CLINICAL GENOME CONFERENCE continued
information is at hand. Some information may not be useful now, but she is confident it will be more useful as our experience grows.
She also reported that WGS has many advantages over whole exome sequencing (WES) in a clinical setting. WGS provides better coverage (~96%). This leads to discovery and explanation of rare diseases. One example involved a splice mutation that would have been invisible by WES. Plus, WGS is one protocol that standardizes laboratory workflow and avoids interruptions for updates, which was common with WES pro- tocols. Turnaround time is about the same. Costs of WGS and WES are coming down rapidly. A group in Australia is now offering clinical-grade WGS for $1500 (including interpretation).
Prof. Jennifer Erwin of the Salk Institute (La Jolla, CA) presented a supporting lecture on mosaicism of single neural cells. Mosaicism is caused by rearrangement (transpositions) or later in development intracellular somatic insertion of DNA changes the sequence in cells. The rearrangement or insertion leads to diversity in propagating cells. Dr. Erwin used deep sequencing of single cells to study the location and structure of transposons. A study of five individuals showed the number of insertions varied from 10 to 40. Somatic insertions are rare, usually with one copy number. However, healthy individuals have a large number of somatic mutations, most of which are silent. It seems that transposons need supportive flanking sequences for activation. Since responsible variants can be positional or sequence, whole ge- nome sequencing is essential.
Copy number variants of DNA segments are frequent in humans. In the past, it was suspected they were strongly associated with various dis- eases including some cancers, but other correlations with diseases have not been confirmed. Dr. Alex Kaplun of BIOBASE GmbH (Wolfenbüttel, Germany) lectured on detecting copy number variants (CNVs) with WGS. CNVs have been implicated in about 14,000 diseased patients. Compared to various probes, CNV with WGS is more reliable. Small CNVs stand out. Translocations and inversions are not ambiguous as they are with exome sequencing. BIOBASE curates data on CNV. Dr. Kaplun reported that about 50 software tools are available for CNV analysis.
Indeed, there is a general consensus that WGS is the best technical choice for clinical genomics. Cost is the only issue, and the differential is declining rapidly.
Benchmarking the WGS laboratory Dr. Worthey provided some benchmarks for laboratory performance in whole genome sequencing. The lab started with exome sequencing, but in 2009, began shifting to WGS. Today, the lab “sequences, analyzes, and interprets a whole genome in under a week.” Typically, for a sample that arrives on Monday, the report is out on Thursday. The report included IF. The key is the HiSeq2500 (Illumina, San Diego, CA), which reduced sequencing, including verification, from 245 hr to 20. Interpretation is now complete in 3.5 hr, down from 7.5.
Reporting actionable incidental findings Prof. Julianne O’Daniel described the policy of treating IF at UNC Lineberger (University of North Carolina at Chapel Hill). First, the lab uses a list of types of IF that may be reported to the physician or patient. Then, the age of onset is compared to the age at actionability. Once eli- gibility based on age is established, actionability is evaluated. The goal is to define the options for patients and parents. “Medically actionable” is defined as a disease that 1) exhibits a strong genotype–phenotype causative relationship, 2) has a serious risk of morbidity, 3) has existing guidelines for prevention and treatment, and 4) demonstrates success in improving patient outcome. Using the actionable criteria, one list has only 120 reportable variants.
Dr. Worthey reported that, when using WGS, the chance of IF is nearly 100%.2
But how many are actionable? Dr. Gail Jarvik (University of
Washington, Seattle) pointed out that only 3.4% of European descen- dants had actionable pathogenic variants. The percentage for African Americans dropped to 1.2%. Both are small numbers, but this also prob- ably reflects the state-of-the-art in oncology.
Data and diagnosis quality As you might expect, data quality was a major concern, since genomics relies upon many external databases, generated by even more laboratories.
Dr. Jarvik addressed the conflicted clinical utility of genomics. Classification of tumors based on genomics was inconsistent, especially between cells of severity, particularly “pathogenic” and “likely patho- genic.” One study showed that of 128 variants, 52% were misclassified. Upon rereading, 63% changed classification, usually to a lower patho- genic class.
As part of the session on references and standards, Valerie Schneider of the National Cancer Institute (Rockville, MD) described the most recent build of the human reference genome, which was issued on 12/24/2013 by the Genome Reference Consortium (GRC) as GRCh38. This adds about 2% to the prior reference genome (GRCh37). In addition to cleaning up various items spread over nearly all chromosomes, Build 38 focuses on centromeres, which are difficult to sequence since they are rich with alpha satellite repeats. These account for about 2.6% of the human ge- nome. The repeats are organized into higher-order repeats of unknown significance, but they are conserved, and hence probably important.
Databases contribute to uncertainty Some caveats according to Dr. Worthey: Some of the “ultrarare” disorders are actually common. The choice of reference data is critical. Databases often fail to update changes in annotations. Dr. Worthey focused on RYR2 p.G1885E, which was first published in 2002 and identified as a polymorphism. It is now recognized as common, with a prevalence of about 5%. Dr. Worthey noted that recuration of databases is a huge, never-ending task.
AMERICAN LABORATORY • 32 • SEPTEMBER 2014
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