Evaluation of Single Exon Deletions in DMD/BMD: Technical and Analytical Concerns
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.355142
Source of Support: None, Conflict of Interest: None
Keywords: Becker muscular dystrophy, Duchenne muscular dystrophy, MLPA, single exon deletions
Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive neuromuscular disorders caused by mutations in the dystrophin (DMD) gene located on Xp21.2 (the largest known human gene). Mutations in the DMD gene may cause alterations in the structure or function of dystrophin causing BMD or may prevent the production of the protein completely causing DMD, leading to muscle weakness and cardiac myopathies. The occurrence of DMD and BMD worldwide is 1:3500 and 1:18500 male births, respectively.
Because of its large size, the mutation spectrum of the DMD gene is very complex with over 5000 reported mutations and several sporadic mutations. Among these, about 65% of DMD cases and 85% of BMD cases consist of large deletions, duplications are present in 5–10% of DMD/BMD cases, and point mutations are present in 15–30% of DMD and 10–15% of BMD cases. It has been observed that 33% of all cases are because of de novo mutations or germline mosaicism. Also, in such cases, point mutations or duplications arise preferentially during spermatogenesis, whereas deletions mostly arise in oogenesis.
A simple and cost-effective method still in regular use for diagnosis of DMD involves the use of conventional polymerase chain reaction (PCR) amplifying 18 hotspot exons in a multiplex and detects 90–98% of all deletions. However, because of its inability to detect duplications, deletion breakpoints, and carrier females, the multiplex ligation-dependent probe amplification (MLPA) technique has emerged as a more effective method for the detection of both deletions and duplications in all 79 exons of the DMD gene (detection rate >75–80%). About 20% cases may have point mutations which may not be visible as whole exon deletions/duplications using MLPA. Sanger sequencing or next-generation sequencing (NGS) may be used to further characterize such cases. Muscle biopsy can be used as a complementary method wherever warranted as it provides fast and reliable results.
Although MLPA is one of the most widely used techniques and has proven its utility in the diagnosis of DMD/BMD, studies have suggested that false positives may arise because of the presence of sequence variations within 8 bp of the MLPA probe-binding site. It has thus also been suggested that such cases with single exon deletions be confirmed using an alternate method: PCR or sequencing., Thus, the aim of this study was to evaluate such single exon deletions in the study population. Exon skipping therapies targeting specific exons of the DMD gene have been approved and are in use. It, thus, becomes even more important to distinguish between true and false positives in order to help make therapy decisions.
Our laboratory observed 25.12% (49/195) males with single exon deletions by MLPA during the period from 2015 to 2019 [Table 1]. Out of these, 46 samples were proved to carry single exon deletion as confirmed by an alternate method (conventional PCR or Sanger's sequencing), that is, no amplification was observed with an alternate primer set. However, amplification was observed by alternate primers for the remaining three samples, S7, S8, and S10 (single exon deletion of exons 13, 18, and 42), which were then sequenced to identify the variations at the primer-binding site. These variations were further characterized to assess their pathogenic nature [Table 2].
Sample S7 when sequenced demonstrated c.1602 G >A variation at the splice site of the exon which was also the first base of the left probe oligonucleotide (LPO) sequence of the MLPA probe for exon 13. On further investigation, it was noted that the c.1602 G >A variant was reported as a benign variant; however, a different nucleotide substitution at the same position (c.1602 G >T) had been reported in an individual with Becker muscular dystrophy. The variant was also not reported to be observed in large population cohorts. The c.1602 G >A nucleotide change results in a synonymous amino acid substitution (p.Lys534=). In silico tools predict that c.1602 G >A changes the natural splice site of intron 13 and leads to abnormal gene splicing. However, because there are no RNA/functional studies carried out, the actual effect of c.1602 G >A on splicing in this individual remains unknown.
On sequencing sample S8, c.2227C>T variation was observed, which was the second base of the RPO sequence of the MLPA probe for exon 18. This variation leads to the generation of a premature stop codon. This variant has been reported in the literature (Clinvar) as pathogenic owing to the formation of a truncated protein. A study carried out on a cohort of the Spanish population reported this variant in patients with DMD. Another study also observed this variant in an individual with DMD where an ambiguous MLPA ratio was observed. Our laboratory also observed a relative peak ratio of 0.41, which may be attributed to the nucleotide mismatch between the patient sequence and the probe sequence, resulting in sub-optimal annealing of the primer to the target sequence during MLPA.
A deletion of two bases involving the last base of the LPO sequence and the first base of the right probe oligonucleotide (RPO) sequence of the exon 42 probe of MLPA was observed on sequencing sample S10: c. 5951_5952del TG. This variant has not been described in the literature; however, it leads to premature termination (Val1984Aspfs*3) which may probably have deleterious effects.
Based on the observations made in our laboratory, we suggest that evaluation of single exons in DMD be performed with caution. We observed single exon deletions in 25.12% cases, which is similar to that observed by other studies., We detected a false positive rate of 6.12% by MLPA. Thus, all single exon deletions should be confirmed with an alternate method/technique because point mutations may be present near the probe-binding site which may hamper subsequent hybridization/ligation steps and may lead to false positive results. Other parameters such as peak ratio, peak calling, ratio chart, fragment electropherograms, and so on need to be carefully evaluated and analyzed as default bin settings may cause ambiguous peak calling, leading to improper analysis of MLPA data.
The suggested algorithm for the diagnosis of DMD is the use of MLPA to detect deletions/duplications, followed by the use of sequencing to detect point mutations where no deletions/duplications are detected and further negative cases with a strong suspicion of dystrophinopathies can be characterized using muscle-derived mRNA analysis. For evaluation of single exon deletions, in particular, we suggest using a combination of MLPA along with PCR and sequencing for clearer resolution. Additionally, NGS can be performed for cases negative by any of the aforementioned methods. Careful evaluation of single exon deletions needs to be carried out in order to avoid false positives, and characterization of point mutations, if present, with the help of in silico tools and database searches is important to know the pathogenic nature of the mutation.
This material is the author's original work which has not been previously published elsewhere.
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[Table 1], [Table 2]