Increased prime edit rates in KCNQ2 and SCN1A via single nicking all-in-one plasmids.

BACKGROUND: Prime editing (PE) is the most recent gene editing technology able to introduce targeted alterations to the genome, including single base pair changes, small insertions, and deletions. Several improvements to the PE machinery have been made in the past few years, and these have been tested in a range of model systems including immortalized cell lines, stem cells, and animal models. While double nicking RNA (dncRNA) PE systems PE3 and PE5 currently show the highest editing rates, they come with reduced accuracy as undesired indels or SNVs arise at edited loci. Here, we aimed to improve single ncRNA (sncRNA) systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids driven by an EF-1α promoter, which is especially suitable for human-induced pluripotent stem cell (hiPSC) models. RESULTS: pAIO-EF1α-PE2 and pAIO-EF1α-PE4max were used to edit the voltage gated potassium channel gene KCNQ2 and voltage gated sodium channel gene SCN1A. Two clinically relevant mutations were corrected using pAIO-EF1α-PE2 including the homozygous truncating SCN1A R612* variant in HEK293T cells and the heterozygous gain-of-function KCNQ2 R201C variant in patient-derived hiPSC. We show that sncRNA PE yielded detectable editing rates in hiPSC ranging between 6.4% and 9.8%, which was further increased to 41% after a GFP-based fluorescence-activated cell sorting (FACS) cell sorting step. Furthermore, we show that selecting the high GFP expressing population improved editing efficiencies up to 3.2-fold compared to the low GFP expressing population, demonstrating that not only delivery but also the number of copies of the PE enzyme and/or pegRNA per cell are important for efficient editing. Edit rates were not improved when an additional silent protospacer-adjacent motif (PAM)-removing alteration was introduced in hiPSC at the target locus. Finally, there were no genome-wide off-target effects using pAIO-EF1α-PE2 and no off-target editing activity near the edit locus highlighting the accuracy of snc prime editors. CONCLUSION: Taken together, our study shows an improved efficacy of EF-1α driven sncRNA pAIO-PE plasmids in hiPSC reaching high editing rates, especially after FACS sorting. Optimizing these sncRNA PE systems is of high value when considering future therapeutic in vivo use, where accuracy will be extremely important.

Clinical and genetic aspects of PCDH19-related epilepsy syndromes and the possible role of PCDH19 mutations in males with autism spectrum disorders.

Epilepsy and mental retardation limited to females (EFMR), caused by PCDH19 mutations, has a variable clinical expression that needs further exploration. Onset of epilepsy may be provoked by fever and can resemble Dravet syndrome. Furthermore, transmitting males have no seizures, but are reported to have rigid personalities suggesting possible autism spectrum disorders (ASD). Therefore, this study aimed to determine the phenotypic spectrum associated with PCDH19 mutations in Dravet-like and EFMR female patients and in males with ASD. We screened 120 females suffering from Dravet-like epilepsy, 136 females with EFMR features and 20 males with ASD. Phenotypes and genotypes of the PCDH19 mutation carriers were compared with those of 125 females with EFMR reported in the literature. We report 15 additional patients with a PCDH19 mutation. Review of clinical data of all reported patients showed that the clinical picture of EFMR is heterogeneous, but epilepsy onset in infancy, fever sensitivity and occurrence of seizures in clusters are key features. Seizures remit in the majority of patients during teenage years. Intellectual disability and behavioural disturbances are common. Fifty percent of all mutations are missense mutations, located in the extracellular domains only. Truncating mutations have been identified in all protein domains. One ASD proband carried one missense mutation predicted to have a deleterious effect, suggesting that ASD in males can be associated with PCDH19 mutations.

Clinical spectrum of early-onset epileptic encephalopathies associated with STXBP1 mutations.

OBJECTIVES: Heterozygous mutations in STXBP1, encoding the syntaxin binding protein 1, have recently been identified in Ohtahara syndrome, an epileptic encephalopathy with very early onset. In order to explore the phenotypic spectrum associated with STXBP1 mutations, we analyzed a cohort of patients with unexplained early-onset epileptic encephalopathies. METHODS: We collected and clinically characterized 106 patients with early-onset epileptic encephalopathies. Mutation analysis of the STXBP1 gene was done using sequence analysis of the exon and intron-exon boundaries and multiplex amplification quantification to detect copy number variations. RESULTS: We identified 4 truncating mutations and 2 microdeletions partially affecting STXBP1 in 6 of the 106 patients. All mutations are predicted to abolish STXBP1 function and 5 mutations were proven to occur de novo. None of the mutation-carrying patients had Ohtahara syndrome. One patient was diagnosed with West syndrome at disease onset, while the initial phenotype of 5 further patients did not fit into a specific recognized epilepsy syndrome. Three of these patients later evolved to West syndrome. All patients had severe to profound mental retardation, and ataxia or dyskinetic movements were present in 5 patients. CONCLUSION: This study shows that mutations in STXBP1 are not limited to patients with Ohtahara syndrome, but are also present in 10% (5/49) of patients with an early-onset epileptic encephalopathy that does not fit into either Ohtahara or West syndrome and rarely in typical West syndrome. STXBP1 mutational analysis should be considered in the diagnostic evaluation of this challenging group of patients.

Progressive myoclonic epilepsy as an adult-onset manifestation of Leigh syndrome due to m.14487T>C.

BACKGROUND: m.14487T>C, a missense mutation (p.M63V) affecting the ND6 subunit of complex I of the mitochondrial respiratory chain, has been reported in isolated childhood cases with Leigh syndrome (LS) and progressive dystonia. Adult-onset phenotypes have not been reported. OBJECTIVES: To determine the clinical-neurological spectrum and associated mutation loads in an extended m.14487T>C family. METHODS: A genotype-phenotype correlation study of a Belgian five-generation family with 12 affected family members segregating m.14487T>C was carried out. Clinical and mutation load data were available for nine family members. Biochemical analysis of the respiratory chain was performed in three muscle biopsies. RESULTS: Heteroplasmic m.14487T>C levels (36-52% in leucocytes, 97-99% in muscle) were found in patients with progressive myoclonic epilepsy (PME) and dystonia or progressive hypokinetic-rigid syndrome. Patients with infantile LS were homoplasmic (99-100% in leucocytes, 100% in muscle). We found lower mutation loads (between 8 and 35% in blood) in adult patients with clinical features including migraine with aura, Leber hereditary optic neuropathy, sensorineural hearing loss and diabetes mellitus type 2. Despite homoplasmic mutation loads, complex I catalytic activity was only moderately decreased in muscle tissue. INTERPRETATION: m.14487T>C resulted in a broad spectrum of phenotypes in our family. Depending on the mutation load, it caused severe encephalopathies ranging from infantile LS to adult-onset PME with dystonia. This is the first report of PME as an important neurological manifestation of an isolated mitochondrial complex I defect.