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Exome Sequencing Study Narrows in on New Cushing's Syndrome Gene Culprit

NEW YORK (GenomeWeb News) – In a study published online this week in the New England Journal of Medicine, an international team described new mutations involved in a severe form of Cushing's syndrome caused by adrenal adenoma tumors or overgrowths rather than excess pituitary gland activity.

Through exome sequencing on samples from 10 individuals with a form of Cushing's syndrome attributed to cortisol hormone-producing adrenal adenomas, the team narrowed in on recurrent activating mutations in the gene PRKACA, which codes for a protein kinase A (PKA) enzyme subunit.

The group's targeted gene sequencing or copy number profiling experiments indicated that PRKACA alterations are present in more than one-third of Cushing's syndrome cases involving cortisol release from non-cancerous adrenal gland tumors or from thicker-than-usual adrenal tissues known as hyperplasias. On the other hand, such glitches weren't detected in individuals with high cortisol levels who did not show Cushing's-like clinical symptoms or in those with other types of adrenal tumors.

"The mutation appears to spur the activity of this enzyme," co-first author Constantine Stratakis, director of the Eunice Kennedy Shriver National Institute of Child Health and Human Development's intramural research division and head of the institute's developmental endocrinology and genetics program, said in a statement. "The result appears to be an increase in cell growth and division in adrenal tissue, and an overproduction of cortisol."

Stratakis said the findings point to "a clear path forward for investigating medications that might block the production of excess cortisol."

Cushing's syndrome results from exaggerated cortisol hormone production by the body, which can lead to symptoms ranging from high blood pressure and metabolic problems to osteoporosis, fragile skin, and muscle weakness. In some instances, the excess cortisol is due to unusually high production by the pituitary gland, for example.

But severe Cushing's cases can also occur due to corticotropin-independent cortisol release — namely, cortisol secreted by adrenal overgrowths or benign adenoma tumors.

The researchers focused on that corticotropin-independent type of Cushing's syndrome for their current study, starting with tumor samples from 10 individuals with Cushing's syndrome and adenomas affecting one of their adrenal glands.

The team used Agilent SureSelect kits to capture protein-coding sequences and subsequently sequenced the exomes using Illumina's HiSeq 2000.

From this sequence data, the researchers found that samples from eight of the 10 individuals contained mutations affecting the PRKACA gene, which codes for a catalytic subunit of the PKA enzyme.

Next, the team did targeted PRKACA sequencing in samples from another 89 individuals with cortisol-producing adenomas and dozens more individuals with adenomas that were inactive or that produced the hormone aldosterone. That search unearthed another 14 instances of PRKACA mutation, all in individuals diagnosed with Cushing's syndrome.

In the unilateral adenoma cases — those affecting just one adrenal gland — the study's authors noted that the mutated form of PRKACA appeared to have consistently arisen somatically in the affected tissue, often neighboring tissues that were mutation-free.

But the team also found examples of PRKACA mutations present across both affected and unaffected tissues in the same individual. Array-based comparative genomic hybridization experiments on almost three-dozen individuals with hyperplasia affecting both adrenal glands revealed germline PRKACA duplications in five individuals.

Through follow-up experiments in vitro, the study's authors found evidence that the sort of mutations detected in individuals with somatic or germline mutations all seemed to dial up the activity of the resulting kinase enzyme, leading to surplus cell growth and inappropriately plentiful cortisol production.

Likewise, they noted that "[a]ll of the patients with PRKACA defects, whether germline or somatic, had overt Cushing's syndrome, and none of the patients with sub-clinical Cushing's syndrome or other adrenal tumors had genetic PRKACA alterations."

In a letter to the editor in NEJM, some of the same researchers involved in the Cushing's syndrome study described mutations in another PKA subunit gene called PRKACB in individuals with a rare condition called Carney Complex, another disease marked by the presence of tumors and a jump in levels of another hormone, cortisone.

"It’s likely that the extra copy of this gene also increases the activity of protein kinase A, essentially setting the stage for increased cell proliferation and higher production of hormones," said Stratakis, who also co-authored the Carney Complex letter.