Unveiling a novel hemoglobinopathy classification

Eye on Innovation

As human life commences, one essential constituent makes its debut: hemoglobin, a fundamental protein. Taking up residence within red blood cells from the very moment of birth, this pivotal molecule embarks on a lifelong journey to transport oxygen and expel metabolic waste. At the core of its importance, hemoglobin drives energy production and maintains cellular functionality throughout the body. When the structure or quantity of this vital protein is altered due to genetic mutations or other factors, various health consequences or effects can occur. These include but are not limited to anemias, organ damage, and complications in pregnancy.

In a recent discovery by Mayo Clinic Laboratories, a novel hemoglobinopathy category was identified and termed epsilon gamma thalassemia. The first instance of the disorder was found in 2017 when an obstetric patient underwent a routine screening for blood-related illnesses such as sickle cell disease and thalassemia. After the patient’s sample arrived at Mayo Clinic’s Metabolic Hematology Laboratory, directed by Jennifer Herrick, M.D., and Aruna Rangan, M.B.B.S., protein studies including hemoglobin electrophoresis were performed. In addition, the sample was shared with the Division of Laboratory Genetics and Genomics (LGG), led by Benjamin Kipp, Ph.D. There, the LGG team and Linda Hasadsri, M.D., Ph.D., performed the multiplex ligation-dependent probe amplification (MLPA) test, a molecular genetic technique used to detect and analyze DNA copy number variations (CNVs) in a person’s genome. Upon the test’s completion, the team detected an abnormality they had never seen before.

“One of the things that we look to as a sign or a surrogate marker for a beta thalassemia is whether or not the minor adult hemoglobin (Hb A2) is elevated,” said Dr. Herrick. “The patient’s Hb A2 percentage was elevated, but the rest of her clinical picture didn't quite fit beta thalassemia.”

A closer look at hemoglobin

Hemoglobin consists of four protein chains, each called a globin chain, which are bound together. Different hemoglobin types are found in certain medical conditions and at various stages of human development. While conducting the MLPA test, the lab meticulously examined the sample for which portions of the beta globin gene cluster were altered and correlated with three specific parameters: Hemoglobin A (Hb A), Hemoglobin A2 (Hb A2), and Hemoglobin F (Hb F).

In adults, the most common type of hemoglobin is Hemoglobin A, which consists of two alpha globin chains and two beta globin chains. The second most prevalent type of hemoglobin found in adults is the minor adult form, Hemoglobin A2. This type is present in small amounts in the bloodstream and is composed of two alpha globin chains and two delta globin chains. Hb A2 levels are usually higher in individuals with certain hemoglobinopathies, such as the beta thalassemia trait.

In developing fetuses and newborns, the most common type of hemoglobin is fetal hemoglobin, also known as Hemoglobin F. In contrast to Hb A and Hb A2, Hb F consists of two alpha globin chains and two gamma globin chains. The presence of gamma globin chains in Hb F gives it a higher affinity for oxygen, which allows efficient oxygen extraction from the mother's bloodstream to the developing fetus during pregnancy. As individuals grow and develop after birth, the production of gamma globin decreases, and the production of beta globin increases, leading to the transition from Hb F to Hb A.

“She did not have the microcytosis we would expect, so we performed DNA Sanger sequencing to see whether there was a beta thalassemia, and it was negative. We could not explain the Hb A2 by the first genetic [Sanger] test,” Dr. Herrick said. “The second genetic test [MLPA] looks for deletions which are big, and you can't find them by [Sanger] sequencing. Through this test, we found a novel deletion that we had never seen before.”

This discovery raised concerns about the potential risk of the fetus developing severe neonatal anemia; however, doctors were not entirely certain. To further assess these findings, the sample was then passed to Eric Wieben, Ph.D., of the Biochemistry and Molecular Biology department and the Advanced Diagnostics Laboratory for long-read sequencing. Long-read sequencing is an emerging technology that allows better characterization of genetic mutations. In contrast to MLPA, this test looks more precisely at the ends of mutations and can detect major deletions.

“Long-read sequencing can identify all types of variants: single nucleotide variants (SNVs), small indels, and large structural variants, and may eventually replace the currently used diagnostic methods,” said Dr. Rangan. “This case was novel because we saw a large deletion only in the epsilon and the gamma genes, which hasn't been described before in literature.”

Answering a complex clinical question

In 2021, Mayo Clinic encountered a specimen from a patient (unrelated to the earlier patient) who had given birth to identical twins whose newborn screen tests, performed at the Minnesota Department of Health, had come back with a confusing result. Their Hb A levels were greater than Hb F. Although these infants didn’t show signs of severe neonatal anemia as feared, their distinctive profile leaned more toward that of an adult than an infant, a remarkably uncommon outcome.

The three patients were seen at a follow-up appointment by Mira Kohorst, M.D., a pediatric hematologist/oncologist at Mayo Clinic’s Rochester campus, who sent the samples to the Metabolic Hematology Laboratory for evaluation. The samples were once again shared with the LGG lab for MLPA analysis, and the doctors were amazed to come across mutations like the findings discovered years earlier. Subsequently, the samples were shared with Dr. Wieben’s lab, where a gap-PCR test confirmed that the large deletion was identical to the previous case. All three samples showed elevated levels of Hb A2 but no microcytosis or significant anemia, and the Hb F had disappeared in the infants as expected.

“We were lucky because we got to see how this mutation behaves in the mother and in the babies. What was really interesting and unexpected was that the babies were fine, and this is reassuring for anxious parents to know,” Dr. Herrick said. “Whenever we get a sample from a baby and the adult hemoglobin is higher than the fetal hemoglobin, we assume that they've either been transfused with adult blood right to the fetus, or this possibly could be an accidental misassignment of a sample. There are all sorts of reasons, but this mutation happens to result because the epsilon and the gamma genes are lost. This turns on the adult hemoglobin early, pre-birth and that is unusual.”

The emergence of this new hemoglobinopathy category is of great importance, as it empowers Mayo Clinic's clinical and laboratory teams to improve patient diagnosis and outcomes.

When interpreting findings like these, it's crucial for the labs to determine which diagnostic assessment, Sanger sequencing, MLPA, gap-PCR, or long-read sequencing, is best to identify such deletions for proper genetic counseling.

“By going the extra step and evaluating these patient specimens with newer technologies, our teams worked together to delineate a complex genomic finding into a new hemoglobinopathy category that appears to associate with a specific phenotype,” said Dr. Kipp, chair of the Division of Laboratory of Genetics and Genomics. “It portrays the need to continually test and adopt new technologies allowing us to provide more accurate results for our patients.”

For newborn samples displaying higher Hb A levels compared to Hb F, the differential diagnosis should now encompass epsilon gamma thalassemia alongside factors like sample misassignment, notable maternofetal bleed, and in-utero transfusion. However, according to the research, patients can take comfort in knowing epsilon gamma thalassemia will not lead to severe neonatal anemia in newborns. In the case of older patients exhibiting elevated Hb A2 levels, considerations should also extend to epsilon gamma thalassemia in addition to factors like thyroid disease, specific medication usage and B12 deficiency.

In 2021, this study was also recognized by the American Society of Hematology (ASH). Dr. Christineil Thompson, M.D., a fellow who worked closely with Dr. Kohorst and Dr. Herrick, received the ASH Abstract Achievement Award for her abstract on epsilon gamma thalassemia. The findings were also presented at the 63rd ASH Annual Meeting and Exposition and were published in Clinical Chemistry.

“This discovery is highlighted because of the extraordinary teamwork between members of Hematopathology, Hematology, Advanced Diagnostics Laboratory, the Laboratory of Genetics and Genomics, Computational Biology, Information Technology, and Genome Analysis Core,” said Dr. Kipp. “The teams comprised a mix of collaborative pathologists, hematologists, geneticists, researchers, bioinformaticians, IT analysts, and extremely talented allied health staff who all worked together to answer a complex clinical question.”

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Nicole Holman

Nicole Holman joined Mayo Clinic Laboratories in 2023. She currently serves as communications writer on the marketing team. Nicole enjoys feature writing and storytelling focused on employees, patients, and company culture.