Establishment of a Highly Sensitive Allele Specific Wild Type Blocker Polymerase Chain Reaction Followed by Pyrosequencing for Detection of the p.D816V Point Mutation.
Authors: Siebolts Udo , Al-Ali Haifa Kathrin , Niederwieser Dietger , Wickenhauser Claudia 
Published: October 2019

Abstract
Abstract 4975

Background
The vast majority of mastocytosis patients carry the p.D816V activating point mutation in exon 17 leading to imatinib resistance. In addition, several less common activating kit mutations, at least partially accessible to imatinib, have been described in a minority of systemic and cutaneous mastocytosis. Therefore, exact molecular identification of patients with mastocytosis of uncertain dignitiy is a precondition for diagnostic and therapeutic purposes. However, atypical mast cell infiltrates within the bone marrow biopsies often are very small and microdissection may be hampered by the inconspicuousness of the atypical mast cells. Hence, an appropriate molecular test should exhibit an exceedingly high sensitivity.

Methods
In an attempt to combine high sensitivity and robustness we created an approach where we adapted the principle of wild-type blocker PCR employing LNA-substituted oligonucleotides and pyrosequencing technique. First, the unique properties of LNA substituted oligonucleotides were employed. As a consequence of their 2′-O, 4′-C methylene bridge and their characteristic bicyclic structure binding affinity is significantly increased, resulting in high melting points. One base mismatch between an LNA oligonucleotide and the complementary strand can lower the melting point 20-30°C, thereby allowing a LNA oligonucleotide to discriminate a one base pair difference between templates and, in contrary, a single base pair mismatch in the normal DNA octomer decreases Tm only by 10°C. In order to allow LNA-substituted oligonucleotides to block amplification of wild-type a mutated form of Taq polymerase, termed the Stoffel fragment was applied. Stoffel fragment is a modified form of AmpliTaq® DNA polymerase lacking intrinsic 5' to 3' exonuclease activity.
Employing this methodological setting including the Stoffel fragment, the LNA oligonucleotides and appropriate primer 20 rounds of PCR were performed and an amplicon of 185 bp was received. As demonstrated employing mixtures of assembled DNA sequences keeping the WT or the p.D816V point mutation the WT/D816V ratio turned in favour of the mutated region performing the LNA – Stoffel PCR. Unique in our approach was a second step in which the suchlike amplified PCR product then was adopted in a pyrosequencing assay (Pyromark 24, Qiagen, Hilden, Germany).

Results
First of all, employing WT DNA sequences and the L1236 cell line, the ability of the blocking LNA oligonucleotides to prevent primer extension on WT-DNA by AmpliTaq® DNA polymerase was excluded. To determine the minimum concentration of blocker necessary to prevent amplification of WT oligonucleotides, we used serial dilutions of the blocking oligonucleotides and revealed that a minimum of 50 mM of blocker was necessary to prevent a highly competitive WT-amplicon. We then analyzed the sensitivity of the assay and spiced assembled DNA sequences keeping the WT or the D816V point mutation. Performing this experiment, 1:105 (0,001%) mutant DNA sequences were definitely detectable performing this assay but not performing conventional pyrosequencing without specific preamplification. In none of the control experiments employing WT DNA a false positive amplicon of the exon 16 D816V region was seen indicating an excellent specifity of the assay. In the pyrograms only light emission peaks >10% were evaluated.
To determine the utility of our technique for detecting minority mutations in bone marrow specimen containing few cells or mixed cell populations, we performed the here presented assay on genomic DNA extracted from formalin-fixed, EDTA decalcified paraffin-embedded trephine biopsies. In all 20 trephine biopsies under study, comprising a morphologically established atypical mast cell density from 1 – 15% a clear cut decision of the genomic status was possible.

Conclusions
Wild-type blocker PCR employing nonextendable LNA oligonucleotides represents an exceedingly sensitive, allele-specific method. In combination with the pyrosequencing technique the here presented assay is highly reliable and reproducible, simple and fast to perform (3 h), facile to interpret and should be equally applicable to other single-base mutations. In bone marrow biopsies mRNA fragments often are highly degradated. Therefore, in contrast to PCR assays performed on cDNA, the here presented assay is ideal suited for the molecular diagnosis of systemic mastocytosis on formalin-fixed tissues.

Disclosures
Siebolts: Novartis: Research Funding. Al-Ali:Novartis:. Niederwieser:Novartis: Research Funding. Wickenhauser:Novartis: Research Funding.