When the development of the gonads or genitalia follows an alternative course, the result is sometimes called a disorder of sex development (DSD) or sex reversal. In either case, the anatomical or gonadal sex determination is poorly understood, even though a few gene variants have been identified that appear to lead to DSDs. Such variants are cited in just 39% of DSD diagnoses. An explanation for many other DSD diagnoses, scientists have surmised, may involve alterations in regulatory segments of DNA celled enhancers.

Following up on this possibility, researchers based at the University of Melbourne explored the regulatory landscape upstream of a crucial gene called SOX9. By analyzing new patient data with bioinformatic tools such as luciferase tiling, the researchers identified three enhancers for SOX9 that could lead to a baby being born with a DSD.

“We discovered three enhancers that, together ensure the SOX9 gene is turned on to a high level in an XY embryo, leading to normal testis and male development,” said Andrew Sinclair, a professor of pediatrics at the University of Melbourne. “Importantly, we identified XX patients who would normally have ovaries and be female but carried extra copies of these enhancers (high levels of SOX9) and instead developed testes. In addition, we found XY patients who had lost these SOX9 enhancers (low levels of SOX9) and developed ovaries instead of testes.”

Dr. Sinclair is the senior author of an article that appeared December 14 in the journal Nature Communications. The article, titled “Human sex reversal is caused by duplication or deletion of core enhancers upstream of SOX9,” suggests that unexplained cases of DSD might be better understood if their causes were sought in the 90% of DNA that is made up of so-called junk DNA or genomic dark matter, which contains no genes but does carry important regulators that increase or decrease gene activity.

Before the current study, variants in two key genes, SRY and its target SOX9, had been established as a cause of 46,XY DSD, but the genetic basis of many DSDs had remained unclear. Now, the molecular network underlying DSDs may be a little clearer.

“Here, we identified four DSD patients with overlapping duplications or deletions upstream of SOX9,” wrote the authors of the current study.” Bioinformatic analysis identified three putative enhancers for SOX9 that responded to different combinations of testis-specific regulators. All three enhancers showed synergistic activity and together drive SOX9 in the testes.

“This is the first study to identify SOX9 enhancers that, when duplicated or deleted, result in 46,XX or 46,XY sex reversal, respectively. These enhancers provide a hitherto missing link by which SRY activates SOX9 in humans, and establish SOX9 enhancer mutations as a significant cause of DSD.”

“This study is significant because in the past, researchers have only looked at genes to diagnose these patients, but we have shown you need to look outside the genes to the enhancers,” said the study’s first author, Brittany Croft, a Ph.D. student at the Hudson Institute and a researcher at Melbourne’s Murdoch Children’s Research Institute.

Dr. Sinclair added that across the human genome there were about one million enhancers controlling about 22,000 genes. “These enhancers lie on the DNA but outside genes, in regions previously referred to as junk DNA or dark matter,” he said. “The key to diagnosing many disorders may be found in these enhancers which hide in the poorly understood dark matter of our DNA.”