Newborn screening for Pompe disease: An update, 2011^†

INTRODUCTION

Newborn screening is a public health initiative designed to identify conditions in the newborn infant for which the institution of interventions prior to clinical diagnosis may improve outcome. Newborn screening programs are conducted within state health departments and each state determines the conditions that will be included and the methods that will be utilized for their detection. Historically, newborn screening has been reserved for those conditions for which the screened infant would likely benefit directly from presymptomatic treatment. Increasingly, newborn screening advocates are arguing that other benefits of newborn screening, such as genetic counseling for the parents with prevention of the birth of affected siblings and avoidance of the diagnostic odyssey that often accompanies rare disorders, should be considered. In addition to consideration of the benefits of early diagnosis, disorders are generally considered for inclusion in newborn screening only if they are sufficiently common in the general population to justify screening all newborns and there is a test that can be applied to dried blood spot newborn screening specimens with acceptable cost. Until fairly recently, there was considerable variability from state to state in the number of conditions included in the newborn screening panel. This began to change in the early 1990s with the development of tandem mass spectrometry (MS/MS), a technique that allows for the detection of over 30 different inherited metabolic disorders [Millington et al., 1990]. In the 2000s, an expert panel was convened by the American College of Medical Genetics (ACMG) in response to a request from the Health Resources and Service Administration (HRSA) to evaluate a large number of conditions and make recommendations regarding those disorders that should be included in newborn screening programs. The report, which was subjected to considerable public and professional scrutiny and recommended a uniform panel of 29 disorders, was accepted and endorsed in September 2005 by the Advisory Committee on Heritable Disorders in Newborns and Children formed in 2003 to advise the Secretary of the US Department of Health and Human Services in areas related to heritable conditions in children, especially newborn screening [http://www.hrsa.gov/heritabledisorderscommittee/reports]. The report was published in 2006 [Watson et al., 2006] and, within a very short period of time, all 50 states had adopted the recommendations and were moving toward implementation of screening for all 29 conditions. During their deliberations, the ACMG expert panel considered Pompe disease along with several other lysosomal storage disorders. None of these disorders were considered suitable for inclusion at the time of the report, primarily because of the lack of a testing method suitable for implementation in the public health setting using dried blood spots.

POMPE DISEASE AND BENEFITS OF TREATMENT

Pompe disease is an autosomal recessive lysosomal storage disorder resulting from a deficiency of the enzyme acid α-glucosidase (GAA). The estimated incidence of the disorder is 1 in 40,000 births [Martiniuk et al., 1998; Ausems et al., 1999]. It is associated with progressive accumulation of glycogen within the lysosomes of cells, most notably in skeletal muscle and heart. Although the disorder exhibits a broad spectrum of severity and variable age of onset, it is typically divided into the infantile and later onset forms. The natural history of the infantile form has been well characterized [Kishnani et al., 2006]. Infants typically develop symptoms by 2 months of age, are diagnosed at about 5 months and die at a median age of 9 months.

Hypertrophic cardiomyopathy is the dominant feature, although hypotonia and muscle weakness are common. The natural history of later onset Pompe disease is less well defined. It is highly variable in age of onset, presentation, and rate of progression [Winkel et al., 2005]. The disorder is characterized by progressive muscle weakness and respiratory insufficiency with cardiomyopathy not typically being observed.

Alglucosidase alfa, a recombinant form of human GAA (rhGAA, Myozyme®, Genzyme Corporation, Cambridge, MA) was approved by the US Food and Drug Administration (FDA) in 2006 for the treatment of Pompe disease. It is clear from published trials that treatment with rhGAA prolongs survival and ventilator-free survival and improves motor function in infantile Pompe disease

Alglucosidase alfa, a recombinant form of human GAA (rhGAA, Myozyme®, Genzyme Corporation) was approved by the US Food and Drug Administration (FDA) in 2006 for the treatment of Pompe disease. It is clear from published trials that treatment with rhGAA prolongs survival and ventilator-free survival and improves motor function in infantile Pompe disease.

[Amalfitano et al., 2001; Kishnani et al., 2007]. Response to therapy is highly variable, however, and appears to be suboptimal in patients who produce no cross-reacting material to the GAA enzyme (CRIM) and in those who have persistently high antibody titers to rhGAA [Kishnani et al., 2010]. Immunomodulation has been reported to be effective in some CRIM-negative patients in preventing the development of high titer antibodies and it is likely that this will result in improved outcomes [Mendelsohn et al., 2009].

In 2010, the FDA approved a second form of rhGAA (Lumizyme®, Genzyme Corporation) for treatment of patients 8 years of age or older with later onset Pompe disease who do not have evidence of cardiac hypertrophy. Clinical trials of rhGAA therapy in patients with later onset Pompe disease have documented stabilization of neuromuscular deficits and mild functional improvement [Strothotte et al., 2010]. There have been no published studies addressing the impact of treatment in presymptomatic patients with later onset Pompe disease.

LABORATORY METHODS POTENTIALLY APPLICABLE TO NEWBORN SCREENING FOR POMPE DISEASE

The possibility of performing newborn screening for Pompe disease was first suggested by Chamoles et al. [2004], who described a method for the enzymatic diagnosis of the disorder using a dried blood spot. Based on this concept, Gelb et al. [2006] developed a tandem mass spectrometry based assay for Pompe disease that could be performed simultaneously with assay of four other lysosomal enzymes in separate reaction mixtures, with the reaction products pooled for analysis on a single MS/MS run. Studies using normal controls, patients with Pompe disease and carriers suggest that affected patients can be accurately separated from unaffected individuals using this approach. Subsequent modifications of this approach have been reported which allow for both multiplex assay in a single buffer and simultaneous analysis of enzyme products for either three [Spacil et al., 2011] or six [Metz et al., 2011] lysosomal storage disorders, including Pompe disease. These modifications have reduced analytical time and reagent costs while maintaining a high degree of sensitivity and specificity in validation studies.

A second method potentially applicable to newborn screening for Pompe disease is the immunologic approach described by Parkinson-Lawrence et al. [2006]. Instead of enzyme activity, this method quantitates the amount of enzyme protein present in the sample using a multiplex microbead array technology. Although this method can identify a number of other lysosomal storage disorders in dried blood samples, Pompe disease could only be identified in the published series using protein ratios. If the disorder results in a given patient in a normal or near normal amount of mutant enzyme protein, despite deficient activity, it could not be detected using this method.

The third method for the detection of Pompe disease in dried blood spots is the traditional fluorometric method. This is the method originally utilized by Chamoles in his early reports and relies on the use of the same fluorometric substrate used in diagnostic assays in whole blood or tissues. It is not suitable for simultaneous assay of multiple lysosomal enzymes in the same reaction mixture. More recently, a novel approach to newborn screening for Pompe disease and other lysosomal storage disorders based on the use of the fluorometric substrate has been described. This is the digital microfluidic method which utilizes a disposable microchip programmed to dispense, transport, mix, wash, and incubate individual microdroplets from dried blood spot extracts and reagents under software control

More recently, a novel approach to newborn screening for Pompe disease and other lysosomal storage disorders based on the use of the fluorometric substrate has been described. This is the digital microfluidic method which utilizes a disposable microchip programmed to dispense, transport, mix, wash, and incubate individual microdroplets from dried blood spot extracts and reagents under software control.

[Millington et al., 2010]. There are no published data on the use of this method for the newborn screening of Pompe disease in dried blood spots but validation studies suggest that patient samples can be accurately separated from those of unaffected individuals.

At present, only the fluorometric method has been used in a prospective newborn screening program for Pompe disease so the sensitivity and specificity of the other methods is unknown [Chien et al., 2008, 2011]. A study is currently in progress at the Mayo Clinic in which all of the available screening methods will be compared in a series of 100,000 anonymized newborn screening blood spots in order to determine the most effective strategy for newborn screening for Pompe disease and other lysosomal storage disorders [Matern, 2008]. Abnormal test results will be confirmed by second tier enzymatic assay and/or GAA sequencing.

TAIWANESE NEWBORN SCREENING PROGRAM FOR POMPE DISEASE

Data have been reported thus far from only one prospective newborn screening program for Pompe disease. In 2008, Chien et al. [2008] reported the results of a screening program conducted between October 2005 and March 2007 involving about 45% of all infants born in Taiwan during that time period. Enzymatic activity was assayed in dried blood spots using the fluorometric method. Of 132,538 infants screened, 1,093 (0.82%) required a repeat sample for dried blood spot assay and 121 (0.091%) were referred for further evaluation. This relatively high false positive rate was the result, at least in part, of a high frequency of a pseudodeficiency allele in the Taiwanese population [Labrousse et al., 2010] and of the study design. In 2005, the assay cut-off was set to make sure that no patients were missed; it was adjusted in January 2007 to the current recall rate of 0.4% [Paul Wuh-Liang Hwu, personal communication]. Pompe disease was confirmed in four infants in this reported series. Among unscreened infants born in Taiwan during the same time period, three cases of Pompe disease were diagnosed. The age of diagnosis in the screened infants was less than 1 month as compared to 3–6 months in the unscreened infants. Subsequent data from the same screening program demonstrated that patients with infantile Pompe disease diagnosed through newborn screening and treated for 14–32 months with enzyme replacement therapy had normalization of cardiac size and muscle pathology with normal physical growth and age-appropriate motor development [Chien et al., 2009]. Survival in this screened cohort was significantly improved as compared with an untreated historical cohort (P = 0.0011) and improved but not significantly different from that observed in clinically diagnosed infants during the same time period (P = 0.4795). A recent report from the Taiwan screening program indicated that, among 344,056 infants screened, 13 were diagnosed with later onset Pompe disease (defined as deficient GAA activity and two identifiable GAA mutations but no cardiomyopathy) [Chien et al., 2011]. These infants were followed every 3–6 months with physical and neurological exams and serial creatine kinase (CK) determinations. During a follow-up period of up to 4 years, four patients were started on enzyme replacement therapy because of hypotonia, muscle weakness, delayed development, or elevated CK levels, beginning at 1.5, 14, 34, and 36 months, respectively. The screening program in Taiwan is ongoing and it is anticipated that further data will be generated to assess the benefits of newborn screening in improving prognosis in infantile and later onset Pompe disease.

POMPE DISEASE NEWBORN SCREENING IN THE UNITED STATES

A number of pilot screening programs for Pompe disease are currently underway or are in the planning stages in the United States. A pilot project funded by the National Institutes of Health is being conducted at the Washington State Newborn Screening Laboratory using anonymized samples. A triplex MS/MS assay for Pompe and Fabry diseases and mucopolysaccharidosis type 1 is being utilized. Positive results are confirmed by molecular testing. From the first 50,000 dried blood spots analyzed, two cases of Pompe disease have been identified [Scott, 2011]. Three states have passed legislation mandating the addition of Pompe disease to their newborn screening panels. The first to do so was Illinois where a bill was signed into law in November 2007, requiring the addition of five lysosomal storage disorders, including Pompe disease, to the newborn screening panel within 3 years. In November 2010, a pilot screening program was initiated using the digital microfluidic method to identify three of the five mandated lysosomal storage disorders simultaneously (Pompe, Fabry, and Gaucher diseases). Among the first 8,012 samples analyzed, only two screen positive samples for Pompe disease were identified. Both infants were found to be normal on confirmatory testing. The trial in Illinois was suspended in April 2011, when difficulties were encountered in scaling up the microfluidic assay for high throughput and statewide screening. A second pilot is anticipated, beginning in early 2012, using the multiplex MS/MS assay. Following the lead of Illinois, legislation was passed in 2009 in the state of Missouri mandating newborn screening for the same five lysosomal storage disorders included in the Illinois legislation. The law in Missouri requires screening to begin by July 2012 and a pilot project is scheduled to begin in January 2012. The final state to pass legislation mandating newborn screening for lysosomal storage disorders is New Mexico in which a law was passed in 2010 also requiring newborn screening for Pompe disease and four other lysosomal storage disorders. It is not clear when newborn screening will begin in New Mexico, however, since the state of New Mexico currently contracts with Oregon for the laboratory aspects of newborn metabolic screening and Oregon does not currently perform testing for Pompe disease.

SUMMARY

In 2007, Kemper et al. [2007] reviewed the evidence supporting newborn screening for Pompe disease. They concluded that newborn screening was likely to be of substantial benefit because of the clear benefit of alglucosidase alfa for children with infantile Pompe disease. However, they noted significant concerns regarding screening accuracy, the management of cases of later onset Pompe disease and the challenges of informing parents regarding the potential benefits and harms of newborn screening. They recommended pilot studies to address these concerns. Since that time, the Taiwanese screening program has provided substantial additional data regarding the benefits of newborn screening and the management of later onset cases. More accurate methods of screening for Pompe disease with lower false positive rates have been described. Within the next several years, the emerging pilot programs in the US will provide data on the sensitivity and specificity of newborn screening for this disorder and on benefits to the screened population. There are challenges to be met, to be sure, but challenges have been encountered many times before in the newborn screening arena. All of the potential concerns that have been raised related to Pompe disease newborn screening have been voiced previously with regard to other newborn screening disorders.

Within the next several years, the emerging pilot programs in the US will provide data on the sensitivity and specificity of newborn screening for this disorder and on benefits to the screened population. There are challenges to be met, to be sure, but challenges have been encountered many times before in the newborn screening arena. All of the potential concerns that have been raised related to Pompe disease newborn screening have been voiced previously with regard to other newborn screening disorders.

They are not insurmountable and they do not negate the potential of screening to save the lives of children with infantile Pompe disease and potentially improve the outcome of patients with later onset Pompe disease by facilitating earlier diagnosis and treatment. The time has come for universal screening for Pompe disease. If the developing screening programs in the US perform well and share their experiences quickly with the newborn screening community, this goal may soon be realized.