Neurogenetics and Gene Therapy*

Date/Time: Sunday, September 10, 2023 - 3:30 PM – 5:00 PM
Track: Cross-Cutting Special Interest Group (SIG)
Room: Salons A-B (5th Floor)
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Co-Chair: Suman Jayadev, MD

Co-Chair: Andrea Gropman, MD, FANA

Biomarkers offer a way to accelerate biomedical research by uncovering the pathophysiological 
mechanisms of disease. Biomarkers can also be novel tools for monitoring disease progression, prognosis, and response to drugs, especially in clinical trials, where they can be used to assess the efficacy, efficiency, and side effects of novel drugs or therapies.

Although rare genetic diseases may be less appealing targets for pharmaceutical companies, they are nevertheless in urgent need to evaluate ways for rapid diagnosis, treatment, and establish care guidelines. Due to the small numbers of patients with a particular rare disease, standard methods of clinical trials may not be appropriate. It is important for rare disease groups and FDA to discuss challenges and strategies to move forward to drug approval for these conditions. This SIG will meet this gap in knowledge.

Learning Objectives:

  • Recognize how FDA qualifies biomarkers and how they may be used for clinical trials, 
  • Understand the FDA process towards IND and clinical trials, 
  • Better equipped to assemble a phase 1 study, be aware of what biomarkers are acceptable and understand primary and safety endpoints.

Discovery of Multi-Omic Plasma Biomarkers for Adrenomyeloneuropathy

Speaker: Ali Fatemi, MD, MBA

Adrenomyeloneuropathy (AMN), the slow progressive phenotype of adrenoleukodystrophy (ALD), has no clinical plasma biomarker for disease progression. This feasibility study aimed to determine whether metabolomics and micro-RNA in blood plasma provide a potential source of biomarkers for AMN disease severity. Metabolomics and RNA-seq were performed on AMN and healthy human blood plasma. AMN showed strong disease-severity-specific metabolic and miRNA clustering signatures. Strong, significant clinical correlations were shown with several metabolites and miRNAs. KEGG pathway comparison of mild versus severe disease identified affected downstream systems: GAREM, IGF-1, CALCRL, SMAD2&3, glutathione peroxidase, LDH, and NOS. This feasibility study demonstrates that miRNA and metabolomics are a source of potential plasma biomarkers for disease severity in AMN, providing both a disease signature and individual markers with strong clinical correlations.

Clinical Trial Readiness: Rare Inherited Neuropathies

Speaker: Michael E. Shy, MD, FANA

Rare inherited neuropathies offer the potential to develop rational therapies based on the known biology of the causal gene. However bringing therapies to clinical trial requires careful natural history and biomarker data to determine if the therapy is altering the natural history of the disease in question in a time frame that is feasible for clinical trials. This presentation will review the efforts of the Inherited Neuropathy Consortium (INC) to develop biomarkers that predict meaningful clinical changes through functional and patient reported outcome measures.

Somatic Variants Activating Ras-MAPK Signaling Cause a Spectrum of Focal Lesions Associated with Mesial Temporal Lobe Epilepsy

Oral Abstract Presenter: Sattar Khoshkhoo, MD

Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy subtype and often refractory to anti-seizure medications. Although it has long been known that seizures in MTLE originate in the hippocampus, the cause of this unique predisposition is unknown. Recently, post-zygotic (i.e., somatic) variants, which are well-known causes of neoplasms, have emerged as a major cause of pediatric focal epilepsies associated with focal cortical dysplasia (FCD). Interestingly, mesial temporal sclerosis (MTS) which is the major histopathologic finding in MTLE may co-occur with FCD or low-grade epilepsy-associated tumors (LEATs), suggesting a possible causal role for somatic variants in MTLE. To test whether somatic variants in the hippocampus contribute to MTLE pathogenesis, we performed high-coverage whole-exome sequencing (WES, depth >500X) of hippocampus-derived DNA from 104 surgically-treated patients with MTLE and 30 neurotypical donors. We detected 9 pathogenic somatic variants activating Ras-MAPK signaling in patients with MTLE and none in the controls. All variant-positive patients were seizure-free >2 years after surgery with significantly increased likelihood of Engel class IA outcome. Given the limited sensitivity of WES, to determine the true burden of Ras-MAPK variants in the hippocampus in MTLE relative to the neurotypical donors, we designed a gene-panel and performed duplex sequencing (depth >1000X) on the same samples. We detected pathogenic somatic Ras-MAPK variants in 28.8% of MTLE hippocampi and none of the neurotypical controls (p<0.001). We also observed a correlation between variant allele frequency and the size and regional distribution of the lesion, such that low abundance variants were associated with MTS alone whereas high abundance variants also caused an FCD or a LEAT. Since PTPN11 had the highest number of recurrent variants, we performed molecular assays to investigate the mechanisms of some of these variants. All the tested PTPN11 variants increased Erk1/2 phosphorylation indicating increased Ras-MAPK signaling, but the PTPN11 variants associated with LEATs demonstrated the greatest degree of pathway overactivation. Additionally, Shp2 (protein encoded by PTPN11) variants overexpressed in HEK293T cells demonstrated increased liquid-liquid phase separation behavior, which provides a possible dominant gain-of-function mechanism through which these variants cause pathway overactivation. Overall, our findings strongly suggest that somatic Ras-MAPK variants give rise to a spectrum of temporal lobe lesions depending on the developmental time point at which they were acquired and their specific molecular mechanisms, but all are associated with drug-resistant MTLE.

Inducible Genetically-Encoded Voltage Indicator for Non-Invasive Functional Analysis of Human Stem Cell-Derived Neurons

Oral Abstract Presenter: Scott Adney, MD, PhD

Ascertaining an electrophysiological phenotype from human stem cell-derived neurons (iPSC) can be time-consuming and is subject to considerable variability due to the need to examine one cell at a time with the gold standard of patch clamp. This technique also requires disruption to the cell membrane which could result in the loss of critical unknown components, and does not easily scale for examination of neuronal population properties. Here we employ a well-characterized genetically-encoded voltage indicator called ASAP3Kv under a doxycycline-inducible promoter, which allows labeling of neurons with the rapid lentiviral-mediated NGN2 differentiation protocol widely used. Simultaneous patch clamp with high-speed fluorescent imaging demonstrates the fidelity of this reporter system with evoked action potentials in human neurons. Importantly, neurons in populations can be assessed for spontaneous and synchronized activity with single-neuron resolution. This platform further supports the perfusion of exogenous drugs; we were able to demonstrate the effect of glutamate and GABA on populations of neurons without relying on indirect measurements like local field potentials or multi-electrode arrays (MEAs). To demonstrate the impact of non-invasive population analysis using the inducible GEVI, a gain-of-function SCN2A variant patient iPSC line was compared to its isogenic control line, allowing for comparison of spontaneous firing properties. Future applications include the ability to analyze specific neuronal populations in co-culture and investigation of novel therapeutics on spontaneous firing properties.

Mendelian Randomization of Inflammatory Markers to Show Their Causal Role in Multiple Sclerosis Pathophysiology

Oral Abstract Presenter: Sara Seyedroudbari, BA

Intro: While observational studies can be limited by sample size and confounding variables, Mendelian Randomization (MR) is an analytic tool that uses genetic variation as a natural experiment to investigate causal relationships between exposures and outcomes in observational data. Our study explores the association between well-known serum inflammatory markers and Multiple Sclerosis (MS). Observational studies have revealed associations between several serum markers and MS, but there is little known about causal relationships. We aim to elucidate the causal association between MS and inflammatory markers using 2 sample MR. Methods: We developed genetic instruments for 134 inflammatory markers by mapping them to a genome wide association study (GWAS) that included 47,429 subjects with MS and 68,374 controls. Our inflammatory markers were selected based on literature studies of common serum inflammatory biomarkers in a variety of disease processes, as well as availability of data that could be used for genetic instruments. We employed Wald ratio and inverse-variance weighted MR. Weighted median and weighted mode methods were used for sensitivity analyses. Results: We found that 7 inflammatory markers were nominally associated with MS (p < 0.05). After applying a false discovery rate (FDR < 0.05), an analytic tool for multiple experiments that identifies significant results while maintaining a low false positive rate, we found that 2 markers remained significant and were positively associated with MS (p < 0.05): IL-6 and TLR-4. Discussion: Multiple studies have shown that serum levels of IL-6 are elevated in patients with MS, and our results show that this relationship is indeed causal. Regarding TLR-4, the literature does not agree on its importance in MS pathophysiolgy, yet our results showing causation suggest there is more to be understood about TLR-4. Observational studies have shown that d-dimer, homocysteine, hs-CRP, IFN-γ, and TNF-α are positively correlated with MS pathophysiology; our results show no causative relationship. IL-17 and ESR have also shown correlational significance but were not included in our study, highlighting the need for further investigation using genetic instruments. Conclusion: This study highlights IL-6 and TLR-4 as causal inflammatory markers in MS pathophysiology. Additionally, we reveal key differences between observational and genetic studies. As many patients with MS still do not have a robust response to current treatments, MR may help to augment our understanding of the disease process and guide future therapies.