Use of an automated pyrosequencing technique for confirmation of sickle cell disease
Autoři:
Camila Cruz de Martino aff001; Cecilia Salete Alencar aff002; Paula Loureiro aff003; Anna Barbara de Freitas Carneiro-Proietti aff004; Claudia de Alvarenga Máximo aff005; Rosimere Afonso Mota aff006; Daniela Oliveira Werneck Rodrigues aff007; Nelson Gaburo Junior aff001; Shannon Kelly aff008; Ester Cerdeira Sabino aff001;
Působiště autorů:
Instituto de Medicina Tropical de São Paulo, Laboratório de Parasitologia, LIM 46, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, Brazil
aff001; Laboratório de Investigacao Medica, LIM 03, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, São Paulo, Brazil
aff002; Hemope, Recife, Pernambuco, Brazil
aff003; Hemominas, Belo Horizonte, Minas Gerais, Brazil
aff004; Hemorio, Rio de Janeiro, Rio de Janeiro, Brazil
aff005; Hemominas, Montes Claros, Minas Gerais, Brazil
aff006; Hemominas, Juiz de Fora, Minas Gerais, Brazil
aff007; Vitalant Research Institute, San Francisco, California, United States of America
aff008; UCSF Benioff Children’s Hospital Oakland, Oakland, California, United States of America
aff009
Vyšlo v časopise:
PLoS ONE 14(12)
Kategorie:
Research Article
doi:
https://doi.org/10.1371/journal.pone.0216020
Souhrn
Background
The diagnosis of sickle cell disease (SCD) is made by hemoglobin assays such as high-performance liquid chromatography (HPLC), isoelectric focusing and cellulose acetate or citrate agar electrophoresis. These assays are easy to perform and used in large-scale newborn screening in many countries. These tests however may not easily differentiate Sβ0 thalassemia from SS or identify other hemoglobin variants, and in this case, hemoglobin (HBB) gene sequencing may be necessary.
Objectives
To develop a high throughput DNA based confirmatory assay for SCD and to detect mutations in the HBB gene
Methods
We developed an automated pyrosequencing technique (PyS) based on QIAGEN technology (Hilden, Germany) to detect homozygous or heterozygous hemoglobin S mutations as well as hemoglobin C mutations. The technique was tested on 2,748 samples from patients enrolled in a multi-center SCD cohort in Brazil. Patients were previously tested using HPLC to diagnose SCD as part of routine clinical care. Any subjects with discrepant results between HPLC and PyS or with heterozygous hemoglobin S detected had Sanger sequencing of the HBB gene.
Results
We identified 168 samples with discrepant results between HPLC and PyS and 100 with concordant PyS = heterozygous S and HPLC, which would suggest SB-thalassemia or other heterozygous S variants. The PyS assay correctly identified 1906 (98.7%) of the 1930 HbSS and 628 (98.7%) of the 636 HbSC samples. Of the 179 remaining samples, PyS correctly indicated S heterozygosis in 165 (92.2%). Of the 165 heterozygous S samples confirmed by Sanger as consistent with Sβ thalassemia genotype, 84 samples were classified as Sβ0 thalassemia and 81 as Sβ+ thalassemia. The most frequent beta thalassemia mutations of Sβ0 and Sβ+ were HBB: c.118C>T (Gln40Stop) and HBB c.92 + 6T> C, respectively.
Discussion
The PyS proved to be satisfactory for large-scale confirmatory testing of hemoglobin mutation. Moreover, with this study we were able to describe the most common β+ and β0 mutations in SCD patients with Sβ-thalassemia in a large multi-institutional SCD cohort in Brazil.
Klíčová slova:
Beta-thalassemia – Brazil – Dideoxy DNA sequencing – Hemoglobin – High performance liquid chromatography – Polymerase chain reaction – Sickle cell disease – Thalassemia
Zdroje
1. Kawar N, Alrayyes S, Compton A-A, Aljewari H, Baghdan D, Yang B, et al. Sickle cell disease; An overview of the disease and its systemic effects. Dis Mon. 2018;0(0):1–7.
2. Soares LF, Lima EM, Silva JA da, Fernandes SS, Silva KM da C, Lins SP, et al. Prevalência de hemoglobinas variantes em comunidades quilombolas no estado do Piauí, Brasil. Cien Saude Colet. 2017;22(11):3773–80. doi: 10.1590/1413-812320172211.04392016 29211182
3. Makani J, Ofori-Acquah SF, Nnodu O, Wonkam A, Ohene-Frempong K. Sickle cell disease: new opportunities and challenges in Africa. ScientificWorldJournal. 2013;2013:193252. doi: 10.1155/2013/193252 25143960
4. Colella MP, de Paula E V., Machado-Neto JA, Conran N, Annichino-Bizzacchi JM, Costa FF, et al. Elevated hypercoagulability markers in hemoglobin SC disease. Haematologica. 2015;100(4):466–71. doi: 10.3324/haematol.2014.114587 25596272
5. Rezende P V., Santos M V., Campos GF, Vieira LLM, Souza MB, Belisário AR, et al. Perfil clínico e hematológico em uma coorte neonatal com hemoglobina SC. J Pediatr (Rio J). 2018;(xx).
6. Kato GJ, Piel FB, Reid CD, Gaston MH, Ohene-Frempong K, Krishnamurti L, et al. Sickle cell disease. Nat Rev Dis Prim. 2018;4:1–22. doi: 10.1038/s41572-018-0001-z
7. Harrington CT, Lin EI, Olson MT, Eshleman JR. Fundamentals of pyrosequencing. Arch Pathol Lab Med. 2013;137(9):1296–303. doi: 10.5858/arpa.2012-0463-RA 23991743
8. Viprakasit V, Ekwattanakit S. Clinical Classification, Screening and Diagnosis for Thalassemia. Hematol Oncol Clin North Am. 2018;32(2):237–45. doi: 10.1016/j.hoc.2017.11.001
9. Carneiro-Proietti ABF, Kelly S, Miranda Teixeira C, Sabino EC, Alencar CS, Capuani L, et al. Clinical and genetic ancestry profile of a large multi-centre sickle cell disease cohort in Brazil. Br J Haematol. 2018;128(6):895–908.
10. Vrettou C, Traeger-Synodinos J, Tzetis M, Palmer G, Sofocleous C, Kanavakis E. Real-Time PCR for Single-Cell Genotyping in Sickle Cell and Thalassemia Syndromes as a Rapid, Accurate, Reliable, and Widely Applicable Protocol for Preimplantation Genetic Diagnosis. Hum Mutat. 2004;23(5):513–21. doi: 10.1002/humu.20022 15108284
11. Singh PJ, Shrivastava AC, Shrikhande A V. Prenatal Diagnosis of Sickle Cell Disease by the Technique of PCR. Indian J Hematol Blood Transfus. 2015;31(2):233–41. doi: 10.1007/s12288-014-0427-8 25825564
12. Sutton M, Bouhassira EE, Nagel RL. Polymerase chain reaction amplification applied to the determination of beta-like globin gene cluster haplotypes. Am J Hematol. 1989 Sep;32(1):66–9. doi: 10.1002/ajh.2830320113 2757004
13. Kimura EM, Grignoli CRE, Pinheiro VRP, Costa FF, Sonati MF. Thalassemia intermedia as a result of heterozygosis for β0-thalassemia and αααanti-3.7/αα genotype in a Brazilian patient. Brazilian J Med Biol Res. 2003;36(6):699–701.
14. HbVar Menu [Internet]. [cited 2018 May 6]. Available from: http://globin.bx.psu.edu/hbvar/menu.html
15. Chan OTM, Westover KD, Dietz L, Zehnder JL, Schrijver I. Comprehensive and efficient HBB mutation analysis for detection of β-hemoglobinopathies in a pan-ethnic population. Am J Clin Pathol. 2010;
16. Trans-Omics for Precision Medicine (TOPMed) Program. p. https://www.nhlbi.nih.gov/science/trans-omics-prec.
17. Ronaghi M. Pyrosequencing Sheds Light on DNA Sequencing. Genome. 2018;11:3–11.
18. Bravo-Urquiola M, Arends A, Gómez G, Montilla S, Gerard N, Chacin M, et al. Molecular spectrum of β-Thalassemia mutations in the admixed venezuelan population, and their linkage to β-Globin Gene Haplotypes. Hemoglobin. 2012;36(3):209–18. doi: 10.3109/03630269.2012.674997 22563936
19. Sanctis V De, Kattamis C, Canatan D, Soliman AT, Elsedfy H, Karimi M, et al. β-thalassemia distribution in the old world: An ancient disease seen from a historical standpoint. Mediterr J Hematol Infect Dis. 2017;9(1):e2017018. doi: 10.4084/MJHID.2017.018 28293406
20. Kalleas C, Anagnostopoulos K, Sinopoulou K, Delaki E, Margaritis D, Bourikas G, et al. Phenotype and genotype frequency of β-thalassemia and sickle cell disease carriers in Halkidiki, Northern Greece. Hemoglobin. 2012;36(1):64–72. doi: 10.3109/03630269.2011.642489 22188117
21. Murad H, Moassas F, Jarjour R, Mukhalalaty Y, Al-Achkar W. Prenatal molecular diagnosis of β-thalassemia and sickle cell anemia in the Syrian population. Hemoglobin. 2014;38(6):390–3. doi: 10.3109/03630269.2014.978455 25405916
22. Ouali F, Siala H, Bibi A, Hadj Fredj S, Dakhlaoui B, Othmani R, et al. Prenatal diagnosis of hemoglobinopathies in Tunisia: An 18 years of experience. Int J Lab Hematol. 2016;38(3):223–32. doi: 10.1111/ijlh.12457 26993054
23. Monni G, Peddes C, Iuculano A, Ibba RM. From Prenatal to Preimplantation Genetic Diagnosis of β-Thalassemia. Prevention Model in 8748 Cases: 40 Years of Single Center Experience. J Clin Med. 2018;7(2):35.
24. Rocha LB da SM, Freitas M. Distribuição das mutações da β -talassemia em Fortaleza, Ceará. J Bras Patol Med Lab. 2010;46(6):437–41.
25. Fernandes AC, Azevedo Shimmoto MM, Furuzawa GK, Vicari P, Figueiredo MS. Molecular analysis of β-thalassemia patients: First identification of mutations HBB:c.93-2A>G and HBB:c.114G>A in Brazil. Hemoglobin. 2011;35(4):358–66. doi: 10.3109/03630269.2011.588354 21797703
26. Reichert VCD, Castro SM, Wagner SC, Albuquerque DM, Hutz MH, Leistner-Segal S. Identification of β thalassemia mutations in South Brazilians. Ann Hematol. 2008;87(5):381–4. doi: 10.1007/s00277-007-0418-z 18071703
27. Carrocini GCS, Venancio LPR, Pessoa VLR, Lobo CLC, Bonini-Domingos CR. Mutational Profile of Homozygous β-Thalassemia in Rio de Janeiro, Brazil. Hemoglobin. 2017;41(1):12–5. doi: 10.1080/03630269.2017.1289958 28366028
28. Cabral CHK, Serafim ESS, de Medeiros WRDB, de Medeiros Fernandes TAA, Kimura EM, Costa FF, et al. Determination of β haplotypes in patients with sickle-cell anemia in the state of Rio Grande do Norte, Brazil. Genet Mol Biol. 2011;34(3):421–4. doi: 10.1590/S1415-47572011005000027 21931513
29. Silveira ZML, Barbosa M das V, Fernandes TAA de M, Kimura EM, Costa FF, Sonati M de F, et al. Characterization of beta-thalassemia mutations in patients from the state of Rio Grande do Norte, Brazil. Genet Mol Biol. 2011;34(3):425–8. doi: 10.1590/S1415-47572011005000032 21931514
30. Khan J, Ahmad N, Siraj S, Hoti N. Genetic determinants of β-thalassemia intermedia in Pakistan. Hemoglobin. 2015;39(2):95–101. doi: 10.3109/03630269.2014.1002136 25707679
31. Warghade S, Britto J, Haryan R, Dalvi T, Bendre R, Chheda P, et al. Prevalence of hemoglobin variants and hemoglobinopathies using cation-exchange high-performance liquid chromatography in central reference laboratory of India: A report of 65779 cases. J Lab Physicians. 2018;10(1):73–9. doi: 10.4103/JLP.JLP_57_17 29403210
32. Patel AP, Patel RB, Patel SA, Vaniawala SN, Patel DS, Shrivastava NS, et al. β-thalassemia mutations in Western India: Outcome of prenatal diagnosis in a hemoglobinopathies project. Hemoglobin. 2014;38(5):329–34. doi: 10.3109/03630269.2014.951889 25222044
33. Silva FR. O Tráfico de Escravos para O Portugal Setecentista: Uma Visão A Partir do “Despacho dos Negros Da Índia, De Cacheo e de Angola” Na casa da Índia de Lisboa. Revista de História. 2013;47–73.
34. Araújo AS, Silva WA, Leão SAC, Bandeira FCGM, Petrou M, Modell B, et al. A Different Molecular Pattern of β-Thalassemia Mutations in Northeast Brazil. Hemoglobin. 2003;27(4):211–7. doi: 10.1081/hem-120026045 14649311
35. Uludaǧ A, Uysal A, Uludaǧ A, Ertekin YH, Tekin M, Kütük B, et al. Prevalence and mutations of β-thalassemia trait and abnormal hemoglobins in premarital screening in Çanakkale province, Turkey. Balk J Med Genet. 2016;19(1):29–34.
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