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Genotyping Two Promoter Polymorphisms in the MIF Gene: A −794 CATT5–8 Microsatellite Repeat and a −173 G/C SNP

  • Lin Leng
  • Edwin Siu
  • Richard BucalaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2080)

Abstract

Macrophage migration inhibitory factor (MIF) is an upstream proinflammatory cytokine encoded by a functionally polymorphic locus. The promoter region of the human MIF gene contains two polymorphisms. A variable nucleotide tandem repeat at position −794 comprises five to eight CATT repeats (referred to henceforth by numbers from 5 to 8, rs5844572). Gene reporter assays show a proportional increase in transcription with CATT repeat number; the 5-repeat allele leads to low expression, and the 6-, 7-, and 8-repeat alleles lead to correspondingly higher expression of MIF. A second MIF promoter polymorphism comprises a G-to-C single nucleotide polymorphism (SNP) at position −173 (rs755622), which is in strong linkage disequilibrium with −794 7-CATT and is associated with arthritis clinical severity and higher serum and synovial fluid MIF levels. This allele also has been reported to confer improved survival in patients with outpatient pneumonia. In this chapter, we will introduce the methods of genotyping CATT5–8 repeats and the MIF −173 G/C from human samples.

Key words

MIF polymorphisms CATT repeats −173SNP Capillary electrophoresis Real-time PCR 

Notes

Acknowledgements

This work was supported by National Institutes of Health grants AR049610 and HL130669 to R.B. We are grateful to the staff of the W. M. Keck Foundation DNA Sequencing Facility at Yale School of Medicine for their assistance in developing the microsatellite fragment analysis and TaqMan assay protocols. We are also grateful to Bruce Liberi for his assistance in developing and optimizing the PCR-RFLP protocol.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Internal MedicineYale School of MedicineNew HavenUSA

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