Updated Test Promises Fewer False Positives in Newborn Screening for Krabbe Disease

Mayo Clinic’s Biochemical Genetics Laboratory has announced an updated second-tier test to detect Krabbe disease (KD) that uses psychosine (PSY) as a disease marker. The new test method has significantly higher sensitivity to detect this devastating disease in infants and allows identification of KD patients with minimal psychosine elevations. This high-sensitivity psychosine test will continue to help reduce the false-positive rate for this disease in newborn screening (NBS). The test will also further improve the specificity of screening. A false-positive result usually means costly, time-consuming follow-up testing, which can inflict high emotional stress to parents while they await definitive results.

The current first-tier newborn screening method looks at markers for a variety of different genetic conditions. For KD, the newborn screening test measures galactocerebrosidase (GALC) enzyme activity in an infant’s dried blood spot sample on filter paper (collected via a heel prick on day two of life). However, relying on GALC activity for screening has poor specificity, which means more false positives.

Unlike the first-tier newborn screening method, the PSY assay is specific to Krabbe disease when GALC activity is reduced. Thus, as a second-tier test, psychosine can “markedly improve the positive predictive value in newborn screening for Krabbe disease,” according to the Mayo Clinic study results, published recently in Genetics in Medicine, the official journal of the American College of Medical Genetics and Genomics.

The study, which provides data from a large number of patients with pathogenic variants of Krabbe disease, validated this highly sensitive PSY assay in dried blood spots and red blood cells, using liquid chromatography-tandem mass spectrometry.

Dietrich Matern, M.D., Ph.D.

“Overall, we believe our findings enable the prevention of unnecessary follow up of unaffected infants while allowing rapid identification of affected patients so that they can be treated as early as possible,” says Dietrich Matern, M.D., Ph.D., Professor of Laboratory Medicine, Medical Genetics, and Pediatrics at Mayo Clinic, and senior author on the study. “We can also better say whether the patient, if they have the disease, has severe Krabbe disease, with onset in infancy, or a later-onset variant. That information is very important when deciding the timing of treatment.”  

Anatomy of a deadly disease

Krabbe disease occurs when a child inherits two pathogenic gene variants (mutations known to be disease-causing in other patients). About 1 out of every 400,000 babies is born with this rare condition. If the variants are severe, the newborn doesn’t produce any GALC enzyme, which causes the destruction of the protective coating of nerve cells in the brain and nervous system.

Amy L. White, LCGC

“In these severe cases, called early infantile Krabbe disease, the patients’ neurologic symptoms usually begin within three to six months after birth, with a life expectancy of two years, on average,” says Amy L. White, LCGC, Genetic Counselor in the Biochemical Genetics Laboratory. “Infantile Krabbe disease symptoms include the progressive loss of mental and physical abilities, leading to the inability to swallow and breathe in the late stage of disease. In other cases, newborns inherit two ‘milder’ pathogenic variants and can still produce a small amount of GALC enzyme, which means they appear normal and healthy over their first year of life. After that, symptoms can appear anytime between childhood and adulthood. These milder cases are known as late-onset Krabbe disease (LOKD).”

Then there are “carriers”: individuals who carry only a single pathogenic variant along with one normal GALC gene, who can generally live a normal life without symptoms. However, if their partner or spouse also has a single variant, there is a 25 percent chance their children could be born with KD.

Meanwhile, because there has been neither an efficient newborn screening test nor a cure for KD, the disease is currently not included in the Recommended Uniform Screening Panel (RUSP). The RUSP currently comprises 35 core conditions that the U.S. Secretary of Health and Human Services recommends every baby should be screened for.

But things are changing, in large part due to patient advocacy groups and, in turn, state politicians. Over seven states have now added KD to their screening programs, and others are in process of adding it. More and more parents are advocating for KD screening as news coverage of stem cell transplantation gains more traction. The treatment, via a bone marrow transplant from a compatible donor, must be initiated in the first month of life, before the infant shows neurological symptoms, if it is to be of benefit.

“When transplanted early, patients can develop more developmental skills than patients without a transplant,” says White. “This is why newborn screening is critical for early, accurate detection of the disease, so that treatment can occur when it is most effective.”

However, medical opinions on whether or not stem cell transplantation is sufficiently effective as a treatment for KD are mixed. While the treatment may significantly prolong life, it cannot reverse symptoms; instead, it can delay them and/or make them less severe. Also, the transplant process is quite invasive, with risk of complications and death for infants. And even if there are no complications from transplantation, the disease may still progress to death. Still, some parents are willing to accept the risks and pursue this treatment, as it is the only one currently available.   

Making sense of GALC activity test results 

The problem with GALC enzyme testing is that the results can confuse the issue of whether or not a patient has KD. Why? Because some low enzyme results reflect disease and others are “pseudodeficient,” which means that they appear low with the enzyme test but aren’t really low in the patient. Thus, it has been hard to determine, from enzyme testing of GALC alone, whether an infant truly has KD. To clarify the enzyme results, a genetic DNA test may be performed on a patient with suspected KD. The DNA test result can show 0, 1, or 2 variants. A variant(s) is categorized as one of the following:

  • Pathogenic – known in other patients to be disease-causing
  • Likely pathogenic – likely to be disease-causing
  • Benign
  • Pseudodeficiency – the GALC enzyme test result appears as if the patient has disease, but it is an “artifact” of the test and the patient’s actual GALC levels in their cells are sufficient 
  • Variant of uncertain significance (VUS) – this gene change is unknown and there is no reported evidence as to whether it causes disease or not

Making sense of such results can be a conundrum for physicians: “If a patient has a genetic test that shows one VUS and one pathogenic variant, or two VUSs, then the physician still doesn’t know if the patient has Krabbe disease because the DNA test result is inconclusive—VUSs have unknown effects in the body,” says White. “But with a biochemical test like our PSY assay, we can check a psychosine level that will be elevated when GALC enzyme isn’t present in enough quantity to break it down. In this way, we can tell a physician if there is truly disease occurring in the body, because the psychosine level is a functional measure of the disease.”

Potential use as a monitoring tool

The new assay can also be used as a follow-up test for patients identified with early infantile Krabbe disease or LOKD by monitoring psychosine levels. 

Coleman Turgeon

“When the GALC enzyme is not working properly, it’s unable to effectively clear psychosine that would otherwise be metabolized,” says Coleman Turgeon, Development Technologist with Mayo Clinic’s Biochemical Genetics Laboratory, who developed the PSY assay. “And so elevated psychosine is an indicator that the GALC enzyme is not working as well as it should. With lower enzyme activity, a higher psychosine level is expected, providing a direct measurement of disease status.”

In other words, regular PSY testing could be used to decide if and when the patient is a candidate for a bone marrow transplant, based on their psychosine levels.

“If you have a patient whose psychosine levels are high but, ideally, they don’t have symptoms yet, then you know you need to intervene with a bone marrow transplant,” says Turgeon, also an author on the study paper. “After transplantation, you can then monitor psychosine levels to determine if the transplant is effective at providing a supply of healthy cells. If it works, we’ll see psychosine levels start to fall as transplanted cells provide the source for functioning enzymes. This reduces the toxic compound in their system.”  

Again, such an intervention is perilous to any child, and clinicians are especially prudent before going ahead with a transplant on an infant. “For infants with late-onset KD, the longer we can wait before subjecting the patient to that degree of medical intervention, the less chance there will be of other complications,” says White. “Timing is critical, and there are only a few select transplant centers and physicians who make that determination.”

If their psychosine levels can be monitored, LOKD patients can ideally be allowed to grow in size and become less fragile before a transplant is initiated, to reduce the risk of mortality. It’s a delicate balance, because the procedure must also be performed before symptoms occur.

To transplant or not to transplant

Obviously, a bone marrow transplant should never be done if the child doesn’t have the disease. The procedure is extremely costly, requiring a host of blood work, neurologic evaluation, and physiological studies that precede a transplant. “All these things help the transplant experts determine whether they should transplant or not, and whether it is the right time,” says Dr. Matern. “So the psychosine test should be very helpful information to them.”

When the first case of KD was identified through newborn screening in Kentucky, this assay was part of the testing (Mayo Clinic provides screening for KD and three other conditions for the Kentucky Newborn Screening Program). The infant, identified with early infantile Krabbe disease, had reduced GALC activity and very high psychosine, which prompted immediate referral to a transplant center. The genotype, once determined, revealed a pathogenic variant as well as a variant of uncertain significance.

“In a state not using psychosine, they would likely have sat on this and said, ‘Well, let’s just follow this child,’ or possibly ordered other tests, and then at some point would’ve realized that this child needed a transplant,” says Dr. Matern. “And they would have probably realized this too late for a transplant to do much good. So because we measured his psychosine level as part of newborn screening, this child immediately received a transplant on the twenty-fourth day of life.” 

Or, consider the case of an infant at a hospital center in New York, identified through newborn screening as having low GALC activity and a genotype that contained a known mutation, and a variant of uncertain significance. But this was not enough data for the clinician to know if the child had Krabbe disease or not. So the baby’s blood sample was sent to Mayo Clinic with a request for a quick turnaround.

“Of course, we wanted to help,” says Dr. Matern. “So we immediately ran our psychosine assay on the child’s blood sample. And we found that the GALC activity was indeed low—in the late-onset Krabbe disease range—and that the psychosine was just a bit elevated. Once we sent the full results back to the child’s clinician, he was very appreciative, because now he had something to tell the family and could explain why he wanted to see the child in two months, to run another psychosine test, evaluate him, and keep an eye on him. Alternatively, if the GALC activity tested in the pseudodeficiency or carrier range, and psychosine levels had been completely normal, we would have suggested that the child did not have Krabbe disease and could go home and enjoy life. So that’s a real example of how our test can be used to clarify uncertain results.”   

Turnaround time and setting up regional testing

Perhaps the question by now is: Why can’t the PSY assay be performed at most hospital laboratories and, thus, avoid the cost of having to send a sample to Mayo Clinic? The short answer is, it just wouldn’t be practical.

“Implementing this test in every hospital and newborn screening lab doesn’t make a lot of sense, especially because you don’t have to run it very often,” says Dr. Matern. “Also, we’re using equipment that is more sensitive than the typical tandem mass spectrometers that are used in a screening lab. And, in front of the instrument, we have a liquid chromatography system that you also don’t find in most screening labs. So this test is not something that you can implement in just any public health lab and expect it to operate efficiently.”  

Turgeon chimes in: “This test is so specialized, requiring high expertise, so we feel this is part of the reason it should be more regionalized to only a handful of screening centers rather than every hospital,” he says. “Because the overhead to maintain a test like this is not going to be practical unless you’re running a lot of samples. This is why regionalization seems to make sense.”

Infantile Krabbe disease is among the critical conditions for which Mayo Clinic Laboratories is committed to a 24-hour turnaround. In fact, if a blood sample were to arrive in the morning, Mayo Clinic Laboratories can usually have results by that afternoon. The test itself is performed very fast. “Once the sample is in our hands, it’s a 30-minute extraction with 15 minutes of processing, followed by a 4.5-minute analysis,” says Turgeon.    

Dr. Matern believes that the importance of the test mitigates cost. “From our perspective, it is cheaper to do a few second-tier tests than the cost of following up on false positives,” he says. “If you look at the overall cost of newborn screening, false positives cost money that could be saved."

Envisioning the future of PSY testing

While the PSY assay is highly sensitive and currently the best test of its kind for Krabbe disease, the clinical interpretation of intermediate psychosine levels still needs further studies.

“For those with an intermediate level of psychosine, we think they should have regular clinical evaluations and repeat testing for psychosine in blood and CSF if a lumbar puncture was performed to measure other, nonspecific indicators such as protein,” says Dr. Matern. “The question is, at what point does psychosine, as a marker, tell us we should transplant, or at what level should it be considered already too late? Those are things we don’t actually know yet. We don’t have enough data yet.” 

One way to help this along is to normalize psychosine levels in a blood sample itself, which is why the study tested red blood cells in addition to dried blood spots. “We believe that measuring red blood cells might be more helpful because, unlike with dried blood spots, we can normalize psychosine in the blood to the hemoglobin concentration,” says Dr. Matern. “This is helpful because psychosine is found in blood, primarily in the red blood cells. Then, if you check psychosine levels over time, we think it should be more reliable in telling us whether those levels are going up or down.”

With the current blood spot process—mainly only used for newborn screening—a needle breaks the skin and the pricked heel area is squeezed by hand until a blood drop forms, which is then put on filter paper and left to dry.

“If you’ve ever watched someone do this, you realize it’s very difficult to standardize,” says Dr. Matern. “Skin perfusion, applied pressure, contamination with skin products, and drying time are all factors that are difficult to control. So it’s not very reliable if you want to do it over and over to compare psychosine levels. That’s why we brought up the assay using red blood cells, so we have a means to normalize this method and, over time, we can rely on the psychosine concentration a patient has based on the amount of hemoglobin. Our hope is that this will help us to better monitor patients.”     

Chris Bahnsen

Chris J. Bahnsen covers emerging research and discovery for Mayo Clinic Laboratories. His writing has also appeared in The New York Times, Los Angeles Times, and Smithsonian Air & Space. He divides his time between Southern California and Northwest Ohio.