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BRIEF REPORT
Year : 2022  |  Volume : 70  |  Issue : 4  |  Page : 1615-1617

Evaluation of Single Exon Deletions in DMD/BMD: Technical and Analytical Concerns


1 Department of Molecular Pathology, Metropolis Healthcare Ltd., Mumbai, Maharashtra, India
2 Department of Medical Genetics, Metropolis Healthcare Ltd., Mumbai, Maharashtra, India

Date of Submission21-May-2021
Date of Decision06-Apr-2022
Date of Acceptance09-Jun-2022
Date of Web Publication30-Aug-2022

Correspondence Address:
Tavisha Dama
156, Shera Villa, Dr. M. B. Raut Road No. 2, Shivaji Park, Mumbai – 400 028, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0028-3886.355142

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 » Abstract 


Background: Oftentimes, a variation at the multiplex ligation-dependent probe amplification (MLPA) probe binding site leads to improper hybridrization/ligation of the probe showing up as a deletion of an exon leading to false positive results for the detection of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD).
Objective: Investigating cases with single exon deletion using an alternate method [polymerase chain reaction (PCR) or sequencing] for confirmation of the deletion.
Methods: We evaluated males with single exon deletion (n = 49) in our study population (2015-2019). Forty-six were confirmed by an alternate method (conventional PCR/Sanger's sequencing) to confirm the deletion.
Results: We observed 25.12% single exon deletions in our study cohort. Further evaluation determined a false positive rate of 6.12%. Three out of 49 single exon deletions had a point mutation near the probe-binding site, indicating a false positive result. Single exon deletions, thus, need to be evaluated with extreme caution, and point mutations, if any, need to be characterized to determine the nature of their pathogenicity.


Keywords: Becker muscular dystrophy, Duchenne muscular dystrophy, MLPA, single exon deletions
Key Message: Cautious evaluation of single exon deletions in DMD/BMD is crucial in order to avoid false negative results when using MLPA as a method for detection because of the presence of mutations at the probe-binding site.


How to cite this article:
Dama T, Chheda P, Limaye S, Pande S, Vinarkar S. Evaluation of Single Exon Deletions in DMD/BMD: Technical and Analytical Concerns. Neurol India 2022;70:1615-7

How to cite this URL:
Dama T, Chheda P, Limaye S, Pande S, Vinarkar S. Evaluation of Single Exon Deletions in DMD/BMD: Technical and Analytical Concerns. Neurol India [serial online] 2022 [cited 2022 Oct 2];70:1615-7. Available from: https://www.neurologyindia.com/text.asp?2022/70/4/1615/355142




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.[1] 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.[2]

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.[3] 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%).[4] About 20% cases may have point mutations which may not be visible as whole exon deletions/duplications using MLPA.[5] 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.[6]

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.[7] It has thus also been suggested that such cases with single exon deletions be confirmed using an alternate method: PCR or sequencing.[8],[9] 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.[10] 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].
Table 1: Single exon deletions observed in the study population

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Table 2: Variations detected on sequencing single exon deletions (by MLPA)

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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.[11] The variant was also not reported to be observed in large population cohorts.[12] 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.[13] 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.[14]

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.[7],[8] 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.[15] 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.

Ethical statement

This material is the author's original work which has not been previously published elsewhere.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 » References Top

1.
Forrest SM, Cross GS, Flint T, Speer A, Robson KJ, Davies KE. Further studies of gene deletions that cause Duchenne and Becker muscular dystrophies. Genomics 1988;2:109-14.  Back to cited text no. 1
    
2.
Grimm T, Kress W, Meng G, Müller CR. Risk assessment and genetic counseling in families with Duchenne muscular dystrophy. Acta Myol 2012;31:179-83.  Back to cited text no. 2
    
3.
Nouri N, Fazel-Najafabadi E, Salehi M, Hosseinzadeh M, Behnam M, Ghazavi MR, et al. Evaluation of multiplex ligation-dependent probe amplification analysis versus multiplex polymerase chain reaction assays in the detection of dystrophin gene rearrangements in an Iranian population subset. Adv Biomed Res. 2014;3:72.  Back to cited text no. 3
    
4.
Mercuri E, Muntoni F. Muscular dystrophies. Lancet Lond Engl 2013;381:845-60.  Back to cited text no. 4
    
5.
Aartsma-Rus A, Ginjaar IB, Bushby K. The importance of genetic diagnosis for Duchenne muscular dystrophy. J Med Genet 2016;53:145-51.  Back to cited text no. 5
    
6.
Nilipour, Y. The art of muscle biopsy in new genetic era: A Narrative Review. Iran J Child Neurol 2019;13:7-17.  Back to cited text no. 6
    
7.
Kim MJ, Cho SI, Chae JH, Lim BC, Lee JS, Lee SJ, et al. Pitfalls of multiple ligation-dependent probe amplifications in detecting DMD exon deletions or duplications. J Mol Diagn 2016;18:253-9.  Back to cited text no. 7
    
8.
Varga RE, Mumtaz R, Jahic A, Rudenskaya GE, Sánchez-Ferrero E, Auer-Grumbach M, et al. MLPA-based evidence for sequence gain: Pitfalls in confirmation and necessity for exclusion of false positives. Anal Biochem 2012;421:799-801.  Back to cited text no. 8
    
9.
Santos R, Gonçalves A, Oliveira J, Vieira E, Vieira JP, Evangelista T, et al. New variants, challenges and pitfalls in DMD genotyping: Implications in diagnosis, prognosis and therapy. J Hum Genet 2014;59:454-64.  Back to cited text no. 9
    
10.
Aartsma-Rus A, Straub V, Hemmings R, Haas M, Schlosser-Weber G, Stoyanova-Beninska V, et al. Development of Exon skipping therapies for duchenne muscular dystrophy: A critical review and a perspective on the outstanding issues. Nucleic Acid Ther 2017;27:251-9.  Back to cited text no. 10
    
11.
Hagiwara Y, Nishio H, Kitoh Y, Takeshima Y, Narita N, Wada H, et al. A novel point mutation (G-1 to T) in a 5' splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy. Am J Hum Genet 1994;54:53-61.  Back to cited text no. 11
    
12.
Lek M, Karczewski K, Minikel E, Samocha KE, Banks E, Fennell T, et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature 2016;536:285–91.  Back to cited text no. 12
    
13.
Vieitez I, Gallano P, González-Quereda L, Borrego S, Marcos I, Millán JM, et al. Mutational spectrum of Duchenne muscular dystrophy in Spain: Study of 284 cases. Espectro mutacional de la distrofia muscular de Duchenne en España: estudio de 284 casos. Neurologia 2017;32:377-85.  Back to cited text no. 13
    
14.
Niba ET, Tran VK, Tuan-Pham le A, Vu DC, Nguyen NK, Ta VT, et al. Validation of ambiguous MLPA results by targeted next-generation sequencing discloses a nonsense mutation in the DMD gene. Clin Chim Acta 2014;436:155-9.  Back to cited text no. 14
    
15.
Fratter C, Dalgleish R, Allen SK, Santos R, Abbs S, Tuffery-Giraud S, et al. EMQN best practice guidelines for genetic testing in dystrophinopathies. Eur J Hum Genet 2020;28:1141–59.  Back to cited text no. 15
    



 
 
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