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Editorials |
National Human Genome Research Institute, NIH, Bethesda, MD
Address correspondence to the author at:, National Human Genome Research Institute, NIH, Rm. 10C-103, MSC 1851, Bldg. 10, 10 Center Dr., Bethesda, MD 20892-1851, Fax 301-402-2740, E-mail bgahl{at}helix.nih.gov
Detailed criteria for newborn screening programs have been formulated (1), but the basic principles include requirements that the screened disorder be medically serious, that effective therapy be available, and that the cost of detection plus treatment be justifiable (2). Wilson disease, a metal-storage disorder in which copper cannot be properly excreted in the bile and fails to be incorporated into ceruloplasmin (3), meets these criteria. Wilson disease is caused by biallelic mutations in ATP7B1 (ATPase, Cu++ transporting, beta polypeptide), which encodes a copper-transporting ATPase located in the trans-Golgi network (4). Typically, Wilson disease manifests in adulthood with liver disease, neurologic impairment, or both (3). These complications impart chronic morbidity and mortality to affected individuals; in addition, intravascular hemolysis induced by overly aggressive chelation can cause acute renal failure and death. Therapy with a copper-restricted diet and chelation has proved efficacious; early treatment provides additional benefit, given that the hepatic and neurologic damage eventually becomes irreversible. Finally, with an approximate incidence of 1 in 30 000 live births, Wilson disease appears economically suitable for newborn screening, particularly in populations with high frequencies of the disorder (5).
The key issue in newborn screening for Wilson disease has been the method of detection. Theoretically, screening could rely on blood copper measurements, because 90% of circulating copper is bound to ceruloplasmin (3) and ceruloplasmin concentrations are low in Wilson disease. Copper is assayed by atomic absorption, however, and this methodology is not widely available. Therefore, investigators have chosen to screen for Wilson disease by measuring ceruloplasmin directly by ELISAs that use antibodies to ceruloplasmin. A complicating factor is that ceruloplasmin concentrations in infants and newborns are considerably lower than in adults. Hence, initial attempts to use ceruloplasmin concentrations to screen newborns in Japan were unsuccessful (6), and the Korean experience indicated a recommendation for screening at 3 months of age (7). Subsequently, however, Kroll et al. demonstrated that ceruloplasmin values in newborn blood spots were lower for Wilson disease patients than for healthy control individuals (8). Nevertheless, the degree of variation in both ceruloplasmin antibody binding and ceruloplasmin values in healthy controls has made these detection techniques less than satisfying.
deWilde et al. (9) have pursued an antibody- independent approach and in this issue of Clinical Chemistry present a method that uses liquid chromatography–tandem mass spectrometry (LC-MS/MS)2 to quantify ceruloplasmin by its tryptic-digest fingerprint. These authors performed all of the requisite control experiments, including determination of signature peptides, optimization of tryptic digestion, standardization of sample preparation, use of internal standards, establishment of LC-MS/MS conditions, preparation of a calibration curve, determination of imprecision, and validation with samples from patients with Wilson disease. The results resemble those previously obtained with the ELISA assay, i.e., ceruloplasmin values of 0–2 mg/dL (0–20 mg/L) for Wilson disease patients, compared with values >20 mg/dL for Wilson disease carriers (7).
What are the advantages of this new LC-MS/MS method for detecting patients with Wilson disease? First, it frees screening from the variation produced by the use of ceruloplasmin antibodies. Second, it uses blood spots without enrichment or modification. Third, it uses equipment already largely available in newborn-screening laboratories. The method is also relatively rapid.
Yet some reservations remain. The mettle of the LC-MS/MS methodology must be tested in the hands of other investigators. Larger trials should reveal how often the authors’ signal-to-noise ratio threshold of >10 fails to be met. Finally, screeners will have to address the 5% of Wilson disease patients who have typical ceruloplasmin concentrations typical for healthy controls (10).
This report has additional implications. For example, the LC-MS/MS method should be able to detect individuals with Menkes disease, an X-linked disorder in which the ceruloplasmin concentration is low because a deficiency in another copper-transporting ATPase reduces the amount of all copper-containing proteins (3)(11). Newborn screening for Menkes disease could be important, because it is a devastating and fatal neurologic disorder and a promising treatment exists (12). On the other hand, Menkes disease is relatively easy to diagnose clinically, unlike Wilson disease.
Perhaps the greatest contribution of this study, however, is its demonstration of the efficacy of LC-MS/MS analysis of peptides for newborn screening of proteins that are deficient in specific disorders. The method has limitations, of course, including the inability to detect proteins of low abundance. Ceruloplasmin is present in concentrations of tens of milligrams per deciliter, allowing ceruloplasmin quantification from a filter-paper blood spot. Other proteins that could reflect the basic defect in a metabolic disease, however, may be present only in blood leukocytes, making their concentrations several orders of magnitude lower. Hence, one could not use tryptic-peptide analysis to directly measure, for example, the ATP7B gene product, which could be present in Wilson disease patients anyway, if they had missense mutations.
Nevertheless, for circulating proteins at sufficiently high abundance, the LC-MS/MS method of detection provides a potentially important advance for the field of newborn screening. We await the application of this method to other disorders. Perhaps its limit of detection can be improved to detect less abundant proteins that serve as markers of disease by virtue of either their absence or their presence.
Acknowledgments
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors’ Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: This work was supported by the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland.
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played a direct role in the preparation and approval of the manuscript.
Footnotes
1 Human genes: ATP7B, ATPase, Cu++ transporting, beta polypeptide. 
2 Nonstandard abbreviations: LC-MS/MS, liquid chromatography–tandem mass spectrometry.
References
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