Familial Adult Myoclonic Epilepsy (FAME) is a genetically heterogeneous disorder characterized by cortical tremor and seizures. Intronic TTTTA/TTTCA repeat expansions in SAMD12 (FAME1) are the main cause of FAME in Asia. Using genome sequencing and repeat-primed PCR, we identify another site of this repeat expansion, in MARCH6 (FAME3) in four European families. Analysis of single DNA molecules with nanopore sequencing and molecular combing show that expansions range from 3.3 to 14 kb on average. However, we observe considerable variability in expansion length and structure, supporting the existence of multiple expansion configurations in blood cells and fibroblasts of the same individual. Moreover, the largest expansions are associated with micro-rearrangements occurring near the expansion in 20% of cells. This study provides further evidence that FAME is caused by intronic TTTTA/TTTCA expansions in distinct genes and reveals that expansions exhibit an unexpectedly high somatic instability that can ultimately result in genomic rearrangements.

Disease gene discovery on chromosome (chr) X is challenging owing to its unique modes of inheritance. We undertook a systematic analysis of human chrX genes. We observe a higher proportion of disorder-associated genes and an enrichment of genes involved in cognition, language, and seizures on chrX compared to autosomes. We analyze gene constraints, exon and promoter conservation, expression, and paralogues, and report 127 genes sharing one or more attributes with known chrX disorder genes. Using machine learning classifiers trained to distinguish disease-associated from dispensable genes, we classify 247 genes, including 115 of the 127, as having high probability of being disease-associated. We provide evidence of an excess of variants in predicted genes in existing databases. Finally, we report damaging variants in CDK16 and TRPC5 in patients with intellectual disability or autism spectrum disorders. This study predicts large-scale gene-disease associations that could be used for prioritization of X-linked pathogenic variants.

Familial adult myoclonus epilepsy (FAME) results from the same pathogenic TTTTA/TTTCA pentanucleotide repeat expansion in six distinct genes encoding proteins with different subcellular localizations and very different functions, which poses the issue of what causes the neurobiological disturbances that lead to the clinical phenotype. Postmortem and electrophysiological studies have pointed to cortical hyperexcitability as well as dysfunction and neurodegeneration of both the cortex and cerebellum of FAME subjects. FAME expansions, contrary to the same expansion in DAB1 causing spinocerebellar ataxia type 37, seem to have no or limited impact on their recipient gene expression, which suggests a pathophysiological mechanism independent of the gene and its function. Current hypotheses include toxicity of the RNA molecules carrying UUUCA repeats, or toxicity of polypeptides encoded by the repeats, a mechanism known as repeatassociated non-AUG translation. The analysis of postmortem brains of FAME1 expansion (in SAMD12) carriers has revealed the presence of RNA foci that could be formed by the aggregation of RNA molecules with abnormal UUUCA repeats, but evidence is still lacking for other FAME subtypes. Even when the expansion is located in a gene ubiquitously expressed, expression of repeats remains undetectable in peripheral tissues (blood, skin). Therefore, the development of appropriate cellular models (induced pluripotent stem cell-derived neurons) or the study of affected tissues in patients is required to elucidate how FAME repeat expansions located in unrelated genes lead to disease.

Disease gene discovery on chromosome (chr) X is challenging owing to its unique modes of inheritance. We undertook a systematic analysis of human chrX genes. We observe a higher proportion of disorder-associated genes and an enrichment of genes involved in cognition, language, and seizures on chrX compared to autosomes. We analyze gene constraints, exon and promoter conservation, expression and paralogues, and report 127 genes sharing one or more attributes with known chrX disorder genes. Using a neural network trained to distin-guish disease-associated from dispensable genes, we classify 235 genes, including 121 of the 127, as having high probability of being disease-associated. We provide evidence of an excess of variants in predicted genes in existing databases. Finally, we report damaging variants in CDK16 and TRPC5 in patients with intellectual disability or autism spectrum disorders. This study predicts large-scale gene-disease associations that could be used for prioritization of X-linked pathogenic variants.

Transcriptional interference and transcription through regulatory elements (transcriptional read-through) are implicated in gene silencing and the establishment of DNA methylation. Transcriptional read-through is needed to seed DNA methylation at imprinted genes in the germ line and can lead to aberrant gene silencing by DNA methylation in human disease. To enable the study of parameters and factors influencing transcriptional interference and transcriptional read-through at human promoters, we established a somatic cell culture system. At two promoters of imprinted genes (UBE3A and SNRPN) and two promoters shown to be silenced by aberrant transcriptional read-through in human disease (MSH2 and HBA2) we tested, if transcriptional read-through is sufficient for gene repression and the acquisition of DNA methylation. Induction of transcriptional read-through from the doxycycline-inducible CMV promoter resulted in consistent repression of all downstream promoters, independent of promoter type and orientation. Repression was dependent on ongoing transcription, since withdrawal of induction resulted in reactivation. DNA methylation was not acquired at any of the promoters. Overexpression of DNMT3A and DNMT3L, factors needed for DNA methylation establishment in oocytes, was still not sufficient for the induction of DNA methylation. This indicates that induction of DNA methylation has more complex requirements than transcriptional read-through and the presence of de novo DNA methyltransferases.