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Altering the human epigenome with gene-editing technology in attempt to treat a variety of diseases and conditions seems scientifically feasible. We explore some of the ethical and regulatory issues related to the clinical translation of human epigenetic editing arguing that such approaches should be considered akin to somatic therapies.

Background and purpose Most spinal muscular atrophy (SMA) patients are homozygous for survival of motor neuron 1 gene (SMN1) deletion. However, some SMA patients carry an intragenic SMN1 mutation. Such patients provide a clue to understanding the function of the SMN protein and the role of each domain of the protein. We previously identified mutations in the Tudor domain and C-terminal region of the SMN protein in three Japanese SMA patients. To clarify the effect of these mutations on protein stability, we conducted expression assays of SMN with mutated domains. Patients and methods Patients A and B carried a mutation in SMN1 exon 3, which encodes a Tudor domain, c.275G>C (p.Trp92Ser). Patient C carried a mutation in SMN1 exon 6, which encodes a YG-box; c.819_820insT (p.Thr274Tyrfs). We constructed plasmid expression vectors containing wild-type and mutant SMN1 cDNAs. After transfection of HeLa cells with the expression plasmids, RNA and protein were isolated and analyzed by reverse-transcription PCR and western blot analysis. Results The abundance of wild-type and mutant SMN1 transcripts in HeLa cells was almost the same. However, western blot analysis showed lower levels of mutant SMN proteins compared with wild-type SMN. In mutant SMN proteins, it is noteworthy that the level of the p.Thr274Tyrfs mutant was much reduced compared with that of the p.Trp92Ser mutant. Conclusions SMN mutations may affect the stability and levels of the protein.

We describe a gold nanoparticle-based technique for the detection of single-base mutations in the glucose-6-phosphate dehydrogenase (G6PD) gene, a condition that can lead to neonatal jaundice and hemolytic anemia. The aim of this technique is to clearly distinguish different mutations frequently described within the Asian population from their wild-type counterparts and across different mutant variants. Gold nanoparticles of different sizes were synthesized, and each was conjugated with a single-strand DNA (ssDNA) sequence specific for a particular mutation in the G6PD gene. It was found that only mutant targets presented a characteristic band on the agarose gel, indicating the successful formation of dimeric nanostructures. No such dimer bands were observed for the wild-type targets. The difference in the relative dimer band levels allowed different mutant variants to be distinguished from one another. The technique was further validated using G6PD-deficient patient samples. This simple mutation detection method with direct result readout is amenable for rapid and mass screening of samples.

Association studies are widely seen as the most promising approach for finding polymorphisms that influence genetically complex traits, such as common diseases and responses to their treatment. Considerable interest has therefore recently focused on the development of methods that efficiently screen genomic regions or whole genomes for gene variants associated with complex phenotypes. One key element in this search is the use of linkage disequilibrium to gain maximal information from typing a selected subset of highly informative single-nucleotide polymorphism (SNP) markers, now often called “tagging SNPs” (tSNPs). Probably the most common approach to linkage-disequilibrium gene mapping involves a three-step program: (1) characterization of the haplotype structure in candidate genes or genomic regions of interest, (2) identification of tSNPs sufficient to represent the most common haplotypes, and (3) typing of tSNPs in clinical material. Early definitions of tSNPs focused on the amount of haplotype diversity that they explained. To select tSNPs that would have maximal power in a genetic association study, however, we have developed optimization criteria based on the r2 measure of association and have compared these with other criteria based on the haplotype diversity. To evaluate the full program and to assess how well the selected tags are likely to perform, we have determined the haplotype structure and have assessed tSNPs in the SCN1A gene, an important candidate gene for sporadic epilepsy. We find that as few as four tSNPs are predicted to maintain a consistently high r2 value with all other common SNPs in the gene, indicating that the tags could be used in an association study with only a modest reduction in power relative to direct assays of all common SNPs. This implies that very large case-control studies can be screened for variation in hundreds of candidate genes with manageable experimental effort, once tSNPs are identified. However, our results also show that tSNPs identified in one population may not necessarily perform well in another, indicating that the preliminary study to identify tSNPs and the later case-control study should be performed in the same population. Our results also indicate that tSNPs will not easily identify discrepant SNPs, which lie on importantly discriminating but apparently short genealogical branches. This could significantly complicate tagging approaches for phenotypes influenced by variants that have experienced positive selection.

Heteroduplex formation, required for the complete detection of hemi/homozygotes using high-resolution melting analysis, can be induced either by pre-PCR mixing of genomic DNAs or by post-PCR mixing of PCR products from unknown and reference samples. This study investigates the effects of both methods using two single nucleotide polymorphisms in X-linked DMD gene. The results show that both methods resulted in the same effect when mixing samples with the same gene copy number. Mixing samples with different gene copy numbers has not been previously explored and we show that post-PCR mixing is insensitive to gene copy number differences as compared to pre-PCR mixing.

Antisense oligonucleotides-mediated exon skipping to restore dystrophin expression for DMD therapy is promising in human clinical trials. Current AON design methods are semi-empirical involving either trial-and-error and/or preliminary experimentations. To increase the productivity of developing efficient AONs, a rational AON design approach is desirable. While pioneering retrospective studies have extracted many AON design variables, they were not tested prospectively to design AONs for skipping DMD exons. Here, we proposed a recipe for the design of efficient AONs that is based on the biophysical and biochemical mechanisms involved in the exon splicing process. The recipe was applied to design 23 novel AONs, each to skip one of nine DMD exons, and their exon skipping efficiencies were subsequently validated in wet experiments. 19 AONs were found to be efficacious in inducing specific exon skipping (83% of total), of which 14 were considered efficient (61% of total), i.e. induced exon skipping in >25% of total transcripts. The validated design variables facilitate the rational design of AONs that could expedite development of optimal AONs for therapy.