A team led by scientists from Tufts Medical Center has demonstrated the ability of a method called standardized nanoarray PCR to assess fetal gene expression in amniotic fluid, according to recently published research.
Standardized nanoarray PCR, or SNAP, a gene expression profiling system originally developed by Biotrove (now Life Technologies), could be used to create a gene expression assay to examine fetal organ system function, the authors wrote in their paper, which was published online this week in The Journal of Molecular Diagnostics.
Although the initial study evaluated age and sex, the researchers reported that the method shows promise as a possible diagnostic test for fetal organ system disorders. In addition, some genes used in the study may prove useful as members of an eventual test panel for organ health.
Lauren Massingham, a researcher at the Floating Hospital for Children at Tufts Medical Center and principal investigator of the study, told PCR Insider this week that the researchers were contacted by Tom Morrison, who helped develop SNAP while at Biotrove and was interested in collaborating on a pilot study to see if the technology "would even work to look at some of the genes in the amniotic fluid."
"We wanted to make sure it would work first before we went on to picking specific genes that might be helpful" for a diagnostic test, Massingham said.
SNAP technology allows for the simultaneous quantitative assessment of tens to hundreds of genes from reduced and degraded nucleic acid samples, overcoming the quality concerns of processing primary human samples. Gene expression that varies by up to five orders of magnitude can be quantified using a single assay, the researchers said.
The technology has been in development since at least December 2008, when BioTrove and Gene Express inked an agreement to co-market a test using SNAP technology for profiling certain indicators of lung cancer risk.
BioTrove said at the time that the SNAP system combined the OpenArray nanofluidic PCR platform; Gene Express’ standardized RT-PCR, or StaRT-PCR, measurement standards; and TaqMan fluorogenic-labeled probes. The company also said that the technology was designed to provide researchers with a dynamic range of standard real-time quantitative PCR while simplifying workflow and reducing test sample quantity.
Then, in September 2009 BioTrove began a collaboration with the US Food and Drug Administration's Center for Biologics Evaluation and Research to investigate using SNAP to rapidly and specifically detect pathogens in the nation’s blood supply.
Life Tech acquired BioTrove in November 2009 (PCR Insider, 11/17/2009). A Life Tech spokesperson said this week that Morrison no longer works for the company; but that Life Tech's collaboration with CBER is ongoing.
Diana Bianchi, the lab's leader and a co-author on the study, told PCR Insider that her team was interested in SNAP because they were looking for "a way to do multiple PCR analyses" of genes they had identified as important for fetal health. She said the researchers have not yet been in contact with Life Technologies, but that the group was impressed with the platform's performance in this proof-of-concept study.
In their evaluation, the researchers used a 21-gene panel made up of a previously developed panel of lung cancer genes with five additional genes to test SNAP's ability to measure gene expression changes associated with fetal age and sex in cell-free amniotic fluid supernatant.
"The reason we picked the cancer panel was because in our previous research we found that a lot of time cancer genes are up-regulated in fetal samples, [which] we think is probably just normal fetal gene expression," Massingham said. "We wanted to see if any of these genes we thought might have potential to show differences, whether they would."
First the group demonstrated the dynamic range and accuracy of the SNAP technology on RNA extracted from amniocytes. The platform's performance in this initial test led them to move on to measure the expression of the 21 test genes using 19 supernatant samples from routine amniocentesis.
Statistically significant differences in expression were observed using SNAP in 5 of 21 genes — ANXA5, GUSB, PPIA, CASC3, and ZNF264 — as a function of advancing age. Two other genes, MTOR and STAT2, showed different expression in female and male fetuses.
"We sort of went into it not quite knowing what to expect," said Massingham. "Once we got into it the [results] weren't really surprising but it was good in confirming some of our thoughts, like [some of] the cancer genes really did change, indicating that the fetus is growing and changing."
In the paper, the authors wrote that SNAP technology may be "an advance over existing approaches, including global gene expression profiling using microarrays." Though high initial development costs are a drawback, benefits like "intrinsic quality assurance, quality control, and unbiased PCR" make SNAP an attractive platform for assessment of a "sufficient number of genes to evaluate fetal organ system function."
Massingham said that one area with potential for a cell-free AF supernatant diagnostic test is a condition called echogenic bowel, which can be caused by chromosomal anomalies, cystic fibrosis, infections, fetal growth restriction, and intra-amniotic bleeding.
"The idea is to hopefully develop a panel that would be helpful in elucidating … if the cause of the echogenic bowel might be an infection [as opposed to] the baby is having trouble growing or there is blood," she said. "A panel like this could help elucidate what the cause is, and if they need to do anything to treat that it might be very helpful."
Additionally, Massingham said, there is a range of prenatal issues that clinicians call "soft markers" for aneuploidies, for which an amniotic fluid cell-free test might be similarly illuminating.
"We often think about choroid plexus cysts, echogenic focus of the heart, kidney anomalies in prenatal clinics," she said. "Sometimes they're associated with Down's syndrome or trisomy 21, but most of the time they're not, and we don't necessarily have an explanation as to why these ultrasounds are finding this compared to ones that don’t find them."
In the JMD study, the researchers established that SNAP could be an effective method for examining cell-free amniotic fluid for gene expression changes. Next they hope to identify a panel of genes with diagnostic potential in these disorders, Massingham said, though a definite plan or timeline has not yet been set.
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