Joseph J. Orsini

Newborn screening for Krabbe disease: the New York State model.

Krabbe disease is a rare inherited neurologic disorder affecting the central and peripheral nervous systems. The disease has four phenotypes: early infantile, later onset, adolescent, and adult. The only known treatment is hematopoietic stem cell transplantation, which is, in the early infantile form of the disease, most beneficial if performed before onset of clinical symptoms. In August 2006, New York State began screening all newborns for Krabbe disease. A rapid and accurate technique for assessing galactocerebrosidase activity and performing DNA mutation analysis had been developed. Interpreting these results was limited, however, because neither enzyme activity nor genetic mutation reliably predicts phenotype. A series of initiatives were therefore developed by a multidisciplinary group of neurologists, geneticists, metabolic pediatricians, neurodevelopmental pediatricians, and transplant physicians (the Krabbe Consortium of New York State) to enhance the effectiveness of the newborn screening program. A standardized clinical evaluation protocol was designed based on the available literature, criteria for transplantation for the early infantile phenotype were formulated, a clinical database and registry was developed, and a study of developmental and functional outcomes was instituted. This multidisciplinary standardized approach to evaluating infants who have positive results on newborn screening may serve as a model for other states as they begin the process of screening for Krabbe disease and other lysosomal storage disorders.

Development and Evaluation of Quality Control Dried Blood Spot Materials in Newborn Screening for Lysosomal Storage Disorders

BACKGROUND Lysosomal storage disorders (LSDs) comprise more than 40 genetic diseases that result in the accumulation of products that would normally be degraded by lysosomal enzymes. A tandem mass spectrometry (MS/MS)-based method is available for newborn screening for 5 LSDs, and many laboratories are initiating pilot studies to evaluate the incorporation of this method into their screening panels. We developed and evaluated dried blood spot (DBS) QC materials for LSDs and used the MS/MS method to investigate their suitability for LSD QC monitoring. METHODS We incubated 3.2-mm punches from DBS controls for 20-24 h with assay cocktails containing substrate and internal standard. Using MS/MS, we quantified the resulting product and internal standard. Samples were run in triplicate for 3 consecutive days, and results were reported as product-to-internal standard ratios and enzyme activity units (micromol/L/h). RESULTS Enzyme activity interday imprecision (CV) for the high, medium, and low series were 3.4%-14.3% for galactocerebroside alpha-galactosidase, 6.8%-24.6% for acid alpha-galactosidase A, 7.36%-22.1% for acid sphingomyelinase, 6.2%-26.2% for acid alpha-glucocerebrosidase, and 7.0%-24.8% for lysosomal acid alpha-glucosidase (n = 9). In addition, DBS stored at -20 degrees and 4 degrees C showed minimal enzyme activity loss over a 187-d period. DBS stored at 37 degrees and 45 degrees C had lower activity values over the 187-day evaluation time. CONCLUSIONS Suitable QC materials for newborn screening of LSDs were developed for laboratories performing DBS LSD screening. Good material linearity was observed, with goodness-of-fit values of 0.953 and higher. The QC materials may be used by screening laboratories that perform LSD analysis by MS and/or more conventional fluorescence-based screening methods.

Newborn screening for Fabry disease by measuring GLA activity using tandem mass spectrometry

BACKGROUND Fabry disease (FD) is an X-linked lysosomal storage disorder caused by the deficiency of alpha-galactosidase A (GLA). We evaluated a tandem mass spectrometry method to measure GLA activity. METHODS One 3.2mm punch from a dried blood spot sample (DBS) was incubated with substrate and internal standard in the reaction buffer for 22 h. The resulting product was quantified against internal standard using MS/MS. RESULTS The median GLA activity of male newborn DBS (N=5025) was 9.85 + or - 6.4 micromol/h/l (CI 95% is 9.67-10.02 micromol/h/l); The median GLA activity of female newborns (N=4677) was 10.2 + or - 6.3 micromol/h/l (CI 95% is 10.02-10.38 micromol/h/l). The difference between the two subgroups is within assay analytical variation. The GLA activities in the DBS samples from 9 juvenile and adult males with previously identified FD were below 1.64 micromol/h/l. The GLA activities from 32 juvenile and adult females with confirmed FD were below 4.73 micromol/h/l. In 5 (16%) females GLA activities were above the 0.5th percentile of lower limit of CI 95% at 3.18 micromol/h/l. CONCLUSIONS The MS/MS method for Fabry disease newborn screening is robust and can be readily multiplexed with other lysosomal disorders such as Pompe, Gaucher, Niemann-Pick, and Krabbe diseases.

Implementation of newborn screening for Krabbe disease: Population study and cutoff determination

OBJECTIVE The aim of this study was to develop a newborn screening algorithm for Krabbe disease. DESIGN AND METHODS We measured the galactocerebrosidase activity of 139,074 anonymous newborns, 56 known carriers, and 16 Krabbe patients using a tandem mass spectrometry method. The activities were converted to percentages of daily mean activity (%DMA), and the results from diseased and normal populations were used to establish cutoffs. RESULTS The absolute activities for the newborns ranged from 0.17 to 355 micromol/L h (N=139,074) and activities for Krabbe-positive controls ranged from 0.08 to 0.48 micromol/L h (N=16, n=91 measurements) while activities for carriers ranged from 0.28 to 2.71 micromol/L h (N=56, n=72 measurements). Cutoffs were set based on results from Krabbe-positive and carrier controls and the newborn population distribution. CONCLUSIONS The algorithm and cutoffs we propose provided 100% detection of all positive controls with 60/100,000 screen positive results predicted. In the course of this study, one anonymous newborn was predicted to have Krabbe disease based on enzyme activity and subsequent DNA analysis.

Simplified Newborn Screening Protocol for Lysosomal Storage Disorders

BACKGROUND Interest in lysosomal storage disorders, a collection of more than 40 inherited metabolic disorders, has increased because of new therapy options such as enzyme replacement, stem cell transplantation, and substrate reduction therapy. We developed a high-throughput protocol that simplifies analytical challenges such as complex sample preparation and potential interference from excess residual substrate associated with previously reported assays. METHODS After overnight incubation (16-20 h) of dried blood spots with a cassette of substrates and deuterated internal standards, we used a TLX-2 system to quantify 6 lysosomal enzyme activities for Fabry, Gaucher, Niemann-Pick A/B, Pompe, Krabbe, and mucopolysaccharidosis I disease. This multiplexed, multidimensional ultra-HPLC-tandem mass spectrometry assay included Cyclone P Turbo Flow and Hypersil Gold C8 columns. The method did not require offline sample preparation such as liquid-liquid and solid-phase extraction, or hazardous reagents such as ethyl acetate. RESULTS Obviating the offline sample preparation steps led to substantial savings in analytical time (approximately 70%) and reagent costs (approximately 50%). In a pilot study, lysosomal enzyme activities of 8586 newborns were measured, including 51 positive controls, and the results demonstrated 100% diagnostic sensitivity and high specificity. The results for Krabbe disease were validated with parallel measurements by the New York State Screening Laboratory. CONCLUSIONS Turboflow online sample cleanup and the use of an additional analytical column enabled the implementation of lysosomal storage disorder testing in a nationwide screening program while keeping the total analysis time to <2 min per sample.

Lysosomal storage disorder 4 + 1 multiplex assay for newborn screening using tandem mass spectrometry: Application to a small-scale population study for five lysosomal storage disorders

BACKGROUND We sought to modify a previously published tandem mass spectrometry method of screening for 5 lysosomal storage disorders (LSDs) in order to make it better suited for high-throughput newborn screening. METHODS Two 3-mm dried blood spot (DBS) punches were incubated, each with a different assay solution. The quadruplex solution was used for screening for Gaucher, Pompe, Krabbe and Fabry diseases, while a separate solution was used for Niemann-Pick A/B disease. RESULTS The mean activities of acid-β-glucocerebrosidase (ABG), acid sphingomyelinase (ASM), acid glucosidase (GAA), galactocerebroside-β-galactosidase (GALC) and acid-galactosidase A (GLA) were measured on 5055 unidentified newborns. The mean activities (compared with their disease controls) were, 15.1 (0.35), 22.2 (1.34), 16.8 (0.51), 3.61 (0.23), and 20.7 (1.43) (μmol/L/h), respectively. The number of specimens that fell below our retest level cutoff of <20% daily mean activity (DMA) for each analyte is: ABG (6), ASM (0), GAA (5), GALC (17), and GLA (2). CONCLUSIONS This method provides a simplified and reliable assay for screening for five LSDs with clear distinction between activities from normal and disease samples. Advantages of this new method include significant decreases in processing time and the number of required assay solutions and overall decreased complexity.

Development of a bile acid–based newborn screen for Niemann-Pick disease type C

A newborn screen for Niemann-Pick disease type C was developed on the basis of discovery of a bile acid marker. Expanding the newborn screen Niemann-Pick disease type C (NPC) is a fatal neurologic disorder caused by the deficiency of an enzyme involved in cholesterol storage. Although this disease was untreatable in the past, new therapeutics are now in clinical trials, but they are most likely to be effective if treatment is started as early as possible, before neurodegeneration has occurred. Jiang et al. identified three bile acids that are greatly increased in the blood of patients with NPC compared to healthy controls. The authors also demonstrated that one of these bile acids can be reliably measured in dried blood spots using mass spectrometry, suggesting that this bile acid test should be evaluated for potential addition to neonatal screening programs. Niemann-Pick disease type C (NPC) is a fatal, neurodegenerative, cholesterol storage disorder. With new therapeutics in clinical trials, it is imperative to improve diagnostics and facilitate early intervention. We used metabolomic profiling to identify potential markers and discovered three unknown bile acids that were increased in plasma from NPC but not control subjects. The bile acids most elevated in the NPC subjects were identified as 3β,5α,6β-trihydroxycholanic acid and its glycine conjugate, which were shown to be metabolites of cholestane-3β,5α,6β-triol, an oxysterol elevated in NPC. A high-throughput mass spectrometry–based method was developed and validated to measure the glycine-conjugated bile acid in dried blood spots. Analysis of dried blood spots from 4992 controls, 134 NPC carriers, and 44 NPC subjects provided 100% sensitivity and specificity in the study samples. Quantification of the bile acid in dried blood spots, therefore, provides the basis for a newborn screen for NPC that is ready for piloting in newborn screening programs.

Determination of psychosine concentration in dried blood spots from newborns that were identified via newborn screening to be at risk for Krabbe disease

BACKGROUND New York State has screened over 1.2 million newborns for Krabbe disease, and we identified 4 newborns with infantile Krabbe disease. In addition, 6 other newborns were identified with very low galactosylcerebrosidase (GALC) activity. Because these patients remain asymptomatic, we investigated whether psychosine levels could be a useful marker for disease. METHODS HPLC-MS/MS methodology was used to determine the psychosine concentrations in dried blood spots (DBS) collected from the following cohorts: known Krabbe patients, screened babies that were determined to have infantile Krabbe disease, asymptomatic infants with low GALC activity, and normal controls. RESULTS The psychosine concentrations from the known Krabbe patients ranged from 7 to 50 ng/ml. Newborns identified by screening who were confirmed with infantile Krabbe disease ranged from 23 to 73 ng/ml. Asymptomatic individuals with low GALC activity had concentrations ranging from 1.7 to 5.7 ng/ml. Concentrations in newborns with normal GALC activity were all <3 ng/ml. CONCLUSIONS The psychosine concentrations in DBS from confirmed infantile patients are at least four times higher than the asymptomatic newborns and nearly an order of magnitude greater than normal newborns. Further studies are needed to determine if psychosine can be used as a predictor of disease status/progression in screen positive newborns.

Newborn screening for Krabbe disease in New York State: the first eight years' experience

Purpose:Krabbe disease (KD) results from galactocerebrosidase (GALC) deficiency. Infantile KD symptoms include irritability, progressive stiffness, developmental delay, and death. The only potential treatment is hematopoietic stem cell transplantation. New York State (NYS) implemented newborn screening for KD in 2006.Methods:Dried blood spots from newborns were assayed for GALC enzyme activity using mass spectrometry, followed by molecular analysis for those with low activity (≤12% of the daily mean). Infants with low enzyme activity and one or more mutations were referred for follow-up diagnostic testing and neurological examination.Results:Of >1.9 million screened, 620 infants were subjected to molecular analysis and 348 were referred for diagnostic testing. Five had enzyme activities and mutations consistent with infantile KD and manifested clinical/neurodiagnostic abnormalities. Four underwent transplantation, two are surviving with moderate to severe handicaps, and two died from transplant-related complications. The significance of many sequence variants identified is unknown. Forty-six asymptomatic infants were found to be at moderate to high risk for disease.Conclusions:The positive predictive value of KD screening in NYS is 1.4% (5/346) considering confirmed infantile cases. The incidence of infantile KD in NYS is approximately 1 in 394,000, but it may be higher for later-onset forms.Genet Med 18 3, 239–248.

Newborn screening for X-linked adrenoleukodystrophy in New York State: diagnostic protocol, surveillance protocol and treatment guidelines.

PURPOSE To describe a diagnostic protocol, surveillance and treatment guidelines, genetic counseling considerations and long-term follow-up data elements developed in preparation for X-linked adrenoleukodystrophy (X-ALD) newborn screening in New York State. METHODS A group including the director from each regional NYS inherited metabolic disorder center, personnel from the NYS Newborn Screening Program, and others prepared a follow-up plan for X-ALD NBS. Over the months preceding the start of screening, a series of conference calls took place to develop and refine a complete newborn screening system from initial positive screen results to long-term follow-up. RESULTS A diagnostic protocol was developed to determine for each newborn with a positive screen whether the final diagnosis is X-ALD, carrier of X-ALD, Zellweger spectrum disorder, acyl CoA oxidase deficiency or D-bifunctional protein deficiency. For asymptomatic males with X-ALD, surveillance protocols were developed for use at the time of diagnosis, during childhood and during adulthood. Considerations for timing of treatment of adrenal and cerebral disease were developed. CONCLUSION Because New York was the first newborn screening laboratory to include X-ALD on its panel, and symptoms may not develop for years, long-term follow-up is needed to evaluate the presented guidelines.