Multiplex ligation dependent probe amplification - A useful, fast and cost-effective method for identification of small supernumerary marker chromosome in children with developmental delay and congenital heart defect

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Small supernumerary marker chromosome (sSMC) is a rare chromosomal abnormality and is detected in about 0.3% in cases with multiple congenital anomalies (MCA) and/or developmental delay. Different techniques for investigation of cases with MCA and/or developmental delay are available ranging from karyotyping to molecular cytogenetic technique and ultimately multiplex ligation dependent probe amplification (MLPA). Here we present a patient with multiple congenital anomalies for which classical cytogenetic technique was used as a first step in diagnosis and the results being confirmed by MLPA. The karyotype disclosed a sSMC considered to be a fragment of chromosome 22. The MLPA analysis using SALSA MLPA probemix P064-C2 Microdeletion Syndromes-1B confirmed the karyotype results, and according to the manufacturer’s recommendation we performed another confirmation analysis with MLPA probemix P311-B1 Congenital Heart Disease and MLPA probemix P250-B2 DiGeorge. We also suspected an Emanuel syndrome and performed another MLPA analysis with SALSA MLPA probemix P036-E3 Subtelomeres Mix 1 and probemix P070-B3 Subtelomeres Mix 2B for investigation of subtelomeric region that revealed a duplication of 11q25 region and the confirmation was performed using SALSA MLPA probemix P286-B2 Human Telomere-11.

In conclusion, we consider that MLPA is a valuable method for identification of sSMC in children with developmental delay and congenital anomalies. Genetic diagnosis using different molecular techniques, such as MLPA, for increasing accuracy in identification of chromosomal structural aberrations has an important role in clinical diagnosis and in genetic counselling and our case explain the importance of using a specific laboratory technique for each stage of diagnosis.

1. Osoegawa K, Iovannisci DM, Lin B, Parodi C, Schultz K, Shaw GM, et al. Identification of Novel Candidate Gene Loci and Increased Sex Chromosome Aneuploidy among Infants with Conotruncal Heart Defects. Am J Med Genet A. 2014 Feb;0(2):397–406. DOI: 10.1002/ajmg.a.36291

2. Bernstein D. Epidemiology and genetic basis of congenital heart disease. Kliegman R, Stanton B, St Geme JW, Schor NF, Behrman RE, Nelson Textbook of Pediatrics, Philadelphia, 2016, pps 2182-2187.

3. Murray LE, Smith AH, Flack EC, Crum K, Owen J, Kannankeril PJ. Genotypic and phenotypic predictors of complete heart block and recovery of conduction after surgical repair of congenital heart disease. Heart Rhythm. 2017 Mar;14(3):402-9. DOI: 10.1016/j.hrthm.2016.11.010

4. Muntean I, Şuteu C, Togănel R, Bănescu C. Association between MDR1 gene polymorphism and clinical course of pediatric pulmonary arterial hypertension. Rev Romana Med Lab. 2018;26(3):305-12. DOI: 10.2478/rrlm-2018-0025

5. Cowan JR, Ware SM. Genetics and genetic testing in congenital heart disease. Clin Perinatol. 2015 Jun;42(2):373-93. DOI: 10.1016/j.clp.2015.02.009

6. Gelb BD, Chung WK. Complex Genetics and the Etiology of Human Congenital Heart Disease. Cold Spring Harb Perspect Med. 2014 Jul;4:a013953. DOI: 10.1101/cshperspect.a013953

7. Soares G, Alvares S, Rocha C, Teixeira MF, Mota MC, Reis MI, et al. Congenital heart defects and chromosomal anomalies including 22q11 microdeletion (CATCH 22). Rev Port Cardiol. 2005 Mar;24(3):349-71.

8. Rachel S, Ian JW, Jamie B, Rebecca D, Ana T, Diana Z, et al. Contribution of Global Rare Copy-Number Variants to the Risk of Sporadic Congenital Heart Disease. Am J Hum Genet. 2012 September;91(3):489-501. DOI: 10.1016/j.ajhg.2012.08.003

9. Pânzaru M, Rusu C, Voloşciuc M, Braha E, Butnariu L, Gramescu M, et al. Benefits of cytogenetic testing in diagnosis of plurimalformative syndromes with congenital heart defects. Rev Romana Med Lab. 2012;20(3):265-72.

10. Monteiro RAC, Freitas ML, Vianna GS, de Oliveira VT, Pietra RX, Ferreira LCA, et al. Major Contribution of Genomic Copy Number Variation in Syndromic Congenital Heart Disease: The Use of MLPA as the First Genetic Test. Mol Syndromol. 2017 Aug;8:227–35. DOI: 10.1159/000477226

11. Lander J, Ware SM. Copy Number Variation in Congenital Heart Defects. Curr Genet Med Rep. 2014;2(3):168–78. DOI: 10.1007/s40142-014-0049-3

12. Karen RS, Rafaella M, Janaina H, Lucia CP, Mariluce R. Cytogenomic Evaluation of Subjects with Syndromic and Nonsyndromic Conotruncal Heart Defects. Biomed Res Int. 2015; 2015:401941.

13. 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 Jun;30(12):e57. DOI: 10.1093/nar/gnf056

14. Slavotinek AM. Novel microdeletion syndromes detected by chromosome microarrays. Hum Genet. 2008 Aug;124(1):1-17. DOI: 10.1007/s00439-008-0513-9

15. Sireteanu A, Popescu R, Braha EE, Bujoran C, Butnariu L, Caba L, et al. Detection of chromosomal imbalances using combined MLPA kits in patients with syndromic intellectual disability. Rev Romana Med Lab. 2014;22(2):157-64. DOI: 10.2478/rrlm-2014-0019

16. Lam AC, Lam ST, Lai KK, Tong TM, Chau TC. High rate of detection of subtelomeric aberration by using combined MLPA and subtelomeric FISH approach in patients with moderate to severe mental retardation. Clin Biochem. 2006 Feb;39(3):196-202. DOI: 10.1016/j.clinbiochem.2006.01.003

17. Zou PS, Li HZ, Chen LS, Ma M, Chen XH, Xue D, et al. A rare case of trisomy 11q23.3-11q25 and trisomy 22q11.1-22q11.21. Genetic and Molecular Research. 2016;15(2):gmr15028140. DOI: 10.4238/gmr.15028140

18. Liehr T, Chapter 5 Small Supernumerary Marker Chromosomes known to be correlated with specific syndromes, Small Supernumerary Marker Chromosomes (sSMC), Springer-Verlag Berlin Heidelberg, 2012, pp 47-52. DOI: 10.1007/978-3-642-20766-2_5

19. Trifonov V, Fluri S, Binkert F, Nandini A, Anderson J, Rodriguez L, et al. Complex rearranged small supernumerary marker chromosomes (sSMC), three new cases; evidence for an underestimated entity?. Mol Cytogenet. 2008 Apr;1:6. DOI: 10.1186/1755-8166-1-6

20. Sadek AA, Ashry MM. Yield of karyotyping in children with developmental delay and/or dysmorphic features in Sohag University Hospital, Upper Egypt. Egyptian J Med Hum Genet. 2018;19(3):253-9. DOI: 10.1016/j.ejmhg.2017.12.007

21. Hochstenbach R, Nowakowska B, Volleth M, Ummels A, Kutkowska-Kaźmierczak A, Obersztyn E, et al. Multiple Small Supernumerary Marker Chromosomes Resulting from Maternal Meiosis I or II Errors. Mol Syndromol. 2016 Feb;5(6):210–21.

22. Jehee FS, Takamori JT, Vasconcelos MPF, Pordeus AC, Latini FR, Bertola DR, et al. Using a combination of MLPA kits to detect chromosomal imbalances in patients with multiple congenital anomalies and mental retardation is a valuable choice for developing countries. Eur J Med Genet. 2011;54:425-32. DOI: 10.1016/j.ejmg.2011.03.007

23. 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 Apr;158A(4):720-5. DOI: 10.1002/ajmg.a.35214

24. Wentzel C, Fernström M, Ohrner Y, Annerén G, Thuresson AC. Clinical variability of the 22q11.2 duplication syndrome. Eur J Med Genet. 2008;51(6):501-10. DOI: 10.1016/j.ejmg.2008.07.005

25. Woori J, Hyojin C, Jiyeon K, Jung-Ok S, Seok CK, Bo Kyung K, et al. Identification of small marker chromosomes using microarray comparative genomic hybridization and multicolor fluorescent in situ hybridization, Mol Cytogenet. 2016;9:61. DOI: 10.1186/s13039-016-0273-5

Revista Romana de Medicina de Laborator

Romanian Journal of Laboratory Medicine

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