New Study in Nature Genetics uses SPARK database to investigate variants that are less damaging than de novo mutations, but contribute almost as much risk

Researchers have identified a rare class of genetic differences transmitted from parents without autism to their affected children with autism and determined that they are most prominent in “multiplex” families with more than one family member on the spectrum. These findings are reported in Recent ultra-rare inherited variants implicate new autism candidate risk genes, a new study published in Nature Genetics.

The hunt is on in earnest for the genes involved in autism, now that technology and vastly lower costs allow the aggregation of thousands of genomes of people with autism and their family members. Knowing precisely which genes are at play will enable greater understanding of the condition known as autism and may ultimately lead to treatments for those who desire them.

This new study is notable because the majority of autism genes discovered to date have been identified through studies of de novo mutations, genetic differences that first arise in the person with autism but are not present in either of their parents. The findings indicate that researchers should not assume that the set of autism genes altered by de novo mutations are the same genes as these newly identified inherited rare variants.

According to lead author Amy B. Wilfert, Ph.D., of the University of Washington, in an analysis of 10,905 people with autism, researchers identified and replicated a rare class of genetic variants that are passed (over-transmitted) from parents without autism to children with autism.

“While most autism studies focus on de novo mutations, this study focuses on rare inherited mutations, which are often understudied in autism,” says Dr. Wilfert. “We find that these variants are individually less damaging than de novo mutations but have the potential to contribute almost as much risk and impact the same molecular pathways, through a distinct set of genes. These variants, however, are only able to persist in the general population for a few generations before being selected out by evolution.”

“It is widely understood that de novo mutations cannot and do not explain all of the genetic causes of autism, a phenomenon sometimes referred to as ‘missing heritability,’ ” says Pamela Feliciano, Ph.D., Scientific Director, SPARK (Simons Powering Autism Research). The SPARK Consortium contributed more than 50 percent of the genetic data analyzed in this study, including exomes from 21,331 SPARK participants — 6,539 of them individuals with autism spectrum disorder (ASD). The number of genomes accessible to scientists at this time enables the search for certain categories of genetic changes — such as de novo changes and ultra-rare inherited variants — but not all of them. As more genomes come online, larger categories of variants will be accessible for analysis.

“Interestingly, the vast majority of those variants (95%) are not found in genes already known to be autism genes, indicating that there is much more to be learned about autism genetics,” says Dr. Feliciano, noting that this study is the first step in a much larger investigation. “While the current study is not large enough to confidently identify individual genes that have these rare inherited variants, we are learning more about these genes. Future research that focuses on multiplex families is increasingly important to yield novel insights.”

The researchers also said the study confirmed their expectations that this class of rare inherited variants is more prominent in families with multiple members with autism than in families with only one affected individual. Consistent with this finding, children with ASD in these families are more likely to carry two of these variants as compared with their unaffected siblings.

The study also highlighted the need for greater diversity when conducting research of this kind, because investigators were less able to detect rare variants in people who belong to ancestral groups that are underrepresented in genomic research, including people of African, East Asian and South Asian descent.

H3K9 methylation levels in the cerebellum were lower in the Suv39h2-deficient mice than in control mice controls. CREDIT RIKEN

New research from the RIKEN Center for Brain Science (CBS) in Japan shows that a deficit in histone methylation could lead to the development of autism spectrum disorders (ASD). A human variant of the SUV39H2 gene led researchers to examine its absence in mice. Published in Molecular Psychiatry, the study found that when absent, adult mice exhibited cognitive inflexibility similar to what occurs in autism, and embryonic mice showed misregulated expression of genes related to brain development. These findings represent the first direct link between the SUV39H2 gene and ASD.

Genes are turned on and off throughout our development. But genetic variation means that what is turned off in some people remains turned on in others. This is why, for example, some adults can digest dairy products and others are lactose intolerant; the gene for making the enzyme lactase is turned off when some people become adults, but not others. One way that genes can be turned on and off is through a process called histone methylation in which special enzymes transfer methyl groups to histone proteins that are wrapped around DNA.

Variations in genes related to methylation during brain development can lead to serious problems. One such variation occurs in a rare disorder called Kleefstra Syndrome, in which a mutation prevents methylation of H3K9–a specific location on histone H3. Because Kleefstra Syndrome resembles autism in some ways, RIKEN CBS researchers led by Takeo Yoshikawa looked for autism-specific variations in genes that can modify H3K9. Among nine such genes, they found one variant in an H3K9 methyltransferase gene–SUV39H2–that was present in autism, and the mutated SUV39H2 prevented methylation when tested in the lab. Similar loss-of-function results were found for the mouse version of the variant.

The next step was to see what happens in mice that lack the Suv39h2 gene. Behaviorally, the researchers found that the mice could learn a simple cognitive task, but had difficultly when the task required cognitive flexibility. In the simple task, mice learned to get a reward by poking a door at alternating diagonal corners of a cage. After they could do this well, the possible reward locations switched to the other two diagonal corners. The genetically modified mice did this as well as wild-type mice. In another task, after learning to alternate between the two diagonal corners, only the location of one reward was switched. When the mice were challenged to alternate randomly between these two tasks, wild-type mice could adapt quickly, but the Suv39h2-deficient mice took much longer. “This serial reversal-learning task was essential,” says first author Shabeesh Balan. “Cognitive inflexibility is a core symptom of ASD, and our new task was able to address this behavioral feature in ways that previous mouse studies could not.”

When the researchers examined what happened in the mouse brain when H3K9 methylation failed to occur, they found that important genes that are usually silenced in early development were turned on in the experimental mice. “Suv39h2 is known to be expressed in early neurodevelopment and to methylate H3K9,” explains Yoshikawa. “This keeps a check on genes that should be switched-off. But without it, genes in the protocadherin β cluster were abnormally expressed at high levels in embryonic mice.” Because protocadherins are critical for the formation of neural circuits, the researchers believe they have found an important biological pathway that could be central to several neurodevelopmental disorders.

The team then verified the importance of SUV39H2 in human ASD by finding that its expression was lower in the postmortem brains of people with ASD than of controls. “What began with a loss-of-function mutation in only one person with ASD,” says Yoshikawa, “has led to a general causal landscape for ASD that culminates in brain circuit abnormality.”

Protocadherins have already been proposed to be related to a broad range of mental disorders. This study shows that activating the SUV39H2 gene is a potential therapy for mental disorders–including ASD–that should be investigated more thoroughly in future studies.

Researchers at Johns Hopkins Bloomberg School of Public Health have shown in a brain organoid study that exposure to a common pesticide synergizes with a frequent autism-linked gene mutation.

The results represent one of the clearest pieces of evidence yet that genetic and environmental factors may be able to combine to disturb neurodevelopment. Researchers suspect that genetic and environmental factors might contribute to the increased prevalence of autism spectrum disorder, a developmental disorder characterized by cognitive function, social, and communication impairments.

The study’s use of brain organoids also points the way towards quicker, less expensive, and more human-relevant experimentation in this field when compared to traditional animal studies.

The brain organoid model, developed by the Bloomberg School researchers, consists of balls of cells that are differentiated from human stem cell cultures and mimic the developing human brain. The researchers found in the study that chlorpyrifos, a common pesticide alleged to contribute to developmental neurotoxicity and autism risk, dramatically reduces levels of the protein CHD8 in the organoids. CHD8 is a regulator of gene activity important in brain development. Mutations in its gene, which reduce CHD8 activity, are among the strongest of the 100-plus genetic risk factors for autism that have so far been identified.

The study, which appears online July 14 in Environmental Health Perspectives, is the first to show in a human model that an environmental risk factor can amplify the effect of genetic risk factor for autism.

“This is a step forward in showing an interplay between genetics and environment and its potential role for autism spectrum disorder,” says study lead Lena Smirnova, PhD, a research associate in the Department of Environmental Health and Engineering at the Bloomberg School.

Clinically rare as recently as 40 years ago, autism spectrum disorder now occurs in roughly two percent of live births, according to the Centers for Disease Control and Prevention.

“The increase in autism diagnoses in recent decades is hard to explain–there couldn’t have been a population-wide genetic change in such a short time, but we also haven’t been able to find an environmental exposure that sufficiently accounts for it,” says study co-author Thomas Hartung, MD, PhD, professor and Doerenkamp-Zbinden Chair in the Bloomberg School’s Department of Environmental Health and Engineering. Hartung is also director of the Center for Alternatives to Animal Testing at the Bloomberg School. “To me, the best explanation involves a combination of genetic and environment factors,” says Hartung.

How environmental factors and genetic susceptibilities interact to increase risk for autism spectrum disorder remains mostly unknown, in part because these interactions have been difficult to study. Traditional experiments with laboratory animals are expensive and, especially for disorders involving the brain and cognition, of limited relevance to humans.

Advances in stem cell methods in the past decades have allowed researchers to use human skin cells that can be transformed first into stem cells and then into almost any cell type and studied in the lab. In recent years, scientists have expanded beyond simple lab-dish cell cultures to make cultures of three-dimensional organoids that better represent the complexity of human organs.

For their study, the researchers used brain organoids to model the effects of a CHD8 gene disruption combined with exposure to chlorpyrifos. A group led by co-author Herbert Lachman, MD, professor at Albert Einstein College of Medicine, engineered the cells that make up the organoids to lack one of the two normal copies of the CHD8 gene. This modeled a substantial, but less-than-total, weakening of the CHD8 gene’s activity, similar to that seen in people who have CHD8 mutations and autism. The researchers then examined the additional effect of exposure to chlorpyrifos, which is still widely used on agricultural produce in the U.S. and abroad.

“High-dose, short-term experimental exposures do not reflect the real-life situation, but they give us a starting point to identify genetic variants that might make individuals more susceptible to toxicants,” says Smirnova. “Now we can explore how other genes and potentially toxic substances interact.”

The researchers found that the brain organoids with just one copy of the CHD8 gene had only two-thirds the normal level of CHD8 protein in their cells, but that chlorpyrifos exposure drove CHD8 levels much lower, turning a moderate scarcity into a severe one. The exposure demonstrated clearly how an environmental factor can worsen the effect of a genetic one, likely worsening disease progression and symptoms.

As part of their study, the researchers compiled a list of molecules in blood, urine, and brain tissue that prior studies have shown to be different in autism spectrum patients. They found that levels of several of these apparent autism biomarkers were also significantly altered in the organoids by CHD8 deficiency or chlorpyrifos exposure, and moreso by both.

“In this sense, we showed that changes in these organoids reflect changes seen in autism patients,” Smirnova says.

The findings, according to the researchers, pave the way for further studies of gene-environment interactions in disease using human-derived organoids.

“The use of three-dimensional, human-derived, brain-like models like the one in this study is a good way forward for studying the interplay of genetic and environmental factors in autism and other neurodevelopmental disorders,” Hartung says.

A: Illustration of the experiment. Green fluorescent proteins were introduced so that the target gene and the entire neuron could be observed. B: Each target gene was separately introduced into the cerebral cortex and dendritic spine dynamics were tracked for a period of 2 days (on the 21st and 22nd days after birth). Yellow arrows indicate newly formed spines and red arrowheads indicate spine elimination. C: The quantified results of B in graph form. D: Spine morphology classification data on the spines that formed when Ndn was introduced. Filopedia, a type of immature spine formation, significantly increased upon Ndn introduction. CREDIT Takumi et al., Nature Communications, 2021

A research group including Kobe University’s Professor TAKUMI Toru (also a Senior Visiting Scientist at RIKEN Center for Biosystems Dynamics Research) and Assistant Professor TAMADA Kota, both of the Physiology Division in the Graduate School of Medicine, has revealed a causal gene (Necdin, NDN) in autism model mice that have the chromosomal abnormality (*1) called copy number variation (*2).

The researchers hope to illuminate the NDN gene’s molecular mechanism in order to contribute towards the creation of new treatment strategies for developmental disorders including autism.

These research results were published in Nature Communications on July 1, 2021.

Main Points

• The research group identified Ndn as a causal gene of autism by conducting a screening based on synaptic expression in an animal model of the disorder (15q dup mouse).
• The Ndn gene regulates synapse development during the developmental stage.
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Research Background

Even though the number of patients diagnosed with autism (autism spectrum disorder) has been greatly increasing, many aspects of this developmental disorder are still not well understood. Its causes are divided into genetic factors and environmental factors. Within these genetic factors, particular copy number variations have been found in autistic patients; for example, chromosome 15q11-q13 duplication. These abnormalities in the 15q11-q13 region are divided into maternally derived and paternally derived chromosomal duplication cases. It is understood that the Ube3a gene drives maternally derived chromosomal duplication. However, it is not known which gene is vital for paternally derived duplication.

This research group previously succeeded in developing a mouse model of 15q11-q13 duplication (15q dup mouse). Using this mouse model, they identified numerous abnormalities in paternally derived chromosomal duplication cases, including autism-like behaviors, and abnormalities in dendritic spine (*4) formation. However, the researchers were unable to identify which gene is responsible for autism-like behavior because this region contains many non-coding RNA molecules and genes that code proteins.

Research Methodology

In 15q dup mice, there are a great number of genes as the duplication extends to the 6Mb region. Previous research showed that behavioral abnormalities were not induced by maternally derived chromosomal duplication, therefore around 2Mb was excluded. As for the remaining 4Mb, the researchers first created a new 1.5Mb duplication mouse model and investigated behavior abnormalities. From the results, they were unable to identify any autism-like behavioral abnormalities in 1.5Mb duplication mice. Consequently, the researchers excluded this 1.5Mb, leaving them with three protein-encoding genes as possible candidates.

Next, these three genes were individually introduced into the cerebral cortex of mice via in utero electroporation (*5). The researchers measured the spine turnover rate (formation and elimination of dendritic spines over a 2 day period) in vivo using a two-photon microscope (*6) and discovered that the number of spines drastically increased when the Ndn gene was introduced (Figure 1A-C). Furthermore, morphology classification of these spines indicated that the majority were immature. This reveals that the Ndn gene regulates the formation and maturation of dendritic spines during the developmental stage (Figure 1D).

Using CRISPR-Cas9 (*7), the researchers subsequently removed the one copy of the Ndn gene from the 15q dup mouse model to generate mice with a normalized genomic copy number for this gene (15q dupΔNdn mouse). Using this model, they demonstrated that the abnormalities observed in 15q dup mice (abnormal spine turnover rate and decreased inhibitory synaptic input) could be ameliorated (Figure 2).

Lastly, the researchers investigated whether the previously observed autism-like behaviors in 15q dup mice (including increased anxiety in a new environment, reduced sociability and increased perseveration) were evident in 15q dupΔNdn mice. They showed that in the majority of behavioral test results for 15q dupΔNdn mice, abnormal behaviors related to sociability and perseveration were ameliorated (Figure 3).

Further Research

This research study revealed that in 15q dup autism model mice, the NDN gene does not only play an important role in autism-like behaviors, but also affects aspects such as excitation/inhibition imbalance in synaptic dynamics and the cerebral cortex. Next, the research team hopes to clarify the NDN gene’s functions. By artificially regulating these functions or identifying and controlling their downstream factors, the researchers hope to understand the onset mechanism of developmental disorders like autism, and develop new treatment strategies.

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Glossary

1. Chromosomal abnormality: Chromosomes are structures that contain genes and are located inside the cell nucleus. Abnormalities such as duplications or deletions in specific regions of the chromosome are often seen in those with autism.

2. Copy number variation: A chromosomal abnormality where the number of copies of a region on the genome is abnormal (duplications or deletions). Normally there should be 2 copies (diploid). If there are 3 copies or just one copy, then this is a copy number variation.

3. Synapse: A junction between two nerve cells (neurons), which is involved in neuronal activities such as transmitting signals.

4. Dendritic spine: These are protrusions on the dendrites of excitable neurons that receive input from synapses.

5. in utero electroporation: A method of introducing outside DNA into the brain. DNA is injected through the wall of the uterus and is induced to enter the cells using electric waves.

6. Two-photon microscope: A microscope with powerful laser, which enables the brains of mice to be observed while they are still alive. In this study, it was used to track changes in dendritic spines over a 2 day-period.

7. CRISPR-Cas9: Technology that enables a specific region of a genome to be precisely cut and altered. This genome editing method won the 2020 Nobel Prize in Chemistry. In this study, it was used to remove the Ndn gene from the genome.

Coronal section of a mouse brain, with several major axonal tracts stained in green. Image courtesy of Dr. Ahlem Assali. CREDIT Dr. Ahlem Assali, Medical University of South Carolina

After reviewing a database of gene mutations in children with autism , a team of Medical University of South Carolina (MUSC) researchers decided to study a specific gene mutation that likely caused autism in a girl. They demonstrated that the mutation was damaging to the gene, and that female, but not male, mice lacking a working copy of the gene also showed autism -associated symptoms. Better understanding the interplay between genetics and sex in autism could set the stage for developing sex-specific treatments for autism.

The MUSC team was led by Christopher Cowan, Ph.D., the William E. Murray SmartState Endowed Chair in Neuroscience and chair of the Department of Neuroscience, and Ahlem Assali, Ph.D., research assistant professor in the same department. Their findings are published in Nature Neuropsychopharmacology.

One in 54 children is diagnosed with an autism . Of the children with autism, four boys are diagnosed for every girl. Individuals with autism typically have deficits in communication and social interaction and exhibit restricted, repetitive patterns of behavior, activities, or interests. Many people with autism also present with associated symptoms, such as hyperactivity, attention deficits, epilepsy and intellectual abilities that can range from severely disabled to gifted.

Cowan and Assali investigated the effect of a mutation in the gene, EPHB2, detected in a female patient with autism. EPHB2 is important for forming connections, or synapses, in the brain. The patient had a version of EPHB2 that caused the protein to be cut short. “It’s as if a sentence had a period in the middle instead of the end,” said Cowan. The shortened protein can no longer serve its function, leaving this autism individual with less functional protein than neurotypical people.

To confirm that this gene can cause autism, Cowan and Assali created mice that had only one of two working copies of EPHB2. They found that these animals showed repetitive behaviors, hyperactivity and learning and memory problems as well as changes in brain cell function.

Cowan and Assali went a step further and divided the animals based on sex. They did this because the child with ASD and the EPHB2 mutation was a female. They found that the female mice showed much stronger behavior symptoms and brain cell dysfunction than the male mice. Understanding the interplay between genetics and biological sex could be important for understanding autism risk and eventually for developing therapeutics.

“We know that 80% to 90% of autism risk is genetic, but this is a very clear-cut case where the gene and the sex of the animal are interacting to alter neurotypical development,” said Cowan.

Historically, autism has been diagnosed mostly in boys, so research on autism has often been biased toward male subjects. The work of Cowan and Assali highlights the importance of sex-specific differences in autism and the need to examine those differences in research studies that include both sexes. This could set the stage for developing sex-specific treatments for autism .

“That’s the only way we’re going to start to change research inequalities that have happened in the past,” said Assali.

In future studies, Cowan and Assali hope to explore more deeply the mechanisms of EPHB2 actions in the developing brain. They want to understand why this gene causes symptoms predominantly in female subjects and how hormones might affect autism risk. Their aim is to improve the understanding of the interplay between genetics and biological sex in autism, with a view to informing future personalized, sex-specific treatments for autism.