Rapid Genome Sequencing Diagnoses Rare Diseases in NICU

Ricki Lewis, PhD

October 03, 2012

October 3, 2012 — A "clinicopathological correlation tool" developed at Children's Mercy Hospitals and Clinics in Kansas City, Missouri, sequences the genome of an infant in the neonatal intensive care unit (NICU) in just 50 hours, analyzing genes implicated in nearly 600 monogenic disorders matching the patient's symptoms. The report appears in the October 3 issue of Science Translational Medicine.

"Of 4.1 million babies born in the U.S. each year, 1 in 20 are admitted to the NICU, and the cause is likely genetic. The definitive way to diagnose a genetic illness is to sequence the gene affected and identify mutations. But how does a physician pick the right gene to sequence out of the more than 3500 known genetic diseases?" asked Stephen Kingsmore, MB, DSc, director of the Center for Pediatric Genomic Medicine at Mercy, during a news conference.

The new technology offers precision that can clarify the genetic heterogeneity and pleiotropy common in rare monogenic diseases. For example, the researchers write, dystonia due to mutations in the sepiapterin reductase gene responds to drugs that are ineffective against similar movement disorders. Whole genome sequencing (WGS) can also assign genes to atypical cases, such as the Crohn's-like disease in the boy with the mutation in the X-linked inhibitor of apoptosis (XIAP) gene that was the first case of diagnosis via exome sequencing.

Ilumina HiSeq 2500 genome sequencer

The team combined faster WGS using the Illumina HiSeq 2500 system with 2 software programs. The first, symptom- and sign-assisted genome analysis, finds genes relevant to a specific symptom set.

The second software program, Rapid Understanding of Nucleotide Variant Software, characterizes each of the 4 million or so single-base variants in an individual by using existing databases and software that predicts consequences of altering a protein.

For the technique to work, mutations must also be of low frequency in the population because most monogenic disorders are rare. Only 14% of the 4 million variant sites meet this criterion, the researchers write.

Practical Matters

To initiate the process, a physician would send a blood sample to the team and select symptoms from among 227 clinical terms. Up to 11 terms describe each of 591 monogenic diseases. For example, a boy with Tay-Sachs disease presented with hypotonia, developmental regression, and leukodystrophy-like areas on magnetic resonance imaging of the brain. Although 350 candidate genes were associated with 1 of the symptoms, only 9 were associated with all 3.

The cost of WGS plus validation resequencing is about $13,500, Dr. Kingsmore said, which must be balanced against the average $8000-a-day-stay in the NICU and the costs of other diagnostic tests and possibly inappropriate treatment.

For 500 diseases, faster diagnosis can affect morbidity and mortality. A child with untreated severe Pompe (glycogen storage type 2) disease, for example, will die within a year. "But with enzyme replacement therapy, he or she will live at least 4 years and won't need a ventilator," Dr. Kingsmore said. To validate the protocol, the researchers retrospectively diagnosed 533 newborns on the basis of symptoms and found a sensitivity of 99.3% (529 correct diagnoses). The failures arose from incorrect descriptions, such as "recurrent infections" for a boy with cystic fibrosis who actually had "recurrent respiratory infections."

Next, the researchers validated the test protocol on DNA from 2 babies who had had molecular diagnoses: the boy with Tay-Sachs and a child with a Menkes disease variant. "These results encouraged us that the technology was suitable to be used on live babies," said Dr. Kingsmore.

The investigators applied the technology to 5 prospective cases, finding answers for 4: a boy with severe epidermolysis bullosa caused by a mutation in connexin 26; a girl in a consanguineous family with a seizure disorder traced to BRAT1; and 2 brothers with congenital heart disease and heterotaxy, both compound heterozygotes from parents who carried different nonsynonymous variants of the B cell chronic lymphocytic leukemia/lymphoma 9-like (BCL9L) gene.

Only 1 case proved an enigma — for now, pointed out first author and Clinical Laboratory Director Carol Jean Saunders, PhD. "That baby died at day 5 with signs of a suspected mitochondrial disease. There were hundreds of genes on the differential. We were able to cross a lot of things off that list and recognize the limitations in interpreting a genome. Important variants can lie outside the regions that we can analyze," she said. Sequencing is also blind to copy number variants.

Patients have already gained actionable knowledge. Dr. Saunders described a recently sequenced family who had had 2 stillborn births due to nemaline myopathy. They underwent preimplantation genetic diagnosis to select an unaffected embryo.

The researchers suggest that until WGS in the NICU earns Clinical Laboratory Improvement Amendments compliance, results be initially verbally disclosed to the neonatologist and told to parents after resequencing confirms results, which takes at least 4 days.

The team plans to offer the testing more widely next year. Because fewer than 2% of physicians have training in genomics, the researchers point out, genetic counselors will be critical in delivering the technology.

"WGS is a powerful tool for diagnosis of rare genetic disorders. This study demonstrates that rapid turn-around time is possible, which would be necessary for implementation in NICUs. Parents should have thorough counseling prior to performing WGS, so that they can fully understand the scope, benefits, limitations, and risks of this testing in order to make an informed decision and be prepared for possible results and implications," Julie Cohen, ScM, CGC, a genetic counselor at the Kennedy Krieger Institute, Baltimore, Maryland, told Medscape Medical News. Ms. Cohen was not involved in the study.

Nine of the study's authors are employees of Illumina Inc. The other authors and the commentator have disclosed no relevant financial relationships.

Sci Transl Med. Published online October 3, 2012.