SAN FRANCISCO (GenomeWeb) – A noninvasive test for recessive single-gene disorders could be used to screen prenatally for rare, inherited diseases for which one of the parents is a known carrier, according to researchers at Stanford University.
The researchers developed the test for metabolic and skeletal disorders using droplet digital PCR and demonstrated that from nine at-risk pregnancies, it could accurate classify affected fetuses by screening cell-free DNA in maternal blood, work that they published this month in Clinical Chemistry.
They are now working to validate it on a larger cohort of patients at Stanford's Lucile Packard Children's Hospital where they hope to eventually run it as a clinical test. In addition, the team has filed for a patent on the technology and is considering licensing it to a company.
According to Louanne Hudgins, a professor of pediatrics at Stanford and director of perinatal genetics at Lucile Packard Children's Hospital, such a test would be especially valuable for women living in more rural areas, since it could help them decide whether they should deliver at a tertiary care center or whether they could stay local.
It could also help prospective parents and physicians better prepare to care for a child with a disorder. For example, for some metabolic disorders treatment can be started immediately after birth. And for others, the pregnancies themselves can be managed differently.
For instance, Hudgins said, if a woman is at risk for having a male affected by ornithine transcarbamylase deficiency, a disorder that causes ammonia to accumulate in the blood, physicians would want to make sure she is getting plenty of fluids with dextrose. Physicians would also potentially give her ammonia scavenging agents to keep ammonia levels in the normal range, she said.
Hudgins added that the Stanford team is now "working closely with the hospital to put in place a pilot project in order to validate the test for clinical use."
Joan Camunas-Soler, lead author of the study and a postdoctoral research fellow in Stephen Quake's lab at Stanford, said that designing a noninvasive test for single-gene disorders was challenging because "you have to see one single spot in the fetal genome in a background of mostly maternal DNA. So there's a lot of noise."
The researchers first developed a droplet digital PCR assay to quantify the amount of cell-free DNA in a maternal plasma sample derived from the fetus. Knowing fetal fraction is critical for the accuracy of the test, Camunas-Soler said. "You need to tell whether the amount of a mutation you observe agrees with the fetus having inherited that mutation," he explained.
For instance, if a woman is a carrier for a given disorder, half of her alleles should be wildtype and half mutated, he said. In a pregnant woman, some fraction of circulating cell-free DNA will be from the fetus, so that ratio of wildtype to mutant allele will differ depending on whether the fetus inherited the mutant copy or not. "Knowing how much DNA comes from the fetus allows you to know if the balance of mutations you see means that the fetus is affected or not," Camunas-Soler said.
To calculate fetal fraction, the researchers selected a set of 47 high-variability SNPs, as well as both X and Y chromosome markers, that would represent individuals from many different ancestral backgrounds, in order to ensure that the assay would be relevant for different populations, Camunas-Soler said. The SNPs had minor allele fractions of greater than 0.4 for the five subpopulations included in the 1,000 Genomes Project. In addition, the SNPs were selected to fall outside of highly repetitive areas and regions of structural variation.
The assay for determining fetal fraction is run separately from the assay to test for the inherited disorder and uses a portion of blood from the initial draw. In addition, the researchers ran a separate assay to quantify the total amount of cell-free DNA.
They tested whether their method could calculate the likelihood of whether known carriers of a disease-causing mutations had passed on the mutant variant to their fetus, evaluating nine carriers of seven disorders: hemophilia, ornithine transcarbamylase deficiency, cystic fibrosis, beta-thalassemia, mevalonate kinase deficiency, acetylcholine receptor deficiency, and DFNB1 nonsyndromic hearing loss.
After running the initial cfDNA quantification and fetal fraction assays on the samples, the researchers then use a ddPCR assay to test for the known pathogenic mutation in the cfDNA sample.
Ultimately, the team correctly classified all nine pregnancies, two of which were affected, as confirmed by neonatal testing. The method was performed as early as 11 weeks gestation and at a fetal fraction as low as 3.7 percent.
The researchers also calculated the theoretical accuracy of the assay, determining that for a fetal fraction of 4 percent, they would need to draw between 25 and 30 milliliters of blood and analyze 20,000 molecules to achieve a false positive and false negative rate of 0.2 percent to 1 percent.
"That would need to be confirmed with a larger study," Camunas-Soler said. However, he said, the results are so far consistent with NIPT for chromosomal aneuploidies.
Aside from doing a larger study with the hospital to develop a clinical test as a service, Camunas-Soler said the group is also looking at the possibility of licensing the technology to a company. So far, he said, companies have not developed technology to screen for inherited single-gene disorders because tests have to be individualized, rather than one broad test for everyone, so the market is much smaller.
Natera and Baylor Genetics have both launched noninvasive prenatal tests for de novo single-gene disorders, but those are panel tests that analyze 30 genes for pathogenic mutations that are not present in either parent, and thus do not have to be customized to the patient.
Camunas-Soler said that one possible commercialization route would be to develop a panel that includes the most common mutations for a set of inherited disorders. However, the advantage of ddPCR-based bespoke testing is that it would keep the costs lower as compared to a sequencing-based panel, which would require high sequencing coverage to be able to identify the pathogenic variant and determine whether it came from fetal DNA or maternal DNA.
Regardless of the testing methodology, Camunas-Soler said noninvasive testing for single-gene disorders would be especially compatible with expanded carrier screening, since those tests would identify at-risk couples who may ultimately want prenatal testing.