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Story of discovery: newborn screening for lysosomal storage diseases

For most people, having a baby is one of the greatest joys in life. When an infant is born with a rare genetic disorder, parental joy can be mixed with fear; but fortunately, recent decades have also brought good news for many families of children born with an inherited disease, as progress in the treatment of some of these disorders is improving health and quality of life for many affected infants.

In some cases, therapies achieve the best possible health outcomes if they are initiated before outward signs of the disorder develop. For this reason, it is critically important that advances in treatment for genetic disorders be matched with development of newborn screening programs that help quickly identify children who need them.

Newborn screening programs in most U.S. states test for dozens of genetic disorders, including several that are within the NIDDK’s mission area. These include sickle cell anemia, cystic fibrosis, as well as some lysosomal storage diseases, a constellation of rare diseases whose detection in newborn screening has undergone a revolution in recent years.

What Are Lysosomal Storage Diseases?

The body’s cells recycle many of the substances they no longer need by digesting them with proteins inside cellular compartments called lysosomes. The proteins that are missing or damaged in people with lysosomal storage diseases are considered “enzymes,” meaning that each functions to promote the conversion of one chemical compound into another. Specifically, lysosomal enzymes convert toxic cellular waste products into materials that the cell can recycle or safely excrete. When one of these enzymes is missing or inactive, toxic waste products are not properly degraded. Instead, they build up in the lysosomes where they can lead to severe organ damage. Diseases caused by such enzyme deficiencies—lysosomal storage diseases—are individually rare, but collectively affect about 1 in 7,700 infants born in the United States. Symptoms vary, and are often not apparent at birth; however, as the undigested materials accumulate, they can cause serious problems such as weakness, severe pain, brittle bones, intellectual disability, corneal clouding, organ failure, and death. Dozens of lysosomal storage diseases have been characterized, including Gaucher disease; Pompe disease; Fabry disease; and several forms of mucopolysaccharidosis (known as MPS I, MPS II, etc.), a subset of the lysosomal storage diseases resulting when someone lacks any of several enzymes needed to recycle one particular class of biological molecules.

Built on the discoveries of scientists supported by the NIDDK and other NIH Institutes and Centers and product development by pharmaceutical companies, lysosomal storage disease research is a classic story of translating remarkable findings from basic research into Food and Drug Administration (FDA)-approved treatments for many of these diseases. To take the greatest advantage of these developments, the NIDDK has also invested in developing improved methods for identifying infants who would benefit from these therapies.

Blood Spot Screening: The Beginning of the Revolution

The first widely utilized newborn screen for a genetic disease did not detect a lysosomal storage disease; rather, it tested for phenylketonuria (PKU), a genetic disorder caused by the inability to break down an amino acid called phenylalanine. Amino acids are the building blocks of proteins. In PKU, phenylalanine can build up to harmful levels in the body, causing intellectual disability and other serious health problems. By the 1950s, physicians understood that the serious symptoms of PKU could be greatly reduced by carefully limiting the amount of phenylalanine in the diet of people with the disease. However, PKU can have serious, lasting effects on a child’s development if the problem is not detected—and the dietary intervention initiated—early enough.

Thus, it was a landmark development when, in 1959, researchers developed a straightforward method to test for PKU. First, they saturated a small piece of paper with a drop of blood, usually collected by pricking the heel of an infant to be tested. After the spot had dried, they placed the paper onto a petri dish containing bacteria that can only grow in the presence of supplemental phenylalanine. Since healthy people store very little phenylalanine, and people with PKU accumulate significant amounts of the amino acid, this bacterial growth test made it much easier to identify babies with PKU during their first few days of life. First tested on a large scale in 1962 using blood spots from 400,000 babies, the approach was a tremendous success, allowing early dietary intervention that greatly improved outcomes for children with PKU. Within a few years, states throughout the Nation had adopted the PKU screen; a 1968 World Health Organization report led to screening in many foreign countries as well.

A Method To Identify Several Diseases at Once

Similar tests using dried blood spots and bacterial growth soon followed for other genetic diseases, but there were limits to the number of such tests that could be performed with a single blood spot, and limits to the number of blood spots that could reasonably be collected from every infant. Researchers found a solution to this problem by utilizing “mass spectrometry,” a method for simultaneously measuring the amounts of multiple chemical components from a small sample.

Early models of mass spectrometers, developed more than 120 years ago, relied on differences in the mass and charge of chemicals in a sample in order to separate them. Modern mass spectrometry methods take advantage of other chemical differences, and are powerful enough to separate and quantify thousands of molecules at once. This technology has been widely adopted by clinical laboratories to perform a wide variety of diagnostic tests. Importantly, these tests can include simultaneous screens for dozens of genetic diseases, provided that each of those diseases has a measurable effect on the chemical components of a dried blood spot or other easily obtained sample.

The Development of Treatments for Lysosomal Storage Diseases Creates a Need for Early Detection

Although mass spectrometry was utilized to detect a growing number of genetic diseases in newborns, for at least two reasons, lysosomal storage diseases were not initially among them. First, it turns out that the levels of the metabolites that accumulate in lysosomal storage diseases can be quite variable in both healthy babies and those with lysosomal storage diseases, which can lead to test inaccuracy. A second reason was that in the late 1990s, when mass spectrometry screening of dried blood spots was becoming more common, there were no effective treatments for lysosomal storage diseases. It is not considered useful or cost-effective to test for a condition if a positive result would only make parents worry that their children are likely to develop a disease when there is no treatment their pediatrician can suggest to keep them healthy.

Fortunately, research in the early 2000s—supported by the NIDDK, other components of NIH, and voluntary groups—led to significant improvements in the treatment of many lysosomal storage diseases. Scientists studying the basic biology of the lysosome discovered that healthy cells do not simply synthesize the enzymes and deposit them directly into their lysosomes. Rather, each enzyme is initially synthesized in an inactive, precursor form, which the cell excretes. Either the same cell or another cell nearby reabsorbs the precursor and directs it to a lysosome, where it is converted into its mature, active form. To treat a lysosomal storage disease, therefore, clinicians can periodically inject some of the needed enzyme in its precursor form, taking advantage of these final processing steps to deliver it to lysosomes and activate it.

While by no means a cure, “enzyme replacement therapy,” as the treatment approach is called, significantly improves health for people with a variety of lysosomal storage diseases. Several such therapies are FDA-approved, while still more are under consideration. Importantly, just as with PKU, best results are obtained when treatment begins early, before organ damage has occurred. So once treatments were available, there was a need for corresponding improvements in methods to screen for lysosomal storage diseases in newborns.

Fluorescence Tests for Lysosomal Storage Disorders

Unlike with PKU, researchers found that the most reliable way to test for lysosomal storage diseases was to test for the activities of the lysosomal enzymes in a tissue sample from the person being tested. Early forms of such tests were both invasive—requiring a muscle or skin biopsy—and labor intensive, so they were not used unless there was a clear reason to suspect a child had a lysosomal storage disease (because he or she had developed the symptoms, or had an older sibling with the disease.) Other tests were developed that measured the amount of the enzyme in the blood sample relative to the amount of other proteins, for example; but their usefulness was limited by the fact that some lysosomal storage disease-causing mutations do not eliminate the relevant enzyme, but rather make it unable to perform its chemical function.

A major step forward came in 2001, when researchers described a simple process whereby the activity of an enzyme called α-L-iduronidase (IDUA), which is missing or inactive in people with the lysosomal storage disease MPS-I, could be reconstituted from a dried blood spot. Just as importantly, they developed a straightforward test to check for that enzymatic activity. The test relied on a modified form of the chemical that IDUA normally acts on in the lysosome: IDUA splits the modified form of the chemical into two parts, one of which is highly fluorescent, and can be detected and quantified by a light sensor. The diagnostic test for MPS-I takes advantage of this fluorescent component—samples from people who lack normal levels of IDUA activity produce much less fluorescence than those from people without the disease. The principle utilized in the MPS-I test was extended to develop related fluorescence-based strategies for diagnosing Pompe, Gaucher, Fabry, and other lysosomal storage diseases.

Special tools allowed laboratories to perform these tests on blood spots from dozens of babies at once, suggesting that wide-scale screening might be possible. Therefore, other researchers conducted pilot studies in which they used these methods to screen for one or more lysosomal storage diseases in large numbers of children. One such study took place in Taiwan, where Pompe disease is more common than in the United States. Testing dried blood spots from over 200,000 newborns born in one region of the country, researchers diagnosed 6 babies with the disease. Compared to babies born in other regions of Taiwan at about the same time, those diagnosed earlier were also able to begin enzyme replacement therapy sooner and had better health outcomes. Taiwanese researchers later performed an analogous pilot study to diagnose Fabry disease with similar results. Other researchers supported these findings through pilot studies conducted in Europe, South America, and Missouri. The fluorescence assays were therefore the first practical approaches to screening for lysosomal storage diseases using dried blood spots from large numbers of newborns. The FDA has therefore approved tests using this technology for newborn screening to detect MPS-I, Pompe, Gaucher, and Fabry diseases.

The Next Generation of Lysosomal Storage Disease Testing Methods

While these fluorescence tests were a critical stride toward allowing widespread newborn screening for lysosomal storage diseases, they are not perfect. For example, for reasons that are not fully understood, some people have low (but non-zero) IDUA activity, yet never develop MPS-I symptoms. And because the tests have a limited capacity to detect small differences in the enzyme activities, it was sometimes unclear whether babies with intermediate levels of IDUA activity were perfectly healthy or would go on to develop a mild form of MPS-I. A similar problem exists for the enzymes missing in some of the other lysosomal storage diseases. Another important limitation of the fluorescence approach is that all of the lysosomal storage disease tests utilize the same fluorescent product, meaning each must be performed separately.

Fortunately, researchers have identified a significantly more sensitive method for measuring the activities of lysosomal enzymes, and it allows testing for multiple lysosomal storage diseases at once. Their insight was to combine the enzyme reconstitution methods pioneered in the development of the fluorescence tests with use of mass spectrometry technologies to detect their activities—without the need for fluorescence. NIDDK-supported researchers first developed such an approach to diagnose a lysosomal storage disease known as Krabbe disease, in which people lack the enzyme galactosylceramidase (GALC). As described in a 2004 scientific publication, the scientists allowed enzymes extracted from a blood sample to mix with a normal, unmodified form of a chemical GALC splits into two products in lysosomes. With a few additional steps, they were able to use mass spectrometry to separate those products in the mixture, and measure how much of one of them had been created, finding little or none if the sample was from someone with Krabbe disease.

Initially, the method relied on GALC extracted from a fresh blood sample, but later the researchers found they could reconstitute the enzyme from a dried blood spot, based on the same principles utilized for the fluorescence tests. And importantly, they also found that if they included chemicals acted on by IDUA and other enzymes missing in various lysosomal storage diseases, the mass spectrometry process could separate out each of the different reaction products. Thus, they were able to use just a small part of a single blood spot to check for at least six different lysosomal storage diseases simultaneously. In addition, the mass spectrometry screening methods are notably better than the fluorescence approach at reliably measuring small differences in the enzyme activities, making it easier for clinical testing labs to distinguish infants likely to develop lysosomal storage diseases from those with enough enzymatic activity to keep them healthy.

Moving Innovations in Lysosomal Storage Disease Testing into Widespread Clinical Use

Although there are tests for hundreds of other genetic diseases, in addition to the lysosomal storage diseases, it would be difficult and costly to screen every child for each of them. Some tests may be difficult, requiring a more invasive sample procedure than the use of a dried blood spot, or requiring expensive, unusual testing equipment, for example. Others may often yield ambiguous results, or cause needless worry among parents by misdiagnosing a disease in infants who are actually healthy. Even if the test is accurate and simple to perform, if there is no way to treat for the disease, or if it can be treated effectively even after symptoms develop, routine screening of most newborns would not be useful.

For these reasons, the U.S. Health Resources and Services Administration assembled a group of experts in diagnostic testing called the Advisory Committee on Heritable Disorders in Newborns and Children, which carefully considers evidence from clinical research to determine whether states throughout the Nation should routinely screen newborns for various genetic diseases. The Committee publishes and periodically updates a list, the Recommended Uniform Screening Panel, that now includes tests for MPS-I and Pompe disease among the dozens of diseases infants should be screened for. The bar is high for adding additional diseases, given limited resources, but the availability of reliable tests and treatments for several other lysosomal storage diseases suggest they may be added in the future. Indeed, many states require testing for lysosomal storage diseases not currently listed on the Uniform Panel.

The Future of Research and Treatment for Lysosomal Storage Diseases

In 2018, the FDA approved a simultaneous mass spectrometry test for Gaucher, Niemann-Pick A/B, Pompe, Krabbe, Fabry, and MPS I Diseases. The availability of reliable testing means many children with lysosomal storage diseases may soon be diagnosed earlier, and more accurately. This fact, combined with the therapeutic options now available, is reducing the burden of these serious diseases for affected children and families. Earlier diagnosis not only allows therapy to begin before irreversible organ damage may have occurred, it also potentially helps avoid costly diagnostic efforts to determine the cause of a child’s health issues, as well as potentially ineffective therapies that might precede an eventual correct diagnosis. While lysosomal storage disease therapeutics currently in clinical use are not perfect and well short of a cure, further research to develop improved methods to treat these diseases is currently in development.

In addition, researchers have developed and are testing diagnostic methods for many more lysosomal storage diseases, including other forms of MPS (such as types II, IIIA, IIIB, IVA, and VI). Such developments would provide the opportunity to screen for many more lysosomal storage diseases, administer available therapy early, and greatly improve the lives of those affected by these disorders.

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