ANALYSIS OF GENE COPY NUMBER VARIATIONS IN PATIENTS WITH CARDIAC SEPTAL DEFECTS USING MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION
CNVs analysis in CSDs
Objective: Cardiac septal defects (CSDs), the most common human congenital heart malformations are complex and heterogeneous. Progress in molecular biology has helped to identify many genes responsible for cardiac morphogenesis. However, etiologic factors in familial as well as isolated syndromes are being identified; the root genetic cause still needs to be resolved and its mechanism is yet to be revealed. The objective of this study is to identify DNA copy number variations (CNVs) and their possible association with septal defects.
Methods: Multiplex ligation-dependent probe amplification (MLPA) was used to detect DNA copy number in non-syndromic CSDs using the P311-A1 Kit consisting of probes for the key genes, namely, NKX2-5 (NK2 transcription factor related, locus 5), GATA4 (GATA binding protein 4), TBX5 (T-box transcription factor), bone morphogenetic protein 4, and CRELD1 (cysteine rich with EGF-like domains 1).
Results: We studied 124 clinically diagnosed CSD subjects, of which 111 (89.5%) had atrial septal defects and 13 (10.5%) had ventricular septal defects. MLPA assay was carried out in all these patients after a thorough clinical and cytogenetic screening. CNVs were identified in 16 (12.9%) cases, of which heterozygous deletions and heterozygous duplications were detected (8 patients each) with apparent phenotypes.
Conclusion: MLPA could be a useful assay for the detection of CNVs and to be adopted as the first line of screening in patients with congenital heart diseases.
2. Saxena A, Mehta A, Sharma M, Salhan S, Kalaivani M, Ramakrishnan S, et al. Birth prevalence of congenital heart disease: A cross-sectional observational study from North India. Ann Pediatr Cardiol 2016;9:205-9.
3. Dietz H. Genetic epidemiology. In: Ferencz C, Rubin JD, Loffredo CA, Magee CA, editors. Epidemiology of Congenital Heart Disease: The Baltimore Washington Infant Study 1981-1989. Vol. 11. Mount Kisco, NY: Futura Publishing Company; 1994. p. 455-6.
4. Pierpont ME, Basson CT, Benson DW Jr., Gelb BD, Giglia TM, Goldmuntz E, et al. Genetic basis for congenital heart defects: Current knowledge: A scientific statement from the American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young: Endorsed by the American Academy of Pediatrics. Circulation 2007;115:3015-38.
5. Granados-Riveron JT, Pope M, Bu’lock FA, Thornborough C, Eason J, Setchfield K, et al. Combined mutation screening of NKX2-5, GATA4, and TBX5 in congenital heart disease: Multiple heterozygosity and novel mutations. Congenit Heart Dis 2012;7:151-9.
6. Moorhead PS, Nowell PC, Mellman WJ, Battips DM, Hungerford DA. Chromosome preparations of leukocytes cultured from human peripheral blood. Exp Cell Res 1960;20:613-6.
7. 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 Acids Res 2002;30:e57.
8. Iafrate AJ, Feuk L, Rivera MN, Listewnik ML, Donahoe PK, Qi Y, et al. Detection of large-scale variation in the human genome. Nat Genet 2004;36:949-51.
9. Sebat J, Lakshmi B, Troge J, Alexander J, Young J, Lundin P, et al. Large-scale copy number polymorphism in the human genome. Science 2004;305:525-8.
10. Miller DT, Adam MP, Aradhya S, Biesecker LG, Brothman AR, Carter NP, et al. Consensus statement: Chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. Am J Hum Genet 2010;86:749-64.
11. Cooper GM, Coe BP, Girirajan S, Rosenfeld JA, Vu TH, Baker C, et al. A copy number variation morbidity map of developmental delay. Nat Genet 2011;43:838-46.
12. Edwards JJ, Gelb BD. Genetics of congenital heart disease. Curr Opin Cardiol 2016;31:235-41.
13. Stuppia L, Antonucci I, Palka G, Gatta V. Use of the MLPA assay in the molecular diagnosis of gene copy number alterations in human genetic diseases. Int J Mol Sci 2012;13:3245-76.
14. John N, Rajasekhar M, Girisha KM, Sharma PS, Gopinath PM. Multiplex ligation-dependant probe amplification study of children with idiopathic mental retardation in South India. Indian J Hum Genet 2013;19:165-70.
15. Sørensen KM, El-Segaier M, Fernlund E, Errami A, Bouvagnet P, Nehme N, et al. Screening of congenital heart disease patients using multiplex ligation-dependent probe amplification: Early diagnosis of syndromic patients. Am J Med Genet A 2012;158A:720-5.
16. Liao J, Kochilas L, Nowotschin S, Arnold JS, Aggarwal VS, Epstein JA,et al. Full spectrum of malformations in velo-cardio-facial syndrome/ DiGeorge syndrome mouse models by altering Tbx1 dosage. Hum Mol Genet 2004;13:1577-85.
17. Borkar Y, Nayak K, Shetty RK, Moka R. A TBX5 nonsense mutation in siblings with divergent phenotypes associated with isolated septal defects. Asian J Pharm Clin Res 2017;10:126-30.
18. Liu Z, Wang J, Liu S, Deng Y, Liu H, Li N, et al. Copy number variation of GATA4 and NKX2-5 in Chinese fetuses with congenital heart disease. Pediatr Int 2015;57:234-8.
19. Guida V, Lepri F, Vijzelaar R, De Zorzi A, Versacci P, Digilio MC, et al. Multiplex ligation-dependent probe amplification analysis of GATA4 gene copy number variations in patients with isolated congenital heart disease. Dis Markers 2010;28:287-92.
20. Soemedi R, Wilson IJ, Bentham J, Darlay R, Töpf A, Zelenika D, et al. Contribution of global rare copy-number variants to the risk of sporadic congenital heart disease. Am J Hum Genet 2012;91:489-501.
21. Barber JC, Bunyan D, Curtis M, Robinson D, Morlot S, Dermitzel A, et al. 8p23.1 duplication syndrome differentiated from copy number variation of the defensin cluster at prenatal diagnosis in four new families. Mol Cytogenet 2010;3:3.
This work is licensed under a Creative Commons Attribution 4.0 International License.
The publication is licensed under CC By and is open access. Copyright is with author and allowed to retain publishing rights without restrictions.