of neural crests. In 2013, Yu et al²⁸ showed that reduced FGF8 expression, which is a critical signal for early cerebellar development, results from CHD7 haploinsufficiency and is responsible for cerebellar vermis hypoplasia, a common finding in CHARGE. Moreover, during earlier stages of cerebellar development, CHD7 regulates the accessibility, histone acetylation, and RNA polymerase II binding at gene enhancers implicated in cerebellar morphogenesis.²⁷ Collectively, these data suggest that CHD7 governs multiple phases of cerebellar development through the accurate regulation of gene transcription; folding and migration anomalies may arise from these deregulated cellular processes that consequently reorganize themselves. Thus, unsurprisingly, CH is well-represented in this genetic condition, together with vermian hypoplasia and cerebellar dysgenesis, which are other common findings.

In the present study, when CH was associated with other complex brain malformations outside the CHARGE spectrum, it was coarse, localized in the deep white matter, and distributed both in the superior and inferior portions of the cerebellum. Associated malformations involved both infra- and supratentorial brain.

A relevant contribution of the present study is the description of patients with isolated CH or CH combined with minor malformative/dysmorphic findings. In these cases, CH consistently showed peripheral subcortical localization in the inferior portion of cerebellar hemispheres, with either unilateral or bilateral distribution. Up to 35% of these patients had CC dysmorphisms, and a similar percentage showed vermian hypoplasia, mainly involving the inferior vermis. CC dysgenesis or dysmorphisms can often be found in association with other minor malformations or brain dysmorphisms, such as periventricular heterotopia²⁹ in patients with neurodevelopmental disorders and variable clinical presentation.

Inferior vermian hypoplasia (IVH) is characterized by a volumetric reduction of the inferior portion of the cerebellar vermis. Patients with isolated IVH have been reported to show delayed development, gross and fine motor disabilities, as well as social-communication deficits and behavioral problems.¹⁷

In our cohort, patients with isolated CH showed a high prevalence of developmental delay, with an even greater occurrence of language development delay. Unlike isolated cerebral heterotopias, which can be completely devoid of clinical correlates, CHs are thus likely to be associated with at least some degree of developmental delay.

Neurodevelopmental disorders were the most represented clinical diagnoses, including intellectual disability, ASD, and specific learning disorders. Recurring features in the cohort were also behavioral problems, including social skills impairment, and motor difficulties. Of note, specific cerebellar signs were observed only in 2 patients, and only 1 patient showed extra-neurologic malformations. Indeed, specific cerebellar signs were not expected to be determined by the presence of CH; conversely, the imaging finding of CH can provide further insight to comprehensively assess neurodevelopmental disorders, considering the impairment of specific cerebro-cerebellar circuits that may be relevant, for instance, to the development of ASD, language, and behavioral issues. According to the presented results, CHs are most often associated with at least some degree of developmental delay.

A direct comparison of clinical features of group A and group B was not carried out for a 2-fold reason. First, considering the complexity, localization, and extent of variability of associated brain malformations of group B as opposed to the recurrent finding of almost isolated CH in group A, we assume that in the former, major events of disruption are involved in the developmental process, thus unsurprisingly leading to composite and heterogeneous syndromic phenotypes. Moreover, group B was mainly represented by the group of CHARGE patients, who have a well-known spectrum of clinical features. The remaining 4 patients were then few, and with such a heterogeneous neuroimaging finding, a mere comparison with group A was deemed trivial.

The genetic etiology of many brain malformations remains poorly understood, and the yield of genetic testing has been widely reported to be low in patients with minor cerebellar malformative findings.³⁰ Nevertheless, access to standardized extensive genetic testing is not always available, and this could negatively bias this outcome. The same is applicable to our cohort, in which 76% of patients with isolated CH underwent some genetic testing, but a detailed assessment inclusive of whole exome sequencing was performed only in 35%, representing one considerable limitation of this study, being retrospective. Overall, a genetic diagnosis was reached in 23% of patients.

A novel de novo frameshift variant in the KDM6B gene was found in a patient with peripheral subcortical monolateral inferior CH and neurodevelopmental disorder. KDM6B (MIM *611577) pathogenic variants have been recently described as associated with a rare “neurodevelopmental disorder with coarse facies and mild distal skeletal abnormalities” syndrome.²⁹ Neuroradiologic features in only 3 single patients have been reported, showing mild cerebellar cortical and subcortical atrophy and/or paracerebellar ventricular enlargement.³¹ KDM6B is highly expressed in cerebellar neurons, where it plays an important role in neuronal migration during development, as well as in non-neuronal cells such as Bergmann glia, which constitute the scaffold for neuronal migration^32,33; moreover, the clinical features associated with this syndrome (eg, hypotonia, ASD, and attention deficit/hyperactivity disorder traits) represent a link to possible cerebellar circuits dysfunction. A de novo pathogenic variant in the ANKRD11 gene was found in 2 patients showing peripheral subcortical, bilaterally symmetric inferior CH associated with IVH and thick CC in one case and dysmorphic CC in the other. Pathogenic variants in ANKRD11 are responsible for KBG syndrome (MIM *611192), typically characterized by developmental delay, short stature, and characteristic dysmorphic findings. To date, only unspecific neuroradiologic defects have been reported in patients with KBG, such as white matter abnormalities, CC defects, cerebellar vermis hypoplasia,³⁴ cortical abnormalities including periventricular nodular heterotopia, and, only recently, CH.¹⁵ ANKRD11 encodes for a protein mainly expressed in neurons and glial cells of the developing brain, playing a crucial role in proliferative processes of cortical neural precursor cells.^35,36 Moreover, ANKRD11 contributes to the global regulation of transcription, possibly modulating the expression of other genes playing a role in the regulation of cortical development.³⁵ Among these genes, NCOR2 (MIM *600848) was reported to be co-expressed in Purkinje cells with ANKRD11 and CHD7. As for CHD7, ANKRD11 might also be implicated in potential molecular and cellular mechanisms by which chromatin remodelers contribute to brain morphogenesis during development and disease.³⁴

A third patient with peripheral subcortical, bilateral, inferior CH, who featured intellectual disability and epilepsy, was found to carry a de novo pathogenic variant in the PAK1 gene (MIM *618158) gene, which encodes a member of serine/threonine p21-activating kinase family that regulates cell motility and morphology and is expressed in the cerebellum as well.¹³ Previous reports on associated brain MRI findings included single descriptions of periventricular and subcortical white matter abnormalities. All these observations, along with the emerging literature linking cerebellar functions to neurodevelopmental disorders such as ASD,^13,37 prompt further studies to better understand the role of the above-mentioned genes in cerebellar development.

Three of the 4 non-CHARGE patients belonging to group B received a genetic diagnosis. One patient carried a de novo variant in the DYNC1H1 gene (MIM *614563), which encodes for a cytoplasmic dynein ubiquitously expressed in the brain and functions in intracellular motility. Neuroradiologic findings in patients with DYNC1H1 mutations include cerebellar hypoplasia and dysplasia.³⁸ A second patient had a complex clinical phenotype associated with trisomy 21. Cerebellar abnormalities are frequently observed in association with Down syndrome; trisomy 21 is linked with a delay in ciliogenesis, recognized as a cause of dysregulation of neuron outgrowth and cell migration.³⁹ The third patient had a complex chromosomal rearrangement involving chromosomes 4 and 13, leading to both neurologic and extra-neurologic multiple malformations.

A clear male predominance (78%) has been observed; nevertheless, as of this date, no X-linked mutations have been found. Even though it’s speculative, a possible explanation could be the presence of unknown regulatory genes on X chromosomes linked to cerebellar development.

In the overall cohort, CH showed a higher prevalence of inferior cerebellum localization, with a clear predominance in group A. Superior CH has been described in 4 patients, 3 of whom belong to group B. The paucity of patients may reflect the relative inferior prevalence of such a condition, although a precise mechanism for such an occurrence remains unknown. Patients with superior CH show a more severe neurodevelopmental phenotype, although genotype-phenotype correlations cannot be drawn given the reduced number of patients and the frequent association with other structural brain anomalies. One possible explanation of this finding is that in light of the different origins of superior and inferior cerebellum, distinct cell types arising from diverse subregions of a primordium can be affected by a developmental disruption, thus leading to a different contribution to the assembly of a complex 3D structure according to long fate genetic mapping of cell movements.⁴⁰ Alternatively, the clinical phenotype may be predominantly dependent on the genetic cause of the condition. Further studies investigating functional consequences of CH on brain function are needed. The genetic diagnostic yield was predictably higher in patients presenting with CH and other major brain malformations compared with isolated CH with or without associated brain dysmorphisms. Nevertheless, overall, considering the limited access to complete genetic testing for patients with isolated CH, who underwent WES in 35% of cases, the diagnostic yield registered in the latter group of patients appears to be relevant. Given the presented results, the presence of CH in association with developmental delay and or syndromic features, might thus represent a negative prognostic sign. Moreover, the presence of a superiorly located CH seems to correlate with a more severe clinical picture: this element might be relevant for counseling. Regarding the imaging protocol for detecting CH, we believe that T1-weighted sequences in the coronal plane often raise the initial suspicion of heterotopia, which should then be confirmed in at least 1 other plane. Therefore, 3D T1-weighted sequences with a maximum voxel size of 1 mm are highly beneficial for diagnosis. Additionally, assessing signal intensity on other sequences (particularly T2-weighted and, if available, FLAIR) is essential to establish isointensity of the anomalies relative to the cerebellar cortex. DWI sequences are especially helpful in differentiating CH from small gliotic lesions that may affect the cerebellum. While the morphology and signal characteristics of heterotopias on standard morphologic sequences are often highly indicative of CH, DWI can provide additional specificity, for instance, in cases of gliosis where T1 and T2 signal characteristics might overlap with those expected for gray matter. It is important to emphasize that due to the often millimetric size of CHs, evaluation in multiple planes is essential to avoid missing these anomalies.

One of the main limitations of this study is indeed the incomplete availability of genetic testing in the overall cohort. Furthermore, the small sample size and retrospective study design preclude a detailed correlation of CH with neurologic deficits, and therefore, this association does not necessarily imply causation. Another potential limitation is the lack of advanced MRI techniques to help explore the impact of CH on the overall architecture of the brain. The acquisition of high-resolution DWI data and the use of advanced modeling methods (like constrained spherical deconvolution) have provided interesting information on the reorganization of white matter bundles in many supratentorial malformations⁴⁰ and could potentially be applied to this cohort to unveil structural modifications of the white matter, which were not detected by standard anatomic sequences.