RHD/RHCE paralogous misalignment at exon 4 causes false-negative whole-exome sequencing result in a serologically confirmed DFR partial- D individual: Case report

Every time a person receives a blood transfusion, their immune system checks the incoming cells for a specific set of markers, much like a security guard checking IDs at a gate. The most critical of these markers belongs to the Rh system, a group of proteins on the surface of red blood cells that is second only to the ABO system in importance for safe transfusions. The genes that build these proteins are twins in a very literal sense; two of them, called RHD and RHCE, sit side by side in our DNA and are nearly identical copies of each other, sharing about 97 percent of their genetic code. Because they are so similar, the body's immune system can sometimes get confused. If a person has a slightly altered version of the RHD gene, their immune system might not recognize the protein as fully "self," leading to a dangerous reaction if they receive standard Rh-positive blood. This condition, known as a partial-D phenotype, is a serious medical concern because it can cause the body to attack future transfusions.

To prevent these reactions, doctors often try to determine a patient's genetic makeup using a technique called whole-exome sequencing. This method reads the parts of our DNA that contain instructions for making proteins, allowing scientists to look for specific changes that might cause disease. It is a powerful tool used in hospitals and research labs around the world. However, when these genetic instructions are nearly identical twins, the technology faces a unique challenge. The short snippets of DNA that the machine reads can look so much alike that the computer cannot tell which twin they came from. A recent case report from researchers in Pakistan and China highlights exactly how this confusion can lead to a dangerous mistake, where a patient with a known, risky blood type is incorrectly told they are safe.

The story begins with a thirty-four-year-old man who had already been tested by traditional blood typing methods. These older, physical tests, which use antibodies to see how blood cells react, confirmed that he had a specific partial-D type known as DFR. This is a well-documented condition where a small section of the RHD gene has been swapped with a piece from its twin, RHCE. This swap changes three specific letters in the genetic code, altering the shape of the protein just enough to make it dangerous for the man to receive standard Rh-positive blood. Despite this clear serological evidence, the medical team decided to run a whole-exome sequencing test, hoping to confirm the genetic cause. They used three different computer programs to analyze the data, including one specifically designed to find blood group genes.

The results were a complete failure. All three computer programs looked at the man's genetic data and reported that his RHD gene was perfectly normal. They found no errors, no swaps, and no signs of the partial-D condition. In fact, the software was so confident that the gene was normal that it missed the very specific change that had been identified by the blood test. The researchers realized that the computer had not simply missed the error; it had been tricked by the DNA itself. The region of the gene where the swap occurred is so similar to the twin gene that the computer, trying to be efficient, assigned all the DNA snippets from that area to the wrong twin. It placed the reads onto the RHCE gene, leaving the RHD gene looking completely empty in that specific spot.

Because the computer saw no DNA at the RHD location, it assumed the gene was missing or normal, rather than altered. The researchers confirmed this by looking at the data from the twin gene, RHCE. They found that the RHCE gene was covered in a massive amount of DNA data, far more than expected, because it had accidentally absorbed the reads that belonged to RHD. This pattern proved that the DNA was not missing from the body; it was simply being read and filed in the wrong place. The man's body was producing the altered protein, as the blood test showed, but the sequencing machine was blind to it because the genetic instructions were being misfiled. Even a specialized tool built to solve these exact problems could not fix the error, because the data it needed to make a decision was not sitting in the right file.

This case demonstrates a fundamental limitation in using standard short-read sequencing for this specific part of the human genome. The problem is not that the machine did not look hard enough or that the data was not deep enough; adding more data would not help because the error happens at the very moment the computer tries to sort the DNA. The reads are physically present, but they are assigned to the wrong gene, making the true genetic variation invisible to the software. The researchers concluded that for patients with this type of blood group, relying on whole-exome sequencing to rule out dangerous conditions is unsafe. The technology, in its current form, cannot distinguish between a normal gene and a swapped one in this specific region.

The findings serve as a stark reminder that even our most advanced tools have blind spots. For this patient, the standard genetic test would have falsely reassured doctors that he was safe to receive standard blood, a conclusion that could have led to a severe immune reaction. The only way to correctly identify his condition was through the traditional blood test that looked at the protein itself, or by using newer, long-read sequencing technologies that can read longer stretches of DNA and tell the twins apart. This case does not suggest that genetic sequencing is useless, but it does show that for certain complex genetic twins, the old-fashioned methods of looking at the physical protein remain essential for patient safety. The science of blood typing continues to evolve, but in this corner of the genome, the twins remain too similar for the short-read machine to tell them apart.