New genomic study tracks rare blood disease in children

Aplastic anemia is a rare, life-threatening blood disorder where patients are unable to make enough blood cells due to the immune system's attack on blood stem cells. The condition can progress to myelodysplastic syndrome (MDS) and leukemia. A study led by scientists at St. Jude Children's Research Hospital, with multiple collaborating institutions, used state-of-the-art genomic techniques to profile 619 children and adults with aplastic anemia.

They found that different blood stem cells within the same person independently acquire gene mutations that allow cells to escape the immune attack. In some patients, these "rescuing" stem cell clones were enough to restore blood production and provide long-term remission. The work, which includes the largest pediatric cohort of its kind reported to date, was published today in Nature Genetics.

In aplastic anemia, immune cells called autoreactive T cells target and destroy blood stem cells that display peptides on a specific protein on their surface. These are encoded by the human leukocyte antigen (HLA) gene. Each person inherits one copy of this gene from each parent, which can have different variations.

People with aplastic anemia often carry a particular "risk" HLA allele (gene variant) that is thought to trigger the disease. Some blood stem cells evade the immune attack by acquiring changes that silence the risk HLA allele. This can happen via loss-of-function HLA mutations, or through uniparental isodisomy 6p (UPD6p), where the risk allele is replaced with a non-risk allele.

Two other types of escape in blood stem cells are known: paroxysmal nocturnal hemoglobinuria (PNH) or mutations in clonal hematopoiesis (CHIP) genes. However, it was unclear if all these changes arise in a single stem cell or arise independently to help the blood stem cells hide from the immune system. It was also unclear how this process of immune evasion impacted clinical outcomes and cancer risk.

We found that each patient with aplastic anemia that escapes autoimmunity has multiple, independent genetic events in different blood stem cells that allow those cells to escape autoimmunity. Stem cells silence the risk HLA allele through several mechanisms, and our data show that these events are protective, benign events that don't cause progression to MDS or leukemia, even when the rescued clones grow and dominate the bone marrow."

Marcin Wlodarski, MD, PhD, Study Corresponding Author and Associate Member, Department of Hematology, St. Jude Children's Research Hospital

Assessing the risk of blood stem cell 'clones'

Blood stem cells give rise to all other blood cells, meaning their progeny are genetically identical, including any mutations gained over time. The relative abundance of a specific stem cell's genetic "clones" measures the genetic diversity of these blood-making cells. Using single-cell analyses, the researchers showed that protective mutations happen independently in different blood stem cells and not sequentially within a single cell. These independent clones then repopulate the marrow without being found and killed by the immune system.

"We saw that patients with blood stem cell clones that escape autoimmunity can improve their blood counts," Wlodarski said. "We also learned that these clones do not indicate an increased risk for leukemia. On the contrary, they often indicate the possibility of long-lasting remission."

To assess these clones, the scientists analyzed bone marrow and blood samples from 619 patients with aplastic anemia (256 children and 363 adults). Overall, 69% of patients carried at least one acquired change: HLA mutations or UPD6p clones were found in 16%, PNH clones in 44%, and CHIP mutations in 21%.

First author Masanori Yoshida, MD, PhD, St. Jude Department of Hematology, then established and applied a single-cell DNA sequencing assay to simultaneously profile mutations and cell-surface proteins of 304,902 single cells from 48 samples. The study was complemented by long-read whole-genome sequencing and single-cell whole-genome sequencing. 

The experiments showed that acquired mutations are just as common in children as in adults, but in pediatric patients, 65% of the CHIP mutations occurred in just three genes (BCOR, BCORL1 and ASXL1), compared with 27% in adults. Because age-related CHIP mutations are not expected to preexist in children, these mutations seem to be immune-escape events acquired in response to the autoimmune attack.

HLA alleles are lost multiple times in aplastic anemia, beginning early in life

To understand how these protective events arise and to count them precisely, the authors performed whole-genome sequencing on many single blood stem cells. They expected to see one to three events per individual; instead, they found a median of three per patient, and in one patient 15 independent clones, all resulting in the loss of the risk HLA allele, showing convergent evolution to escape a strong immune attack.

That extreme diversity pointed to an unusual, convergent evolutionary process, so the scientists reconstructed a phylogenetic "family tree" of individual blood stem cells by reading all mutations acquired throughout life in single whole genomes. This method enabled them to pinpoint each clone's origin.

"We had expected that these mutations occur right before disease onset," Wlodarski said. "But we found some of these HLA-loss clones arose many years before clinical diagnosis."

The team also showed that long-lived, rescued clones had higher expression of a surface marker for blood stem and progenitor cells: CD34. This suggests that CD34 enrichment could serve as a biomarker of long-lasting recovery. In addition, clones with loss of HLArisk alleles and CHIP mutations almost never co-occurred in the same cells, indicating that HLA loss provides enough of a proliferative advantage on its own that additional CHIP mutations, which can predispose to MDS, are not selected.

So, they appear to act as protective events against their MDS and leukemia evolution. These results challenge prior assumptions about when and how protective clones arise in aplastic anemia, and their presence can be a factor in restoring blood formation.

"Aplastic anemia shows us convergent evolution in miniature: Multiple independent mutational events arise in different cells, all leading to the same escape from autoimmunity," Wlodarski said. "It shows the amazing nature of human hematopoiesis to cure itself from bad actors, like the autoimmune T cells, and reconstitute the bone marrow."