Newborn Screening: What s New?

Transcription

1 Newborn Screening: What s New? Patricia M. Jones, PhD (Department of Pathology, University of Texas Southwestern Medical Center and Children s Medical Center of Dallas, Dallas, TX) DOI: /LMOOXWVPSM5FC3B6 Abstract Screening newborn infants for inborn errors of metabolism (IEM) began in the United States almost 50 years ago with the introduction of testing for phenylketonuria (PKU) in the state of Maine. As the understanding of genetics and metabolism grew, and as testing methodologies became more automated and sensitive, both After reading this paper, readers should be able to describe the rationale for performing newborn screening and discuss the history and development of the newborn screening process. The readers should also be able to describe the expanded newborn screening The knowledge of the presence of genetic defects that alter the activity of enzymes and result in disorders of basic metabolism began with the finding of excess intermediate metabolites in urine. 1 The earliest recognition was that, in certain patients, a urine sample darkened on standing. Dr. Archibald E. Garrod termed these disorders inborn errors of metabolism (IEM) in With the ability to identify the specific metabolite present, whether it was an abnormal metabolite or simply a normal metabolite in excess, came a growing understanding of biochemical pathways and how defects in a pathway affected the human organism. Phenylketonuria (PKU) was one of the earliest disorders of this type recognized and, although its incidence is only about 1:14,000, PKU is also one of the more common disorders of this type. The adverse symptoms seen in PKU were determined to be the result of a block in the metabolism of phenylalanine with the subsequent build up of excess phenylalanine and phenylalanine metabolites in body tissues. It was realized that early detection of the disorder and early dietary intervention would be necessary. The prevention of the toxic buildup of metabolites was imperative in order for the effects of this type of disorder to be prevented or mitigated. The basic purpose of a newborn screening (NBS) program is to detect devastating disorders for which there are treatments before the onset of adverse and usually irreversible conditions. The intent is not just to save lives that would otherwise not be saved, but to improve the quality of life by preventing or lessening the adverse conditions resulting from the disorder. Phenylketonuria is an excellent example of an NBS success story. Like many IEM, individuals with PKU are normal at birth because the mother s phenylalanine metabolic pathways perform the necessary functions for the developing fetus as well. After birth, when the infant begins to consume protein in breast milk the number of states performing newborn screening (NBS) and the number of disorders for which patients can be screened began to grow. During the 1990s and the first decade of the 2000s, a new technology was introduced called tandem mass spectrometry (MS/MS) into the clinical laboratory and NBS arenas, which spurred the growth of screening to include as many as 30 disorders by almost all 50 states and Washington, DC. This technology gave laboratories the ability to rapidly and accurately test for multiple IEM simultaneously. Newborn screening programs became an integral part of assessing the health and well-being of every infant born in the United States. process, including the technologies involved, and discuss possible future directions in newborn screening. Chemistry questions and corresponding answer form are located after this CE Update article on page 742. or formula, phenylalanine builds up rapidly to toxic concentrations. A PKU individual identified at birth via newborn screening and receiving the correct diet avoids this toxic damage; the mental retardation that high concentrations of phenylalanine cause is prevented. Increasing numbers of individuals affected with PKU and diagnosed shortly after birth are reaching reproductive age and having children of their own. An individual with classic PKU who is missed at birth is neurologically devastated, has a low IQ, and usually will be institutionalized. The monetary cost of a $5 test and a restricted diet formula is considerably less than the cost of institutionalization. The quality of life cost difference cannot be estimated. Newborn screening began in the state of Maine in 1960 after Dr. Robert Guthrie developed a simple bacterial growth inhibition test that accurately screened for PKU by detecting a zone of inhibition of bacterial growth around a blood spot sample from patients with excess phenylalanine metabolites in their blood. 2,3 New York rapidly followed Maine in setting up newborn screening for PKU, and by 1970 all 50 states and the District of Columbia had ongoing NBS programs. Newborn screening programs are not federally mandated or controlled but are entirely dependent upon the state in which they reside. Even though all 50 states have programs, the programs are as extensive or as limited as the individual state can afford to support. Since 2000, however, several working groups and panels, including the American Academy of Pediatrics and the American College of Medical Genetics (ACMG), have evaluated the newborn screening programs in the United States and made recommendations regarding all aspects of the NBS process. 4-8 These recommendations include such items as what disorders should be included in the screening, what methodologies are recommended for use in screening, and what level of follow-up support should be available. labmedicine.com December 2008 j Volume 39 Number 12 j LABMEDICINE 737

2 Table 1_Critera for Inclusion of a Test in a Newborn Screening (NBS) Program Criterion Rationale 1. Treatable Disease morbidity/mortality tempered by early detection 2. Common Disease is common enough to be a significant health issue if untreated 3. Cost effective Cost of program (NBS, notification, confirmatory testing) is less than cost of undetected/ untreated disease 4. Easily identifiable Able to be detected with a simple, fast screening test 5. Easy sample acquisition Requires the least volume of sample; easily transportable 6. Highly sensitive technique Minimal false-positive rate with no false-negatives 7. Follow up available Ability to confirm or rule out screening results Initially, progress on adding disorders to screening programs was slow. Inborn errors of metabolism had to meet a list of criteria for inclusion in a newborn screening program. Table 1 lists the general criteria that has been included in the list over the years and the rationale for each. Recently, the ACMG Newborn Screening Expert Group, at the behest of the Maternal and Child Health Bureau of the Health Resources and Service Administration of the U.S. Department of Health and Human Services refined this list to 3 basic criteria: 1) the disorder can be Table 2_Changes in Newborn Screening Disorder Number of States Screening Phenylketonuria (PKU) Congenital hypothyroidism Galactosemia Sickle cell disease Congenital adrenal hyperplasia Biotinidase deficiency Maple Syrup Urine Disease Homocystinuria Medium chain acyl-coa dehydrogenase (MCAD) deficiency Tyrosinemia Cystic fibrosis Figure 1_Representative tandem mass spectrometry (MS/MS) schematic with amino acid mass transitions from precursor to product masses. Panel A represents a neutral loss multiple reaction monitoring (MRM) with each compound losing a mass of 102. Panel B represents an MRM where the m/z transitions would be set separately for each compound (ie, for phenylalanine and for alanine). identified at a phase (24 to 48 hrs after birth) at which it would not ordinarily be clinically detectable; 2) a test is available for it that has appropriate sensitivity and specificity; and 3) there are demonstrated benefits of early detection, timely intervention, and efficacious treatment of the disorder. 9 Other reasons for the initial slow growth of NBS programs include: 1) the need for sensitive and specific assays that could be performed on dried blood spots and limited sample volumes; 2) confirmatory tests were not available for many of the disorders; 3) treatment was not available for the disorders; and 4) states could not afford to expand the existing programs. By 1996, all 50 states were screening for 2 disorders, and most states were screening for about 5 (Table 2). Current NBS The Expanded Newborn Screen The advent of tandem mass spectrometry (MS/ MS) into the clinical laboratory resulted in an explosion in the addition of IEM to the growing field of newborn screening. The technology was first proposed for newborn screening in and began appearing in the clinical arena around the time of the development of crucial methods for the measurement of amino acids and acylcarnitines using the instrument The testing was developed for simultaneous identification of these analytes using dried blood spots, and suddenly approximately 30 IEM could 738 LABMEDICINE j Volume 39 Number 12 j December 2008 labmedicine.com

3 be tested for in a single assay without drawing additional blood from an infant. In 1999, the New England Newborn Screening Program, which performs NBS for Massachusetts, Maine, New Hampshire, Vermont, and Rhode Island, began implementing expanded newborn screening using this technology; by 2008, 40 states had the expanded newborn screen in place and 5 more had it mandated but not yet available. 22 Table 2 shows the change in disorders screened for since As can be seen, very slow progress in adding tests was made between 1996 and Between 2002 and 2008, however, MS/MS became increasingly available and most states now use it to test for multiple disorders. A map of the U.S. with clickable states to access each state s individual NBS program is available at uthscsa.edu. Tandem mass spectrometry works as a mass filter system using 3 mass spectrometers arranged in a series. Pretreated samples are introduced into the first mass spectrometer (MS1), fragmented in the second mass spectrometer (often called a collision cell ), and then the fragments are introduced into the third mass spectrometer (MS3). The instrument is set up to select for fragments with specific masses. The versatility of the instrument lies in its ability to be used to select only specific precursor (parent) masses in MS1, only product (daughter) masses in MS3, or a combination of masses of the precursor and products using MS1 and MS3. This last modality is often referred to as multiple reaction monitoring (MRM) and is a true tandem mass spectrometry method. An example is shown in Figure 1, which depicts 2 representative types of MRM used in MS/MS. Panel A depicts a neutral loss MRM where the amino acids all lose the same size neutral fragment (102). In this scenario, MS1 and MS3 are set to detect precursor and product masses that are 102 mass units apart. Panel B depicts an MRM scheme where the fragmentation in the collision cell results in different sizes of fragments lost. In this type of method, MS1 and MS3 are set up to detect specific mass to charge (m/z) transitions that the molecule undergoes between the 2 mass spectrometers. For example, for phenylalanine the transition would be Newborn screening is performed on 5 blood spot samples collected on filter paper cards specific for this purpose. The current list of tests for most states still includes those separate tests that have been performed before the introduction of MS/MS, including TSH measurement for congenital hypothyroidism, hemoglobin electrophoresis for sickle cell disease, 17-OH-progesterone for congenital adrenal hyperplasia, and galactose- 1-phosphate uridyl-transferase activity for galactosemia. In addition to these disorders and the disorders that can be screened for by MS/MS, 43 states are also screening for cystic fibrosis (CF). Cystic fibrosis is generally screened for by means of a dual screen. An initial test for immunoreactive trypsinogen (IRT) is run. Elevated IRT results are followed by DNA testing via polymerase chain reaction (PCR) for the 25 most common CF mutations. With the introduction of MS/MS into the NBS field, 1 more test has been added: the combined amino acid/acylcarnitine analysis. Due to the nature of the abilities of MS/MS, this additional test has the capability of detecting approximately 30 more IEM. Table 3 delineates the disorders shown to be detectable by a combination amino acid/acylcarnitine screen on the MS/MS and also shows those that are currently recommended to be included on an expanded newborn screen. Those tests which are not currently recommended for inclusion may be detected on the initial screen or as part of a diagnostic follow up, Table 3_Disorders That Can Be Detected by MS/MS Disorder Amino Acids Phenylketonuria (PKU) Maple Syrup Urine Disease (MSUD) Homocystinuria (HCY) Citrullinemia, type 1 (CIT) Argininosuccinic aciduria (ASA) Tyrosinemia, type 1 (TYR I) Hyperphenylalaninemia (H-PHE) Tyrosinemia, types 2 and 3 (TYR II, III) Argininemia (ARG) Citrullinemia, type 2 (CIT II) Methioninemia (MET) Biopterin metabolic defects (BIOPT, BS, and REG) Organic Acids Isovaleric acidemia (IVA) Glutaric acidemia, type 1 (GA I) 3-hydroxy, 3-methylglutaric aciduria (HMG) Multiple carboxylase deficiency (MCD) Methylmalonic acidemia (mutase deficiency) (MUT) Methylmalonic acidemia (B12 disorders) (Cbl A, B) 3-methylcrotonyl CoA carboxylase deficiency (3MCC) Propionic acidemia (PROP) Beta ketothiolase deficiency (BKT) Cobalamin C, D defects (Cbl C,D) Malonic acidemia (MAL) Isobutyryl CoA dehydrogenase deficiency (IBG) 3-methyl, 3-hydroxybutyric acidemia (2M3HBA) 2-methylbutyryl CoA dehydrogenase deficiency (2MBG) 3-methylglutaconic aciduria (3MGA) Fatty Acid Oxidation Medium chain acyl-coa dehydrogenase deficiency (MCAD) Very long chain acyl CoA dehydrogenase deficiency (VLCAD) Long chain L-3-hydroxyacyl CoA dehydrogenase deficiency (LCHAD) Trifunctional protein deficiency (TFP) Carnitine uptake defect (CUD) Short chain acyl CoA dehydrogenase deficiency (SCAD) Medium/short chain L-3-hydroxyacyl CoA dehydrogenase deficiency (M/SCHAD) Medium chain ketoacyl-coa thiolase deficiency (MCKAT) Carnitine palmitoyltransferase 1 and 2 (CPT I and II) Carnitine acylcarnitine translocase deficiency (CACT) Dienoyl-CoA reductase deficiency (DE RED) Glutaric acidemia, type 2 (GA2) Recommended for Inclusion but they may not have a current treatment or the disease process may not be well understood. In general, if one of these disorders is picked up on the screen or follow up, the information is supplied to health care providers; however, they are not officially included as part of an NBS. Tandem MS, with all its capabilities, is still not the magic bullet for identifying all IEM. Problems associated with its use for NBS are recognized and carefully monitored and include issues surrounding amino acids that have identical masses, the setting of cutoff values for abnormal results in different populations and age groups, and the realization that mild defects with some enzyme activity may not be detected and some disorders are detected only when the infant is metabolically decompensated As with any screening test, the purpose is not to diagnose, but to screen for the possible presence of a disorder. Specificity is sacrificed for the sake of sensitivity. A good screening test has a low (ideally 0) false-negative rate so that a normal test means a healthy child. affected children should be missed labmedicine.com December 2008 j Volume 39 Number 12 j LABMEDICINE 739

4 by the test (false-negative), even if many healthy children also have a positive test (false-positive). The rationale for this is that a false-positive result can be sorted out with additional tests. A false-negative result could have devastating consequences. Most newborn screening programs strive to keep their false-positive rate <0.5%. Although the initial cost of MS/MS instruments is fairly high, the greatest cost involved in a newborn screening program is related to the follow up of infants with false-positive test results, both in terms of monetary and emotional costs. The availability of both timely and accurate follow-up testing with the ability to confirm or rule out the diagnosis of any disorder picked up in the screen is absolutely necessary. The responsibility of the NBS program does not end with the report of positive results. They must have the ability to direct the parents to centers where follow up is available. Probably the most contentious criterion for inclusion of a disorder in a NBS program is whether or not to report disorders that can currently be detected but are not currently treatable. Before the advent of a technology allowing even extremely rare disorders to be detected with no additional expense on the front end, these disorders would not have been considered for inclusion in an NBS program. w that the disorders can be detected as part of an expanded screen, the issue becomes whether to report and follow up on an untreatable disorder, taking into consideration the monetary and emotional costs and the fact that nothing can be done. The major arguments in favor of reporting these disorders are that with increasing technological and genetic advances, treatment may become available. Also, genetic counseling about the current state of knowledge could be vital, especially if future pregnancies are planned. The Future of Newborn Screening A secondary driver of this explosion in the field of NBS was the increasingly computer-literate public and the ability to use the Internet to learn about IEM and testing for IEM. 26 As the general public has become able to easily find information about IEM, including such issues as testing, treatment, signs and symptoms, and prognosis, the demand for expanded NBS has gone public. To a large extent, the rapid expansion of the NBS programs to incorporate MS/MS technology and provide better IEM screening has been driven by demand from the private sector. Overall, the expanded NBS can be considered a success story. Texas began an expanded NBS program in January 2007 using MS/MS technology. In the first 9 months the program operated, our hospital, which is a center for follow-up testing, had diagnosed 13 IEM, 11 of them presymptomatically. Before the use of the expanded screen, 9 of these disorders would not have been diagnosed before the infant presented with symptoms. The ability to detect and diagnose IEM presymptomatically has affected disease management. Genetics departments must now manage and monitor patients who have not demonstrated any symptoms, and who now may never show any. Paradigms used in the past with very sick, symptomatic infants must be adapted for asymptomatic, well infants. New treatment and monitoring paradigms must be instituted. The benefits achieved by beginning treatment before the infant gets sick, however, cannot be overstated. Lives are saved or made better by having this system in place. Tandem MS techniques and analyte tests continue to be developed and suggested for use as part of the NBS program. The future of NBS may lie in a marriage of MS/MS and molecular testing as the specific gene defects are being elucidated. Cystic fibrosis is the first IEM to be screened for using molecular techniques, specifically PCR. However, with the increasing introduction of molecular techniques into the clinical laboratory, and the technological advances being made in laboratory testing, the likelihood will be that MS/MS technology will continue to be used for initial screens, but confirmation will be done via DNA sequencing for the defective gene. Also, the continuing growth and development of such fields as proteomics and genomics may lead to new innovations in screening methods. Multiplex testing of any kind, with its ability to look at patterns and multiple analytes rather than single disorders and single analytes has immense potential as a screening technique for NBS. The future may allow the interpretation of a pattern of protein expression to be used as a screening tool for IEM. Technology continues to drive our understanding of metabolism as new testing techniques more clearly elucidate metabolic pathways, discover new IEM, and suggest new ways to treat them. Newborn screening will probably continue to expand beyond the expanded newborn screen and into the arena of genetic wellness newborn screening. As with all new technologies, the advent of the MS/MS expanded newborn screen has brought many issues to be resolved, but the benefits far outweigh the disadvantages. Continuing advances will no doubt prove to be as beneficial as MS/MS has been. LM 1. Garrod AE. The incidence of Alkaptonuria: A study in chemical individuality. Lancet. December 13,1902: Guthrie R, Susi A. A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics. 1963;32: Guthrie R. The origin of newborn screening. Screening. 1992;1: American Academy of Pediatrics Newborn Screening Task Force. Serving the Family from Birth to the Medical Home. 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