In a surprising result, a new clinical genomic trial in neonates in the NICU setting found no major difference in the diagnostic rate between whole genome sequencing (WGS) and whole exome sequencing (WES). The randomized clinical study, A Randomized Controlled Trial of the Analytic and Diagnostic Performance of Singleton and Trio Rapid Whole Genome and Exome Sequencing in Ill Infants, found that while WGS was more technically more complete and accurate than data provided by WES, the two methods were equivalent in terms of the diagnostic rate. Approximately 20% of the neonates tested in each group received a genetic diagnosis. Additionally, the time to diagnosis was the same, approximately 11.8 days. The paper was published September 26, 2019 in the American Journal of Human Genetics.
“We are at an early stage right now in terms of international implementation of genome sequencing as part of the care of acutely ill infants who are hospitalized and might have a genetic disease,” says first author Stephen Kingsmore, MD, President and CEO of the Rady Children’s Institute for Genomic Medicine (RCIGM),established as a pediatric research institute within Rady Children’s Hospital in San Diego, CA. “People have been doing it in different ways and there has not been a consensus if there is a single best method nor how best to use this new exciting technology.”
This paper is the first report of the second study in the NIH Initiative called Newborn Sequencing Genomic Medicine and Public Health(NSIGHT). NSIGHT is composed of four separate research projects, including RCIGM’s project, “Clinical and Social Implications of 2-day Genome Results in Acutely Ill Newborns.”
This trial, known as NSIGHT2, focused on babies immediately admitted to NICU centers within the Rady Children’s Hospital San Diego (RCHSD) system. “In this study, we wanted to use a genome test as early in the hospital course as possible,” Kingsmore add. “If you’re using a genome as a test of last resort it’s not going to change treatment that much, so we wanted to test babies very soon after admission to the NICU.”
In the study, all babies within the RCHSD NICU system were screened for inclusion as an attempt to generalize any findings to the NICU setting at large. For that purpose, over a 15-month period between 2017-2018, all babies admitted to the RCHSD NICUs were screened for trial inclusion. In total, 1,248 babies were screened during that time and 578 infants (46%) were deemed eligible for study inclusion. To be included, infants had to have been admitted to a NICU within the past 96 hours or within 96 hours of a new clinical finding.
Nearly one-third of eligible participants, or 213 infants, were enrolled in the trial. However, 24 babies (11% of the total enrolled) were deemed extremely ill and received ultra-rapid WGS instead of randomization. The remaining 89% of the enrolled infants were tested with WGS (94) or WES (93).
In the study, genome DNA was prepared for WGS with TruSeq DNA LT kits (Illumina) or Hyper kits (KAPA Biosystems). Sequencing libraries were analyzed with a Library Quantification Kit (KAPA Biosystems) and High Sensitivity NSG Fragment Analysis Kit (Advanced Analytical). Nucleotide WGS was performed with Illumina HighSeq 2500, HiSeq 4000, or NovaSeq 6000.
For WES, samples were prepped and sequenced by GeneDx. Exome enrichments was done with the xGen Exome Research Panel v1.0 (Integrated DNA Technologies) followed by amplification with the Hercules II Fusion polymerase (Agilent). FASTQ files were provided to RCIGM for interpretation.
Superior rWGS Analytic Performance
By multiple measures, data provided by WGS was significantly more complete and accurate than that from WES. Whole genome sequencing compared to whole exome sequencing resulted in 12% more coding variants, 37% more variants of types likely to have a functional affect or is the cause of disease, and 6.5-fold more variants with splice-altering consequences. “And we found twice as many variants as were in the database of variants that we all use of known pathogenic or likely pathogenic variants,” says Kingsmore referring to ClinVar P and LP variants. “It’s important to note the WGS was superior across all measures compared with WES in this study because there has been debate whether they are the same or is one superior over the other.”
Genetic Disease Diagnosis
In total, 24% of the 213 enrolled infants (52) were found to have a simple genetic disease. In all, 54 genetic diseases were identified. If one assumes these 213 infants were representative of the 578 babies eligible for the trial, the team suggests that the prevalence of genetic diseases in infants in regional NICUs to be 14%. “This figure is higher than the proportion currently tested, and a lot higher than people would have suggested,” says Kingsmore.
Diagnosis Rate and Time Comparable
Both WGS and WES had a nearly identical time to diagnosis of 11.8 days. But despite its superiority in terms of technical performance, the diagnostic rates were similar between those randomized to WGS and those who received WES. Eighteen of 94 babies (19%) tested with WGS had a genetic diagnosis compared with 19 of 95 infants (20%) receiving WES. “This finding was surprising to us because previous studies have found genomes have a higher diagnostic rate than an exome, and our own study found WGS was superior in terms of analytic accuracy,” says Kingsmore, who attributes this finding to a small study size. The authors reference four other studies comparing WES and WGS finding a 4-7% increased diagnostic difference with WGS. “Our study was not powered to identify such a change,” he said. It should be noted that the overall trial’s primary endpoints are not diagnosis rate or time to diagnosis but acute clinical utility and outcome (1-year survival) which will be reported in a later manuscript.
Other researchers in the field are not as surprised by the comparable diagnosis rates, though. “I’m not terribly surprised that exome and genome sequencing were similar in this context,” says Robert Green, MD, of the Brigham and Women’s Hospital and co-leader of the BabySeqProject, another NSIGHT project, which focuses on genetic testing of healthy newborns. “Most of the population we are dealing with mostly have Mendelian disease which involve changes in the genes, and the exome sequences all of the genes; it’s the working part of the genome.”
He does, however, find it impressive that the percentage of babies diagnosed in either arm is around 20%. “That is remarkable that using either exome or genome they were able to get clarification of molecular diagnosis in one-fifth of the sick babies in the study,” he says. “If you think of all the diagnostic testing we do, if you had a test that gives you the answer in one-fifth of the time that’s incredible.”
Singleton and Trio Testing Roughly Equivalent
The study also compared singleton testing of the infant and trio testing of infant, moth, and fat. Earlier studies from the Kingsmore group suggested that genetic testing in the neonatal setting should include trio testing. In this study, however, they discovered only a 1% difference between singleton infant testing and trio testing. “From this study, it’s probably not worthwhile in most children to do a trio. The additional cost is not going to be matched with a higher diagnostic rate,” says Kingsmore.
Ultra-rapid Whole Genome Sequencing Superior
In those 24 critically ill infants tested with urWGS, 11 (46%) were found with a simple genetic disease, which is more than double the diagnostic rate in WGS or WES, though this was not unexpected since these babies were sicker, more were receiving antibiotics and they had a higher short-term death rate than other babies in the trial. The time to result was also much faster; 2.3 days compared with almost 12 days.
“The ultra-rapid WGS is the way to go in our opinion; focus on that technology and get a 2-3 day turnaround for every baby,” says Kingsmore who added his own institution’s data within the RCHSD community includes an average turnaround time in July and August of this year of 60 hours and 58 hours, respectively. “We have demonstrated that ultra-rapid WGS, when performed as a first-tier test completed in about 58 hours provides early diagnostic information that may lead to improvements in clinical management of seriously ill babies very early in life when it could yield significant, or life-saving outcomes.”
But in the everyday NICUs around the country, cost remains the reason why exomes are mostly ordered over genomes. “They are just about as good and they are about cheaper, says Green. “In general, an interpreted exome is somewhere around $800-1,200. An interpreted genome is $1,000-2,500; a step up for sure.” And regardless of the type of sequencing used, Green attributes cost as “one more piece of evidence in a medical care system that has been extraordinarily slow to acknowledge the power of genomic sequencing and to make arrangements for reimbursement.” In this trial, Kingsmore confirms that the cost of WES to be $1,500 less costly than WGS.
Although it was not possible at the time of the study to perform urWGS on all infants, the Illumina technology used has progressed to where the cost of an urWGS is the same as a WES in Kingsmore’s experience. While this study did not involve babies randomized either to urWGS or WES, Kingsmore suggests this type of direct comparison is warranted.