title: Development of a highly resolutive method, using a double quadruplex tetra-primer-ARMS-PCR coupled with capillary electrophoresis to study CD40LG polymorphisms.
abstract: Polymorphisms in the CD40 ligand gene (CD40LG) are associated with various immunological disorders such as tumors, autoimmune and infectious diseases. The aim of this study was to develop a highly optimized double quadruplex tetra-primer amplification refractory mutation system PCR (double quadruplex T-ARMS-PCR) coupled with capillary electrophoresis to allow genotyping of eight relevant candidate CD40LG SNPs and to establish haplotypes. After conducting the double quadruplex T-ARMS-PCR, the genotypes obtained through agarose electrophoresis were compared with those obtained through capillary electrophoresis. This strategy was applied to analyze the genetic patterns of CD40LG in two distinct cohorts of blood donors (211 French and 274 Tunisian). The T-ARMS-PCR method was rapid, inexpensive, reproducible and reliable for SNP determination. Regarding the separation technique, capillary electrophoresis allows traceable and semi-automated analysis while agarose electrophoresis remains a cost-effective technique that does not require specialized or costly equipment. Using these methods, we identified significantly different genetic heterogeneity between the two investigated populations (p ≤ 0.0001) and we also extensively characterized their haplotypes. The obtained genotype distribution and the optimized quadruplex T-ARMS-PCR technique coupled with capillary electrophoresis provides valuable information for studying pathologic inflammation leading to various diseases in which CD40LG might be a candidate gene.
authors.affiliation: GIMAP-EA3064, University of Lyon, Saint-Etienne, 42270 Saint-Priest-en-Jarez, France; French Blood Establishment, EFS Auvergne-Loire, 42023 Saint-Etienne, France.
authors.forename: Chaker
authors.initials: C
authors.lastname: Aloui
authors.affiliation: GIMAP-EA3064, University of Lyon, Saint-Etienne, 42270 Saint-Priest-en-Jarez, France; French Blood Establishment, EFS Auvergne-Loire, 42023 Saint-Etienne, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Sut
authors.affiliation: GIMAP-EA3064, University of Lyon, Saint-Etienne, 42270 Saint-Priest-en-Jarez, France; French Blood Establishment, EFS Auvergne-Loire, 42023 Saint-Etienne, France.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Cognasse
authors.affiliation: Department of Molecular Genetics, Saint-Etienne University Hospital, 42270 Saint-Priest-en-Jarez, France.
authors.forename: Viviana
authors.initials: V
authors.lastname: Granados
authors.affiliation: Blood Bank of Monastir, F. Bourguiba University Hospital, 5000 Monastir, Tunisia.
authors.forename: Mohsen
authors.initials: M
authors.lastname: Hassine
authors.affiliation: Regional Centre of Transfusion of Sousse, F. Hached University Hospital, 4000 Sousse, Tunisia.
authors.forename: Tahar
authors.initials: T
authors.lastname: Chakroun
authors.affiliation: GIMAP-EA3064, University of Lyon, Saint-Etienne, 42270 Saint-Priest-en-Jarez, France; National Institut of Blood Transfusion, 75739 Paris, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Garraud
authors.affiliation: GIMAP-EA3064, University of Lyon, Saint-Etienne, 42270 Saint-Priest-en-Jarez, France; French Blood Establishment, EFS Auvergne-Loire, 42023 Saint-Etienne, France. Electronic address: Sandrine.laradi@efs.sante.fr.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Laradi
mesh_terms: D023201:CD40 Ligand / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D005260:Female; D005602:France / Q000208:ethnology; D054856:G-Quadruplexes; D005838:Genotype; D006239:Haplotypes; D006801:Humans; D008297:Male; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D014416:Tunisia / Q000208:ethnology
chemical_list: D017931:DNA Primers; D023201:CD40 Ligand
keywords: CD40LG; Haplotype; Inflammation; Population; Quadruplex tetra-primer ARMS-PCR; SNP