NEW YORK (GenomeWeb) — A team led by researchers at France's University of Montpellier has repurposed a qPCR-based mutant-enrichment technique originally developed for cancer analysis and applied it to analyzing circulating cell-free fetal DNA for non-invasive prenatal diagnosis of monogenic disorders.
In a paper published last week in Clinical Chemistry and Laboratory Medicine, the researchers demonstrated that the method — mutant enrichment with 3'-modified oligonucleotides (MEMO) qPCR — could detect a common cystic fibrosis mutation in wild-type maternal DNA with 100 percent accuracy at a detection threshold of 5 percent.
According to the researchers, the test is being implemented when possible at the Regional University Hospital Center of Montpellier (CHRU Montpellier) prior to invasive procedures to detect CF in the fetuses of couples carrying non-identical mutations. In addition, the approach may serve as an inexpensive, simple, and time-efficient way to provide NIPD of a broad range of monogenic disorders, the study's authors said.
Over the last several years, clinical researchers in general have been investigating a number of ways to perform NIPD based on circulating cell-free fetal DNA in maternal blood as an alternative to invasive testing procedures.
Monogenetic disorders are diseases caused by a mutation in a single gene on either one or both chromosomes, and include disorders such as CF, sickle cell disease, polycystic kidney disease, Tay-Sachs disease, Huntington's disease, and Marfan syndrome.
Several methods have been proposed or developed for NIPD of single-gene disorders, and most methods are based on detecting a single-based substitution, insertion, or deletion using modified PCR methods or direct DNA sequencing. And while many of these methods show promise, in general they have proven to be overly complicated, too expensive, and too prone to contamination to perform routinely in clinical labs.
The French researchers, some of which offer routine diagnostic services at CHRU Montpellier, sought a simpler method to provide NIPD for these monogenic disorders, and, in searching the literature, homed in on the MEMO-qPCR method, which was originally described by Korean researchers in a 2011 Journal of Molecular Diagnostics paper.
MEMO-qPCR is based on the use of a 3'-modified oligonucleotide primer that blocks extension of the mutated allele. The Korean researchers originally developed and evaluated the method to detect trace mutant alleles for cancer research and molecular diagnostics, but the technique can be applied broadly to real-time PCR and subsequent melt curve analysis.
Claire Guissart, first author on the recent CCLM paper and a researcher in the University Institute of Clinical Research at Montpellier University and CHRU Montpellier, told PCR Insider in an email this week that her team chose the method for NIPD because it had several advantages over other current methods.
"We found several proof[s] of concept of NIPD techniques, but all have limitations," Guissart said. "We wanted an approach that would overcome these limitations. That is why we decided to expand our bibliographic research on how to detect mutant DNA in oncology. In fact, cell-free fetal DNA characteristics are close to DNA from tumor-derived cell[s]: both are minor alleles which are present in a DNA mixture."
Guissart said that the method appeared to be suitable because of its high sensitivity; ability to detect trace mutant alleles, potentially at ratios of less than 1 percent; compatibility with other downstream analysis methods; and the fact that it can be implemented using existing laboratory infrastructure.
"Sanger sequencing requires a threshold of at least 20 percent of trace mutant allele for detecti[on]," Guissart said. Meantime, "massively parallel sequencing is much more powerful but very expensive [to apply] for point mutations and small deletions/insertions," she added. The MEMO-qPCR method, she noted, "can be performed in a closed tube and in one step … [and] allows [one] to limit the risk of contamination and to secure the process."
To test MEMO-qPCR for NIPD of monogenic disorders, Guissart and colleagues first evaluated its performance on chimeric DNA samples at 100 picograms/µL with different ratios of the p.Gly542* mutation and wild-type DNA to mimic maternal plasma. This mutation, found in the cystic fibrosis trans-membrane regulator gene, is common in French populations, comprising up to 6.7 percent of CF alleles.
The researchers showed that in the range of 2 percent to 50 percent of the minor mutant allele, the method produced melting curve profiles for the mutant allele that were distinguishable from wild-type, with correct identification of 100 percent of the chimeric samples at a 5 percent mutant ratio.
In addition, Guissart and colleagues used MEMO-qPCR to perform a blind test of 10 chimeric DNA samples containing 5 percent of different CFTR genotypes, and correctly diagnosed the genotype in all cases.
Next, they used the method to analyze three maternal plasma samples collected from mothers undergoing CFTR genotyping with an increased risk of CF based on fetal ultrasound, and correlated the results with previously performed amniocenteses. None of the tested maternal plasmas contained the p.Gly542* mutation, and MEMO-qPCR correctly reported no false positives.
Finally, they evaluated their approach in a clinical context by applying it to two other mutations: a splicing mutation of the CFTR gene from a couple at increased risk for CF based on ultrasound findings; and a missense mutation from a couple with familial CF history. MEMO-qPCR correctly classified the healthy and CFTR-positive controls and showed that the mutations were present in the fetus from maternal plasma samples.
One major caveat to the method is a general issue with detecting cell-free fetal DNA in maternal plasma: It is often difficult to distinguish true negative results from false negative results due to insufficient amounts of free fetal DNA. As such, an independent test is needed to validate the presence of fetal DNA in the plasma sample.
To address this, Guissart and colleagues used Thermo Fisher Scientific's Life Technologies AmpFlSTR MiniFiler PCR amplification kit — an assay frequently used in forensic STR analysis — to confirm the presence of cell-free fetal DNA in the maternal plasma and safeguard against false negatives.
This approach was successful in validating the two aforementioned negative results obtained from the three maternal plasma samples from mothers undergoing CFTR genotyping. However, when testing the method independently on a number of maternal plasma and sera samples, the researchers found that it performed suitably in plasma samples but yielded inconclusive results in sera samples.
Nevertheless, the STR analysis method showed promise as an independent method for validating the presence of cell-free fetal DNA in maternal plasma, and based on these initial results, the researchers plan to further evaluate its usefulness as a quality control.
Guissart said that the group is also now beginning clinical validation of the MEMO-qPCR method on a larger group of maternal plasma samples. Although it does not have plans to commercialize the method, "we will continue to offer NIPD test[ing] for couples carrying non-identical mutations before invasive procedures whenever it is possible," she said.