Skip to main content
Premium Trial:

Request an Annual Quote

Pair of Exome Sequencing Studies of Similar Syndrome Converge on Variants in One Gene

NEW YORK (GenomeWeb News) – A pair of exome sequencing studies searching for the genetic basis of two similar pediatric syndromes homed in on an overlapping set of variants affecting the same gene. The two studies, one from a team of Israeli researchers and one from a group at the US National Institutes of Health, appeared in the online early edition of the New England Journal of Medicine yesterday evening.

The Israeli team performed exome sequencing on six families of Georgian Jewish or German ancestry with multiple cases of pediatric polyarteritis nodosa, a condition marked by fever and tissue ischemia, including of the skin and cardiovascular and nervous systems. They also conducted targeted sequencing on an additional 17 people.

The NIH team, meanwhile, sequenced the whole exomes of five patients, who appeared to be sporadic cases of a syndrome marked by fever, skin rash, and recurrent stroke, and their families. They, too, performed targeted sequencing on additional patients from the UK and Turkey.

"While no other incidence of this condition occurred in the families who came to our clinic, the early age of onset suggested to us that the patients were born with a genetic condition," said Daniel Kastner, the scientific director of the US National Human Genome Research Institute and a co-senior author of the NIH study, in a statement. "We used a whole-­exome screen to locate genetic variants in each of the patients."

Both groups found that variants in the CECR1 gene, which encodes the adenosine deaminase 2, or ADA2, protein and is involved in the immune system, appeared to be behind the blood vessel inflammation syndromes they were investigating.

While loss-of-function mutations in CECR1 can lead to a suite of symptoms like those exhibited by the patients in these two studies, the NIH researchers suggested that they could also be involved in other conditions like early onset recurrent stroke and vasculitis.

"The role of ADA2 in such serious human disease is important and suggests that ADA2 variants may contribute to other more common illnesses," Kastner added.

To search for the genetic basis of pediatric polyarteritis nodosa, the Israeli team, led by Ephrat Levy-Lahad at the Shaare Zedek Medical Center in Jerusalem, sequenced the exomes of 20 patients from six families — five of Georgian ancestry and one of German ancestry — with polyarteritis nodosa to uncover variants that were potentially damaging to protein function. They also sequenced patient family members and a set of controls. The condition, the researchers noted, occurs with some frequency among Israeli Jewish people of Georgian Caucasus ancestry.

The patients exhibited a range of symptoms. For instance, 18 of the 19 Georgian Jewish patients had cutaneous manifestations of the disease, typically livedo reticularis or mottled skin, 10 of the 19 had visceral involvement, including gastrointestinal symptoms and renal hypertension, and about half had neurological disease.

An examination of their exomes indicated that all the Georgian Jewish patients were homozygous for a Gly47Arg mutation in CECR1; their unaffected family members were either heterozygous for the mutation or lacked it entirely. The researchers noted that the mutation was not present in an internal database of 864 exome sequences or in the more than 7,500 exome sequences included in public databases.

The affected members of the German family, by comparison, were compound heterozygotes for the CECR1 variants Arg169Gln and Pro251Leu — the parents were heterozygous for one allele and an unaffected sibling lacked either variant.

Levy-Lahad and her colleagues also performed targeted sequencing on a further 14 unrelated Turkish patients and three of Georgian Jewish descent, and uncovered additional CECR1 variants — including another set of heterozygous damaging variants encoding Gly47Val and Trp264Ser — linked to polyarteritis nodosa.

Similarly, NHGRI's Kastner and his colleagues sequenced the exomes of their three initial cases as well as the patients' healthy parents and siblings.

These patients, too, had varied phenotypes, though all had recurrent fevers, mottled skin, and strokes beginning before age 5. All were of European ancestry, though not from founder populations.

Trio exome sequencing of the first two patients and their parents uncovered compound heterozygous missense mutations affecting CECR1, including a shared Tyr453Cys variant. Exome sequencing of the third patient and parents revealed a paternally inherited Arg169Gln variant and a maternally inherited CECR1 deletion.

Two additional patients from the NIH were examined through candidate gene sequencing, from which the researchers uncovered the three mutations they already identified plus a new His112Gln variant.

Kastner and his colleagues also performed candidate gene sequencing on four more patients, one from the UK and three from Turkey, with related, though not fully overlapping, phenotypes. These patients, the researchers reported, were homozygous for the Gly47Arg mutation — one of the mutations noted by the Israeli group.

The CECR1 gene encodes ADA2, a 511 amino acid protein that is secreted into the plasma. It is an adenosine deaminase and an adenosine deaminase–related growth factor. In people, ADA1 and ADA2 catalyze the degradation of adenosine to inosine and deoxyadenosine to deoxyinosine. Recessive ADA1 mutations, Levy-Lahad and her colleagues said, are one cause of severe combined immune deficiency.

"The identification of mutations altering ADA2 as one cause of polyarteritis nodosa is an initial step in the gene-based definition of disease and may contribute to molecular classification of the vasculitides," they said.

Levy-Lahad and her colleagues noted that the variants they observed in their patients occur in highly conserved regions, and, in particular, the Gly47Arg and Gly47Val likely disrupt the stability of the protein homodimers or their subunits while the Pro251Leu and Trp264His variants likely affect the enzyme active site.

Similarly, Kastner and his colleagues said that the missense mutations they uncovered are likely loss-of-function mutations that affect protein stability and dimerization.

Serum samples from the Israeli patients showed that patients homozygous for the Gly47Arg mutation had ADA2 activity that was reduced by more than four fold, as compared to controls. Heterozygous carriers of the mutation, though, had ADA2 activity similar to controls. Kastner and his colleagues, too, found lower plasma levels and activity of ADA2, though not of ADA1, in patients.

In mammalian cells, Levy-Lahad and her colleagues found that when mutant and wild type ADA2 were expressed exogenously, the constructs were present at similar levels, but in transfected cells, the amount of mutant ADA2 secreted into the media was much lower than non-mutant, suggesting that there could be impaired secretion of the proteins, unstable proteins, or both.

By expressing recombinant mutant and non-mutant proteins in Drosophila cells and purifying them from the media, the researchers found that the proteins that yield enough sample to be analyzed were unstable and partially unfolded.

Kastner and his colleagues also examined immune system function in their ADA2 deficient cohort, finding that they largely had normal immune responses. However, they also saw that, in culture, there was a higher rate of spontaneous B-cell death in the samples from patients as compared to controls.

By modeling the disease in zebrafish, the NIH researchers found that zebrafish with the mutant ADA2 have increased cranial bleeding — as did some NIH patients — and that ADA2 is involved in blood vessel development. However, they noted that ADA2 is not expressed in endothelial cells, suggesting the involvement of other cell types in vasculopathy.

"Data from zebrafish and patients suggest that ADA2 deficiency may compromise endothelial integrity while polarizing macrophage and monocyte subsets toward proinflammatory cells, establishing a vicious circle of vasculopathy and inflammation," they added.

They also suggested, based on the range of symptoms their patients experienced, that ADA2 could be involved in other disorders. For instance, they speculated that it could be involved in lacunar stroke, vasculitis, and possibly Sneddon's syndrome.

The groups' findings offer some insight into how the syndromes may be treated.

The Israeli researchers suggest that anti-TNF agents could be used, especially in severe cases. Both teams said that ADA2-replacement therapy through, for instance, fresh-frozen plasma or recombinant ADA2 should be considered. The NIH researchers also said that bone marrow transplants may help such patients.

The NIH team also named their syndrome, dubbing it 'deficiency of ADA2.'