Researchers identify FAH mutation without elevated succinylacetone

Nov. 12, 2016

Tyrosinemia type 1 is a rare genetic disorder of tyrosine metabolism that results from mutations in fumarylacetoacetate hydrolase (FAH), the last enzyme in the tyrosine degradation cascade. Improper breakdown of tyrosine causes metabolites such as succinylacetate and fumarylacetoacetate to accumulate in the liver and kidneys, potentially affecting DNA repair and leading to genomic instability in hepatocytes.

Normally, the disease is detected during newborn screening by measuring blood or urine levels of succinylacetone, the pathognomonic marker of the disorder. Treatment consists of the hydroxyphenylpyruvate dioxygenase inhibitor, nitisinone, combined with a phenylalanine- and tyrosine-reduced diet. Infants who don't receive treatment frequently develop liver and renal dysfunction in the first months of life, and the disorder often progresses to cirrhosis and hepatocellular carcinoma and, ultimately, to the need for liver transplantation.

A novel FAH mutation

In 2013, a Kuwaiti family was referred to Mayo Clinic's campus in Rochester, Minnesota. Three of the family's eight children had developed liver disease and jaundice during infancy, and the older daughter, who was lost to follow-up, eventually died of hepatocellular carcinoma. Her younger sister also developed liver cancer, received chemoembolization followed by curative liver transplantation at Mayo Clinic. A male sibling, then 9 years old, presented with a stable liver lesion. Despite classic symptoms and disease progression in these children, extensive testing in Kuwait and at Mayo Clinic found no biochemical evidence of tyrosinemia type 1.

In collaboration with Mayo medical geneticists, several family members, including the mother, an unaffected older brother and the sister who received the liver transplant, underwent whole-exome sequencing. An analysis of the test results by Patrick R. Blackburn, Ph.D., a researcher at Mayo Clinic's campus in Minnesota, revealed a novel mutation in FAH now named FAH R142G. The young transplant patient was homozygous for the mutant enzyme, and her mother and brother were carriers.

Although Sanger sequencing confirmed that FAH R142G was a true mutation, the approximately 40 known variations of FAH all cause elevated levels of succinylacetone. Dr. Blackburn's aim was to determine why the Kuwaiti family had what appeared to be tyrosinemia without the classic biochemical signature that is the gold standard for its detection.

He began by injecting FAH-null mice with either the wild-type or mutant version of the protein. Over a period of 42 days, the mice with the wild-type protein maintained transgene expression whereas those injected with the mutant version did not, suggesting that the mutant protein provided no rescue of the null phenotype.

Dr. Blackburn then hypothesized a difference in binding affinity between FAH substrates in the mutation. Fumarylacetoacetate has a carbon-carbon double bond that constrains its entrance into the binding pocket of FAH and prevents it from being broken down. Succinylacetoacetate, on the other hand, can fit in the catalytic pocket of the protein, allowing it to be processed and leading to undetectable levels in patients with the FAH R142G variant. These findings appeared in the August 2016 issue of Human Mutation.

The significance of FAH R142G

Mounif El-Youssef, M.D., a pediatric gastroenterologist and hepatologist at Mayo Clinic's campus in Rochester, Minnesota, notes how important the discovery of the novel gene has been to the family.

"They now have a diagnosis," he says, "and we are offering genetic testing to the extended family, including an older brother who recently married his cousin. We would like to treat his younger sibling, who we know is homozygous for the gene, but at age 11, it is really too late for nitisinone alone to be effective, so we are using the drug as a bridge to transplant."

The implications of the discovery extend beyond one family, however. Two other consanguineous families have been described with the mutation, suggesting that sequencing is in order for patients with cryptogenic cirrhosis in whom all other known disorders have been ruled out. Further, the FAH somatic mouse model has proved effective for modeling liver disease by linking rescue of FAH deficiency with transgene expression and could potentially be equally useful for modeling other undiagnosed disorders.

"This mouse model was extremely quick; we got an answer within three to four months, which is definitely within the timeline to impact patient care," Dr. Blackburn says.

Dr. El-Youssef adds: "This case was pursued and the mutation discovered because urine and blood tests were negative, and we were persistently curious as to why children in this family developed cancer at such an early age. Now, when we have patients from consanguineous families, we go straight to whole-exome sequencing. In this day and age, it is essential to have a culture of collaboration because knowledge is advancing so quickly, no one person can do it alone. Only when we collaborate can we move the science forward."

For more information

Blackburn PR, et al. Silent tyrosinemia type I without elevated tyrosine or succinylacetone associated with liver cirrhosis and hepatocellular carcinoma. Human Mutation. 2016;37:1098.