Combined mutation and rearrangement screening by quantitative PCR high-resolution melting: is it relevant for hereditary recurrent Fever genes?

Abstract

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.

Figure 1. Flow chart for the screening of mutations in the genes MVK, NLRP3 and TNFRSF1A.

In our patient cohort, we performed a simultaneous search for punctual mutations and gene rearrangements using qPCR-HRM in the prospective group and qPCR alone in the retrospective group (as these patients had already been assessed for qualitative alterations), then SQF-PCR in both groups as a confirmatory quantitative approach.

Figure 2. Results of MVK, NLRP3 and TNFRSF1A gene dosage analysis by qPCR-HRM (prospective study) and qPCR (retrospective study).

We screened by qPCR-HRM (i) exons 2–8 and 10–11 of the MVK gene in 10 controls, 1 DNA sample with a 12q23.2 deletion, and 49 patients, (ii) the exon 3 of the NLRP3 gene in 9 controls, 10 patients, and the artificial positive p.T348M patient and (iii) exons 2–4 of the TNFRSF1A gene in 13 controls and 54 patients. We screened by qPCR (i) exon 9 of the MVK gene in 10 controls, 1 DNA sample with a 12q23.2q24.11 deletion, and 12 patients and (ii) exons 2–9 of the NLRP3 gene in 9 controls and 38 patients.The red bars indicate the range limits of the control ratios, between 0.75 and 1.28.

Figure 3. Pathological genotypes detected by qPCR-HRM in the prospective study.

Qualitative changes were assessed using the gene scanning module of the LightCycler 480 Software version 1.5. Wild type sequences were used to define baselines. Difference plots revealed disease associated variants.

Figure 4. Results of MVK, NLRP3 and TNFRSF1A gene dosage analysis by SQF-PCR.

We screened by SQF-PCR (i) all coding exons of the MVK gene in 9 controls, 1 DNA sample with a 12q23.2 deletion, and 28 patients (ii) all coding exons of the NLRP3 gene in 9 controls and 86 patients and (iii) all coding exons of the TNFRSF1A gene in 9 controls and 54 patients.The red bars indicate the range limits of the control ratios, between 0.75 and 1.25.