Targeted long-read sequencing enables comprehensive analysis of the genetic and epigenetic landscape of inherited myopathies

Fig. 1. Myopathy cohort characteristics and examples of known genetic variants identified by Oxford Nanopore Technologies long-read sequencing (ONT LRS). a Myopathy cohort summary with participants grouped into positive controls, newly solved cases following ONT LRS, and cases remaining unsolved following ONT LRS. The upper matrix summarises the distribution of muscle weakness. The middle matrix summarises the molecular findings contributing to each diagnosis. The lower matrix shows genetic diagnoses before and after ONT LRS. b Case L124 (known diagnosis of limb-girdle muscular dystrophy [LGMD] 2A/R1) harbours a missense variant (c.1468C>T) in exon 11 and duplication (c.2279dupA) in exon 22 of CAPN3 in trans (red & blue reads representing different alleles). c Case L141 (known diagnosis of congenital myopathy 18) harbours a complex, homozygous, multinucleotide variant (c.27_45delinsGCCATCCTCAC) in exon 1 of CACNA1S. d Case L149 (known diagnosis of a mitochondrial myopathy) harbours m.3243A>G at a heteroplasmy of 26% in blood. e Case L128 (known diagnosis of Duchenne muscular dystrophy) harbours a large deletion of DMD exons 3–7. ONT LRS generated chimeric reads spanning the deletion,…

Fig. 2. Targeted Oxford Nanopore Technologies long-read sequencing characterises the genetic and epigenetic architecture of facioscapulohumeral muscular dystrophy (FSHD). a Chromosome 4 D4Z4 read classification and annotation schematic. Reads are classified as complete, partial distal, partial proximal, or partial internal based on alignment. Reads are annotated with specific sequence features: simple sequence length polymorphism [SSLP], p13-E11, D4Z4 unit, pLAM, and 4qA/4qB markers. 5mC methylation is measured at all CpG sites and per-read distal-most D4Z4 unit average methylation fraction is calculated. b Per-read D4Z4 copy number and distal-most D4Z4 unit methylation fraction plots. Dashed lines represent an arbitrary 50% methylation value (vertical) and the 10-copy diagnostic threshold for FSHD1 (horizontal). Representative cases are shown for FSHD1 (L150: one distally-hypomethylated, 5-copy 4qA D4Z4 array allele and one normomethylated, non-contracted 4qA D4Z4 allele), non-FSHD (L249: normomethylated, non-contracted 4qA D4Z4 arrays), and FSHD2 (L153: distally-hypomethylated, non-contracted D4Z4 arrays on 4qA, 4qB and 10qB alleles). c Per-read 4q D4Z4 copy number distribution of FSHD…

Fig. 3. Targeted Oxford Nanopore Technologies long-read sequencing (ONT LRS) clarifies diagnoses in complex facioscapulohumeral muscular dystrophy (FSHD) cases. a Pedigree for cases L246 and L247. b Per-read 4qA D4Z4 copy number and distal-most D4Z4 unit methylation fraction plot for case L247. c Pile-up of complete 4qA reads from case L247 aligned to the pLAM sequence. d Pedigree of cases L154, L153 and L438. e 5mC methylation profile across the Hifiasm-assembled D4Z4 array duplication contig (bottom schematic), coloured by ‘higher’ (red) or ‘lower’ (blue) methylation with respect to an arbitrary 50% methylation value (dashed line). f Boxplots of distal-most D4Z4 unit methylation fraction in cases L154, L153, and L438. The dashed line represents an arbitrary 50% methylation value. Boxplots show the median (centre line), first and third quartiles (hinges), and most extreme values within 1.5 × the interquartile range of the hinges (whiskers). The number of reads contributing to each boxplot varied between samples (12 to 242). g Historical ‘positive’ 4qA-specific Southern blot (SB) in case L117. h Per-read D4Z4 copy number and distal-most D4Z4 unit methylation fraction plot for case L117…

Fig. 4. Targeted Oxford Nanopore Technologies long-read sequencing (ONT LRS) characterises the genetic and epigenetic architecture of oculopharyngodistal myopathy (OPDM). a Pedigrees of OPDM families diagnosed by ONT LRS. Symbols: square, male; circle, female; filled-in, affected; arrow, proband; diagonal line, deceased. IDs for the proband (P), mother (M), father (F) and brother (B) are shown for family members who underwent ONT LRS. b GGC•CCG short tandem repeat (STR) lengths and CpG methylation fractions across NOTCH2NLC, RILPL1, GIPC1, and ABCD3 in individuals from OPDM families. In the top panels, allele-specific STR repeat lengths are represented separately (yellow = haplotype 1, orange = haplotype 2). In the bottom panels, violin plots represent the density of per-read methylation values. c CpG methylation across the GIPC1 promoter in all OPDM family members. Vertical bars indicate methylation levels at different CpG sites. d Genome browser view of GIPC1 read-level methylation in case L126, a participant with OPDM2 due to a GIPC1 (GGC)217-570 STR expansion, and case L137, the mother of case L126, who has Parkinson’s disease but no myopathy and a GIPC1 (GGC)76-85 STR expansion.…

Fig. 5. Additional new genetic diagnoses made using Oxford Nanopore Technologies long-read sequencing (ONT LRS). a Pedigree of case L236. Symbols: square, male; circle, female; filled-in, affected; arrow, proband; diagonal line, deceased. b Genome browser view of a homozygous intronic LAMA2 variant, c.2749+4_2749+15del, in case L236. c LAMA2 reverse transcription polymerase chain reaction (RT-PCR) confirmed absence of a normal splice product and generation of two novel splice products (in-frame truncation and complete skipping of exon 19) in case L236 but not in a control. RT-PCR was run twice with concordant results. d Genome browser view of CAPN3 variants, c.1453A>G and c.2440-75C>G, in trans in case L323. e SpliceAI predicts CAPN3:c.2440-75C>G may create a new donor splice site (Δ0.28) one base-pair downstream and also activate an upstream cryptic acceptor site (Δ0.33), together resulting in inclusion of a 95 bp pseudoexon into the CAPN3 transcript between the penultimate and final exons. The new pseudo-exon is predicted to translate into a novel 20 amino acid sequence (arrow) followed by a premature stop codon. f CAPN3 RT-PCR using two combinations of forward (F) and reverse (R)…