Combined mutation and rearrangement screening by quantitative PCR high-resolution melting: is it relevant for hereditary recurrent Fever genes?
The recent identification of genes implicated in hereditary recurrent fevers has allowed their specific diagnosis. So far however, only punctual mutations have been identified and a significant number of patients remain with no genetic confirmation of their disease after routine molecular approaches such as sequencing. The possible involvement of sequence rearrangements in these patients has only been examined in familial Mediterranean fever and was found to be unlikely. To assess the existence of larger genetic alterations in 3 other concerned genes, MVK (Mevalonate kinase), NLRP3 (Nod like receptor family, pyrin domain containing 3) and TNFRSF1A (TNF receptor superfamily 1A), we adapted the qPCR-HRM method to study possible intragenic deletions and duplications. This single-tube approach, combining both qualitative (mutations) and quantitative (rearrangement) screening, has proven effective in Lynch syndrome diagnosis. Using this approach, we studied 113 unselected (prospective group) and 88 selected (retrospective group) patients and identified no intragenic rearrangements in the 3 genes. Only qualitative alterations were found with a sensitivity similar to that obtained using classical molecular techniques for screening punctual mutations. Our results support that deleterious copy number alterations in MVK, NLRP3 and TNFRSF1A are rare or absent from the mutational spectrum of hereditary recurrent fevers, and demonstrate that a routine combined method such as qPCR-HRM provides no further help in genetic diagnosis. However, quantitative approaches such as qPCR or SQF-PCR did prove to be quick and effective and could still be useful after non contributory punctual mutation screening in the presence of clinically evocative signs.
Unité Médicale des Maladies Auto-Inflammatoires, Laboratoire de Génétique, Hôpital A de Villeneuve, Montpellier, France.
Nathalie
N
Pallares-Ruiz
Laurent
L
Philibert
Bruno
B
Dumont
Aurélie
A
Fabre
Laurence
L
Cuisset
Elodie
E
Cointin
Cécile
C
Rittore
Stéphan
S
Soler
Isabelle
I
Touitou
D002352:Carrier Proteins / Q000235:genetics; D005260:Female; D015321:Gene Rearrangement; D020022:Genetic Predisposition to Disease / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods; D005838:Genotype; D056660:Hereditary Autoinflammatory Diseases / Q000175:diagnosis / Q000235:genetics*; D006801:Humans; D008297:Male; D009154:Mutation; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D002352:Carrier Proteins; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; D047888:Receptors, Tumor Necrosis Factor, Type I; C506235:TNFRSF1A protein, human; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase