Abstract
Next generation sequencing (NGS) has changed our approach to diagnosis of genetic disorders. Nowadays, the most comprehensive application of NGS is whole genome sequencing (WGS) that is able to detect virtually all DNA variations. However, even after accurate WGS, many genetic conditions remain unsolved. This may be due to the current NGS protocols, based on DNA fragmentation and short reads. To overcome these limitations, we applied a linked-read sequencing technology that combines single-molecule barcoding with short-read WGS. We were able to assemble haplotypes and distinguish between alleles along the genome. As an exemplary case, we studied the case of a female carrier of X-linked muscular dystrophy with an unsolved genetic status. A deletion of exons 16–29 in DMD gene was responsible for the disease in her family, but she showed a normal dosage of these exons by Multiplex Ligation-dependent Probe Amplification (MLPA) and array CGH. This situation is usually considered compatible with a “non-carrier” status. Unexpectedly, the girl also showed an increased dosage of flanking exons 1–15 and 30–34. Using linked-read WGS, we were able to distinguish between the two X chromosomes. In the first allele, we found the 16–29 deletion, while the second allele showed a 1–34 duplication: in both cases, linked-read WGS correctly mapped the borders at single-nucleotide resolution. This duplication in trans apparently restored the normal dosage of exons 16–29 seen by quantitative assays. This had a dramatic impact in genetic counselling, by converting a non-carrier into a double carrier status prediction. We conclude that linked-read WGS should be considered as a valuable option to improve our understanding of unsolved genetic conditions.
Highlights
IntroductionX-linked recessive neuromuscular disorders due to mutations in the dystrophin gene (MIM 300377)
Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD) areX-linked recessive neuromuscular disorders due to mutations in the dystrophin gene (MIM 300377)
We report the case of a 6-year-old with an elevated CKinand a diagnosis of MLPAand analysis of DMD gene identified exons 16–29
Summary
X-linked recessive neuromuscular disorders due to mutations in the dystrophin gene (MIM 300377). Whereas deletions or duplications of one or more exons in DMD gene account for about 75% of cases, small mutations (missense, nonsense, and splice site variations), short insertions/deletions of bases, and small inversion are detected in the remaining 20–25% of cases [1,2,3]. Generation sequencing (NGS) is used to detect the remaining mutations [9,10]. The updated care considerations for DMD suggest NGS after MLPA in the diagnostic pipeline [11], but the technologies are moving very quickly and we cannot exclude that the two-step protocol could be converted into a single NGS-based test [12]
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