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  • Fine mapping and conditional analysis identify a new mutation in the autoimmunity susceptibility gene BLK that leads to reduced half-life of the BLK protein

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Basic and translational research
Extended report
Fine mapping and conditional analysis identify a new mutation in the autoimmunity susceptibility gene BLK that leads to reduced half-life of the BLK protein
  1. Angélica M Delgado-Vega1,
  2. Mikhail G Dozmorov2*,
  3. Manuel Bernal Quirós3,
  4. Ying-Yu Wu2,
  5. Belén Martínez-García3,
  6. Sergey V Kozyrev4,
  7. Johan Frostegård5,
  8. Lennart Truedsson6,
  9. Enrique de Ramón7,
  10. María F González-Escribano8,
  11. Norberto Ortego-Centeno9,
  12. Bernardo A Pons-Estel10,
  13. Sandra D'Alfonso11,
  14. Gian Domenico Sebastiani12,
  15. Torsten Witte13,
  16. Bernard R Lauwerys14,
  17. Emoke Endreffy15,
  18. László Kovács16,
  19. Carlos Vasconcelos17,
  20. Berta Martins da Silva18,
  21. Jonathan D Wren2,
  22. Javier Martin19,
  23. Casimiro Castillejo-López3,
  24. Marta E Alarcón-Riquelme2,3
  1. 1Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, 751 85, Sweden
  2. 2Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, 73104, United States of America
  3. 3Centro de Genómica e Investigación Oncológica GENYO Pfizer-Universidad de Granada-Junta de Andalucía, Granada, 18007, Spain
  4. 4Department of Medical Biochemistry and Microbiology, Uppsala University, 751 23, Uppsala, Sweden.
  5. 5Institute of Environmental Medicine, Unit of Immnology and Chronic diseases, Karolinska Institut, 761 77, Solna, Sweden.
  6. 6Department of Laboratory Medicine, section of M.I.G., Lund University, 221 00, Lund, Sweden
  7. 7Hospital Carlos Haya, 29009, Málaga, Spain
  8. 8Hospital Virgen del Rocío, 41013, Sevilla, Spain
  9. 9Department of Internal Medicine, Hospital Virgen de las Nieves, Granada, Spain
  10. 10Department of Rheumatology, Sanatorio Parque, Rosario, 2000, Argentina
  11. 11Department of Medical Sciences and Institute of Research in Chronic Autoimmune Diseases (IRCAD), University of Eastern Piedmont, 28100, Novara, Italy
  12. 12Unità Operativa Complessa Reumatología, Azienda Ospedaliera San Camillo-Forlanini, Roma, 00152, Italy
  13. 13Hannover Medical School, 30625, Hannover, Germany
  14. 14Cliniques Universitaires Saint-Luc, Université catholique de Louvain, 1200 Bruxelles, Belgium
  15. 15Department of Pediatrics and Health Center, University of Szeged, H-6721, Szeged, Hungary
  16. 16Department of Rheumatology, Albert Szent-Györgyi Clinical Centre, University of Szeged, H-6725, Szeged, Hungary
  17. 17Hospital Santo Antonio and Unidade Multidisciplinar em Investigação Biomédica/IInstituto de Ciências Biomédicas de Abel Salazar – Universidade do Porto, 4099-003, Porto, Portugal
  18. 18Unidade Multidisciplinar em Investigação Biomédica/Instituto de Ciências Biomédicas de Abel Salazar – Universidade do Porto, 4099-003, Porto, Portugal
  19. 19Instituto de Parasitología y Biomedicina López Neyra, CSIC, Armilla, Spain.
  1. Correspondence to Marta E Alarcón-Riquelme, Centro de Genómica e Investigación Oncológica Pfizer-Universidad de Granada-Junta de Andalucía (GENYO), Avenida de la Ilustración 114, Granada, 18007, Spain; marta.alarcon{at}genyo.es

Abstract

Objectives To perform fine mapping of the autoimmunity susceptibility gene BLK and identify functional variants involved in systemic lupus erythematosus (SLE).

Methods Genotyping of 1163 European SLE patients and 1482 controls and imputation were performed covering the BLK gene with 158 single-nucleotide polymorphisms. Logistic regression analysis was done using PLINK and conditional analyses using GENABEL's test score. Transfections of BLK constructs on HEK293 cells containing the novel mutation or the wild type form were analysed for their effect on protein half-life using a protein stability assay, cycloheximide and western blot. CHiP-qPCR for detection of nuclear factor κ B (NFkB) binding.

Results Fine mapping of BLK identified two independent genetic effects with functional consequences: one represented by two tightly linked associated haplotype blocks significantly enriched for NFκB-binding sites and numerous putative regulatory variants whose risk alleles correlated with low BLK mRNA levels. Binding of NFkBp50 and p65 to an associated 1.2 Kb haplotype segment was confirmed. A second independent genetic effect was represented by an Ala71Thr, low-frequency missense substitution with an OR=2.31 (95% CI 1.38 to 3.86). The 71Thr decreased BLK protein half-life.

Conclusions These results show that rare and common regulatory variants in BLK are involved in disease susceptibility and both, albeit independently, lead to reduced levels of BLK protein.

This paper is freely available online under the BMJ Journals unlocked scheme, see http://ard.bmj.com/info/unlocked.dtl

https://doi.org/10.1136/annrheumdis-2011-200987

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    Footnotes

    • Funding Funding provided by FCT (Fundação Ciencia e Tecnologia, Portugal), DFG WI 1031/6-1 (Germany), Grupo Italiano LES and the CVDIMMUNE Project supported by the European Commission.

    • Competing interests None

    • Ethics approval Approval provided by the IRB from each participating institution.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Data sharing statement We will share data related to this article.