title: Nested high-resolution melting curve analysis a highly sensitive, reliable, and simple method for detection of JAK2 exon 12 mutations--clinical relevance in the monitoring of polycythemia.
abstract: JAK2 exon 12 mutations are found in myeloproliferative disorders characterized by erythrocytosis. Lying in a 33-bp region and conserving the open reading frame, they often present a low allelic burden (<10%), which excludes screening with techniques such as allele-specific PCR or different sequencing protocols. High-resolution melting (HRM), a fast in-tube method, seems the most accurate routine technique for that. We describe a reliable and powerful nested HRM technique, independent of DNA preparation and with technical sensitivity of 100% (95% CI, 93% to 100%) and specificity of 96.7% (95% CI, 89.7% to 96.7%). Screening a cohort of 10 idiopathic erythrocytosis, 28 polycythemia vera, and 7 secondary erythrocytosis cases allowed the detection of 15 mutants, including 9 different mutations, of which 3 were unreported, all in the polycythemia vera group, and presented a characteristic profile: pure erythrocytosis associated with low serum erythropoietin. Threshold detection level ranged from 1% to 3% allelic burden, depending on the mutation. All of the HRM positive signals were found mutated by sequencing. Six of them (40%), however, required cloning before sequencing, because of low allelic burden. Classic techniques such as genomic sequencing may therefore miss patients with mutations. Given its sensitivity, HRM (and nested HRM) can be used in routine diagnosis and seems to be the most efficient of current techniques for detection of JAK2 exon 12 mutations.
authors.affiliation: Laboratory of Clinical Cytology, CHU Caremeau, Nîmes, France.
authors.forename: Serge
authors.initials: S
authors.lastname: Carillo
authors.forename: Laurent
authors.initials: L
authors.lastname: Henry
authors.forename: Eric
authors.initials: E
authors.lastname: Lippert
authors.forename: François
authors.initials: F
authors.lastname: Girodon
authors.forename: Isabelle
authors.initials: I
authors.lastname: Guiraud
authors.forename: Céline
authors.initials: C
authors.lastname: Richard
authors.forename: Frédérique
authors.initials: F
authors.lastname: Dubois Galopin
authors.forename: Cedric
authors.initials: C
authors.lastname: Cleyrat
authors.forename: Eric
authors.initials: E
authors.lastname: Jourdan
authors.forename: Robert
authors.initials: R
authors.lastname: Kralovics
authors.forename: Sylvie
authors.initials: S
authors.lastname: Hermouet
authors.forename: Thierry
authors.initials: T
authors.lastname: Lavabre-Bertrand
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D005091:Exons; D005260:Female; D005821:Genetic Techniques; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009154:Mutation / Q000235:genetics*; D011087:Polycythemia Vera / Q000175:diagnosis* / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D055815:Young Adult
chemical_list: D053614:Janus Kinase 2