Not every cell in the body is the same genetically, and disease-causing mutations don't necessarily affect every cell—making these mutations easy to miss even with next-generation genomic sequencing. A study from Boston Children's Hospital used a "deep sequencing" technique and was able to identify subtle somatic mutations—those affecting just a percentage of cells—in patients with brain disorders. The approach, described in the August 21st issue of The New England Journal of Medicine, opens up new possibilities for finding genetic causes for previously mysterious neurologic and psychiatric conditions.
"There are two kinds of somatic mutations that get missed," explains Christopher Walsh, MD, PhD, chief of Genetics and Genomics at Boston Children's Hospital and principal investigator on Boston Children's portion of the Undiagnosed Disease Network. "One is mutations that are limited to specific tissues: If we do a blood test, but the mutation is only in the brain, we won't find it. Other somatic mutations may be in all tissues, but occur in only a fraction of the cells—a mosaic pattern. These could be detectable through a blood test in the clinic but aren't common enough to be easily detectable."
Walsh and postdoctoral fellow Saumya Jamuar, MD, now a clinical geneticist at the KK Women's and Children's Hospital in Singapore, used a technique called "targeted high-coverage sequencing" to search for mutations in 158 patients with brain malformations of unknown genetic cause, who had symptoms such as seizures, intellectual disability and speech and language impairments.
Rather than analyzing the whole genome or exome (protein-coding regions of genes), the investigators focused on a panel of known or suspected genes, but drilled deeper than the traditional genomic sequencing technique. Whole genome or exome sequencing typically breaks the DNA into little fragments, each of which is read multiple times—typically 30—to find the disease-causing mutation. But 30 reads aren't enough to reliably catch mutations that only occur in 15 to 20 percent of our cells—especially given that mutations may affect just one of our two copies of a gene. So Walsh, Jamuar and colleagues scaled up the number of reads, sequencing each candidate gene not 30 times but 200 times or more. This enabled them to find mutations in 27 of the 158 patients (17 percent). Of these, eight mutations (30 percent) occurred in only a proportion of the blood cells (so-called mosaic mutations). Five of the eight were missed by traditional Sanger genomic sequencing. One of the eight had also undergone prior whole-exome sequencing that missed the diagnosis.
"Our findings suggest that serious neuropsychiatric disorders can result from mutations that are detectable in as few as 10 percent of patients' blood cells," says Walsh. "By limiting ourselves to selected genes, but covering them more deeply, we may be able to find more answers for families and better understand these disorders."
Walsh, who is part of Boston Children's Brain Development and Genetics Clinic, which sees patients with brain malformations, thinks the results may possibly help explain other brain-based disorders such as autism, intellectual disability and epilepsy that don't have an inherited cause but occur as a result of de novo mutations—those occurring spontaneously and sometimes, as in these cases, after conception.
"Traditionally, our genes are considered to be the same across all cells of our body, and disease-causing mutations are either inherited from one or both parents or occur in the parent's sperm or egg before conception," Jamuar explains. "Our study creates a paradigm shift, providing evidence that a significant proportion of mutations causing brain disorders occur after conception and would be missed by routine testing. Finding the mutation ends the patient's diagnostic odyssey and allows us to provide more accurate genetic counseling to the family."
"The range of possibilities caused by somatic mutations is vast," says Timothy Yu, MD, PhD, a co-author on the study and a researcher in the Division of Genetics and Genomics at Boston Children's. "The challenge remains in developing technologies to study these mutations in other conditions."
The NEJM study was funded by the National Institute of Neurological Disorders and Stroke (R01NS079277, R01NS035129, K23NS069784), the National Institute of Mental Health (1RC2MH089952), the University of Washington Centers for Mendelian Genomics (HG006493), the Manton Center for Orphan Disease Research, the National Institute of General Medical Sciences (T32GM07753), the Allen Foundation and the Howard Hughes Medical Institute.
Boston Children's Hospital is home to the world's largest research enterprise based at a pediatric medical center, where its discoveries have benefited both children and adults since 1869. More than 1,100 scientists, including seven members of the National Academy of Sciences, 14 members of the Institute of Medicine and 14 members of the Howard Hughes Medical Institute comprise Boston Children's research community. Founded as a 20-bed hospital for children, Boston Children's today is a 395-bed comprehensive center for pediatric and adolescent health care. Boston Children's is also the primary pediatric teaching affiliate of Harvard Medical School.
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