The importance of direct genetic testing for determining female carriers of the mutation in dystrophinopathies

Jasmina Maksić; Nela Maksimović; Lukas Rasulić; Olgica Milankov; Ana Marjanović; Dragana Cvetković; Vidosava Rakočević Stojanović; Ivana Novaković

doi:10.2298/VSP190208030M

Jasmina Maksić University of Belgrade, Faculty for Special Education and Rehabilitation, Belgrade, Serbia
Nela Maksimović University of Belgrade, Faculty of Medicine, Belgrade, Serbia
Lukas Rasulić University of Belgrade, Faculty of Medicine, Belgrade, Serbia
Olgica Milankov Institute for Health Care of Children and Youth of Vojvodina, Novi Sad, Serbia
Ana Marjanović University Clinical Center of Serbia, Clinic for Neurology, Belgrade, Serbia
Dragana Cvetković University of Belgrade, Faculty of Biology, Belgrade, Serbia
Vidosava Rakočević Stojanović University of Belgrade, Faculty of Medicine, Belgrade, Serbia
Ivana Novaković University of Belgrade, Faculty of Medicine, Belgrade, Serbia

DOI: https://doi.org/10.2298/VSP190208030M

Keywords: genes, genetic testing, muscular dystrophies, mutation, prenatal diagnosis, women

Abstract

Background/Aim. Duchenne muscular dystrophy (MD) and Becker MD are caused by mutations in the gene for dystrophin (DMD). They are X chromosome-linked reces-sive diseases where males are affected, and females are healthy carriers of the mutation in most cases. It is estimat-ed that 2/3 of mothers of Duchenne MD probands are car-riers, while 1/3 of probands have de novo mutations. The aim of the study was to confirm the carrier status of female members of the families of Duchenne MD/Becker MD probands using direct genetic testing methods. Methods. The study included 38 females from 31 families of Du-chenne MD/Becker MD probands with dele-tion/duplication in the DMD gene. Moreover, 4 cases of prenatal diagnosis of Duchenne MD/Becker MD were in-cluded. The methods of polymerase chain reaction - PCR and the multiplex ligation-dependent probe amplification - MLPA were applied for detecting deletions, i.e., dele-tion/duplication mutations in the DMD gene. Results. In the total of 31 Duchenne MD/Becker MD probands, 87.1% of deletions and 12.9% of duplications of one or more exons in the DMD gene were detected. Of the 29 tested mothers, mutations were found in 17 of them (14 de-letions and 3 duplications). Mutations were detected in 11 (57.9%) out of 19 mothers of probands with the Duchenne MD phenotype and 6 (60%) out of 10 mothers of Becker MD probands. Furthermore, 14 (56%) out of 25 mothers were carriers in probands with deletions, and 3 (75%) out of 4 mothers were carriers in probands with duplications. In the remaining 9 other female relatives of the patients, muta-tions were found in 4. In prenatal diagnosis, we identified a deletion in one male and one female fetus of one single mother who was confirmed as a carrier. Conclusion. The study showed that mothers were carriers in almost 60% of sporadic cases of Duchenne MD/Becker MD with dele-tions and duplications. In addition, the carrier frequency tended to be higher in mothers of the probands with dupli-cations (75%) compared to mothers of probands with dele-tions (56%).

References

1. Emery A. Duchenne muscular dystrophy or Meryon's disease. Lancet 2001; 357(9267): 1529.

2. Haldane JB. The rate of spontaneous mutation of a human gene. 1935. J Genet 2004; 83(3): 235‒44.

3. Bladen CL, Salgado D, Monges S, Foncuberta ME, Kekou K, Kosma K, at al. The TREAT-NMD DMD Global Database: Analysis of More than 7,000 Duchenne Muscular Dystrophy Mutations. Hum Mutat 2015; 36(4): 395‒402.

4. Muntoni F, Torelli S, Ferlini A. Dystrophin and mutations: one gene, several proteins, multiple phenotypes. Lancet Neurol 2003; 2(12):731–40.

5. Kesari A, Pirra LN, Bremadesam L, McIntyre O, Gordon E, Dubrov-sky AL, et al. Integrated DNA, cDNA, and protein studies in Becker muscular dystrophy show high exception to the reading frame rule. Hum Mutat 2008; 29(5):728–37.

6. Mukherjee M, Chaturvedi LS, Srivastava S, Mittal RD, Mittal B. De novo mutations in sporadic deletional Duchenne muscular dystrophy (DMD) cases. Exp Mol Med 2003; 35(2): 113‒7.

7. Sakthivel Murugan SM, Arthi C, Thilothammal N, Lakshmi BR. Carrier detection in Duchenne muscular dystrophy using mo-lecular methods. Indian J Med Res 2013; 137(6): 1102‒10.

8. Maroni G. Molecular and Genetics Analysis of Human Traits. 1st ed. Hoboken, New Jersey: Blackwell Science Inc; 2001.

9. Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G.. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Ac-ids Res 2002; 30(12): e57.

10. Schwartz M, Duno M. Improved molecular diagnosis of dystro-phin gene mutations using the multiplex ligation-dependent probe amplification method. Genet Test 2004; 8(4): 361‒7.

11. Lalic T, Vossen RH, Coffa J, Schouten JP, Guc-Scekic M, Radivojevic D, et al. Deletion and duplication screening in the DMD gene using MLPA. Eur J Hum Genet 2005; 13(11): 1231‒4.

12. Pikó H, Vancsó V, Nagy B, Bán Z, Herczegfalvi A, Karcagi V. Dystrophin gene analysis in Hungarian Duchenne/Becker muscular dystrophy families - detection of carrier status in symptomatic and asymptomatic female relatives. Neuromuscul Disord 2009; 19(2): 108‒12.

13. Carsana A, Frisso G, Tremolaterra MR, Ricci E, De Rasmo D, Sal-vatore F. A Larger Spectrum of Intragenic Short Tandem Re-peats Improves Linkage Analysis and Localization of Intragen-ic Recombination Detection in the Dystrophin Gene. An

Analysis of 93 Families from Southern Italy. J Mol Diagn 2007; 9(1): 64–9.

14. Delgado-Luengo WN, Borjas-Fuentes L, Zabala-Fernández W, Fer-nández-Salgado E, Solís-Añez E, Chávez C, et al. Carrier detection of Duchenne/Becker muscular dystrophy by analysis of STRs loci linked to the gene of dystrophin in Venezuelan families. Invest Clin 2002; 43(4): 239‒54. 15. Kruyer H, Miranda M, Volpini V, Estivill X. Carrier detection and microsatellite analysis of Duchenne and Becker muscular dys-trophy in Spanish families. Prenat Diagn 1994; 14(2): 123‒30.

16. Taylor PJ, Maroulis S, Mullan GL, Pedersen RL, Baumli A, Elakis G, et al. Measurement of the clinical utility of a combined mu-tation detection protocol in carriers of Duchenne and Becker muscular dystrophy. J Med Genet 2007; 44(6): 368–72.

17. Miller SA, Dykes DD, Polesky HF. A simple salting out proce-dure for extracting DNA from human nucleated cells. Nucleic Acids Research 1988; 16(3): 1215.

18. Chamberlain JS, Gibbs RA, Rainer JE, Caskey CT. Multiplex PCR for the diagnosis of Duchenne muscular dystrophy. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR Protocols: A Guide to Methods and Applications. Academic Press New York 1990; 272‒81.

19. MRC-Holland – Start Page. MLPA protocols. Available from: https://www.mlpa.com [last visit 2018. September 2013].

20. Kohli S, Saxena R, Tomas E, Singh J, Verma IC. Gene changes in Duchenne muscular dystrophy: Comparison of multiplex PCR and multiplex ligation-dependent probe amplification tech-niques. Neurol India 2010; 58(6): 852‒6.

21. Sansović I, Barišić I, Dumić K. Improved detection of deletions and duplications in DMD gene using multiplex ligation-dependent probe amplification (MLPA) method. Biochem Genet 2013; 51(3‒4): 189‒201.

22. Songol A, Saberipour B, Bavarsad A. Comparison of multiplex li-gation-dependent probe amplification (MLPA) analysis versus multiplex PCR assays in the detection/insertion of dystrophin gene. Biotech Res Comm 2016; 9(1): 128‒31.

23. Basak J, Dasgupta UB, Mukherjee SC, Das SK, Senapati AK, Banerjee TK. Deletional mutations of dystrophin gene and carri-er detection in eastern India. Indian J Pediatr 2009; 76(10): 1007–12.

24. Dastur RS, Kachwala MY, Khadilkar SV, Hegde MR, Gaitonde PS. Identification of deletions and duplications in the Duchenne muscular dystrophy gene and female carrier status in western India using combinated methods of multiplex polymerase chain reaction and multiplex ligation-dependent probe amplifi-cation. Neurol India 2011; 59(6): 803‒9.

25. Lee T, Takeshima Y, Kusunoki N, Awano H, Yagi M, Matsuo M, et al. Differences in carrier frequency between mothers of Du-chenne and Becker muscular dystrophy patients. J Hum Genet 2014; 59(1): 46–50.

26. Grimm T, Meng G, Liechti-Gallati S, Bettecken T, Müller CR, Müller B. On the origin of deletions and point mutations in Du-chenne muscular dystrophy: most deletions arise in oogenesis and most point mutations result from events in spermatogene-sis. J Med Genet 1994; 31(3): 183–6.

27. Grimm T, Kress W, Meng G, Müller CR. Risk assessment and ge-netic counseling in families with Duchenne muscular dystro-phy. Acta Myol 2012; 31(3): 179–83.

28. Helderman-van den Enden AT, Madan K, Breuning MH, van der Hout AH, Bakker E, de Die-Smulders CE, et al. An urgent need for a change in policy revealed by a study on prenatal testing for Duchenne muscular dystrophy. Eur J Hum Genet 2013; 21(1): 21‒6.

29. Helderman-van den Enden AT, van den Bergen JC, Breuning MH, Verschuuren JJ, Tibben A, Bakker E, et al. Duchenne/Becker muscular dystrophy in the family: have potential carriers been tested at a molecular level? Clin Genet 2011; 79(3): 236‒42.

30. Massalska D, Zimowski JG, Roszkowski T, Bijok J, Pawelec M, Bed-narska‐Makaruk M. Prenatal diagnosis of Duchenne and Beck-er muscular dystrophies: Underestimated problem of the sec-ondary prevention of monogenetic disorders. J Obstet Gynae-col Res 2017; 43(7): 1111‒21.

31. Schade van Westrum SM, Hoogerwaard EM, Dekker L, Standaar TS, Bakker E, Ippel PF, et al. Cardiac abnormalities in a follow-up study on carriers of Duchenne and Becker muscular dys-trophy. Neurology 2011; 77(1): 62‒6.

32. Mercier S, Toutain A, Toussaint A, Raynaud M, de Barace C, Marco-relles P, et al. Genetic and clinical specificity of 26 symptomatic carriers for dystrophinopathies at pediatric age. Eur J Hum Genet 2013; 21(8): 855‒63.

33. Bushby K, Muntoni F, Bourke JP. 107th ENMC international workshop: the management of cardiac involvement in muscu-lar dystrophy and myotonic dystrophy. 7th–9th June 2002, Naarden, the Netherlands. Neuromuscul Disord 2003; 13(2): 166–72.

34. de Jong R, den Dunnen JT, Houwing-Duistermaat JJ, Kneppers AL, Ginjaar HB, et al. Recurrence risk due to germ line mosaicism: Duchenne and Becker muscular dystrophy. Clin Genet 2009; 75(5): 465‒72.