Simple, simultaneous detection of epigenetic and genetic variants for insights into disease mechanisms

Methylation has been implicated in the pathogenic mechanisms of many genetic diseases, such as Prader-Willi syndrome¹ and fragile X syndrome (FXS)². However, traditional sequencing methods require the use of amplification, which erases all epigenetic modifications. This means that the presence of methylation must be inferred by the use of chemical DNA treatment before PCR amplification, sequencing, and comparison to a paired dataset from an untreated library. In contrast, PCR-free nanopore sequencing of native DNA directly detects methylation at single-nucleotide resolution, with no additional sample preparation. To simultaneously investigate methylation and short tandem repeat (STR) expansions in FXS, Stevanovski et al. used targeted nanopore sequencing to span entire STR expansion regions and directly detected their methylation status¹.

‘a comprehensive understanding of the mechanisms of [fragile X syndrome] requires both STR and DNA methylation profiling’

STRs, typically comprised of repeats of 2–6 bp sequences, can exceed 10 kb in length and exhibit pathogenicity through an array of different mechanisms, including hypermethylation³. In their paper, Stevanovski et al. explained that ‘the large size, low sequence complexity, and high GC content of many pathogenic STR expansions make them refractory to analysis by short-read NGS platforms’, highlighting the ‘growing need for improved methods for the molecular characterization of STRs’. This lead the team to employ adaptive sampling — an on-device enrichment method unique to Oxford Nanopore — to perform targeted sequencing of known neuropathogenic STRs, without the need to perform whole-genome analysis.

‘[Oxford Nanopore's adaptive sampling] function has the potential to enable simple, cost-effective sequencing of all known pathogenic STR loci’

The team utilised adaptive sampling on the handheld MinION and the benchtop GridION nanopore sequencing devices to recognise and target a diverse range of genes featuring pathogenic STR expansions. As adaptive sampling ‘is fully flexible and requires no additional laboratory processes beyond standard library preparation’, the team targeted large regions, including 50 kb flanking sequences, and were able to accurately call base modifications in the same dataset. This contrasts with established molecular techniques for STR expansion disorders, which the authors explain are ‘relatively slow, labour-intensive, and imprecise’.

FXS, a common form of inherited intellectual disability, is caused by large expansions of CGG repeats located in the 5′-untranslated region of FMR1; however, DNA methylation has also been implicated in the pathogenic mechanism of FXS. Previous studies have shown that FXS-affected males exhibit hypermethylation of the FMR1 promoter, leading to gene silencing and reduced or absent levels of the FMR1 protein — essential for normal brain development⁴. Offspring inheriting >200 CGG repeats are affected, while offspring inheriting 55—200 CGG repeats — known as a premutation — are classed as carriers but are still at risk of developing FXS and its associated disorders.

Investigating epigenetic regulation in FXS, the team profiled the methylation status of STR expansions in the FMR1 gene, located on the X chromosome. Stevanovski et al. explained that the significance of DNA methylation in premutation carriers is not fully understood and ‘can only be detected using haplotype-aware methodologies, such as ONT sequencing’. With long nanopore reads, methylation calling could be be performed across large regions of interest, enabling the team to identify and phase methylation to the maternal or paternal chromosome and thereby characterise inheritance patterns.

‘The capacity to obtain haplotype-resolved DNA methylation profiles, in addition to STR size and interruption status, all in a single, simple assay is a clear advantage of our approach.’

Accurate, haplotype-resolved methylation profiling using nanopore sequencing confirmed the hypermethylation of the FMR1 promoter region in FXS-affected male-derived clinical research samples with full STR expansions. In contrast, promoter methylation frequencies were low among male-derived clinical research samples with normal and premutation STR alleles. The team observed low methylation frequencies in female-derived clinical research samples, apart from haplotype-specific hypermethylation of the FMR1 promoter in a female FXS premutation carrier research sample. This highlights the utility of direct nanopore sequencing to shed light on epigenetic regulation in disease mechanisms.

With nanopore sequencing, it also is possible to dig deeper into the association between epigenetic and genetic variation and phenotype. The team's multiomic nanopore data revealed that DNA hypermethylation in FMR1 premutation alleles is correlated with STR repeat size and may account for variability in phenotypic expression in premutation carriers. The team’s simple, targeted approach could provide comprehensive insights into factors predisposing alleles to instability and hyper expansion.

However, Stevanovski et al. explained that little is known about the epigenetic regulation of most other genes on their targeted sequencing panel, highlighting the need for further investigation with nanopore sequencing — a ‘powerful tool for this purpose’ as adaptive sampling ‘permits the flexible inclusion of virtually any additional secondary target [such as methylation] … at no extra cost’.

‘We propose that targeted sequencing with ONT can address the pressing need for improved methods for molecular characterization of STR expansions.’

Stevanovski et al. concluded that their targeted nanopore sequencing approach enabled them to resolve large and complex STR expansions, and DNA methylation profiles that ‘eluded characterization by standard molecular testing and short-read NGS’. They suggested that their work could ‘reveal previously unknown genotype-phenotype correlations, enabling better understanding of the pathomechanisms’ in many unsolved genetic studies.