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Sequencing Could Screen for Mendelian Disorders, Aneuploidy in Preimplantation Genetic Testing

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SAN FRANCISCO (GenomeWeb) – Researchers in Spain have tested the feasibility of using Illumina's TruSight One assay, which targets more than 4,800 genes associated with Mendelian disorders, as a way to screen embryos in preimplantation genetic testing for both disease-specific mutations as well as chromosomal aneuploidies in one assay.

The so-called double factor preimplantation genetic test would help reduce the time it takes to conduct genetic testing of embryos as part of in vitro fertilization cycles, and because the TruSight One assay is so broad, it would not require laboratories to design individual tests depending on the genetic disorder in the family.

Although the group said they will need to test many more samples to determine the performance of the assay, results from a pilot, published this year in PLOS One, were promising, showing that the method can both detect chromosomal abnormalities and identify Mendelian disease-causing mutations. The research was the result of a collaboration between the Universitat Autònoma de Barcelona, the Bank of Blood and Tissues of Catalonia, Clinica Eugin Spain and two hospitals

Joaquima Navarro, senior author of the study and researcher at the Universitat Autònoma de Barcelona in Spain, said that because the study evaluated only nine families, the next steps would be to validate that the method works for a broader range of disorders that are included in the TruSight One panel. Then, she said, it would be necessary to do a clinical trial, running the assay alongside conventional testing, which includes PCR-based tests to analyze specific mutations and often array CGH to screen for aneuploidies.

The goal of the study, Navarro said, was to design a simpler test that could combine aneuploidy and single-gene testing in one. In addition, she said, the team wanted a broader test for single-gene diagnosis so that the molecular genetics laboratory would not need to design new probes for each family specific to the mutation. Instead, the comprehensive assay should be able to do both mutation detection and chromosomal analysis.

In the study, the researchers first had to settle on a whole-genome amplification method to use. The team tested four commercial kits: Yikon Genomics' MALBAC method, Qiagen's Repli-g kit, BlueGnome's SurePlex, and GE Healthcare's Illustra GenomiPhi.

Ultimately, the team settled on the Qiagen kit, which Navarro said provided the best performance for both mutation detection and chromosomal analysis, but they also did additional testing with the GE Healtcare kit.

Next, the researchers analyzed 12 embryos that had been discarded by nine families undergoing in vitro fertilization and who had consented to research. For half, they used the Qiagen Repli-g kit for whole-genome amplification and for half they used a GE Healthcare kit. Both kits rely on multiple displacement amplification.

Then the TruSight One panel was used to target the 4,800 genes and sequencing was done on either the Illumina MiSeq or NextSeq system. Each of the embryos had been discarded as part of the IVF process with most containing known mutations in diseases that included hereditary cancer syndromes, spinocerebellar ataxia, and hereditary bone deformity.

Consistent with the team's previous analysis of whole-genome amplification methods, the Repli-g method again had better coverage. For the six embryos analyzed with that method, five had concordant results with the previous diagnosis. The sixth embryo had not previously been identified to carry a disease mutation but was discarded since it was also not confirmed as healthy.

The targeted sequencing method did not call the known disease allele that was present in the family and that had affected another embryo analyzed in the study that was from the same family. But, because the researchers had also analyzed the parents' DNA, they were able to compare the sequencing results of the embryo to the parents and perform a linkage analysis. That enabled them to tease apart the haplotype blocks surrounding the disease and healthy allele and to confirm that the embryo did indeed have the healthy allele.

Navarro noted that this was one reason why including parental DNA was so important and could help confirm whether the embryo was affected or not.

Finally, the team showed that the targeted sequencing method could identify chromosomal aneuploidy. To do this, they used Bio Discovery's Nexus software.

To call a disease mutation, Navarro said that 10 or more reads covering the region are needed. It's possible to call the mutation at this low depth, she added, since the mutation is already known and they are not looking to discover novel mutations, and because the researchers include parental DNA to which they compare the embryo genome.

Many fertility clinics now offer screening for chromosomal aneuploidies, and, in the case where there is a known risk for passing on a genetic disorder, clinics will also test specifically for that mutation. But, Navarro said that such testing can be time consuming, since there is not one uniform way in which all patients can be screened.

"We want to avoid the need of setting up in each family for each mutation a separate detection system," she said. And, at the same time, the team wanted "a system that informs us about the chromosomal aneuploidy situation for all the chromosomes."

Previously, Navarro said that the so-called double factor preimplantation genetic testing method, which involves screening embryos for both specific disease-causing mutations as well as chromosomal aneuploidy, was performed with separate tests for the mutation detection and chromosomal copy number analysis. For each family, a specific PCR-based assay would have to be designed for the mutation of interest, while chromosomal aneuploidy was analyzed via comparative genomic hybridization.

Recently, a number of researchers have begun looking at low-coverage shotgun sequencing as a way to screen for chromosomal aneuploidies in embryos, similar to the strategy now used in many noninvasive prenatal tests. But, because the sequencing is done at such low coverage it is not possible to also detect point mutations.

However, researchers at Peking University have tested a method that uses the low-coverage whole-genome sequencing but with additional amplification and coverage of the specific region with the disease allele.

While that method combines both point mutation detection and chromosomal analysis in one assay, it still requires separate tests for each family.

Analyzing embryos for both monogenic disease mutations and chromosomal aneuploidies has previously been shown to increase pregnancy rates. A study published last year by a team from Italy, for example, tracked outcomes of more than 1,000 embryos that were first tested for monogenic disorders and then screened via array-CGH for chromosomal aneuploidy. That group found that the combined screening led to pregnancy in 49 percent of transferred embryos, an increase from a previous study of PGD only where 34 percent of transfers resulted in pregnancy.

Like the Peking University group, however, the method used in this study involved two separate tests. Nonetheless, Navarro said that it shows that doing both PGD and PGS can help improve outcomes.

In terms of cost, Navarro said that it's too early to say how much a clinical test would cost, but noted that the cost to run the assay in a research setting was around $350. However, that did not include labor or personnel costs, she said. 

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