September 15, 2015 (Vol. 35, No. 16)

Kate Marusina Ph.D.

Whole Exome Sequencing Is Becoming a Method of Choice for Identifying Mendelian Genetic Disorders

The relatively recent need to sequence entire genomes has driven innovative developments to allow for sequencing technology to be faster, cheaper, and more accurate. 

Many scientists and clinicians are encouraged by the fast pace of innovation in this area, as the first clinical diagnostics applications demonstrate the results not achievable by the traditional sequencing or hybridization techniques.

While the scientific community agrees that many important genetic mutations are found in the noncoding areas, sequencing of the entire exome is a powerful and cost-effective new tool for dissecting the genetic basis of many diseases and traits. Whole exome sequencing (WES) is rapidly achieving the status of an approach of choice for identifying genes that underlie Mendelian disorders, especially when conventional approaches fail.


Freed by Cells, Captured by Libraries

Rubicon Genomics focuses on developing kits for generating sequencing libraries from small amounts of DNA.

“Whole genome and whole exome sequencing from circulating cell-free DNA (cfDNA) from plasma is a new application that is poised to revolutionize clinical diagnostics,” says John P. Jerome, Ph.D. applications scientist. “Only a few nanograms of fragmented and short DNA sequences are recovered from a milliliter of plasma.”

Rubicon’s ThruPLEX® technology was specifically optimized to generate high-quality libraries from clinical samples that are often degraded and difficult to work with. The company invested in optimization of multiple steps required to achieve sensitive library preparation.

The ThruPLEX process begins with repair of damaged DNA fragments, followed by ligation of unique stem-loop adaptors to the ends of the generated double-stranded sequences. Stem-loop structures only anneal to themselves, precluding the rise of the background noise typically seen with other library-generation methods. Next, the loop is cleaved, and the DNA is amplified with primers carrying barcodes specific for that particular sample.

“A combination of the proprietary adaptors with optimized enzyme ratios allows us to prepare the entire library in a single tube, saving precious DNA material,” continues Dr. Jerome. “High-efficiency library preparation is essential for all downstream steps to succeed.” Such efficiency can be achieved with ThruPLEX kits in various settings. The kits, for example, are compatible with each of the commonly used technologies for library enrichment and sequencing.

The ThruPLEX kit was used in a landmark study by the Cambridge University in which genomic evolution of metastatic cancers was tracked by “liquid biopsies.” In this study, WES of circulated tumor DNA was just as effective as repeated tissue sampling in detecting mutations arising in response to chemotherapy. This novel paradigm for noninvasive characterization of genes and pathways conferring chemoresistance may be a turning point in our approach to tumor diagnostics and treatment.

In a multicenter study of sequencing human fetal DNA, ThruPLEX technology was used to generate libraries of fetal DNA derived from maternal plasma. Noninvasive determination of genomic sequences of a fetus will facilitate comprehensive prenatal diagnosis of a variety of genetic disorders.

“Rubicon is launching a new kit with optimized enzyme composition, repair, and ligation steps, specifically to support cfDNA sequencing studies,” adds Dr. Jerome. “Liquid biopsies will change medicine, and we want ThruPLEX Plasma-seq to be the platform of choice for this significant application.”


Rubicon Genomics’ ThruPLEX technology is a three-step workflow that can generate DNA libraries from small amounts of fragmented double-stranded DNA (0.05 ng to 50 ng). Blunt-end ligation occurs with high efficiency, and blocked 5’ ends reduce adaptor-adaptor ligation. Background is reduced by using double-stranded adaptors with no single-stranded tails, and by destroying unused adaptors after ligation.

From Wide Shots to Close-Ups

“If exome sequencing is to reach its full diagnostic potential, a fast and efficient protocol with short turnaround time is essential,” says Michael Brockman, Ph.D., manager of research informatics at NimbleGen (a Roche company). A typical library generation and exome sequencing workflow may take several days, which is not optimal for many diagnostic applications.

The R&D team at NimbleGen is analyzing the entire workflow, and it has recently modified the protocol to decrease the amount of the starting material and the time required for exome capture. NimbleGen was able to decrease the time for capture by hybridization from three days to just overnight.

The company’s capture technology, the SeqCap EZ system, delivers exceptional target enrichment. This system has been shown to aid in discovery of novel variants, including single nucleotide variants, insertions, and deletions. Targeted sequencing enables user-specified enrichment to identify and “capture” only the precise regions of interest that are responsible for disease.

In just one study, one involving nearly 3,000 idiopathic pulmonary fibrosis patients and controls, SeqCap EZ reagents helped to implicate a group of mutations in an exoribonuclease with no prior connection to this disease. The study also established the critical link between lung fibrosis and telomere dysfunction.

In another large-scale study, one in which 2,000 patients were referred for evaluation of suspected genetic conditions, SeqCap EZ reagents were used for WES. This study demonstrated relevant molecular findings for 25% of subjects, establishing WES as a potentially valid approach to clinical diagnosis of suspected genetic conditions.

Dr. Brockman notes that NimbleGen will soon release MedExome, a new kit in the SeqCap EZ family. The kit, called MedExome, has been designed to both capture the whole genome and also provide enhanced sequencing coverage for regions defined as medically relevant. For example, the kit will provide additional coverage for relevant genes as identified by ClinVar and GeneTests databases, and by the set of over 4,600 genes assembled by the academic consortium of the Emory Genetics Lab, Harvard Laboratory of Molecular Medicine, and Children’s Hospital of Philadelphia.

NimbleGen also has the ability to add new regions of importance to customers. “One of our competitive advantages is the ability to quickly customize existing catalog items by request,” asserts Dr. Brockman. MedExome claims 95% coverage at 20× or higher for all target bases, and 98–99% coverage for medically relevant exons.


Low-Cost, High-Throughput Approach

The Greenwood Genetic Center (GGC), a genetic diagnostic center based in Greenwood, SC, is different from other genetic diagnostic centers, says Julie Jones, Ph.D., director of the GGC’s Clinical Genomic Sequencing Program. “We are a private, nonprofit organization that is not affiliated with a university or medical center,” she points out. “In addition to conducting diagnostic tests, our clinical geneticists and genetic counselors provide clinical services around the state.”

The GGC recently established and validated WES for clinical diagnostics. Dr. Jones explains that whole genome sequencing is undoubtedly a preferred methodology for identifying variants and decreasing the percentage of false-positive results. “However, whole genome sequencing is still cost-prohibitive for most diagnostic labs and still has a fairly low throughput,” she declares. “WES proved to be an affordable addition to our established menu of diagnostic tests. Unfortunately, there are still many reimbursement issues that must be addressed.”

WES is especially beneficial for patients with complex phenotypes that may not correspond to a specific disorder, or for patients who have had genetic testing in the past and still lack a clear diagnosis.

The GGC successfully utilized WES to provide a rapid clinical diagnosis for a neonate patient with a constellation of symptoms spanning major critical organ systems. WES was performed on the proband and the child’s parents, and results were returned to the clinician in less than three weeks.
“A typical WES turnaround time is four months, with the data analysis presenting a significant bottleneck,” notes Dr. Jones. Bioinformatic tools are able to pick out the variants, but the clinical meaning of these variants is not always clear.

With the aid of the Bench Lab NGS software from Cartagenia, two GGC directors review each case independently, making sure that nothing is missed. In the neonatal case, the de novo mutation in the PURA gene was picked up only by manual review. The association of this gene with developmental disorders was published only two weeks prior to the analysis, and thus was absent from the linked OMIM and HGMD databases.

The GGC continues to work toward improving its WES capabilities using the NextSeq 500 desktop sequencer (Illumina) and newer enrichment techniques. “Increasing the number of exomes in our own reference set and collaborating with other clinical labs will greatly improve our WES diagnostic ability,” asserts Dr. Jones.


Deep Coverage, High Yield

Innovation and optimization of each step in the exome sequencing process is the core of the business model of Personalis. The company’s ACE Clinical Exome™ test augments off-the-shelf components with proprietary innovations to create an augmented exome and comprehensive testing platform. The platform is continuously optimized with a vision to enhance diagnostic yield for clinical care.

“Several major features of the ACE platform—including high levels of gene finishing, detection of large structural variants, and inclusion of nonexonic content—set it apart from the competition,” says Richard Chen, M.D., Personalis’ CSO. “Our test aims to cover every single base, including hard-to-sequence, high-GC regions, to a minimum depth of 20×. We finish 20–50% more exons than other competitive platforms.” These capabilities allow Personalis to make a significant impact on clinical diagnostics, particularly in cases where other genetic diagnostics methods failed.

ACE exome sequencing was performed on a boy who had been diagnosed, along with his identical twin brother, with infantile-onset retinitis pigmentosa. Traditional panel and exome testing had failed to reveal a causative variant, but Personalis was able to identify a pathogenic variant in the RPGR gene, previously linked with this disease. The variant occurred in an exon which is notoriously difficult to sequence and virtually uncovered by other exome capture and sequencing techniques.

Dr. Chen explains that the depth of sequencing provided by ACE is especially significant in cases of genetic mosaicism. Only a few reads may carry genetic alterations on the background of otherwise normal genotype, and these alterations are often dismissed as errors of sequencing.

In one of the more difficult mosaicism cases, the ACE test was able to identify a variant leading to long QT syndrome, a dangerous heart rhythm disorder. A newborn that was severely affected by irregular heartbeats and who had suffered repeated cardiac arrests provided a blood sample that was sent to Personalis after genetic panels and whole genome sequencing failed to reach consistent conclusions. The variant was confirmed in a fraction of reads of approximately 10%.

Personalis is capitalizing on its unique ability to detect low level mosaic variants by expanding in cancer testing with its ACE cancer exome and cancer panel. “Cancer is an extreme form of mosaicism, with multiple low frequency alleles present in one sample due to tumor heterogeneity,” advises Dr. Chen.


A Bridge to Translational Medicine

The Division of Intramural Research of the National Institute for Allergy and Infectious Diseases (NIAID) combines multiple interdisciplinary teams that make scientific discoveries to develop new vaccines, diagnostics, and therapeutics to improve human health. Close collaboration between clinicians and scientists allows for more comprehensive analysis of immunological abnormalities, and can lead to defining new causes of diseases that have stumped other diagnosticians.

“The NIH Clinical Research Center is the largest research hospital in the world,” says Michael Lenardo, M.D., section chief at the NIAID’s Laboratory of Immunology. “Our team encounters many unusual immune problems. Using genomic technologies, we can identity underlying gene mutations, determine the biochemical pathways of disease, and in some cases develop a new treatment.”

In a recently published Nature Immunology article, Dr. Lenardo’s team described patients with persistent infections and expansions of senescent T cells. WES revealed activating mutations in a subunit of a phosphatidylinositol-3′-OH kinase, an inducer of the downstream kinase mTOR, which drove T cells into senescence. Senescent T cells were unable to respond normally, rendering the affected patients unable to suppress viral infections.

“Once the problem was defined,” explains Dr. Lenardo, “we used an already marketed medicine, Rapamycin, a specific mTOR inhibitor, for treatment. As an immunosuppressant it was not previously considered for these immunosuppressed patients.”

Clinical trials with rapamycin and a new Novartis PI(3) kinase inhibitor are currently underway. “Never before has genetic analysis been able to define the molecular basis of disease with such efficiency,” emphasizes Morgan Similuk, a genetic counselor at the NIAID. “Most importantly, having a definitive genetic diagnosis can be beneficial to families, who have been searching for this type of answer for years. Even if treatment options don’t change, genetic diagnosis can help the family and relatives to understand the cause and prognosis of the disorder.”

WES also uncovered the role of magnesium transporter in the pathogenesis of an immunodeficiency called XMEN, which stands for X-linked magnesium deficiency with Epstein–Barr virus and neoplasia. This disease is associated with highly elevated Epstein–Barr virus levels, which can lead to lymphoma. Genetic loss of the transporter was overcome by supplemental magnesium, and EBV levels decreased substantially.

Dr. Lenardo, who not only runs NIAID’s Lymphocyte Molecular Genetics Unit but also directs the organization’s Clinical Genomics Program, notes that fast and precise DNA sequencing gives us power to help people in very specific ways. It will, he emphasizes, likely lead to revolutionary changes in the way medicine is practiced.  


The National Institute of Allergy and Infectious Disease (NIAID) encourages close collaboration between clinicians and scientists. For example, at the NIAID’s Laboratory of Immunology, researchers used whole-exome sequencing to reveal activating mutations that have a role in T-cell senescence.

























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