Genetic diagnosis solves medical mystery for 30 undiagnosed patients

An international team of researchers has provided a genetic diagnosis for 30 individuals whose condition was undiagnosed for years despite extensive clinical or genetic testing. The study, conducted by researchers at Baylor College of Medicine, National University of Singapore and collaborating institutions worldwide, appeared in Genetics in Medicine, the official journal of the American College of Medical Genetics and Genomics.

The story of our findings began with one patient I saw in the clinic presenting an uncommon combination of problems. The patient had severe developmental conditions, epilepsy and complete insensitivity to pain, which was very atypical. The condition had remained undiagnosed despite numerous tests conducted by geneticists and neurologists."

Dr. Daniel Calame, first and co-corresponding author, instructor of pediatric neurology and developmental neurosciences at Baylor

Calame enrolled the patient in the BCM GREGoR (Genomics Research to Elucidate the Genetics of Rare Diseases) research program. "We reanalyzed the patient's genetic and clinical data and that brought us to a gene, FLVCR1, and a medical mystery to solve," Calame said.

One gene, multiple conditions

To try to understand how the rare FLVCR1 mutation in the patient could lead to the condition, Calame and his colleagues researched scientific literature on this gene. Current evidence indicates that the FLVCR1 protein plays a key role in the production of red blood cells and in the transport of choline and ethanolamine in cells. Choline and ethanolamine are important for cells. They are precursors for phosphatidylcholine and for phosphatidylethanolamine, respectively, which are required for cell membrane integrity needed to support cell division and other essential cellular functions.

Other researchers have studied the Flvcr1 gene, the equivalent of the human gene, on animal models. They found that knocking out the gene in mice was lethal at the embryonic stage. "The embryos have many bone malformations in the head and limbs and defective production of red blood cells, which is reminiscent of Diamond-Blackfan anemia (DBA) in people," Calame said. "But this was different from what we saw in our patient."

Patients with DBA also have bone malformations. Interestingly, although the evidence in mice pointed at FLVCR1 as the gene causing DBA, the gene was not identified as playing a role in DBA patients at the time. Other genes were discovered that caused the condition.

In addition, other studies found rare defective copies of the FLVCR1 gene in patients with childhood or adult-onset ataxia, a condition characterized by poor muscle control and incoordination, who also had sensory problems and retinitis pigmentosa, or progressive vision loss. These problems also were not like those Calame was seeing in his patient.

"We were intrigued. On one hand, we had a patient with a rare FLVCR1 mutation and severe developmental conditions, epilepsy and complete insensitivity to pain, but on the other hand there were patients with rare mutations on the same gene that presented with a different set of problems," Calame said. "Could it be that those different mutations of FLVCR1 caused not one set but a spectrum of characteristics we observed in all the patients combined?"

Solving the mystery of FLVCR1 brings answers to patients

The team searched for an answer to this mystery by combining two strategies. One strategy was to increase the number of patients they could study by identifying individuals with undiagnosed neurodevelopmental disorders and FLVCR1 gene variants in large, specialized datasets. They identified the patients either through the Baylor-Hopkins Center for Mendelian Genomics/BCM GREGoR database, the Baylor Genetics clinical diagnostic laboratory database, GeneMatcher or other research and clinical diagnostic laboratories.

"We identified 30 patients from 23 unrelated families with rare FLVCR1 variants," Calame said.

The researchers found 22 gene variants, 20 of which had not been described before. The patients' characteristics range from severe developmental disorders with profound developmental delay, microcephaly (a head much smaller than expected), brain malformations, epilepsy and premature death. Severely affected patients share traits, including anemia and bone malformations, with mice lacking the Flvcr1 gene and DBA, which had not been linked to FLVCR1 before.

The second strategy to find an answer to this medical mystery was to characterize the functional consequences of the FLVCR1 variants in laboratory experiments in collaboration with Dr. Long Nam Nguyen and colleagues at the Yoon Long Lin School of Medicine, National University of Singapore. The team wanted to better understand the effect the different variants they found in patients would have on choline and ethanolamine transport in cells in the lab. They found that FLVCR1 variants significantly reduce choline and ethanolamine transport – up to half of the transport seen with normal FLVCR1 proteins. "We propose that disease severity depends on the residual transport activity of the FLVCR1 variants a patient carries," Calame said.

Other studies have shown that choline is required for normal neurodevelopment and that its deficiency also causes anemia, liver disease, growth retardation and immune deficiency. "Neurodevelopment is also disrupted by defective choline uptake, and we showed that the variants in our patients do reduce choline transport," Calame said.

Altogether, the findings demonstrate that FLVCR1 variants cause a broad spectrum of developmental problems ranging from severe multiorgan developmental disorders resembling DBA to adult-onset neurodegeneration. The variants identified in patients reduce choline and ethanolamine transport in cells in the lab, suggesting that transport of these molecules into the central and peripheral nervous systems is essential to prevent neurodegeneration and required for normal neurodevelopment.

"Our findings also support further studying the potential therapeutic value of choline or ethanolamine supplementation in FLVCR1-related diseases," Calame said. "The 30 patients we identified had not had a diagnosis for years; it was rewarding to be able to provide an explanation for their condition."

This study also underscores the importance of approaching the diagnosis of rare conditions with a wide perspective. "The 30 severely affected individuals reported here had all undergone clinical or research exome or genome sequencing, which identified the reported FLVCR1 variants, yet in each case the variants were previously felt either non-contributory or of uncertain significance given the apparent mismatch of characteristics among patients," Calame said. "Such false assumptions illustrate the importance of incorporating model organism data into personalized genome analysis for rare diseases and the need to anticipate more severe and milder patient characteristics associated with each disease gene to maximize the yield of diagnostic genetic testing."