title: UGT1A1 (TA)n genotyping in sickle-cell disease: high resolution melting (HRM) curve analysis or direct sequencing, what is the best way?
abstract: BACKGROUND
  Minucci et al. have proposed in 2010 a rapid, simple and cost-effective HRM method on the LightCycler 480® apparatus (Roche) for the determination of the 6/6, 6/7 and 7/7 genotypes of the (TA)n UGT1A1 promoter polymorphism. However, they have not studied the n=5 and n=8 alleles which can be quite frequent in sickle-cell disease patients.
  
  
  METHODS
  The aim of our study was to test this HRM protocol to all the 10 possible (TA)n UGT1A1 genotypes (i.e. 5/5, 5/6, 5/7, 5/8, 6/6, 6/7, 6/8, 7/7, 7/8 and 8/8) by using our SCD cohort of patients.
  
  
  RESULTS
  All genotypes could be unambiguously identified except 6/7 and 6/8 which give a similar HRM profile. For those two genotypes, the differentiation necessitates either a direct Sanger sequencing or a second PCR protocol followed by a 3% agarose gel migration.
  
  
  CONCLUSIONS
  For the (TA)n UGT1A1 promoter genotyping of African patients, each lab has to wonder what is the best way between (i) direct Sanger sequencing of all patients and (ii) HRM protocol for all patients followed by a complementary analysis to differentiate the 6/7 and 6/8 genotypes.
authors.affiliation: Laboratoire de Biochimie et Biologie moléculaire, Unité de pathologie moléculaire du globule rouge, Hôpital Edouard Herriot, Hospices Civils & Université Claude Bernard-Lyon 1, Lyon, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Thomas
authors.forename: Blandine
authors.initials: B
authors.lastname: Mazard
authors.forename: Caroline
authors.initials: C
authors.lastname: Garcia
authors.forename: Philippe
authors.initials: P
authors.lastname: Lacan
authors.forename: Marie-Claude
authors.initials: MC
authors.lastname: Gagnieu
authors.forename: Philippe
authors.initials: P
authors.lastname: Joly
mesh_terms: D000483:Alleles; D000755:Anemia, Sickle Cell / Q000175:diagnosis* / Q000235:genetics; D015331:Cohort Studies; D005838:Genotype; D060005:Genotyping Techniques; D014453:Glucuronosyltransferase / Q000235:genetics*; D006801:Humans; D009691:Nucleic Acid Denaturation; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D000099148:UGT1A1 Enzyme
chemical_list: D014453:Glucuronosyltransferase; D000099148:UGT1A1 Enzyme
keywords: Gilbert's syndrome; HRM; Sanger sequencing; Sickle-cell disease; UGT1A1 promoter