Clinically significant exome-based copy number variants detected by re-evaluation of exome sequencing data

Year 2021, Volume: 35 Issue: 1, 1 - 11, 06.04.2021

Fatma Kurt Çolak

https://doi.org/10.5505/deutfd.2021.29053

Abstract

INTRODUCTION: The next-generation sequencing(NGS) method is becoming widespread in many clinical laboratories as a result of this increase in usage is developed some tools to be integrated into analysis platforms. These platforms with dedicated software are increasingly being used to identify copy number variations(CNVs) along with single nucleotide variants(SNVs) simultaneously. Array-based technologies including single nucleotide polymorphism(SNP) genotyping has been be employed in parallel to NGS for further characterization of the CNV event. In this study, exome analysis was performed, no variant was detected and 30 patients who could not be diagnosed were re-evaluated.
METHODS: Clinical Exome Solution(CES) kit by Sophia Genetics was used to IlluminaNextSeq550® platform. The data obtained after sequencing was uploaded to SophiaDDM software for analysis. HumanCytoSNP-12v2.1BeadChip Kit at IlluminaInfinium®SNP-array platform and Affymetrix®Cytoscan Optima chips kit was used for follow-up confirmation of CNV events. The RRM2B gene gains and/or losses were detected by the SALSA MLPAprobemix P089TK2®.
RESULTS: In 4 cases out of 30 patients re-evaluation included RRM2B gene deletion, Xq28 duplication, 22q11.21 deletion, and 15q21.2 deletion.
DISCUSSION AND CONCLUSION: There are many advantages to analysing NGS data with well-developed software. Software that can evaluate both SNV and CNV data together, especially in our cases, help in solving clinical cases. In technology, the steps will lead to the development of software tools that give better results. Such analysis methods are of time and cost benefit in solving challenging cases in medical genetics for recessive conditions, especially when one copy of the gene is deleted and the other copy carries variants that may be pathogenic.

Keywords

Microarray, copy number variations, next generation sequencing

Ekzom dizileme verilerinin tekrar değerlendirilmesi ile saptanan klinik olarak anlamlı ekzom tabanlı kopya sayısı değişimleri

Abstract

GİRİŞ ve AMAÇ: Son zamanlarda yeni nesil dizileme (next generation sequencing-NGS) birçok klinik laboratuarda yaygınlaşmakta ve kullanımdaki bu artış neticesinde hazırlanan özel yazılıma sahip bu platformlar aynı anda tek nükleotid varyantları (single nucleotide variants-SNV) ile birlikte kopya sayısı değişikliklerini(copy number variation-CNV) tanımlamak için giderek daha fazla kullanılmaktadır. Tek nükleotit polimorfizm(single nucleotide polymorphism-SNP) genotiplemesini içeren mikrodizin tabanlı teknolojiler, CNV karakterizasyonu için NGS’e paralel olarak kullanılmaktadır. Bu çalışmada ekzom analizi yapılmış, herhangi bir varyant tespit edilememiş ve tanısı konulamamış 30 hasta tekrar değerlendirmeye alınmış ve tanıya olan katkısına bakılmıştır.
YÖNTEM ve GEREÇLER: Hastalarda NGS testi için Sophia genetics in Clinical Exome Solution (CES) kiti kullanıldı. Illumina Next Seq 550® cihazında çalışıldı. CNV olaylarının teyidi için mikrodizin çalışması olarak Illumina Infinium® HumanCytoSNP-12 v2.1 SNP-array çipleriyle Bluefuse® multi (v4.5) analiz programı ve Affymetrix® Cytoscan Optima çipleri ve Chromosome Analysis Suite (ChAS) 3.1 Thermo Fisher Scientific® programları kullanıldı. Bir hastada teyit için multiplex ligation-dependent probe amplification (MLPA) testi için SALSA MLPA probemix P089 TK2® kiti kullanıldı.
BULGULAR: Tekrar değerlendirilen 30 hastadan 4 vakada sırasıyla RRM2B geni delesyonu, Xq28 duplikasyonu, 22q11.21 delesyonu ve 15q21.2 delesyonu bulunmuştur.
TARTIŞMA ve SONUÇ: NGS verilerini iyi geliştirilmiş yazılımlarla analiz etmenin birçok avantajı vardır. Özellikle bazı durumlarda hem SNV hem de CNV verilerini birlikte değerlendirebilen yazılım, klinik vakaların çözümüne yardımcı olabilmektedir. Teknolojideki gelişmeler daha iyi sonuçlar veren yazılım araçlarının geliştirilmesine yol açmaktadır. Bu tür analiz yöntemleri, özellikle genin bir kopyası silindiğinde, diğer kopya patojenik veya patojenik olabilen varyantlar taşıdığında, resesif koşullar için tıbbi genetikteki zorlu vakaların çözümünde zaman ve maliyet açısından fayda sağlamaktadır.

Keywords

Mikrodizin, kopya sayısı değişikliği, yeni nesil dizileme

References

• Baker SW, Murrell JR, Nesbitt AI, Pechter KB, Balciuniene J, Zhao X, et al. Automated Clinical Exome Reanalysis Reveals Novel Diagnoses. J Mol Diagn. 2019;21:38-48.
• Liu Z, Zhu L, Roberts R, Tong W. Toward Clinical Implementation of Next-Generation Sequencing-Based Genetic Testing in Rare Diseases: Where Are We? Trends Genet. 2019;35:852-67.
• Bruel AL, Nambot S, Quéré V, Vitobello A, Thevenon J, Assoum M, et al. Increased diagnostic and new genes identification outcome using research reanalysis of singleton exome sequencing. Eur J of Hum Genet. 2019;27:1519-31.
• Pang AW, MacDonald JR, Pinto D, Wei J, Rafiq MA, Conrad DF, et al. Towards a comprehensive structural variation map of an individual human genome. Genome Biol. 2010;11:R52.
• 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.
• Hochstenbach R, van Binsbergen E, Engelen J, Nieuwint A, Polstra A, Poddighe P, et al. Array analysis and karyotyping: workflow consequences based on a retrospective study of 36,325 patients with idiopathic developmental delay in the Netherlands. Eur J Med Genet. 2009; 52:161-9.
• Lee C, ve Scherer S W, The Clinical Context of Copy Number Variation in The Human Genome. Expert Rev Mol Med. 2010; 9;12:e8.
• Glessner JT, Bick AG, Ito K, Homsy JG, Rodriguez-Murillo L, Fromer M, et al. Increased frequency of de novo copy number variants in congenital heart disease by integrative analysis of single nucleotide polymorphism array and exome sequence data. Circ Res. 2014;115:884-96.
• Orange JS, Glessner JT, Resnick E, Sullivan KE, Lucas M, Ferry B, et al. Genome-wide association identifies diverse causes of common variable immunodeficiency. J Allergy Clin Immunol. 2011; 127:1360-7.
• de Ligt J, Boone PM, Pfundt R, Vissers LE, Richmond T, Geoghegan J, et al. Detection of clinically relevant copy number variants with whole‐exome sequencing. Human Mutat. 2013; 34:1439-48.
• Tan R, Wang Y, Kleinstein SE, Liu Y, Zhu X, Guo H, et al. An evaluation of copy number variation detection tools from whole‐exome sequencing data. Human Mutat. 2014;35:899-907.
• Krumm N, Sudmant PH, Ko A, O'Roak BJ, Malig M, Coe BP, et al. Copy number variation detection and genotyping from exome sequence data. Genome Res. 2012;22:1525-32.
• Zarrei M, MacDonald JR, Merico D, Scherer SW. A copy number variation map of the human genome. Nat Rev Genet. 2015;16:172-82
• Nussbaum, R, McInnes R, Willard H. Thompson & Thompson Genetics in Medicine, ed 8, Canada, Elsevier Inc, 2016: 1-105.
• Jähn JA, von Spiczak S, Muhle H, Obermeier T, Franke A, Mefford HC, et al. Iterative phenotyping of 15q11. 2, 15q13. 3 and 16p13. 11 microdeletion carriers in pediatric epilepsies. Epilepsy Res. 2014;108:109-16
• Pfundt R, Del Rosario M, Vissers LE, Kwint MP, Janssen IM, De Leeuw N,et al. Detection of clinically relevant copy-number variants by e xome sequencing in a large cohort of genetic disorders. Genet Med. 2017;19(6):667-75.
• Boycott KM, Hartley T, Biesecker LG, Gibbs RA, Innes AM, Riess O, et al. A diagnosis for all rare genetic diseases: the horizon and the next frontiers. Cell. 2019; 177: 32-7.
• Deignan JL, Chung WK, Kearney HM, Monaghan KG, Rehder CW, Chao EC. Points to consider in the reevaluation and reanalysis of genomic test results: a statement of the American College of Medical Genetics and Genomics (ACMG). Genet Med. 2019;21:1267-70.
• Retterer K, Scuffins J, Schmidt D, Lewis R, Pineda-Alvarez D, Stafford A,et al. Assessing copy number from exome sequencing and exome array CGH based on CNV spectrum in a large clinical cohort. Genet Med. 2015;17: 623-9.
• Gorman GS, Taylor RW. RRM2B-Related Mitokondrial Disease. 2014 Apr 17. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mirzaa G, Amemiya A, editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993–2021. [Erişim tarihi: 25.08.2020)] Erişim adresi: https://www.ncbi.nlm.nih.gov/books/NBK195854
• Chen YF, Lin IH, Guo YR, Chiu WJ, Wu MS, Jia W, et al. Rrm2b deletion causes mitochondrial metabolic defects in renal tubules. Sci Rep. 2019;9: 1-12. Göktürk B, Reisli İ. DiGeorge Syndrome. Asthma Allergy Immunol. 2017;14:129-42.
• Ballout RA, El-Hattab AW, Schaaf CP, Cheung SW. Adam MP, Ardinger HH, et al. Xq28 duplication syndrome, Int22h1/Int22h2 mediated. 2016 Mar 10. In: Adam MP,
• Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mirzaa G, Amemiya A, editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993–2021. [Erişim tarihi: 25.08.2020)] Erişim adresi: https://www.ncbi.nlm.nih.gov/books/NBK349624