Michael S. Hildebrand

We describe FoundHaplo, an identity-by-descent algorithm that can be used to screen untyped disease-causing variants using single nucleotide polymorphism (SNP) array data. FoundHaplo leverages knowledge of shared disease haplotypes for inherited variants to identify those who share the disease haplotype and are, therefore, likely to carry the rare [minor allele frequency (MAF) ≤ 0.01%] variant. We performed a simulation study to evaluate the performance of FoundHaplo across 33 disease-harbouring loci. FoundHaplo was used to infer the presence of two rare (MAF ≤ 0.01%) pathogenic variants, SCN1B c.363C>G (p.Cys121Trp) and WWOX c.49G>A (p.E17K), which can cause mild dominant and severe recessive epilepsy, respectively, in the Epi25 cohort and the UK Biobank. FoundHaplo demonstrated substantially better sensitivity at inferring the presence of these rare variants than existing genome-wide imputation. FoundHaplo is a valuable screening tool for searching disease-causing variants with known founder effects using only SNP genotyping data. It is also applicable to nonhuman applications and nondisease-causing traits, including rare-variant drivers of quantitative traits. The FoundHaplo algorithm is available at https://github.com/bahlolab/FoundHaplo (DOI:10.5281/zenodo.8058286).

While it is widely accepted that somatic variants that activate the PI3K-mTOR pathway are a major cause of drug-resistant focal epilepsy, typically associated with focal cortical dysplasia (FCD) type 2, understanding the mechanism of epileptogenesis requires identifying genotype-associated changes at the single-cell level, which is technically challenging with existing methods. Here, we performed single-nucleus RNA-sequencing (snRNA-seq) of 18 FCD type 2 samples removed surgically for treatment of drug-resistant epilepsy, and 17 non-FCD control samples, and analyzed additional published data comprising >400,000 single nuclei. We also performed simultaneous single-nucleus genotyping and gene expression analysis using two independent approaches: 1) a method that we called genotyping of transcriptomes enhanced with nanopore sequencing (GO-TEN) that combines targeted cDNA long-read sequencing with snRNA-seq, 2) ResolveOME snRNA-seq and DNA genotyping. snRNA-seq showed similar cell identities and proportions between cases and controls, suggesting that mosaic pathogenic variants in PI3K-mTOR pathway genes in FCD exert their effect by disrupting transcription in conserved cell types. GO-TEN and ResolveOME analyses confirmed that pathogenic variant-carrying cells have well-differentiated neuronal or glial identities, with enrichment of variants in cells of the neuroectodermal lineage, pointing to cortical neural progenitors as possible loci of somatic mutation. Within FCD type 2 lesions, we identified upregulation of PI3K-mTOR signaling and related pathways in variant-carrying neurons, downregulation of these pathways in non-variant-carrying neurons, as well as associated changes in microglial activation, cellular metabolism, synaptic homeostasis, and neuronal connectivity, all potentially contributing to epileptogenesis. These genotype-specific changes in mosaic lesions highlight potential disease mechanisms and therapeutic targets.

Hypothalamic Hamartoma (HH) Syndrome is a rare condition in which benign subcortical lesion attached to the hypothalamus results in a multitude of symptoms including refractory epilepsy, endocrine dysfunction, developmental delay and psychiatric comorbidities. Gelastic "laughing" seizures are the identifying symptom of HH syndrome, but often remain unidentified until other seizure types occur. In this review we summarize typical presentations that lead to diagnosis. The literature suggests that early MRI imaging, detailed assessment of endocrine function and formal neuropsychological testing are most important after diagnosis. EEG tests are usually less valuable than in other epilepsies and gelastic seizures may occur without ictal EEG changes. Experts agree that most patients with seizures are refractory to antiseizure medications. Therefore, early surgical intervention is recommended. Three main minimally invasive techniques are currently used: Laserinterstitial thermal therapy, radiosurgery and radiofrequency thermocoagulation. Three large meta-analysis collecting data from more than 500 patients treated with these techniques have been published. Safety profiles of all three methods are good and superior to micro-surgical approaches used previously. Most common complications are hypothalamic-endocrine dysfunction and mild neurological symptoms. 70-90% of patients are seizure free after the interventions, around 20-30% requiring multiple treatment to archive this outcome. Overall, minimally invasive approaches have revolutionized long-term outcomes in HH. Meanwhile, new genetic methods are being developed to better understand mechanisms that cause HH. Most importantly knowledge is still lacking about how these localized lesions can cause substantial developmental delay and psychiatric comorbidity that persist even after successful surgical removal.

Cerebral cavernous malformations (CCMs) are intracranial vascular lesions associated with risk of haemorrhages and seizures. While the majority are sporadic and often associated with somatic variants in PIK3CA and MAP3K3, around 20% are familial with germline variants in one of three CCM genes-KRIT1/CCM1, CCM2 and PDCD10/CCM3. We performed comprehensive phenotyping and genetic analysis of nine multiplex families and ten sporadic individuals with CCM. In the familial cases, initial standard analyses had a low yield, we therefore searched for small copy number changes and deep intronic variants. Subsequently, pathogenic germline variants in KRIT1/CCM1 or CCM2 were identified in all 9 multiplex families. Single or multiple exon deletions or splice site variants in KRIT1/CCM1 were found in 3/9 families. Where cavernous malformation tissue was available, second hit somatic PIK3CA variants were identified in 4/7 individuals. These 4 individuals were from separate families with germline KRIT1/CCM1 variants. In 8/10 sporadic cases, we detected recurrent pathogenic somatic PIK3CA, MAP3K3 or CCM2 variants. All familial cases had multiple CCMs, whereas the sporadic cases had a single lesion only, which was in the temporal lobe in 9/10 individuals. Our comprehensive approach interrogating deep intronic variants combined with detection of small copy number variants warrants implementation in standard clinical genetic testing pipelines to increase diagnostic yield. We also build on the established second hit germline and somatic variant mechanism in some CCM lesions. Genetic diagnosis has clinical implications such as reproductive counselling and provides potential eligibility for precision medicine therapies to treat rapidly growing CCMs.

Protein-activated kinases mediate spine morphogenesis and synaptic plasticity. PAK3 is part of the p21-activated kinases (PAKs) family of Ras-signaling serine/threonine kinases. Pathogenic variants in the X-linked gene PAK3 have been described in patients with neurodevelopmental syndromes. We analyzed an Italian family with sleep-related hypermotor epilepsy, intellectual disability, psychiatric and behavioral problems, and dysmorphic facial features. A novel PAK3 c.342_344del (p.Lys114del) inframe deletion was detected in the family. Protein structure analysis supported deleterious impact of p.Lys114 deletion through loss or partial loss of autoinhibition of PAK3 protein kinase activity. The male proband had drug-resistant hypermotor seizures and moderate intellectual disability. His brother had drug-responsive hypermotor seizures and mild intellectual disability. Both brothers were hemizygous and had psychiatric and behavioral problems as well as dysmorphic facial features. Their mother had never had seizures but was shown to be mosaic for the PAK3 pathogenic variant. She had normal intellect but did have short stature and dysmorphic facial features similar to her sons. This is the first reported association of a PAK3 pathogenic variant with sleep-related hypermotor epilepsy. PAK3 testing should be considered in families with suspected X-linked sleep-related hypermotor epilepsy and intellectual disability, including for mosaicism in mildly affected females. PLAIN LANGUAGE SUMMARY: We studied an Italian family with sleep-related hypermotor epilepsy, intellectual disability, psychiatric and behavioral problems, and dysmorphic facial features. A novel PAK3 c.342_344del (p.Lys114del) inframe deletion was detected in the family. Protein structure analysis supported deleterious impact of p.Lys114 deletion through loss or partial loss of autoinhibition of PAK3 protein kinase activity. This is the first reported association of a PAK3 pathogenic variant with sleep-related hypermotor epilepsy. PAK3 testing should be considered in families with suspected X-linked sleep-related hypermotor epilepsy and intellectual disability, including for mosaicism in mildly affected females.