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Cincinnati Children's Hospital Team Describes Clinical Exome Sequencing Experience


NEW YORK (GenomeWeb) – Cincinnati Children's Hospital Medical Center has found that its clinical exome sequencing pipeline can identify the cause of rare pediatric disease in about 30 percent of cases and is often less costly and time consuming and more effective than the litany of genetic tests patients receive.

The findings of their clinical study, which were published this month in Frontiers in Pediatrics, mirror experiences by other clinical labs offering exome sequencing for rare disease, and add to a growing body of evidence that such tests are effective.

In the publication, the researchers described their experience with their first 40 cases. Kejian Zhang, director of the molecular genetics laboratory at Cincinnati Children's Hospital Medical Center, said that the lab has now evaluated around 130 patients, and is still seeing about a 30 percent diagnostic rate.

The lab runs its exome sequencing test using Roche's NimbleGen exome capture and Illumina's HiSeq 2500 with an average turnaround time of three to six months. The lab prefers to do trio sequencing, which helps with interpretation and boosts the likelihood of making a diagnosis, and it does so for about 90 percent of its patients. The test costs $12,000 for a trio and $7,500 for a single patient. In addition, the laboratory requires pre-authorization from the patient's insurer. Zhang said that she did not have exact numbers on how often or how much payors typically reimburse, but said that it varies.

Zhang said that the hospital's patient population is somewhat unique compared to other academic centers that have published findings from their clinical exome sequencing pipeline, including Baylor College of Medicine and the University of California, Los Angeles, which both reported their clinical experiences in the Journal of the American Medical Association last year.

For instance, about 10 percent of Baylor's 2,000 cases were adults and 87 percent had a neurological disorder. By contrast, the Cincinnati cohort consisted solely of pediatric patients. In addition, the most common primary phenotype was multiple congenital anomalies, present in 30 percent of patients. Around 22 percent had a primary phenotype of neurologic disorder, with immunodeficiency and mitochondrial disorders making up 17 percent and 22 percent of patients, respectively.

"It's interesting to see that for this cohort with these disorders, exome sequencing also gives about a 30 percent diagnostic rate," Alexander Valencia, assistant director of the molecular genetics laboratory, told GenomeWeb.

Exome sequencing identified a couple of surprising findings, Valencia said. For instance, there was one patient who presented with cataracts, insulin-dependent diabetes, and hyperglycemia, but "did not fit the classic patterns" of diabetes, the authors wrote. Exome sequencing identified a de novo missense mutation in the insulin gene, which is associated with permanent neonatal diabetes mellitus or type 1b diabetes. Valencia said the mutation explained both the patient's cataracts and his diabetes.

"There were things like this where genetic testing actually reveals more about the biology and the causes of some of these very complex cases," he said.

That molecular diagnosis will also be important for the patient's future treatment. For instance, he should be monitored for long-term complications like retinopathy, neuropathy, and proteinuria. In the future, he could be eligible for beta cell transplantation.

Of the patients that received diagnoses, 47 percent of the causative mutations had previously been unreported.

The laboratory also returns secondary findings in the 56 genes recommended by the American College of Medical Genetics if the families wish to receive such findings. Thirty-six out of 40, or 90 percent, chose to receive such findings and three patients, 8 percent, had medically actionable findings.

For one two-year-old patient, while exome sequencing did not identify the molecular cause of his primary disorder, it did find a mutation in the gene that causes Marfan syndrome. The patient was referred for cardiologic and ophthalmologic evaluation as well as subsequent management for Marfan syndrome.

For about 25 percent of patients, the exome test identified variants of unknown clinical significance. In a few cases, Zhang said the team found some "interesting candidates," and the family has consented to further research.

The study's authors also described a few cases where variants could not be classified as causative due to minimal published literature or higher allele frequency, but that nonetheless may contribute to the patient's phenotype. For example, one patient had two variants in the POLR3B gene. One was a rare variant, while the other was more frequent but with inconsistent predictions on whether it is probably damaging. Both variants however, "support a compound heterozygous model" and "the gene is associated with the phenotype described for this patient," the authors wrote.

In addition, in two cases where a diagnosis was made, the researchers also found compound heterozygous variants of unknown significance that may contribute to the patients' complex phenotypes.

"These cases illustrate the complexity of variant interpretation and that frequency is only one of the variables used to bin them into pathogenicity categories," the authors wrote.

Valencia predicted that diagnostic rates of clinical exome sequencing would rise with additional research, particular into some of the variants of unknown significance. Indeed, Baylor reported that following its first 250 cases, new gene discoveries increased its diagnostic rate from 25 percent to 28 percent. And about 30 percent of patients had mutations in disease genes that had only recently been described. The Cincinnati team also diagnosed 12, or 33 percent, of its patients based on disease-gene discoveries that had occurred in just the past two years.

Although evidence is growing that supports diagnostic exome sequencing as a first-line test for certain patients, the authors reported that nearly half of their patients had received at least four genetic tests before having their exomes sequenced, and of those, three had received more than 10 genetic tests.

In those three cases, the testing combination was more expensive than exome sequencing, and costs became "astronomical" when non-genetic tests were factored in as well. For instance, the authors highlighted one patient that received chromosome and microarray analyses, mitochondrial testing, very long chain fatty acid analysis, brain and spine MRIs, muscle biopsy, and an EEG. Exome sequencing identified a mutation in ASXL3, associated with Bainbridge-Ropers syndrome. The syndrome and disease gene are newly recognized and the diagnosis would not have been made through conventional testing, since no such test existed at the time.

Exome sequencing now has a higher diagnostic rate than karyotype analysis and microarray analysis, which have rates of 5 percent to 15 percent and 15 percent to 20 percent, respectively, the authors reported. Depending on the phenotype, exome sequencing is comparable to gene panels.

Zhang said that, especially in certain cases — for instance, patients that are "very young and very sick" — exome sequencing may make sense as a first-line diagnostic. Often, physicians of those patients want the exome test because it is comprehensive and covers many conditions. In addition, those young patients are especially hard to diagnose by phenotyping alone because often not all of the defining symptoms have presented themselves yet. "Their symptoms are evolving" and are often not present initially, but "by the time we report back, there are more symptoms that have presented that support our diagnosis," Zhang said.

Nonetheless, she acknowledged that there are still a number of challenges to work through, including improving the sensitivity, turnaround time, and cost of exome sequencing.