Abstract:These sensitive and specific qPCRs should be helpful in establishing the diagnosis and in monitoring minimal residual disease in patients during or after therapy.
“…Since the use of our strategy to detect CNV breakpoints in a
hemizygotic deletion in the DMD gene could be considered to be a rather
special case that may not reflect the general applicability of the method, we also used
this approach to detect the breakpoint in autosomal heterozygous deletions. Real-time
qPCR has previously been used to successfully detect heterozygous deletions (Zinke et al , 2015). …”
The routine detection of large and medium copy number variants (CNVs) is well
established. Hemizygotic deletions or duplications in the large Duchenne muscular
dystrophy DMD gene responsible for Duchenne and Becker muscular
dystrophies are routinely identified using multiple ligation probe amplification and
array-based comparative genomic hybridization. These methods only map deleted or
duplicated exons, without providing the exact location of breakpoints. Commonly used
methods for the detection of CNV breakpoints include long-range PCR and primer
walking, their success being limited by the deletion size, GC content and presence of
DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and
large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the
DMD gene and the large autosomal heterozygous
PARK2 deletion were used to demonstrate the workflow that relies
on real-time quantitative PCR to narrow down the deletion region and Sanger
sequencing for breakpoint confirmation. The strategy is fast, reliable and
cost-efficient, making it amenable to widespread use in genetic laboratories.
“…Since the use of our strategy to detect CNV breakpoints in a
hemizygotic deletion in the DMD gene could be considered to be a rather
special case that may not reflect the general applicability of the method, we also used
this approach to detect the breakpoint in autosomal heterozygous deletions. Real-time
qPCR has previously been used to successfully detect heterozygous deletions (Zinke et al , 2015). …”
The routine detection of large and medium copy number variants (CNVs) is well
established. Hemizygotic deletions or duplications in the large Duchenne muscular
dystrophy DMD gene responsible for Duchenne and Becker muscular
dystrophies are routinely identified using multiple ligation probe amplification and
array-based comparative genomic hybridization. These methods only map deleted or
duplicated exons, without providing the exact location of breakpoints. Commonly used
methods for the detection of CNV breakpoints include long-range PCR and primer
walking, their success being limited by the deletion size, GC content and presence of
DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and
large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the
DMD gene and the large autosomal heterozygous
PARK2 deletion were used to demonstrate the workflow that relies
on real-time quantitative PCR to narrow down the deletion region and Sanger
sequencing for breakpoint confirmation. The strategy is fast, reliable and
cost-efficient, making it amenable to widespread use in genetic laboratories.
“…Irrespective of the presence or absence of wildtype or mutant, all 105 MPN patients were subjected to an in-house developed PCR based protocol (ALDA -amplicon length differentiation assay) for simultaneous detection of CALR type-1 and CALR type-2 mutations. Furthermore the ALDA assay was comparatively evaluated with Sanger sequencing and other available real-time PCR methodologies as described by Zinke et.al [13]. Fig.…”
Section: Methodsmentioning
confidence: 99%
“…Furthermore the ALDA assay was comparatively evaluated with Sanger sequencing and other available real-time PCR methodologies as described by Zinke et. al [13]…”
Background
Calreticulin (
CALR
) gene mutations are currently recommended as biomarkers in diagnosis of patients with myeloproliferative neoplasms (MPN) with
Jak2
V617F negative phenotype. Our aim was to establish a rapid, low cost and sensitive assay for identification of
CALR
gene mutations and to validate the diagnostic performance of the established assay in a patient cohort with different clinical MPN phenotypes.
Methods
One hundred five Philadelphia-negative MPN patients, including polycythemia vera (PV), essential thrombocythaemia (ET), and primary myelofibrosis (PMF) were initially screened for
JAK2
mutations by amplification-refractory mutation system (ARMS-PCR) methodology and were further subjected to detection of
CALR
gene mutations by our in-house assay, a PCR based amplicon length differentiation assay (PCR-ALDA). The PCR-ALDA methodology was compared with real time PCR and Sanger sequencing methods. Furthermore, the analytical sensitivity of the assay was established.
Results
PCR - ALDA approach was able to detect and discriminate the pseudo-positive samples containing more than 1%
CALR
mutant alleles.
CALR
mutations were not detected in 63
Jak2
V617F positive cases in all three methods. In contrast, amongst 42
Jak2
V617F negative cases, both PCR-ALDA and Sanger sequencing coherently identified 12
CALR
mutants compared to 10
CALR
mutants detected by real-time PCR method.
Conclusion
PCR-ALDA can be utilized as an easy-to-use, rapid, low cost and sensitive tool in the detection of
CALR
mutations in Philadelphia-negative MPN patients.
Electronic supplementary material
The online version of this article (10.1186/s12881-019-0819-6) contains supplementary material, which is available to authorized users.
“…While semiquantitative, diagnostic fragment length analysis (FLA) has been extensively embraced, qPCR methods have been hampered by the variety of indels observed. However, recent studies focusing on the common type 1 deletion and type 2 insertion mutations that account for approximately 80% of all indels have exhibited improved specificity and sensitivity necessary for MRD purposes [14]. …”
Section: Current Applicationsmentioning
confidence: 99%
“…Initial attempts for qPCR of these common CALR mutations resulted in limited sensitivities of 1-2%, comparable to other molecular screening approaches [54]. Development of alternative qPCR assays has managed to further improve the sensitivity for both type 1 and type 2 CALR mutations [14]. …”
The presence of acquired mutations within the JAK2, CALR, and MPL genes in the majority of patients with myeloproliferative neoplasms (MPN) affords the opportunity to utilise these mutations as markers of minimal residual disease (MRD). Reduction of the mutated allele burden has been reported in response to a number of therapeutic modalities including interferon, JAK inhibitors, and allogeneic stem cell transplantation; novel therapies in development will also require assessment of efficacy. Real-time quantitative PCR has been widely adopted for recurrent point mutations with assays demonstrating the specificity, sensitivity, and reproducibility required for clinical utility. More recently, approaches such as digital PCR have demonstrated comparable, if not improved, assay characteristics and are likely to play an increasing role in MRD monitoring. While next-generation sequencing is increasingly valuable as a tool for diagnosis of MPN, its role in the assessment of MRD requires further evaluation.
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