Lisa-Marie Niestroj

Cytogenic testing is routinely applied in most neurological centres for severe paediatric epilepsies. However, which characteristics of copy number variants (CNVs) confer most epilepsy risk and which epilepsy subtypes carry the most CNV burden, have not been explored on a genome-wide scale. Here, we present the largest CNV investigation in epilepsy to date with 10 712 European epilepsy cases and 6746 ancestry-matched controls. Patients with genetic generalized epilepsy, lesional focal epilepsy, non-acquired focal epilepsy, and developmental and epileptic encephalopathy were included. All samples were processed with the same technology and analysis pipeline. All investigated epilepsy types, including lesional focal epilepsy patients, showed an increase in CNV burden in at least one tested category compared to controls. However, we observed striking differences in CNV burden across epilepsy types and investigated CNV categories. Genetic generalized epilepsy patients have the highest CNV burden in all categories tested, followed by developmental and epileptic encephalopathy patients. Both epilepsy types also show association for deletions covering genes intolerant for truncating variants. Genome-wide CNV breakpoint association showed not only significant loci for genetic generalized and developmental and epileptic encephalopathy patients but also for lesional focal epilepsy patients. With a 34-fold risk for developing genetic generalized epilepsy, we show for the first time that the established epilepsy-associated 15q13.3 deletion represents the strongest risk CNV for genetic generalized epilepsy across the whole genome. Using the human interactome, we examined the largest connected component of the genes overlapped by CNVs in the four epilepsy types. We observed that genetic generalized epilepsy and non-acquired focal epilepsy formed disease modules. In summary, we show that in all common epilepsy types, 1.5–3% of patients carry epilepsy-associated CNVs. The characteristics of risk CNVs vary tremendously across and within epilepsy types. Thus, we advocate genome-wide genomic testing to identify all disease-associated types of CNVs.

Background: Parkinson's disease is the second most common neurodegenerative disorder and affects people from all ethnic backgrounds, yet little is known about the genetics of Parkinson's disease in non-European populations. In addition, the overall identification of copy number variants at a genome-wide level has been understudied in Parkinson's disease patients. Objectives: To understand the genome-wide burden of copy number variants in Latinos and its association with Parkinson's disease. Methods: We used genome-wide genotyping data from 747 Parkinson's disease patients and 632 ancestry matched controls from the Latin American Research Consortium on the Genetics of Parkinson's disease. Results: Genome-wide copy number burden analysis showed no difference between patients vs. controls, whereas patients were significantly enriched for copy number variants overlapping known Parkinson's disease genes compared to controls (Odds Ratio: 3.97 [1.69 - 10.5], P = 0.018). PARK2 showed the strongest copy number burden, with 20 copy number variant carriers. These patients presented an earlier age of disease onset compared to patients with other copy number variants (median age at onset: 31 years vs. 57 years, P = 7.46 x 10-7). Conclusions: We found that Parkinson's disease patients are significantly enriched with copy number variants affecting known Parkinson's disease genes. We also identified that out of 250 patients with early-onset disease, 5.6% carried a copy number variant on PARK2 in our cohort. Our study is the first to analyze genome-wide copy number variants association in Latino Parkinson's disease patients and provides insights about this complex disease in this understudied population.

The triumph of using genetics in precision medicine is the ability to stratify individuals at very high risk of disease and thereby enact specific gene-informed medical management.^1,2 In the case of heritable disease, genetics also allow testing of family members to determine with 100% accuracy whether they are at risk of disease. However, for any single individual carrying a mutation, we can estimate risk by probabilities but not with certainty. For example, a germline mutation in gene X may give an 80% lifetime risk of disease Y, but for the single patient with that germline mutation, the risk of developing disease Y will be 100% or 0%. Searching for genomic modifiers of heritable disease risk at the individual level in humans has proven challenging.^1,3 This makes estimation of clinical outcomes difficult, especially for phenotypically variable disorders. As such, why a mutation in 1 particular gene predisposes to disparate clinical outcomes, including in patients with identical mutations, remains poorly understood.

Iran, despite its size, geographic location and past cultural influence, has largely been a blind spot for human population genetic studies. With only sparse genetic information on the Iranian population available, we pursued its genome-wide and geographic characterization based on 1021 samples from eleven ethnic groups. We show that Iranians, while close to neighboring populations, present distinct genetic variation consistent with long-standing genetic continuity, harbor high heterogeneity and different levels of consanguinity, fall apart into a cluster of similar groups and several admixed ones and have experienced numerous language adoption events in the past. Our findings render Iran an important source for human genetic variation in Western and Central Asia, will guide adequate study sampling and assist the interpretation of putative disease-implicated genetic variation. Given Iran’s internal genetic heterogeneity, future studies will have to consider ethnic affiliations and possible admixture. Based on genome-wide genotype data on over 1000 samples from eleven ethnic groups present in Iran and by comparison to reference data sets of both extant populations and ancient DNA samples, we show that the Iranian population comprises distinct genetic variation with respect to populations in close geographic proximity, a cluster of genetically largely overlapping ethnic groups as well as a number of strongly admixed groups. These observations, also corroborated by f3 migration statistics and other approaches, indicate genetic continuity of and limited influx into the cluster groups over several millennia, despite Iran’s geographic position at a crossroads in West Asia. They also suggest, correspondingly, several instances of language adoption instead of demic replacement in the past. Future human genetic studies, both with a focus on population and medical genetics, will have to consider differences in heterogeneity, consanguinity and degree of admixture between the ethnic groups for an adequate design and interpretation.

It is challenging to estimate genetic variant burden across different subtypes of epilepsy. Herein, we used a comparative approach to assess the genetic variant burden and genotype–phenotype correlations in four most common brain lesions in patients with drug-resistant focal epilepsy. Targeted sequencing analysis was performed for a panel of 161 genes with a mean coverage of >400×. Lesional tissue was histopathologically reviewed and dissected from hippocampal sclerosis (n = 15), ganglioglioma (n = 16), dysembryoplastic neuroepithelial tumors (n = 8), and focal cortical dysplasia type II (n = 15). Peripheral blood (n = 12) or surgical tissue samples histopathologically classified as lesion-free (n = 42) were available for comparison. Variants were classified as pathogenic or likely pathogenic according to American College of Medical Genetics and Genomics guidelines. Overall, we identified pathogenic and likely pathogenic variants in 25.9% of patients with a mean coverage of 383×. The highest number of pathogenic/likely pathogenic variants was observed in patients with ganglioglioma (43.75%; all somatic) and dysembryoplastic neuroepithelial tumors (37.5%; all somatic), and in 20% of cases with focal cortical dysplasia type II (13.33% somatic, 6.67% germline). Pathogenic/likely pathogenic positive genes were disorder specific and BRAF V600E the only recurrent pathogenic variant. This study represents a reference for the genetic variant burden across the four most common lesion entities in patients with drug-resistant focal epilepsy. The observed large variability in variant burden by epileptic lesion type calls for whole exome sequencing of histopathologically well-characterized tissue in a diagnostic setting and in research to discover novel disease-associated genes.

Objective: The cyclin‐dependent kinase like 5 (CDKL5) gene is a known cause of early onset developmental and epileptic encephalopathy, also known as CDKL5 deficiency disorder (CDD). We sought to (1) provide a description of seizure types in patients with CDD, (2) provide an assessment of the frequency of seizure‐free periods and cortical visual impairment (CVI), (3) correlate these features with genotype and gender, and (4) correlate these features with developmental milestones.Methods: This is a cohort study of patients with CDD. Phenotypic features were explored and correlated with gene variant grouping and gender. A developmental score was created based on achieving seven primary milestones. Phenotypic variables were correlated with the developmental score to explore markers of better developmental outcomes. Multivariate linear regression was used to account for age at last visit.Results: Ninety‐two patients with CDD were seen during the enrollment period. Eighteen were male (19%); median age at last visit was 5 years (interquartile range = 2.0‐11.0). Eighty‐one percent of patients developed epileptic spasms, but only 47% of those also had hypsarrhythmia. Previously described hypermotor‐tonic‐spasms sequence was seen in only 24% of patients, but 56% of patients had seizures with multiple phases (often tonic and spasms). Forty‐three percent of patients experienced a seizure‐free period ranging from 1 to >12 months, but only 6% were still seizure‐free at the last visit. CVI was present in 75% of all CDD patients. None of these features was associated with genotype group or gender. CVI was correlated with reduced milestone achievement after adjusting for age at last visit and a history of hypsarrhythmia.Significance: The most common seizure types in CDD are epileptic spasms (often without hypsarrhythmia) and tonic seizures that may cluster together. CVI is a common feature in CDD and is correlated with achieving fewer milestones.

Rare and large copy number variants (CNVs) around known genomic ‘hotspots’ are strongly implicated in epilepsy etiology. But it remains unclear whether the observed associations are specific to an epilepsy phenotype, and if additional risk signal can be found outside hotspots. Here, we present the largest CNV burden and first CNV breakpoint level association analysis in epilepsy to date with 11,246 European epilepsy cases and 7,318 ancestry-matched controls. We studied five epilepsy phenotypes: genetic generalized epilepsy, lesional focal epilepsy, non-acquired focal epilepsy, epileptic encephalopathy, and unclassified epilepsy. We discovered novel epilepsy-associated CNV loci and further characterized the CNV burden enrichment among phenotype-specific epilepsies. Finally, we provide evidence for deletion burden outside of known hotspot regions and show that CNVs play a significant role in the genetic architecture of lesional focal epilepsies.

Summary: Objective: Increasing availability of surgically resected brain tissue from patients with focal epilepsy and focal cortical dysplasia or low‐grade glioneuronal tumors has fostered large‐scale genetic examination. However, assessment of pathogenicity of germ line and somatic variants remains difficult. Here, we present a state‐of‐the‐art evaluation of reported genes and variants associated with epileptic brain lesions.Methods: We critically reevaluated the pathogenicity for all neuropathology‐associated variants reported to date in the PubMed and ClinVar databases, including 101 neuropathology‐associated missense variants encompassing 11 disease‐related genes. We assessed gene variant tolerance and classified all identified missense variants according to guidelines from the American College of Medical Genetics and Genomics (ACMG). We further extended the bioinformatic variant prediction by introducing a novel gene‐specific deleteriousness ranking for prediction scores.Results: Application of ACMG guidelines and in silico gene variant tolerance analysis classified only seven of 11 genes to be likely disease‐associated according to the reported disease mechanism, whereas 61 (60.4%) of 101 variants of those genes were classified as of uncertain significance, 37 (36.6%) as being likely pathogenic, and 3 (3%) as being pathogenic.Significance: We concluded that the majority of neuropathology‐associated variants reported to date do not have enough evidence to be classified as pathogenic. Interpretation of lesion‐associated variants remains challenging, and application of current ACMG guidelines is recommended for interpretation and prediction.

Purpose: Increasing availability of surgically resected brain tissue from Focal Cortical Dysplasia and low-grade epilepsy-associated tumor patients fostered large-scale genetic examination. However, assessment of germline and somatic variant pathogenicity remains difficult. Methods: Here, we critically reevaluated the pathogenicity for all neuropathology-associated variants reported to date in the PubMed and ClinVar databases, including 12 disease-related genes and 88 neuropathology-associated missense variants. We (1) assessed evolutionary gene constraint using the pLI and missense z scores, (2) applied guidelines by the American College of Medical Genetics and Genomics (ACMG), and (3) predicted pathogenicity by using PolyPhen-2, CADD, and GERP. Results: Constraint analysis classified only seven out of 12 genes to be likely disease-associated, while 35 (40%) of those 88 variants were classified as being variants of unknown significance (VUS) and 53 (60%) as being likely pathogenic (LPII). Pathogenicity prediction yielded discrimination between neuropathology-associated variants (LPII and VUS) and rare variant scores obtained from individuals present in the Genome Aggregation Database (gnomAD). Conclusion: We conclude that several VUS are likely disease-associated and will be reclassified by future molecular evidence. In summary, interpretation of lesion-associated gene variants remains complex while the application of current ACMG guidelines including bioinformatic pathogenicity prediction will help improving interpretation and prediction.