Profile view of man with DNA

Two studies published in Nature Neuroscience implicate mosaic mutations arising during embryonic development as a cause of autism spectrum disorder (ASD). The investigations, led by researchers at Boston Children’s Hospital, Brigham and Women’s Hospital (BWH), and Harvard Medical School (HMS), point to new areas for exploring the genetics of ASD and could eventually inform diagnostic testing.

The two reported studies were part of the Brain Somatic Mosaicism Network, funded by the National Institute of Mental Health. One study (“The landscape of somatic mutation in cerebral cortex of autistic and neurotypical individuals revealed by ultra-deep whole-genome sequencing”) exploited deep, ultra-high-resolution whole-genome sequencing to quantify and characterize mosaic mutations in the frontal cortex of people with and without ASD. The research was led by Rachel Rodin, MD, PhD, and Christopher Walsh, MD, PhD, of Boston Children’s, together with Yanmei Dou, PhD, and Peter Park, PhD, of HMS. The other study (“Large mosaic copy number variations confer autism risk”) published in the same issue of Nature Neuroscience, represents the first large-scale investigation of copy number variants (CNVs) in people with ASD that occur in a mosaic pattern. This study was led Maxwell Sherman, MS of BWH, Po-Ru Loh, PhD of BWH, together with Park, and Walsh.

ASD is a complex and heterogeneous neurodevelopmental disease characterized by impairments in communication and social interactions, and repetitive behaviors, Rodin et al wrote. The “genetic architecture” of ASD is also complex, Sherman et al continued, with common variants, rare variants, and germline de novo variants contributing to the risk.

In contrast with germline de novo mutations, mosaic mutations affect only a portion of a person’s cells. “Unlike de novo variants, which occur in parental germ cells and are, thus, present in all cells of the body, mosaic mutations arise after fertilization— sometimes during embryonic development —and are present in only a fraction of cells,” Sherman and colleagues explained. So, rather than being inherited, mosaic mutations arise as a “mistake” that is introduced when a stem cell divides. A mutation in a stem cell will only be passed to the cells that descend from it, producing the mosaic pattern. When mosaic mutations occur during embryonic development, they can appear in the brain and affect the function of neurons. The earlier in development a mutation happens, the more cells will carry it.

The Rodin-led team carried out whole–genome sequencing of donated prefrontcal cortex brain tissue from rom 59 deceased people with ASD, and 15 controls, representing the largest cohort of brain samples ever studied. Their results showed that most of the brains had mosaic single nucleotide, or point mutations. They further calculated that embryos acquire several such mutations with each cell division, and estimated that about half of us carry potentially harmful mosaic mutations in at least 2% of our brain cells. “Our analysis reveals that the first cell division after fertilization produces ~3.4 mutations, followed by 2–3 mutations in subsequent generations. This suggests that a typical individual possesses ~80 somatic single-nucleotide variants present in ≥2% of cells,” they wrote.

In the brains of people with ASD, however, the team found that mosaic mutations were more likely to affect parts of the genome that have a pivotal role in brain function. Specifically, they tended to land in enhancers, which are sections DNA that do not code for genes but regulate whether a gene is turned on or off.

“In the brains of people with autism, mutations accumulate at the same rate as normal, but they are more likely to fall into an enhancer region,” said Rodin, who is first author on the paper. “We think this is because gene enhancers and promoters tend to be in DNA that’s unwound and more exposed, which probably makes them more susceptible to mutations during cell division.”

The authors said the observation of mosaic mutations in brain-specific enhancer regions was “intriguing,” as they represent a mechanism for disrupting gene expression in brain-limited or region-specific ways, in both normal and diseased brains, but without disrupting expression in other tissues. “Hence, mosaic noncoding mutations represent an attractive candidate mechanism to be involved more broadly in ASD and other neuropsychiatric diseases as well,” they wrote.

“Mutations in enhancers are a hidden kind of mutation that you don’t see in typical diagnostic exome sequencing, and it may help explain ASD in some people,” noted Walsh, chief of genetics and genomics at Boston Children’s and co-senior author on the paper with Park, who led the study’s computational analyses. “We also need to better understand the effects of these mutations on neurons.”

The second study reported by Sherman, Park, Walsh, and colleagues is the first large-scale investigation of copy number variants (CNVs) that occur in a mosaic pattern, in people with ASD. As opposed to point mutations in a single gene, CNVs are deletions or duplications of whole segments of a chromosome, which may contain multiple genes.

For their study the team analyzed blood samples from about 12,000 people with autism and 5,500 unaffected siblings provided by the Simons Simplex Collection and the Simons Powering Autism Research for Knowledge (SPARK) datasets. They used blood as a proxy for brain tissue and applied novel computational techniques to sensitively detect mosaic mutations that likely arose during embryonic development.

“People have been interested in CNVs in autism for a long time, and would occasionally notice that some of them were mosaic, but no one had really looked at them in a large-scale study,” said Loh, who is co-senior author on the paper with Walsh and Park. From these large samples, the team identified a total of 46 mosaic CNVs (mCNVs) in the autism group and 19 in siblings. The CNVs affected 2.8% to 73.8% of blood cells sampled from each subject.

Notably, the people with ASD were especially likely to have very large CNVs, with some involving 25% or more of a chromosome. The CNVs spanned a median of 7.8 million bases in the ASD group, versus 0.59 million bases in controls. “Here we demonstrate that large mCNVs contribute a modest but important component to ASD risk,” the authors wrote. “Whereas very large (>4-Mb) germline CNVs are rare in both affected and unaffected individuals, very large mCNVs accounted for a substantial proportion of mosaic chromosomal aberrations that we observed.”

First author, Sherman, a PhD student at MIT, commented, “This is one of the more interesting and surprising aspects of our study. The kids with ASD had very large CNVs that often hit dozens of genes, and likely included genes important for development. If the CNVs were in all their cells, rather than in a mosaic pattern, they would likely be lethal.”

The study also suggested that the larger the CNVs, the greater the severity of autism as assessed with a standard clinical measure. “Large mCNVs significantly increased ASD risk, and increasing mCNV size correlated with increasing ASD severity in affected individuals,” the team wrote. Another surprise was that smaller CNVs already known to be associated with ASD when found in all cells, such as deletions or duplications of 16p11.2 or 22q11.2, were not associated with autism when they occurred in a mosaic pattern.

“This suggests that in order to get autism, you have to mess up a large number of cells in the brain in a pretty substantial way,” said Walsh. “We’re fairly sure that these large CNVs change the behavior of the neurons that carry them.”

“We don’t really know what cell fraction is important, or what particular chromosomes are most susceptible,” Loh further noted. “These events are still very rare, even in people with autism. As larger cohorts are assembled, we hope to get some finer-grained insights.” The authors concluded in their paper, “As efforts to directly assay the genome of the brain expand, we expect the risk contribution and molecular mechanisms of mCNVs to be further refined for both ASD and other neurodevelopmental disorders.”

The findings of both studies could point to the development of diagnostic testing approaches in children with autism. Testing might incorporate the noncoding portions of the genome, such as gene enhancers and promoters, and include higher resolution chromosomal analysis to identify large mosaic CNVs. For now, the findings add to the ever-evolving autism puzzle, deepening the mystery of why so many different genetic mechanisms can lead to the same presentation of autism.

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