Combining Exome and Transcriptome Data to Unravel the Genetic Basis of the Lissencephalies

Malformations of cortical development (MCD) are a heterogenous group of brain malformations including lissencephaly, heterotopia and polymicrogyria. The lissencephaly spectrum (including lissencephaly, pachygyria and subcortical band heterotopia) is a well-defined group of MCD with a strong monogenetic basis. Using current molecular techniques, a causative variant is detected in approximately 80% of individuals with lissencephaly. In a routine diagnostic setting, exome-based gene panels are most frequently used while whole exome sequencing (WES) and whole genome sequencing (WGS) are increasingly being implemented. Both techniques have their shortcomings including the detection of small copy number variants, the identification of pathogenic variants in non-coding regions as well as variant interpretation. The parallel use of quantitative RNA sequencing, measuring differences in RNA expression could be a possible solution for these shortcomings. The proposed research project will for the first time 1) evaluate the added value of WES/WGS combined with quantitative RNA sequencing for the identification of novel genes in individuals with lissencephaly, 2) identify the optimal sampling tissue for RNA sequencing in complex neurological phenotypes and 3) use RNA expression data to provide an evidence base for the current lissencephaly classification.

As mentioned before, lissencephalies have a strong monogenetic base in contrast to other brain malformations. Approximately 80% percent of the lissencephalies can be genetically diagnosed by standard WES or WGS.1 In the remaining 20% percent of unsolved cases quantitative RNA sequencing could make a considerable difference. By identifying RNA expression patterns in lissencephalies we will try to provide unsolved lissencephaly cases with a genetic diagnosis. A genetic diagnosis is im-portant in terms of predicting associated problems, follow-up, prognosis and in some cases family planning (e.g. pre-implantation genetic testing). This study will also investigate the additional diagnostic yield of RNA sequencing in lissencephalies and by extension in the MCD spectrum. And, if indicated, the feasibility of implementing RNA sequencing in the standard diagnostic work-up.

High efficiency in identifying new pathogenic variants and novel gene annotation can be expected because of the strong monogenetic base. These novel variants and gene annotation are indispensable for better understanding the origin and pathophysiology of the lissencephaly spectrum and neuronal migration.

The functional impact of newly discovered genes can be further investigated by the innovative CRISPR-Cas9 method. This gene-editing technique allows researchers to create knock-out or even knock-in genes, as an opportunity to investigate novel annotated genes and their functional consequences. Although it is beyond the scope of this study, it is an interesting item for future joined research projects, within our research group or in collaboration with others. During this study, also genetically diagnosed lissencephaly cases will be subjected to RNA sequencing. The current classifi-cation of lissencephalies is based on pathogenic variants and biological pathways. Alterations in RNA-expression pattern could possibly shine a new light on this classification.

This study will also evaluate which sampling tissues are most suited for RNA extraction and sequencing. Considerations to make include the targeted quality of the extracted RNA and differences in tissue-specific gene expression. Mouth swaps, although non-invasive, are not suited for RNA extraction because of the natural oral flora with multiple viruses and bacteria (exogenous genetic material). Fibroblast-derived RNA is considered to be of good quality, but a skin biopsy is required and considered relatively invasive. Whole blood is obtained by minimal invasive techniques, but gene expression may be of poorer quality compared to fibroblasts.

The first part of the study is performed in a diagnostic setting in unsolved lissencephaly cases. Fibroblasts will be obtained by skin biopsy (punch). RNA seq data will be extracted from fibroblasts and blood. This RNA seq data will be analyzed in search for new pathogenic variants. When new pathogenic variants are identified in the RNA seq data, existing WES data will be reanalyzed. If neces-sary, subsequent WGS will be performed. When a genetic diagnosis is obtained, RNA seq and WES/WGS data will be transferred to the research part of the study.

A second part of the study is performed in a research setting. Lissencephaly patients with a genetic diagnosis will be proposed to donate a skin biopsy and . RNA seq data will be extracted from fibroblasts and blood as in the diagnostic track. In the RNA seq data two items will be observed. Firstly, has RNA seq data extracted from fibroblasts a major advantage over RNA seq data extracted from blood in terms of variant detection? Secondly, can RNA patterns be identified in common affected pathways?

Eligibility Criteria:
Inclusion Criteria:

Overall:

* anomaly on MRI of the lissencephaly spectrum (lissencephaly, pachygyria, subcortical band heterotopia

Diagnostic track:

* No established genetic diagnosis by conventional WES/WGS

Research track:

* An established genetic diagnosis by conventinal WES/WGS

Exclusion Criteria:

* No anomaly of the lissencephaly spectrum on MRI