NEW YORK (GenomeWeb News) – Researchers from Duke University and the University of Michigan successfully diagnosed around half of the individuals that they tested during a pilot study on the use of clinical exome sequencing, according to a study set to come out online this week in the Journal of Medical Genetics.
"There are an awful lot of kids who go through a very, very long process to try to find the genetic diagnosis and don't end up with one," senior author David Goldstein, director of Duke's Center for Human Genome Variation, told GenomeWeb Daily News. "The question is whether a lot of those kids could end up with a genetic diagnosis through either whole-exome or whole-genome sequencing."
Based on their findings so far, Goldstein and his co-authors argued that "the application of [next-generation sequencing] should be strongly considered in all cases where a genetic condition is strongly suspected but traditional clinical genetic testing has proven negative."
The investigators did whole-exome sequencing on individuals from a dozen parent-child trios for the study. In each trio, the child had a condition that included features such as developmental delay, minor to major congenital abnormalities, and or facial dysmorphisms that could not be diagnosed using conventional genetic tests.
In the process, the team pinned down likely causal mutations in six of the individuals, including new mutations in genes linked to Mendelian diseases. The search also led to mutations that seemed to explain some but not all of the clinical features observed in a seventh case.
Such results hint that "many, many cases of certain Mendelian disease are being missed in regular clinical practice because many affected individuals don't have standard [disease presentation]," according to Goldstein.
"I think what's quite striking is that when you take these children who have been tested for genes that clinicians feel are the appropriate genes to test for, a lot of them turn out to have mutations in already known genes," he added.
To get a sense of how exome sequencing might perform as a diagnostic tool in a typical clinical setting, the researchers decided to start with a group of individuals who had unexplained conditions and a range of clinical features and phenotypes.
"[W]e did not seek out patients with similar phenotypes," they explained, "but rather enrolled any undiagnosed proband with an apparently genetic condition when predetermined criteria were met."
All of the children had been seen by pediatricians at Duke Children's Hospital, Goldstein explained, and Duke clinical geneticist Vandana Shashi, co-first author on the study, decided which children best fit the study's selection criteria.
Each child remained undiagnosed following various genetic testing in the past and none of the children had chromosomal abnormalities that could be detected on an Affymetrix 6.0 array.
The 12 parent-child trios included children with symptoms such as intellectual disability or developmental delay, minor or major congenital abnormalities, and/or facial dysmorphia.
For parents and children from each trio, the team isolated DNA from blood samples and used the Illumina HiSeq 2000 to sequence coding sequences nabbed with the Agilent SureSelect Human All Exon 50Mb kit, generating sequences that covered the targeted regions to a depth of 71 times, on average.
In interpreting their exome sequence data, the team looked for mutations patterns that fit de novo mutation, X-linked inheritance, or recessive inheritance models, focusing on alterations in genes known or suspected of contributing to Mendelian diseases that might fit with each child's phenotype.
From the hundreds of single nucleotide variants and small insertions and deletions suspected of being de novo mutations based on their initial analysis, the team narrowed in on around a dozen-and-a-half high-confidence SNVs and two high-confidence indels. Among them: mutations in the TCF4, SCN2A, SMAD4, NGLY1, and EFTUD2 genes that appear to explain six of the 12 cases. For another girl with numerous clinical symptoms, the researchers found an EFEMP1 mutation that appears to explain her macular degeneration but not other features of her condition.
Before information was communicated to families during a second genetic counseling session, researchers had variants that appeared to be pathogenic sequenced and confirmed through a CLIA-certified lab.
Parents were informed during the initial genetic counseling session that "a variant of interest that may be detected may not be definitely proven causal," that "no results may be obtained," and that "variants of uncertain significance would not be reported to them," the study's authors explained.
The ability to make diagnoses from exome sequence data is expected to improve as more and more clinical cases are sequenced, the study's authors noted, adding that such approaches should also help in defining the range of clinical phenotypes associated with some genetic conditions.
In addition, the researchers emphasized the potential importance of doing functional analyses, in some cases, to explore the consequences of candidate mutations — information that is also expected to clarify some of the biological processes affected in various conditions.
"Identifying these mutations will help us to understand human biology a whole lot better," Goldstein said. "So this really is one of the key places to apply next-generation sequencing."