Identification of novel compound heterozygous PFKM variants in a patient with chronic haemolytic anaemia: genotype–phenotype correlation and structural insights and Mutation update

Inside every red blood cell, a tiny but vital factory works around the clock to keep the cell alive. This factory runs on a process called glycolysis, a chemical pathway that breaks down sugar to produce energy. Unlike most cells in the body, red blood cells have no mitochondria, the power plants found in other tissues, so they rely entirely on this sugar-burning process for their energy needs. A key worker in this factory is an enzyme called phosphofructokinase, or PFK. This enzyme acts like a gatekeeper, controlling the speed at which sugar moves through the pathway. If this gatekeeper stops working correctly, the cell cannot make enough energy, and the red blood cell begins to fail and break apart prematurely. When this happens throughout the body, it leads to a condition known as hemolytic anemia, where the blood lacks enough healthy red cells to carry oxygen.

While problems with this enzyme are often associated with muscle weakness and pain during exercise, they can sometimes show up in a different way, affecting only the blood. A recent report from researchers in India describes a patient who lived with chronic anemia and jaundice for years, yet never experienced the muscle cramps typically linked to this disorder. By using advanced genetic sequencing and computer modeling, the team identified two new genetic errors in the patient's DNA that explained her condition. Their work not only solved a long-standing medical mystery for this individual but also added two new pieces to the global puzzle of how genetic changes can alter the shape and function of this essential enzyme.

The patient was a thirty-two-year-old woman who had suffered from recurring yellowing of the skin and eyes since childhood. For years, she endured episodes of vomiting and fatigue, and she had received multiple blood transfusions to manage her low hemoglobin levels. When doctors first examined her, they found clear signs that her red blood cells were being destroyed faster than her body could replace them. Her liver and gallbladder were also under stress, a common consequence of the body trying to process the massive amount of broken-down blood cells. Standard tests ruled out the most common causes of blood cell destruction, such as a lack of the enzyme G6PD or a defect in the enzyme pyruvate kinase. However, a specific test for the PFK enzyme revealed that its activity was severely reduced, dropping to just 2.1 units per gram of hemoglobin, well below the normal range of 5.5 to 10.8. This pointed directly to a rare metabolic disorder known as Tarui disease, or glycogen storage disease type VII, which is caused by a failure in the PFK enzyme.

To find the exact cause, the medical team turned to whole-exome sequencing, a technique that reads the protein-coding instructions in a person's DNA. They were looking for errors in the gene that provides the blueprint for the muscle version of the PFK enzyme, known as PFKM. The search revealed two distinct genetic changes, one on each copy of the gene the patient inherited from her parents. The first change occurred in a section of the gene called exon 6, where a single letter in the genetic code was swapped, changing a building block called arginine into tryptophan at position 184 of the protein. The second change was found in exon 11, where a different swap turned a leucine building block into a proline at position 324. Neither of these specific errors had been seen before in medical literature, making them novel discoveries. The researchers confirmed these findings using a standard laboratory method called Sanger sequencing, which acts as a precise re-check of the genetic code.

Understanding how these tiny changes could cause such a large problem required looking at the three-dimensional shape of the enzyme. The researchers used a computer model based on the known structure of the rabbit PFK enzyme to visualize what was happening inside the patient's cells. They found that the first error, at position 184, occurred in a critical area where the enzyme binds to a molecule called ADP, which helps regulate its activity. In a healthy enzyme, this spot forms a stable connection with a neighboring part of the protein. The new tryptophan building block was too bulky to fit in that space, effectively jamming the mechanism and preventing the enzyme from regulating itself properly. The second error, at position 324, happened in a tightly packed core of the protein that holds the enzyme's shape together. Replacing the flexible leucine with the rigid proline distorted the local structure, much like trying to force a stiff hinge into a flexible joint. This distortion weakened the overall stability of the enzyme, making it prone to falling apart or failing to function.

The patient's case highlights a crucial point about how genetic diseases can appear. While Tarui disease is classically described as a condition causing muscle pain and exhaustion during exercise, this patient showed almost no muscle symptoms. Instead, her body was dominated by the effects of hemolytic anemia. This suggests that the specific combination of genetic errors she carried affected the enzyme in a way that hit the red blood cells hardest, perhaps because these cells rely so heavily on glycolysis for survival. The researchers noted that the two errors likely worked together to destabilize the enzyme, with one disrupting its regulatory switch and the other weakening its structural foundation. This dual attack explains why the enzyme activity was so low and why the red blood cells could not survive.

Following the genetic diagnosis, the medical team focused on treating the complications that had built up over years of chronic anemia. The patient had developed gallstones and inflammation in her bile ducts, a direct result of the high levels of bilirubin produced by the breaking red blood cells. She underwent a procedure to place a stent in her bile duct to relieve the blockage and allow bile to flow freely. Over the course of a few days, her bilirubin levels dropped significantly, and she began to feel better, eventually being discharged with a plan for surgery to remove her gallbladder. The successful treatment of her acute symptoms, combined with the genetic diagnosis, provided a clear path forward for her care and future family planning.

This report adds two new entries to the growing list of genetic variations that cause PFK deficiency. By documenting these specific errors and showing how they alter the enzyme's structure, the study helps other doctors recognize that this rare disease can present with blood problems alone, without the classic muscle symptoms. The work also demonstrates the power of combining modern genetic sequencing with computer modeling to understand how a single letter change in DNA can ripple out to cause disease. For the patient, it meant moving from a state of unexplained illness to a clear understanding of her condition, allowing for targeted management and genetic counseling. As more cases like this are identified, the medical community will gain a better picture of the full range of ways this enzyme can fail, leading to more accurate diagnoses and better care for people with rare metabolic disorders.