A defective gene makes the brain too big or too small

A gene called ZNRF3, known to be involved in cancer, also disrupts the brain. The human brain relies on two copies of this gene to build a properly sized brain. If one copy is defective, the brain will be either too small or too large (known as the mirror effect), leading to a variety of neurological symptoms.

Nearly a decade ago, we saw a patient with a very rare condition, an abnormally small brain, speech delay, and ectodermal dysplasia—a congenital disorder that affects the hair, nails, teeth, and skin. We sequenced some of her DNA and discovered a defect in one copy of the ZNRF3 gene, a gene that had not previously been associated with congenital disorders. This defect results in the production of a harmful protein, so we suspected that was the cause.

Since then, we have collected DNA from 11 other patients from around the world who are thought to carry a harmful mutation in the same gene. Most of them had a defective copy of the ZNRF3 gene and had variable neurodevelopmental signs with abnormally large brains.

We tested the defective versions of the gene in the laboratory and found a correlation between the size of the patients’ brains and the location of the mutations in the gene. After a long diagnostic odyssey, we were finally able to establish a definitive cause of these patients’ disease. Our study is published in the journal American Journal of Human Genetics.

Global collaboration enables rare disease research

As the disease described here is extremely rare, we used a global collaboration through professional network databases where we published our candidate gene and received matches from all over the world. We were able to collect a total of 11 additional patients with suspicious alterations in the same gene. Eight of them had a defective copy of ZNRF3, while four patients had lost one copy.

Of the eight patients with defective copies, seven had varying neurodevelopmental problems with abnormally large brains, while one had profound developmental delay with an abnormally small brain.

The four patients with only one functional copy had no neurological symptoms, but dysfunctions in other organs, such as the heart, adrenal glands or kidneys. We did not observe any patients who had lost both copies, suggesting that the absence of this gene is incompatible with life.

The ZNRF3 gene is often mutated in several cancers

The ZNRF3 gene produces two copies of a protein that prevents the brain from producing too many or too few brain cells. It does the same in many other organs, so mutations in its DNA sequence can lead to uncontrolled cell proliferation and are therefore associated with various tumors, such as colon or adrenal cancer.

One of our analyses revealed that there is a small region of the ZNRF3 gene, called RING, where many of the mutations found in cancers are found compared to the rest of the gene. In fact, most patients with abnormally large brains have their mutations in the RING region. This means that they may have an increased risk of developing tumors later in life.

Two regions of the ZNRF3 gene are essential for brain size

Our analyses showed that almost all of the mutations that lead to abnormal development are located in two distinct regions of the gene: one in the RING region, and the other in a smaller region that is important for interacting with another gene called RSPO. It turned out that almost all of the defects in the RING region came from patients with abnormally large brains, while the defect in the region interacting with RSPO came from the patient with the abnormally small brain.

However, one patient had an abnormality in the RING region, but his brain was abnormally small. We traced his family history and found that his mother had taken a lot of drugs during pregnancy, which could explain his abnormally small, rather than large, brain. Apparently, environmental influences can cancel out the genetic defects in this disease.

Lab experiments and modeling explain molecular defects

The ZNRF3 gene orchestrates the perfect balance of biochemical signals, particularly in the Wnt signaling pathway, needed to produce the right number of brain cells. This gene works in concert with the RSPO gene, which also interacts with Wnt signaling.

In the lab, we created different defective versions of the ZNRF3 gene and measured the signal that represents changes in the Wnt signaling pathway. We found that defects in the RING region (in patients with abnormally large brains) increased Wnt signaling, while mutations in the RSPO-interacting region (in patients with abnormally small brains) decreased Wnt signaling.

These results showed that right brain size depends on balanced Wnt signaling, which, when biased toward too much or too little, can cause the brain to become too big or too small.

Sophisticated modeling of defective versions of the ZNRF3 protein also revealed disrupted enzymatic functions for RING region defects, or impaired binding to the RSPO interacting protein for RSPO interacting region defects.

Better monitoring and treatment of patients at risk of cancer

With FDA-approved Wnt pathway modulators available, these findings open the possibility of using Wnt modulators for therapeutic purposes. However, this intervention should be approached with caution, as a Wnt inhibitor should only be considered for patients with abnormally large brains and not for those with abnormally small brains, even if they have defective copies of the same gene.

ZNRF3 joins a list of dozens of other genes involved in the Wnt signaling pathway that have been linked to brain size. However, it is so far the only one of these genes that leads to opposite brain sizes with a region-specific pattern, known as the mirror effect.

Since the Wnt signaling pathway is linked to cancer when disrupted, monitoring and intervention could be planned and personalized for patients with defective ZNRF3 gene.