Use of an automated pyrosequencing technique for confirmation of sickle cell disease

English version

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.

Use of an automated pyrosequencing technique for confirmation of sickle cell disease

Souhrn

Background

Objectives

Methods

Results

Discussion

Klíčová slova:

Zdroje

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