NEW YORK (GenomeWeb) – A multiplex selector-based sequencing capture assay can be an effective first-line test to diagnose mutations that cause a class of immunodeficiency diseases known as primary immunodeficiencies (PIs), a study published this week in PlosOne said.
Scientists led by Mats Nilsson of Uppsala University created a 179-gene panel using Agilent HaloPlex technology and Illumina sequencing to resolve the mutation status of 33 individuals, of which 18 had at least one known causal mutation at the start of the study. The assay, Nilsson and his colleagues wrote, was able to find the defective gene or mutation in 78 percent of those patients.
"The detection rate for causative variants in the genes we are targeting is as good as Sanger, or NGS exome, or genome sequencing," Nilsson told GenomeWeb in an email.
PIs are a heterogeneous group of over 200 diseases caused by genetic defects of the immune system, and are characterized by severe, recurring, and often life-threatening infections. The current standard of testing involves specialized methods including cytotoxicity assays and flow cytometry.
Genetic testing can help clinicians determine treatment strategies. However it is not always clear which of the 200-plus genes associated with PIs should be tested. Mutations in different genes can result in similar phenotypes, and mutations in different parts of the same gene can present with distinct phenotypes. On top of that, laboratories accredited to test for PIs sometimes only cover a few of the potential disease genes, so testing may need to be divided to different labs.
But with next-generation sequencing, a multiplexed diagnostic assay allows for the simultaneous analysis of multiple genes included on the test. "This is probably the cheapest way of diagnosing variants," Nilsson said. However, he noted that "causative variants outside these genes will not be found."
The researchers extracted DNA from whole blood using the Qiagen FlexiGene DNA kit. For some patients, blood was also collected in Paxgene Blood RNA tubes from PreAnalytix, a Qiagen/Becton Dickinson jointly owned company. Then they extracted RNA using the Paxgene Blood RNA Kit, including 500 ng for cDNA synthesis using Roche's First-strand cDNA synthesis for RT-PCR. The researchers designed the selector assays to cover all exons of the 179 genes related to PIs. High-throughput sequencing was performed at GATC Biotech on the Illumina GAIIx system.
The researchers were able to match several cases with an unknown cause to known gene mutations. Two individuals had mutations in the STAT3 gene, two more had mutations in the lambda 5 gene, and one patient inherited two different mutations in the RNF168 gene.
Based on the results of the study, the authors suggest a two-stage process for diagnosing PIs: using a relatively cheap and fast targeted panel that could explain a majority of disease cases, followed by whole-exon or whole-genome sequencing of the cases that could not be resolved by the assay.
The authors noted that second-generation assay technology could make a multiplexed test even more useful in diagnosing PIs. "Using a second-generation HaloPlex design, 99.5 percent of the target region would be covered in silico, compared to 98.6 percent in the current assay. Combined with better sequence coverage and read depths – thanks to improved capture uniformity – this would greatly advance the assays' performance," the authors wrote.