Advances in technology are rapidly changing the field of medical genetics in both the research laboratory and the clinic. With the use of next-generation, or massively parallel, DNA sequencing, it is possible to determine the sequence of essentially all genes in an individual's genome — referred to as the exome — within a matter of days.
This technology became widely available in 2005, and the first proof-of-principle experiment showing the power of exome sequencing for the discovery of genes associated with disease was published a few years later.
1 Since then, exome analysis has been used in the research setting to identify the genetic cause of dozens of disorders, including intellectual disability.
2-4
A couple of years ago, Veltman and colleagues used exome sequencing to test the hypothesis that sporadic intellectual disability is caused by de novo mutations (genetic changes that are present in affected persons but not in their parents).
2 Now de Ligt, Veltman, and colleagues
5 report in the
Journal results obtained with a similar approach in a diagnostic setting to identify genetic causes of severe intellectual disability in 100 patients.
These patients all had severe intellectual disability with an IQ of less than 50, and each had previously undergone extensive testing, which included the use of chromosome microarrays to detect chromosomal deletions and duplications. For each patient, DNA from both parents was available for analysis. By determining which variants in the patient's exome were absent in the exomes of both parents, the investigators were able to identify de novo sequence variants in the patient. Not every de novo variant is pathogenic, however, and the investigators had to make a determination about each variant in order to issue a clinical report.
For this study, de novo variants in a gene already known to cause intellectual disability were considered pathogenic, provided that the phenotype of the patient was consistent with the phenotypes previously described and that bioinformatics programs predicted the variant to be damaging (i.e., likely to compromise protein function). For de novo variants in other genes, the task was trickier. Several factors were considered, including what is known about the gene's expression and function, whether the change was predicted to be damaging by bioinformatics prediction tools, and whether the change occurred at a spot in the genome that is evolutionarily conserved. The ultimate test, however, was to identify another de novo variant that was predicted to be damaging in the same gene in a similarly affected patient. The authors sequenced five of the candidate genes in a second series of 765 patients with intellectual disability and also examined the previously obtained exomes of 10 patients with severe intellectual disability. They had access to DNA samples from the parents of patients in the replication set so that they could determine whether observed variants in the affected patients were de novo. They observed mutations in three of the five candidate genes that they sequenced.
The authors thus identified the genetic cause of intellectual disability in 16% of patients, with the majority of cases caused by a mutation in a gene that was already known to cause intellectual disability. A diagnostic yield of 16% in patients with intellectual disability is encouraging and similar to the yield that can be obtained by chromosome microarray to detect deletions and duplications (15 to 20%).
6 Another 19 genes carried a de novo variant that might be pathogenic, but a conclusion about the clinical significance of the genetic variant could not be made. The patients in the replication set have yet to be screened for mutations in these 19 genes, suggesting that the diagnostic yield may increase once these genes have been analyzed in this series of patients.
Given the remarkable genetic heterogeneity of intellectual disability (since mutations in a large number of different genes can cause this disorder), exome sequencing is a good alternative to sequential gene sequencing. But is the technique ready for the diagnostic laboratory? The fact that 16% of cases were solved suggests that it is indeed a reasonable test to consider for patients with severe intellectual disability.
On the other hand, the movement of exome sequencing into clinical diagnostic laboratories has been remarkably quick, so there is bound to be a steep learning curve related to implementation of the technology, interpretation of the data, and reporting of the output in a manner that is understandable to both physicians and families of patients. This is also the case for data obtained by chromosome microarrays, in which many variants of uncertain significance are reported and sometimes interpreted differently by different laboratories.
7
It is also important to note that the validation of three variants required sequencing of the gene in an independent set of 765 patients with intellectual disability — a considerable effort. Most diagnostic laboratories will have neither the samples from patients nor the resources to follow up every putative mutation affecting a gene in this fashion, so there must be continued collaboration between researchers and clinicians. A systematic approach to sharing data on sequence variants among laboratories will facilitate the validation process, because large numbers of patients are required to determine whether a variant of undetermined significance is in fact causal. Finally, it will be important to have a method for different researchers to revisit the test results periodically in order to reevaluate variants of uncertain significance and to reannotate those that have been validated. Such a resource would provide a means of concluding the diagnostic odyssey for even more patients and their families.
These are exciting times in medical genetics, and exome sequencing is revolutionizing the field. The study by de Ligt et al. shows how the technology can be used successfully in a diagnostic setting for patients with severe intellectual disability. Genomewide testing for deletions and duplications typically provides a diagnosis in 15 to 20% of cases. Additional studies of the 19 genes in which de novo mutations were identified but were of unclear significance may explain another 10 to 15%. Therefore, with the resources available today, a genetic diagnosis can be made in a substantial portion of affected patients.
Genomic technologies are evolving rapidly. Routine whole-genome sequencing is on the horizon, and with advances in our understanding of regulatory DNA,
8 it is conceivable that noncoding mutations will soon be identified in another subset of patients. Improvements in the detection of deletions and duplications from sequence data
9 will eventually eliminate the need for chromosome microarrays as a separate test. In this era of genomic medicine, we should proceed with both optimism and careful thought as we guide our patients through the diagnostic process ushered in by these new, powerful, and yet imperfect tools.
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