Brain mosaicism of hedgehog signalling and other cilia genes in hypothalamic hamartoma.

Hypothalamic hamartoma (HH) is a rare benign developmental brain lesion commonly associated with a well characterized epilepsy phenotype. Most individuals with HH are non-syndromic without additional developmental anomalies nor a family history of disease. Nonetheless, HH is a feature of Pallister-Hall (PHS) and Oro-Facial-Digital Type VI (OFD VI) syndromes, both characterized by additional developmental anomalies. Initial genetic of analysis HH began with syndromic HH, where germline inherited or de novo variants in GLI3, encoding a central transcription factor in the sonic hedgehog (Shh) signalling pathway, were identified in most individuals with PHS. Following these discoveries in syndromic HH, the hypothesis that post-zygotic mosaicism in related genes may underly non-syndromic HH was tested. We discuss the identified mosaic variants within individuals with non-syndromic HH, review the analytical methodologies and diagnostic yields, and explore understanding of the functional role of the implicated genes with respect to Shh signalling, and cilia development and function. We also outline future challenges in studying non-syndromic HH and suggest potential novel strategies to interrogate brain mosaicism in HH.

WWOX developmental and epileptic encephalopathy: Understanding the epileptology and the mortality risk.

OBJECTIVE: WWOX is an autosomal recessive cause of early infantile developmental and epileptic encephalopathy (WWOX-DEE), also known as WOREE (WWOX-related epileptic encephalopathy). We analyzed the epileptology and imaging features of WWOX-DEE, and investigated genotype-phenotype correlations, particularly with regard to survival. METHODS: We studied 13 patients from 12 families with WWOX-DEE. Information regarding seizure semiology, comorbidities, facial dysmorphisms, and disease outcome were collected. Electroencephalographic (EEG) and brain magnetic resonance imaging (MRI) data were analyzed. Pathogenic WWOX variants from our cohort and the literature were coded as either null or missense, allowing individuals to be classified into one of three genotype classes: (1) null/null, (2) null/missense, (3) missense/missense. Differences in survival outcome were estimated using the Kaplan-Meier method. RESULTS: All patients experienced multiple seizure types (median onset = 5 weeks, range = 1 day-10 months), the most frequent being focal (85%), epileptic spasms (77%), and tonic seizures (69%). Ictal EEG recordings in six of 13 patients showed tonic (n = 5), myoclonic (n = 2), epileptic spasms (n = 2), focal (n = 1), and migrating focal (n = 1) seizures. Interictal EEGs demonstrated slow background activity with multifocal discharges, predominantly over frontal or temporo-occipital regions. Eleven of 13 patients had a movement disorder, most frequently dystonia. Brain MRIs revealed severe frontotemporal, hippocampal, and optic atrophy, thin corpus callosum, and white matter signal abnormalities. Pathogenic variants were located throughout WWOX and comprised both missense and null changes including five copy number variants (four deletions, one duplication). Survival analyses showed that patients with two null variants are at higher mortality risk (p-value = .0085, log-rank test). SIGNIFICANCE: Biallelic WWOX pathogenic variants cause an early infantile developmental and epileptic encephalopathy syndrome. The most common seizure types are focal seizures and epileptic spasms. Mortality risk is associated with mutation type; patients with biallelic null WWOX pathogenic variants have significantly lower survival probability compared to those carrying at least one presumed hypomorphic missense pathogenic variant.

Sporadic hypothalamic hamartoma is a ciliopathy with somatic and bi-allelic contributions.

Hypothalamic hamartoma with gelastic seizures is a well-established cause of drug-resistant epilepsy in early life. The development of novel surgical techniques has permitted the genomic interrogation of hypothalamic hamartoma tissue. This has revealed causative mosaic variants within GLI3, OFD1 and other key regulators of the sonic-hedgehog pathway in a minority of cases. Sonic-hedgehog signalling proteins localize to the cellular organelle primary cilia. We therefore explored the hypothesis that cilia gene variants may underlie hitherto unsolved cases of sporadic hypothalamic hamartoma. We performed high-depth exome sequencing and chromosomal microarray on surgically resected hypothalamic hamartoma tissue and paired leukocyte-derived DNA from 27 patients. We searched for both germline and somatic variants under both dominant and bi-allelic genetic models. In hamartoma-derived DNA of seven patients we identified bi-allelic (one germline, one somatic) variants within one of four cilia genes-DYNC2I1, DYNC2H1, IFT140 or SMO. In eight patients, we identified single somatic variants in the previously established hypothalamic hamartoma disease genes GLI3 or OFD1. Overall, we established a plausible molecular cause for 15/27 (56%) patients. Here, we expand the genetic architecture beyond single variants within dominant disease genes that cause sporadic hypothalamic hamartoma to bi-allelic (one germline/one somatic) variants, implicate three novel cilia genes and reconceptualize the disorder as a ciliopathy.

Bi-allelic SMO variants in hypothalamic hamartoma: a recessive cause of Pallister-Hall syndrome.

Pallister-Hall syndrome, typically caused by germline or de novo variants within the GLI3 gene, has key features of hypothalamic hamartoma and polydactyly. Recently, a few similar cases have been described with bi-allelic SMO variants. We describe two siblings born to non-consanguineous unaffected parents presenting with hypothalamic hamartoma, post-axial polydactyly, microcephaly amongst other developmental anomalies. Previous clinical diagnostic exome analysis had excluded a pathogenic variant in GLI3. We performed exome sequencing re-analysis and identified bi-allelic SMO variants including a missense and synonymous variant in both affected siblings. We functionally characterised this synonymous variant showing it induces exon 8 skipping within the SMO transcript. Our results confirm bi-allelic SMO variants as an uncommon cause of Pallister-Hall syndrome and describe a novel exon-skipping mechanism, expanding the molecular architecture of this new clinico-molecular disorder.

Infantile-onset myoclonic developmental and epileptic encephalopathy: A new RARS2 phenotype.

Recessive variants in RARS2, a nuclear gene encoding a mitochondrial protein, were initially reported in pontocerebellar hypoplasia. Subsequently, a recessive RARS2 early-infantile (<12 weeks) developmental and epileptic encephalopathy was described with hypoglycaemia and lactic acidosis. Here, we describe two unrelated patients with a novel RARS2 phenotype and reanalyse the published RARS2 epilepsy phenotypes and variants. Our novel cases had infantile-onset myoclonic developmental and epileptic encephalopathy, presenting with a progressive movement disorder from 9 months on a background of normal development. Development plateaued and regressed thereafter, with mild to profound impairment. Multiple drug-resistant generalized and focal seizures occurred with episodes of non-convulsive status epilepticus. Seizure types included absence, atonic, myoclonic, and focal seizures. Electroencephalograms showed diffuse slowing, multifocal, and generalised spike-wave activity, activated by sleep. Both patients had compound heterozygous RARS2 variants with likely impact on splicing and transcription. Remarkably, of the now 52 RARS2 variants reported in 54 patients, our reanalysis found that 44 (85%) have been shown to or are predicted to affect splicing or gene expression leading to protein truncation or nonsense-mediated decay. We expand the RARS2 phenotypic spectrum to include infantile encephalopathy and suggest this gene is enriched for pathogenic variants that disrupt splicing.

Identification of a recurrent mosaic KRAS variant in brain tissue from an individual with nevus sebaceous syndrome.

Nevus sebaceous syndrome (NSS) is a rare, multisystem neurocutaneous disorder, characterized by a congenital nevus, and may include brain malformations such as hemimegalencephaly or focal cortical dysplasia, ocular, and skeletal features. It has been associated with several eponyms including Schimmelpenning and Jadassohn. The isolated skin lesion, nevus sebaceous, is associated with postzygotic variants in HRAS or KRAS in all individuals studied. The RAS proteins encode a family of GTPases that form part of the RAS/MAPK signaling pathway, which is critical for cell cycle regulation and differentiation during development. We studied an individual with nevus sebaceous syndrome with an extensive nevus sebaceous, epilepsy, intellectual disability, and hippocampal sclerosis without pathological evidence of a brain malformation. We used high-depth gene panel sequencing and droplet digital polymerase chain reaction (PCR) to detect and quantify RAS/MAPK gene variants in nevus sebaceous and temporal lobe tissue collected during plastic and epilepsy surgery, respectively. A mosaic KRAS c.34G > T; p.(Gly12Cys) variant, also known as G12C, was detected in nevus sebaceous tissue at 25% variant allele fraction (VAF), at the residue most commonly substituted in KRAS Targeted droplet digital PCR validated the variant and quantified the mosaicism in other tissues. The variant was detected at 33% in temporal lobe tissue but was absent from blood and healthy skin. We provide molecular confirmation of the clinical diagnosis of NSS. Our data extends the histopathological spectrum of KRAS G12C mosaicism beyond nevus sebaceous to involve brain tissue and, more specifically, hippocampal sclerosis.

Somatic mutation: The hidden genetics of brain malformations and focal epilepsies.

Over the past decade there has been a substantial increase in genetic studies of brain malformations, fueled by the availability of improved technologies to study surgical tissue to address the hypothesis that focal lesions arise from focal, post-zygotic genetic disruptions. Traditional genetic studies of patients with malformations utilized leukocyte-derived DNA to search for germline variants, which are inherited or arise de novo in parental gametes. Recent studies have demonstrated somatic variants that arise post-zygotically also underlie brain malformations, and that somatic mutation explains a larger proportion of focal malformations than previously thought. We now know from studies of non-diseased individuals that somatic variation occurs routinely during cell division, including during early brain development when the rapid proliferation of neuronal precursor cells provides the ideal environment for somatic mutation to occur and somatic variants to accumulate. When confined to brain, pathogenic variants contribute to the "hidden genetics" of neurological diseases. With burgeoning novel high-throughput genetic technologies, somatic genetic variations are increasingly being recognized. Here we discuss accumulating evidence for the presence of somatic variants in normal brain tissue, review our current understanding of somatic variants in brain malformations associated with lesional epilepsy, and provide strategies to identify the potential contribution of somatic mutation to non-lesional epilepsies. We also discuss technologies that may improve detection of somatic variants in the future in these and other neurological conditions.