Red blood cells carry oxygen throughout the human body, an essential function for survival. Anemia results when someone has fewer red blood cells than normal. The world’s most common blood disorder, anemia comes in many different varieties—mild to severe, acquired or genetic.

Yale pediatrician and geneticist Patrick Gallagher, MD, studies hereditary spherocytosis (HS), an inherited disease associated with hemolytic anemia, when red blood cells are destroyed faster than they are produced due to abnormal membranes. A novel mutation in the gene that encodes alpha-spectrin, a protein essential for normal red blood cell membranes, is responsible for many cases of recessive hereditary spherocytosis (rHS), the most severe form of the disease, reports Gallagher’s team in a paper published in the Journal of Clinical Investigation.

“Red blood cells are unusual compared to many other cells because they travel throughout the circulatory system,” said Gallagher, lead author on the JCI paper. “When red blood cells are rapidly moving in the arteries, their membranes must protect them from shear stress. When they are squeezing through tiny capillaries, the cells deform extensively, so their membranes have to be very flexible.”

Alpha-spectrin provides both strength and flexibility to red blood cell membranes, helping cells maintain their shape and integrity while making their circuit through the body, he explained. Cells without sufficient alpha-spectrin in their membranes suffer membrane damage, losing strength and flexibility.

These damaged, alpha-spectrin-deficient red blood cells are then trapped and destroyed by the spleen. The excessive removal of damaged red blood cells leads to anemia, which in some cases is life-threatening.

“In this study, we studied many people with rHS,” said Gallagher. “It was already known rHS was linked to recessively inherited abnormalities in alpha-spectrin.”

His team started by looking for mutations in the exons of the patients’ alpha-spectrin genes. Exons are the sequences in a gene that provide exact directions for the fabrication of a protein, whereas introns are the sequences of DNA between the exons. Introns are spliced out during the creation of mRNA, so they aren’t part of the mature proteins, which is why geneticists start by looking for exon mutations when trying to figure out where protein manufacture goes awry.

“While many of the rHS patients we studied had exon mutations, in some of them—assuming recessive inheritance—we only found one mutation, not two,” said Gallagher. “We even had a couple of patients with no exon mutations in their alpha-spectrin genes, even though they had rHS with alpha-spectrin-deficient red blood cells.”

A recessively inherited condition is only expressed if a person has mutations on both copies of a gene, he explained, so if some rHS patients had one or no exon mutations, mutations of the exons alone could not explain these rHS cases. But the team had been looking in the wrong place, Gallagher said: The mutation they identified was hidden in the intron of the alpha-spectrin gene.

“So, we did whole genome sequencing,” he continued, “and found a rare variant in an alpha-spectrin gene intron, which had been described only once in the literature. But it was never clear if this rare variant—literally described in a couple of patients before—was just an incidental finding or if it had anything to do with the disease.”

As it turns out, all the patients in this study with one or fewer exon mutations had this rare intron mutation. Through mini-gene splicing assays—where the processing of flawed genes is observed in action—the team demonstrated that the intron mutation was causing an error in splicing, said Gallagher. It found that the intron mutation strengthened an alternate “branch point” in splicing, which resulted in abnormal mRNA, or incorrect instructions for alpha-spectrin assembly. Then, using gene editing to make cell lines with the intronic mutation, the researchers demonstrated that this abnormal mRNA is subject to rapid degradation by the cell’s quality-control machinery, a process known as nonsense mediated decay. Thus, this abnormal mRNA disappears before it can make any alpha-spectrin protein at all. This underproduction of alpha-spectrin results in an increased proportion of alpha-spectrin-deficient red blood cells, which then presents in patients as severe hemolytic anemia.

“HS can be symptomatic in utero,” said Gallagher, “and if left untreated, could lead to fetal death or death in the neonatal period. Some of the patients referred to us have had a sibling who died in utero of the disease with the next child requiring in utero and postnatal blood transfusions.”

Most people with typical HS are cured by splenectomy, the removal of the spleen. “Even though the blood cell membranes are still abnormal for the rest of their life, the spleen is not there to trap those abnormal cells, which can still carry oxygen as they would normally,” he said.

Yet, given the rarity of the condition—about 1 in 2,500–5,000 people have dominantly inherited HS and the recessive form of the disease is even less common—it may be difficult to diagnose. Patients with rHS may therefore not receive appropriate treatment in time, said Gallagher, noting that this is one of the reasons that finding this intron mutation is so exciting.

One of the immediate clinical implications for this finding is that it will update commercially available diagnostic gene panels used to screen DNA for HS mutations, meaning more cases will be correctly diagnosed, he said. These improved gene panels can assist providers in selecting the most effective treatment options, as splenectomy sometimes fails in patients with rHS, who may instead require a hematopoietic stem cell transplant, Gallagher noted. Finally, better gene panels will allow for more accurate genetic counseling for prospective parents with family histories of HS.

Yale is a leader in benign hematology (the study of non-cancerous blood disorders), and Gallagher’s study attracted participants from as far as Hawaii and as near as Guilford, CT. This new work builds on the nearly 50-year history of investigation by Yale researchers into the genetic basis of HS and other blood disorders. Some of the Yale leaders in this area include Drs. Vince and Sally Marchesi, Dr. Sherman Weissman, Dr. Bernard Forget, Dr. Edward Benz, Dr. Howard Pearson, and Dr. Jon Morrow.

This study was supported in part by the National Institutes of Health. An additional donation of equipment to assist this research came from the Arnold J. Alderman family.

Other authors include Yelena Maksimova, Kimberly Lezon-Geyda, Susan J. Baserga, and Vincent P. Schulz of Yale, as well as Peter E. Newburger, Desiree Meideros, Robin D. Hanson, Jennifer Rothman, Sara Israels, Donna A. Wall, Robert F. Sidonio Jr., Colin Sieff, L. Kate Gowans, Nupur Mittal, Roland Rivera-Santiago, and David W. Speicher.