Pompe disease (PD, OMIM #232300) and Mucopolysaccharidosis Type I (MPS I, OMIM #607014) are the first two lysosomal storage disorders to be added to the Recommended Uniform Screening Panel (RUSP) for newborn screening (NBS) in the United States [1
]. Pompe disease is a progressive, autosomal recessive lysosomal storage disorder primarily affecting skeletal and cardiac muscle. It presents with varying degrees of severity, ranging from an infantile form with cardiomyopathy and weakness before 12 months of life to later onset forms which present primarily with proximal muscle weakness, without cardiomyopathy [2
]. MPS I is also a progressive, autosomal recessive lysosomal storage disorder with multiorgan involvement. There is great clinical variability in MPS I, with severe infantile forms that can result in death during the first decade of life and attenuated forms with significant morbidity but a near normal life span [4
]. Enzyme replacement therapy is available for both disorders, and stem cell transplants are utilized to care for patients with severe MPS I. Improved outcomes have been reported with early intervention for both disorders [5
NBS for both disorders is fully underway in many states, however it is not yet universal across the United States. These disorders are identified by measuring the activity of acid alpha-glucosidase (GAA, deficient in PD) and alpha-iduronidase (IDUA, deficient in MPS I) from dried blood spots (DBS) [5
]. From a laboratory perspective, screening for PD and MPS I require the introduction of additional technology into the NBS lab, either MS/MS based enzyme assays [3
] or digital microfluidics [6
]. Primary screening using enzyme assays has been hampered by clinically benign pseudodeficiencies, particularly for MPS I [11
]. Post-analytical tools, using the Collaborative Laboratory Integrated Reports (CLIR; https://clir.mayo.edu/
) and its predecessor, Region 4 Stork (R4S) have been utilized with MS/MS screening for lysosomal storage disorders [1
] and other inherited metabolic disorders [13
] to improve laboratory performance over what had been previously reported. Second tier tests with high specificity, such as the quantification of glycosaminoglycans for MPSI and creatine/creatinine ratios for PD can also improve performance [1
]. As screening panels expand across the country, care must be taken to balance the identification of true positive cases with the burden of false positive (FP) cases, both due to the impact on families who are notified of screening results, and to the health care system which must deal with these cases quickly and thoroughly.
The National Institutes of Health (NIH) have developed a program to fund states to perform pilot studies and provide information that may allow for more efficient implementation around the country. New disorders, and new technologies can be difficult to implement. Information about successful strategies can be invaluable when decisions are being made.
In the United States, Missouri has the longest history of screening for a panel of lysosomal storage disorders, having screened for a panel of five LSDs by digital microfluidics, including Pompe and MPS I, since 2013 [6
]. Published data of approximately 308,000 screened infants, showed 32 cases classified as true positive for PD and 9 genotypes of uncertain significance. For MPS I, there were two confirmed cases and 2 genotypes of uncertain significance [6
]. During the screening, there were 161 positive screens for Pompe disease and 133 for MPS I [15
]. Results have also been recently published for early screening performed on infants born in Illinois [7
], Kentucky [1
], North Carolina [8
], and New York [5
]. Summary data for the performance of each of these states is shown in Table 1
2. Materials and Methods
As part of the task orders for funding, the request was to screen 60,000 infants for each disorder. Initially, the decision was made to screen using digital microfluidics based enzyme assays. There were several delays with this system obtaining the appropriate regulatory approvals, as it was classified as Investigational Use Only and awaiting clearance from the Food and Drug Administration. Proceeding with this assay would have required consent under federal rules in place at the time and this was not feasible for our project and budget. Screening commenced using a laboratory developed test (LDT) with tandem mass spectrometry (MS/MS) as the detection system. This assay has been well described elsewhere [1
]. We chose a custom two tier screening strategy not previously utilized by other states, and obtained custom substrate and internal standard mixes for this scheme (Perkin Elmer). The decision to proceed with the customized two-tier screening strategy was based on optimizing the screening costs and minimizing FP screens. Each enzyme screened in the reagent cocktail adds a fixed, incremental amount to the cost of the assay due to the substrate/internal standard combination (approximately $
1/enzyme). The remainder of the reagents used in the sample preparation are inexpensive, and do not change with the increase in disorders. Using six enzymes in the initial step would have resulted in a 3-fold increase in fixed costs (3X more enzymes in 60,000 samples = ~$
240,000 increase in reagent costs) and raised ethical questions about testing for enzymatic deficiencies but not reporting them, which we wanted to avoid. Including the 6-plex assay as a second-tier test provided a cost-effective testing strategy to reduce FP screens, while minimizing the chances of an off-target finding for one of the other enzyme analyzed.
The initial step of screening utilized a two-plex assay, measuring only GAA and IDUA activities. Any screen positives were re-analyzed using an expanded panel of six enzymes (additional enzymes included: Alpha-galactosidase (Fabry disease), acid sphingomyelinase (Niemann-Pick A/B), beta-glucosidase (Gaucher disease) and galactocerebrosidase (Krabbe disease)). The second-tier test was done on the same DBS sample. The screening algorithm developed for this program is shown in Figure 1
. Data about more advanced second tier tests (dermatan and heparan sulfate in blood spots for MPS I [1
], and creatine ratios for PD [14
]) had not been published at the time this study was designed (2016).
Specimens were punched (⅛” punch) at the state public health laboratory and transported to the testing lab (EGL Genetics, Tucker, GA, USA; CAP/CLIA certified). Decisions about specimen quality were made by state NBS staff, using the same criteria for all other disorders screened for in Georgia. All acceptable specimens with sufficient sample remaining during the study period (January 2017–June 2017) were included in this study.
Post-analytical tools, using the Collaborative Laboratory Integrated Reports platform (CLIR; https://clir.mayo.edu/
) were developed for each tier of testing. Site specific tools were created for Georgia’s screening panel, including single condition tools (PD and MPS I) for the 2-plex and 6-plex assays, and a dual scatter plot for each condition utilizing the 6-plex assay. The post-analytical tool for the first-tier test utilized the enzyme activities for GAA and IDUA, as well as the ratio between the two. The post-analytical tools for the second-tier text (6-plex) utilized the targeted enzyme for the condition, and the ratios of the other five enzymes to the targeted enzyme). Dual scatter plots utilized the same group of analytes and ratios, and the population of FP screens in the database. The interpretation algorithm used for this study is shown in Figure 2
. Dual scatter plots were not used with the first tier of testing during this study to send as many samples as possible for the second-tier test, to evaluate the performance as broadly as possible. Both tiers of testing were completed from the original newborn screening sample. This strategy was designed to minimize FP screens, and reduce undue burden on the NBS system and to avoid unnecessary stress on families.
After review with the Institutional Review Boards of Emory University and the Georgia Department of Public Health, this study was deemed not to be research, as the conditions were already recommended for inclusion in the Uniform Newborn Screening Panel at the federal level and this was a study to evaluate their implementation in Georgia. The review boards determined that informed consent was not required, and the testing was conducted on all specimens submitted for routine NBS testing. Georgia’s Newborn Screening Advisory Committee also reviewed the pilot study proposal and approved.
There are several screening strategies that have been proposed for use with the enzyme assays used for LSDs in NBS. Post-analytical tools, fixed cutoffs and cutoffs based on the daily mean have all been used. Based on previously published data, and the results of our study (Table 1
), any evidence based, appropriately validated screening strategy should detect all true positive cases reliably, with variations in the number of FP results introduced into the screening system. For MPS I, the most effective second tier test is likely quantitation of dermatan and heparan sulfate in blood spots, as used in the screening of infants born in Kentucky [1
]. Due to the high prevalence of pseudodeficiency alleles, and the reduction in enzyme activity associated with them, additional enzymes as a second tier test was not sufficient to reduce FP screens. North Carolina’s MPS I post hoc analysis of their screening showed similar performance to Georgia, showing that sequence analysis of IDUA
as a second tier test did not reduce FP results beyond additional enzymes being analyzed [8
]. For PD, the PPV was significantly better than states who screened using some variation of cutoffs, whether it was a fixed cutoff or the percentage of the daily mean [7
]. The PPV for PD compared favorably to the results of screening Kentucky newborns performed by Mayo Clinic using similar post-analytical tools [1
As new conditions are added to screening panels aggressive management of laboratory performance particularly with respect to PPV, needs to be considered. Our 2-plex approach for the first-tier test offers cost-savings compared to the first tier 6-plex approach utilized for the Kentucky screening, as the reagent cost of the 2-plex is approximately ⅓ of the 6-plex. Other variations of the second-tier test (with 3, 4 or 5 enzymes) are possible, however there is unlikely to be a significant decrease in total costs, if this strategy refers more children for follow-up. The power of post-analytical tools is greatest when multiple sites can collaborate and share data to increase the population of cases for rare disease.
The pilot study provided valuable data for the decision makers involved with Georgia’s NBS program. In May 2018, the Commissioner of Public Health approved the recommendation for these conditions to be added to the state’s NBS screening panel, contingent upon proper funding being provided for the screening and follow-up process, including molecular testing where appropriate. This funding was approved in the budget for fiscal year 2020 (July 2019–June 2020). The difficulty in getting insurance approvals for timely molecular analysis of IDUA was one of the major roadblocks encountered in this study, and the inclusion of funding from the NBS program should resolve this and ensure appropriate follow-up for all infants identified by NBS. Since the conclusion of the pilot study, at least two patients with infantile PD and two patients with early onset MPS I were born in Georgia. All of these cases came to the attention of the medical genetics clinic at Emory University and all had a significant gap between birth and diagnosis. The clinical identification of these patients during the post-pilot period has increased confidence that there were no FN for infantile onset disease during the pilot study. Missed cases of later onset forms may not be ascertained for years.
A two-tiered screening strategy offers several advantages in the NBS setting. The use of a lower specificity test on the first tier allows for aggressive filtering based on these results to identify possible true positive cases, which can be refined by proper use of the second tier test to only report out those cases with the highest probability of being a true positive. This strategy has proven effective for cystic fibrosis, congenital adrenal hyperplasia, maple syrup urine disease, and remethylation disorders [20
]. Given the potential burden to the health system of screen positives for LSDs, reduction of FP screens should be a priority. The screening strategy we utilized for our pilot study combines a lower specificity first tier test with a more expensive, and higher specificity second tier test, and post-analytical tools to take advantage of multiple analytes included in screening. The most effective screening strategy for PD and MPS I differed in this study. Our strategy utilizing an expanded panel of enzymes with post-analytical tools provided good performance for PD, but resulted in a high number of FP results for MPS I. Based on this study, and other published NBS results, the most effective second tier test for MPS I is likely LC-MS/MS analysis of dermatan and heparan sulfate [1
]. These strategies can result in savings in the NBS lab setting, which is significant due to the fact that many NBS labs do not have direct control over their fees. While the pilot studies were successful in Georgia, full screening has not yet been implemented. One of the barriers to implementation was the difficulty in obtaining insurance coverage for molecular testing required to resolve all screen positive cases in an appropriate timeline. We also experienced issues with follow-up by families when presented with uncertain results and possible late onset conditions. This information was valuable for the state NBSAC in making its recommendations to add conditions and provide funding for testing to the Commissioner of Public Health.