Genetic background shapes outcomes in chromosome 16 deletion disorders

Individuals that share the same deletion of a portion of chromosome 16 are at risk of developing neurodevelopmental disorders, but some experience severe intellectual disability or developmental delay, while others may only exhibit milder psychiatric features like depression or anxiety. How can this be?

To answer this, a team led by Penn State scientists has developed methods to evaluate how genetic variants elsewhere in an individual's genome work with the deletion to help determine the features that the individual will manifest. Rather than the conventional focus on single causal variants for neurodevelopmental disorders, the researchers said this study shifts emphasis to the role of interactions among a patient's entire genetic architecture and could inform individualized precision medical interventions for complex disorders.

The paper describing the research is available online in the journal Nature Communications.

We know that many traits have complex genetic underpinnings, meaning that the way they appear is influenced by the interactions among many genes. We've been working for more than a decade with a deletion on chromosome 16, referred to as 16p12.1, that deletes eight genes and is associated with varying neurodevelopmental outcomes, including autism, developmental delay and congenital abnormalities. We've studied the deletion in a fruit fly model and at the population level in humans, but we wanted to see if we could drill down to understand how clinical features of the deletion vary from family to family."

Santhosh Girirajan, T. Ming Chu Professor of Genomics and head of the Department of Biochemistry and Molecular Biology in the Penn State Eberly College of Science, leader of the research team

Unlike many other genetic disorders, which are often caused by a new mutation in an individual, in most cases, patients with the 16p12.1 deletion inherited it from a parent that carried the mutation but may not have been diagnosed with any clinical features, the researchers explained.

"A child that inherits the deletion from one of its parents gets half of its genome from the other parent," Girirajan said. "This new combination of genetic information in the child might expose the deletion to a different set of genetic variants - from the parent without the deletion - that it can interact with to cause different features. It also allows us to compare family members to help identify the secondary genetic variants that might influence the features that appear and their severity."

To try to tease out why individuals from different families differ, the team used two strategies. The first strategy used induced pluripotent stem cells (iPSC) derived from patients with the 16p12.1 deletion, members of their families and healthy donors. The iPSCs were derived from blood samples donated by the patients and their families, and controls from healthy donors were supplied by the National Institute of Neurological Disorders and Stroke.

"Induced pluripotent stem cells (iPSCs), generated by reprogramming blood or skin cells, can be differentiated into many cell types" said Jiawan Sun, graduate student in molecular, cellular and integrative biosciences at Penn State and co-first author of the paper. "By adding certain small molecules, we can make them differentiate into different cell types in the neuronal lineage and then compare gene expression in cells from different families and cells with or without the deletion."

The team converted the iPSCs into neuronal precursors, called neural progenitor cells, as well as immature neurons and mature neurons. They also used CRISPR gene-editing technology to induce the deletion into iPSCs from a healthy donor. Some of these cell lines with the deletion developed abnormal cell proliferation, cell death and premature differentiation that differed among individuals. These abnormalities align with several clinical features of individuals that carry the 16p12.1 deletion, including variation in head size, which have also been reported in studies of autism and schizophrenia, the researchers said.

By completely sequencing the genomes of each cell line used in the study, the researchers identified rare mutations in the genetic background of each individual. They also quantified the expression levels of all genes in each cell type. From this data, the team found that the genetic background of the iPSC lines contributed to specific changes in gene expression among the individual cell lines. They also found differences among the cell lines in accessibility of genome regions that did not code for a gene, but might help control the expression of other genes.

"There have been studies that compare cell lines with a particular mutation to otherwise identical cells without the mutation and studies that look across a group of individuals looking for common genetic features shared by the group," Girirajan said. "What makes our study unique is the ability to identify variation in the genetic background that are family specific."

The team also saw interactions with the genetic background in the cells from the healthy donor with the CRISPR-induced 16p12.1 deletion. This indicates that even otherwise heathy individuals may carry genetic variants that could contribute to neurodevelopmental disorders in a context that includes the deletion and could impact risk for their children, the researchers explained.

The second strategy used by the team to tease out differences in clinical features associated with the 16p12.1 deletion was to use CRISPR gene editing to one-by-one restore the function of genes in the deletion. They found that each restored gene impacted the expression of an independent set of genes that differed between families and cell types in the study.

"We used to think the variation we see among individuals was caused by what we called the 'two-hit' model," said Serena Noss, graduate student in molecular, cellular and integrative biosciences at Penn State and co-first author of the paper. "The deletion was the first hit, and it could interact with a second-hit mutation elsewhere in the genome, but we've moved away from that language because we realize the deletion is interacting with multiple variants across an individual's genome. It's really more of a multi-hit model."

Understanding this multi-hit model, and how an individual's personal genetic architecture contributes to complex neurodevelopmental disorders, can help in the development of potentially individualized therapies for these disorders, the researchers said.

In addition to Girirajan, Sun and Noss, the research team at Penn State included Corrine Smolen and Deepro Banerjee, graduate students in bioinformatics and genomics; Venkata Hemanjani Bhavana, graduate student in biochemistry and molecular biology; Maitreya Das, graduate student in molecular, cellular and integrative biosciences; Belinda Giardine, computer programmer; and Anisha Prabhu, undergraduate student in biochemistry and molecular biology. The team also included David J. Amor, Kate Pope and Paul J. Lockhart at the University of Melbourne in Australia.

The U.S. National Institutes of Health funded the research.