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Rapid Whole-Genome Sequencing Used to Diagnose Infants with Genetic Disease

NEW YORK (GenomeWeb News) – Researchers from Children's Mercy Hospital demonstrated in a proof-of-principle study that whole-genome sequencing in a neonatal intensive care unit could be used to diagnose genetic disease.

Reporting online today in Science Translational Medicine, the team demonstrated that sequencing on Illumina's HiSeq 2500 machine, which can sequence a whole genome in around 26 hours, combined with the hospital's internally-developed automated analysis pipeline could theoretically provide an answer in around 50 hours for an estimated cost of $13,500.

"We can now consider whole-genome sequencing to be relevant for hospital medicine," Stephen Kingsmore, director of Children's Mercy Hospital's Center for Pediatric Genomic Medicine, said in a conference call with the media discussing the study. "We think this is going to transform the world of neonatology, by allowing neonatologists to practice medicine that's influenced by genomes."

In the study, the actual time to result was longer than 50 hours because the sequencing was done at Illumina's UK facility, so data had to be shipped between the Kansas City, Missouri-based hospital and the UK. However, the hospital expects to begin running the test from its own facilities under a research protocol in November, when it receives Illumina's HiSeq 2500 system.

The team tested its protocol retrospectively on two patients who had previously been diagnosed through clinical testing and also prospectively on five undiagnosed newborns.

The protocol correctly diagnosed both retrospective samples, one of which had Tay Sachs disease and the other which had Menkes disease. It also provided a diagnosis for four out of five of the undiagnosed newborns.

Keys to the workflow were reducing the sequencing time and automating much of the informatics, which has been a major hurdle for implementing sequencing in the clinic.

For analysis, the hospital's researchers developed a tool dubbed SSAGA, for symptom- and sign-assisted genome analysis. SSAGA provides a framework for physicians to enter in the patient's clinical phenotype, and then it spits out a candidate list of disease genes associated with that phenotype, thus narrowing analysis of the entire genome.

Currently, the program has a menu of 227 clinical terms associated with 591 genetic diseases, but it is being expanded to include all 3,500 genes that are known to be causative of genetic disease.

The researchers tested the algorithm on 553 children that had already received a molecular diagnosis, and found it had a sensitivity of 99.3 percent and nominated an average of 194 genes per patient.

The team tested the protocol in a research setting on five newborn patients that had been admitted to the neonatal intensive care unit at Children's Mercy Hospital. Because it was done in a research setting, before results were given to the parents confirmatory testing had to be done, which took at least an additional four days.

In one patient with severe epilepsy a molecular basis was identified within just one hour of data analysis, despite the fact that the variant in the disease gene had only recently been reported in two unrelated Amish infants and was not yet in the reference databases.

The patient ultimately passed away after five weeks of failed treatment when the parents chose to end ventilatory support. The decision was made before the parents received the diagnosis, however, the diagnosis was still useful because the family was able to receive genetic counseling and the information was shared with other family members at risk for carrying the mutation.

"As a result of this project, the family has received accurate genetic counseling, and can now pursue carrier testing for at-risk individuals," said Carol Saunders, the clinical lab director, during the call.

In a patient with a severe skin disease, the team identified a novel de novo mutation in a gene known to cause lethal skin disease. However, because it is a novel gene, "we need more evidence," said Saunders, adding that the team is now collaborating with another group to determine if the variant was in fact causative. This patient also passed away.

"This was the first baby for these parents, and they are interested in trying for a healthy baby, but understandably would like to know their risks for this to happen again," Saunders said.

In a third family, the researchers think that they may have identified an entirely new disease gene. In this case, the parents had two affected babies with heart defects and a condition in which some of the internal organs are on the wrong side of the body.

The initial search did not turn up any known disease genes. Because the unaffected parents had two affected children, the researchers also sequenced the parents and looked for variants for which the parents were heterozygous and were shared by the siblings and identified two variants in the gene BCL9L. While it had not previously been reported as a disease gene in humans, previous significant animal modeling further supported the identification of a new disease gene.

Additionally, the parents now know that they are each carriers, which "allows them to have accurate genetic counseling regarding their risk to have another affected baby, and to make informed decisions about their reproductive future," Saunders said.

In the fifth case, no molecular diagnosis was made. The patient died after five days and doctors suspected a mitochondrial disorder. In this case, Saunders said that the causative mutation likely falls outside of their detection abilities.

"There are many important variants that lie outside the regions that we typically know to analyze, and some types of variants aren't captured very well by this technology yet," she said.

Since the study, the team has analyzed another two patients. In one case a molecular diagnosis has led to the parents pursuing pre-implantation genetic screening in order to have a healthy child, and in the second case the researchers believe they may have identified another novel disease gene.

Moving forward, the Children's Mercy Hospital team plans to begin offering the test on a research basis in its own neonatal intensive care unit when it receives its HiSeq 2500 in November.

Under that protocol, preliminary results will be able to be verbally released to the physician, but confirmatory testing will still need to be done. Eventually, Kingsmore said the team would offer the test to other neonatal intensive care units, and also plans to pursue certification to offer it as a CLIA-certified laboratory-developed test.