Nanopore-based single molecule sequencing for D4Z4 array responsible for facioscapulohumeral muscular dystrophy

Abstract

Subtelomeric macrosatellite repeats are difficult to sequence using conventional sequencing methods owing to the high similarity among repeat units and high GC content. Sequencing these repetitive regions is challenging, even with recent improvements in sequencing technologies. Among these repeats, a haplotype carrying a particular sequence and shortening of the D4Z4 array on human chromosome 4q35 causes one of the most prevalent forms of muscular dystrophy with autosomaldominant inheritance, facioscapulohumeral muscular dystrophy (FSHD). Here, we applied a nanoporebased ultra-long read sequencer to sequence a BAC clone containing 13 D4Z4 repeats and flanking regions. We successfully obtained the whole D4Z4 repeat sequence, including the pathogenic gene DUX4 in the last D4Z4 repeat. The estimated sequence accuracy of the total repeat region was 99.8% based on a comparison with the reference sequence. Errors were typically observed between purine or between pyrimidine bases. Further, we analyzed the D4Z4 sequence from publicly available ultra-long whole human genome sequencing data obtained by nanopore sequencing. This technology may be a new tool for studying D4Z4 repeats and pathomechanism of FSHD in the future and has the potential to widen our understanding of subtelomeric regions.Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent adult-onset muscular dystrophies. The genomes of most patients with FSHD have a common feature, i.e., a contracted subtelomeric macrosatellite repeat array called D4Z4 on chromosome 4q35. The D4Z4 array consists of a highly similar 3.3-kb single repeat unit. Normally, the D4Z4 array is highly methylated and forms heterochromatin. Patients with FSHD have less than 11 D4Z4 repeats 1-3 . In Japan, the majority of patients with FSHD have less than 7 repeats 4 . Shortening of the D4Z4 array causes the de-repression of the flanking genes as well as DUX4, located in the last D4Z4 repeat. The ectopic expression of DUX4 is toxic in muscle tissues and is thought to be a causal factor for FSHD [5][6][7][8][9] . In addition to the repeat number, the haplotype of the last D4Z4 repeat is important for the development of FSHD 1,2 . The telomeric flanking region of D4Z4 contains the 3′ UTR of DUX4 and is called the pLAM region. The presence of a polyadenylation signal in this region allows DUX4 expression and disease manifestation 10 . In contrast, individuals without polyadenylation signals do not manifest the disease 2 .

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“…For example, facioscapulohumeral muscular dystrophy has been associated with the D4Z4 repeat that is 3300 bases in length. This repeat was detected by southern blotting and fully sequenced using Nanopore-based sequencing technology [49]. While the cost of long-read sequencing is currently high, several initial large-scale studies have been reported.…”

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“…Long-read technologies that can sequence through repetitive segments are likely to revolutionise diagnostics and gene discovery in STR-related diseases. Several approaches have been developed that use long-read technologies to identify tandem-repeat variants on a genome-wide basis [107][108][109] , and these long reads have been used to identify known pathogenic STR variants and discover novel tandem repeat causes of disease [110][111][112][113][114] .…”

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“…K-mers have the potential to reveal new alleles associated with desirable phenotypic traits in maize [ 100 ]. However, the short-read sequencing which was used produced a high fraction of non-uniquely mapped reads in the maize population [ 100 ]; a challenge that would likely be overcome using long Nanopore reads [ 101 ].…”