Detection of MPLW515L/K mutations and determination of allele frequencies with a single-tube PCR assay.

A gain-of-function mutation in the myeloproliferative leukemia virus (MPL) gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs). The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system)-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5%) of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner

Detection of <i>MPL</i>W515L/K mutations using DARMS-PCR.

<p>(A) The primers used in the DARMS-PCR assay. The two inner primers harbored sequences (underlined) that matched <i>MPL</i>W515L or W515K, but not the wild-type allele. Other mismatches (capital letters) were introduced into the inner primers to reduce the annealing of the mutant-specific primers to the wild-type sequence. The reverse primers were labeled with FAM (5-carboxyfluorescein hydrate) at the 5′ terminus. (B) A schematic representation of DARMS-PCR products. The two outer primers were designed to generate a 169-base-pair (bp) PCR product from all <i>MPL</i> alleles. The F_inner and R_inner primers annealed specifically to the <i>MPL</i>W515L and W515K alleles, respectively; in combination with the outer primers, they generated 84- and 128-bp PCR products, respectively. From a mutant allele, both 169-bp and 84- or 128-bp fragments were amplified, while, only the 169-bp fragment was generated from the wild-type allele. (C) Demonstration of DARMS-PCR. A capillary electropherogram of DARMS-PCR products showing three peaks derived from wild-type <i>MPL</i>, W515L, and W515K. This result was obtained when PCR was performed with a standard DNA mixture containing equal ratios of <i>MPL</i> wild-type, W515L, and W515K alleles with a total copy number of 10⁵. The horizontal axis represents the fragment length, and the vertical axis represents the fluorescence intensity.</p

Comparison of <i>MPL</i> mutant frequencies determined using DARMS-PCR and deep sequencing.

<p>The horizontal axis represents the frequencies of the <i>MPL</i>W515L (A) or W515K (B) mutant alleles determined using deep sequencing, and the vertical axis represents the mutant allele frequencies determined using DARMS-PCR. The specific values are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104958#pone-0104958-t001" target="_blank">Table 1</a>. Filled symbols represent alleles identified using both DARMS-PCR and deep sequencing. Open symbols represent alleles identified only using deep sequencing.</p

Demonstration of DARMS-PCR to determine <i>MPL</i> mutant allele frequencies.

<p>Standard curves for the quantification of <i>MPL</i>W515L (A) and W515K (B) allele frequencies. A series of templates containing different amounts of mutant allele DNA were used for DARMS-PCR. The fluorescence peak value of the mutant allele over the value for all <i>MPL</i> alleles was calculated for each template. The mean values and errors obtained from three independent experiments are shown.</p

<i>MPL</i> allele frequencies determined using DARMS-PCR and deep sequencing.

<p>*Allele frequencies below 1%, as determined using deep sequencing, were defined as negative for the assessed mutation. Note that allele frequencies below 5%, as determined using DARMS-PCR, were also defined as negative based on the results in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104958#pone-0104958-g003" target="_blank">Figure 3</a>. N.D.: the corresponding peak was not detected.</p

Lower limit of <i>MPL</i> mutation detection by DARMS-PCR.

<p>Capillary electropherograms for DARMS-PCR assays with low levels of <i>MPL</i>W515L or W515K mutant alleles are presented. The average fluorescence intensities for nonspecific PCR products were defined as noise. S/N ratios, which were calculated as the fluorescence peak value for a mutant PCR product over the value for noise, are shown. An arrow head in the W515L 0% panel indicates a false PCR product (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104958#s3" target="_blank">Results and Discussion</a>).</p