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Exome Sequencing Comes to the Clinic

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Vincent Pieterse was so eager to enter the world on December 16, 2002, that he couldn’t wait for the hospital. His startled father, Marc, delivered the boy in the family’s home in a small village in the southern Netherlands. Vincent seemed healthy as a baby, but at school he acquired labels of nonverbal learning disability and mild autism. By age 8, his learning disability, autism, hypotonia, extra teeth, and elastic skin prompted a pediatrician to gently suggest the possibility of a genetic syndrome. But tests for select neurological diseases and chromosomal abnormalities were negative, and no relatives shared symptoms with Vincent.

When the diagnostic odyssey ruled out gene after gene, Marc read about exome sequencing and realized that the approach could provide information on many genes, perhaps identifying a treatable problem. Sequencing would also reveal whether Vincent had a new (de novo) mutation or whether his parents had passed the condition on. Finally, in 2012, on Marc’s persistence, Joris Veltman, PhD, an investigator at Radboud University in Nijmegen who was conducting an exome sequencing study of children with severe intellectual disability (de Ligt J et al. N Engl J Med. 2012;367[20]:1921-1929), agreed to sequence the exomes of the boy and his parents.

Ricki Lewis, PhD

Sequencing Vincent’s exome, the 1% of the genome that encodes protein and accounts for 85% of inherited disease, uncovered a de novo mutation in a gene that encodes a specific ribosomal protein, but no information indicated how the mutation could be pathogenic. “So I looked at the literature, and found a publication on Diamond-Blackfan anemia (DBA),” Marc recalled. Based on the publication (Lipton JF, Ellis SR.Curr Opin Pediatr. 2010;22[1]:12-19), some of Vincent’s symptoms matched the ribosomopathy DBA, but he didn’t have anemia and his mutation had never been associated with DBA.

Marc contacted the lead author of the publication, Jeffrey Lipton, MD, PhD, from Schneider Children’s Hospital in New Hyde Park, NY, and by fall 2014, the researchers found that Vincent’s RNA processing was normal. But Marc, convinced that his son had DBA, emailed a dozen researchers studying the disorder, including Alyson MacInnes, PhD, from the Sanquin Research and Landsteiner Laboratory in Amsterdam, who began coordinating studies of Vincent’s ribosomes in several model systems. MacInnes was intrigued by Vincent’s case history, mutation, and the sequence of events.

“Marc’s email turned a corner into the future of genetic research. Instead of a patient going to the clinic with a list of features, getting a diagnosis, doing the exome sequencing, and then enlisting a researcher to figure out the molecular mechanism, it’s going in reverse. Now the patient, armed with exome data, contacts the researcher, who figures out the mechanism, and then with the clinician is able to determine the diagnosis,” she said.

Robert Marion, MD, pediatrician and chief of the division of genetics at The Children’s Hospital at Montefiore, agreed. “It used to be we’d look at a kid and come up with a differential diagnosis based on the features, and then we’d test. Now we do testing first.”

Two dozen laboratories and companies currently provide whole exome sequencing and analysis. “We’re getting samples from community hospitals, but the majority of exome sequencing is still done through academic tertiary care centers,” said Jacob. A family practitioner might request exome sequencing when single-gene tests, chromosome tests, and copy number variant panels are normal, he added.

Patients or their parents may present “raw,” uninterpreted exome data. For $1095, a customer can buy a buccal swab kit from Gene By Gene (, send in saliva, and 10 weeks later receive an incomprehensible string of DNA bases as results.

“The data are useless,” Marion said, “unless you are a scientist with the ability to understand which genes are important in the clinic and which ones not.” Otherwise, making sense of raw exome sequence is like trying to reconstruct the story of Moby Dick from a pile of word-sized pieces cut from the novel. The challenge lies in determining which gene variants, among thousands, could account for the clinical phenotype.

Detailed documentation of clinical features prior to exome sequencing is key, as it can help to subsequently narrow down possible candidates from the gene variants identified. Helpful tools for matching phenotypes to genotypes include Phenomizer ( and the Human Phenotype Ontology (, which compute differential diagnoses and identify possible candidate genes. Another tool, ClinVar (, detects signatures in a DNA sequence that suggest pathogenicity, such as mutations that disrupt protein conformation or halt protein production.

However, sorting through gene variants is time consuming. “Genetics experts at the laboratories search a huge wealth of information for nuggets. Are variants clinically actionable so you can use them to make decisions in patient care?” said Joy Larsen Haidle, MS, president of the National Society of Genetic Counselors ( who practices at the Humphrey Cancer Center in Minneapolis. A colleague of Marion’s with an undiagnosed arrhythmia is analyzing his own 40 000 gene variants. “It took 6 months for this physician-scientist in his spare time to find half a dozen hits,” Marion said. Particularly vexing are “variants of uncertain significance” that have not yet been associated with disease and can confuse and alarm patients.

Genetic counselors can help when sequencing reveals secondary findings, Larsen Haidle said. The American College of Medical Genetics and Genomics lists disorders deemed reportable to patients because they are actionable ( Patients can choose during the informed consent process whether to receive such findings as well as discoveries of carrier status for recessive diseases. Secondary findings arise in up to 10% of sequenced exomes.

When exome results don’t lead to a diagnosis, full genome sequencing may help, said Soden. This was the case in the recent Science Translational Medicine study by Soden and colleagues wherein exome sequencing failed to produce a diagnosis for 2 sisters presenting with hypoglycemia, hypotonia, finger contractures, and dysmorphic features. Although exome sequencing did not reveal a diagnosis, “we still felt strongly that there was something to find,” Soden recalls. Whole genome sequencing subsequently identified a variant in the gene MAGEL2.

Exome sequencing did not identify this variant in MAGEL2 because the gene is mired in a GC-rich stretch of DNA, a feature that disrupts sequencing and contributes to the 5% of genes that exome sequencing misses. Exome sequencing generally cannot detect copy number variants, repetitive DNA sequences such as trinucleotide repeats associated with Huntington disease, long insertion or deletion variants, aneuploidy, or epigenetic alterations (Biesecker LG, Green RC. N Engl J Med. 2014;370[25]:2418-2425). The overall error rate of exome sequencing is 0.1% to 0.6% but can be even greater for rare or unique variants (Wall JD et al. Genome Res. 2014;24[11]:1734-1739).

Insurance coverage for exome sequencing is on a case-by-case basis, and appeals often are required. “It’s frustrating for the physician because the situation feels out of control. We’re the ones sitting in the room with the family saying ‘We’re sorry, but your insurance won’t cover even standard gene tests, let alone exome sequencing,’” said Soden.

Having genetic expertise on a team helps. “If a clinical geneticist says, ‘I need to do testing’ for a child with a group of abnormalities that don’t fit, insurers may cover that,” Marion said. Exome sequencing can be economical, he added. A test panel for 62 autism genes costs $5500, whereas exome sequencing covers 20 000 genes for about the same price.

When insurance says no, families still can find funding. Clinical geneticists may enroll patients in clinical trials or in programs like the New York Genome Center ( or the National Institutes of Health’s Undiagnosed Diseases Program ( The Rare Genomics Institute (RGI) also matches families with clinical trials. “If all else fails, we help with crowdfunding,” said president Jimmy Lin, MD, PhD, referring to web pages on the RGI site ( set up to raise funds for particular families.

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