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Noninvasive Prenatal Single-Gene Disease Testing to Benefit from Universal Method

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NEW YORK (GenomeWeb) – Researchers at the Chinese University of Hong Kong and their collaborators have developed a universal method for noninvasive prenatal testing (NIPT) for single-gene diseases that does not require knowledge of specific disease-causing mutations or DNA from an affected family member.

The approach, published as a proof-of-concept study in Clinical Chemistry last week, uses linked-read sequencing technology from 10x Genomics to resolve the haplotypes of the parental genomes and to identify SNPs surrounding the disease locus. This is followed by maternal plasma DNA sequencing and relative haplotype dosage (RHDO) analysis to infer the mutational status of the fetus.

"We think this is a major achievement in NIPT," Rossa Chiu, Choh-Ming Li professor of chemical pathology at the Chinese University of Hong Kong and the senior author of the study, told GenomeWeb.

NIPT has been widely adopted for fetal chromosomal aneuploidy testing because protocols for this application have been easy to implement, she said. However, for single-gene disease testing, it has not yet taken off because up until now, labs needed to design a new assay for each mutation they wanted to test for. "Now, with this streamlined protocol, we think there is a chance that NIPT for single-gene disease will become more prevalent, because now every lab would just need to learn one protocol," she said.

Many prenatal screening programs determine whether parents are carriers of genetic diseases that frequently occur in their region, Chiu explained, for example thalassemia in Southeast Asia, or cystic fibrosis and sickle cell anemia in the US. If both parents are found to be carriers, prenatal testing is often recommended, which today usually relies on invasive methods, such as amniocentesis or chorionic villus sampling, that carry a small risk of miscarriage. To avoid such risk, researchers have been working on developing noninvasive approaches for diagnosing single-gene diseases prenatally.

In 2008, Chiu and her colleagues, including Dennis Lo, published an approach called relative mutation dosage, which determines the relative amount of mutant and wildtype alleles of a disease-causing gene in maternal plasma. The method, which requires a specific assay for each mutation, can then deduce whether the fetus is affected.

While this approach, which uses digital PCR, is relatively inexpensive, Chiu said, for some mutations, it is difficult to design an assay that works well, and the method requires a certain fetal DNA fraction. The gene for 21-hydroxylase, for example, which is mutated in congenital adrenal hyperplasia, has a highly homologous pseudogene, so a PCR assay that works on highly fragmented maternal plasma DNA is hard to establish. Also, bespoke assays sometimes take too long to develop and optimize. "We needed an assay that doesn't require specific mutations for the assay design and is more sensitive," she said.

In 2010, Chiu and Lo's teams published a sequencing-based method to genotype the entire fetal genome. This relies on maternal plasma DNA sequencing and comparing the data to the parental haplotypes, using so-called relative haplotype dosage (RHDO) analysis. "The concept is similar to relative mutation dosage, but the difference is that this time, we no longer depend on detecting the [specific] mutation," Chiu explained.  They later used a targeted version of this approach that only sequenced a 6-megabase area of the genome to noninvasively diagnose congenital adrenal hyperplasia, a study that was published in 2014.

Apart from avoiding mutation-specific assays, RHDO is more sensitive because it uses more sequencing reads than just those immediately surrounding the mutation site, Chui said. But it still came with a bottleneck that the new method they published last week solves: haplotyping the paternal genomes.

"Haplotyping is not easy to do at all," Chiu said. For their previous studies, she and her colleagues used DNA from a previously affected child to do this, but that restricted the assay to families with an affected member. Her group and others also tried other haplotyping strategies, including Illumina's Moleculo technology, but those "are rather cumbersome" and sometimes left gaps that needed to be filled in with population-based haplotype data, she said.

For their latest study, the researchers haplotyped parental DNA using 10x Genomics' linked-read technology, combined with Illumina sequencing. This method partitions long DNA fragments into gel beads, where the DNA is fragmented and barcoded. After sequencing, the fragments are reassembled into haplotypes based on the barcodes. "Now, we can conveniently haplotype the father's and mother's DNA," Chiu said, and then analyze the maternal plasma DNA to find out whether the fetus is affected.

For their study, the researchers recruited 13 families at risk of having a fetus with a monogenic disease, including congenital adrenal hyperplasia, beta-thalassemia, and hemophilia.

After resolving the parental haplotypes and determining SNPs around the disease locus, they interpreted sequencing data from maternal plasma DNA using the RHDO approach. This enabled them to accurately determine the fetal mutational profiles for 12 of the families — for the last family, they did not have sufficient coverage of informative SNPs in the plasma DNA sample, Chiu said.

Using a targeted rather than a genome-wide sequencing approach lowered the overall cost in some cases, Chiu said, but for some of the diseases, they did not have capture probes for the disease gene locus available, so they relied on genome-wide sequencing.

Right now, the protocol, which takes one to two weeks, is still quite costly, especially the 10x Genomics reagents, Chiu said. Overall, reagents for the un-optimized protocol cost at least $2,000. Another limitation is that it is not applicable to trinucleotide repeat disorders because it does not tell whether repeats inherited from a carrier mother have further expanded in the fetus, which may turn on the disease.

The next step will be a validation study, ideally involving hundreds of cases, to prove the accuracy of the test. Recruiting families for such a study takes time, though, because the incidence of any one single-gene disease is low, Chiu said. For example, 8 percent of individuals in Hong Kong are carriers for thalassemia, and about 120 couples are at risk of having an affected fetus every year, she said. However, collectively, single-gene diseases are a large burden, and "besides aneuploidy, single-gene disease is the other major reason why prenatal diagnosis is considered," she added.

Chiu said she sees several applications for the test: for families with a history of a genetic disorder who want to find out if their fetus is affected without using an invasive test, for couples with no disease history who have been identified as carriers of the same disease, and for families where structural abnormalities on an ultrasound point to a single-gene disorder.

In the future, the parental haplotyping approach could also be used for hypothesis-free carrier screening once it becomes less expensive, she said, and the haplotyping information gained in this way could help with subsequent noninvasive prenatal testing.

While it is too early to develop the test commercially, "I do feel there is commercial potential because it does fill a need in prenatal testing," she said.

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