- Email: [email protected]
34 Newborn screening for Krabbe disease
Abstracts / Molecular Genetics and Metabolism 92 (2007) S11–S34 (HSCT), the developmental trajectory depends on a number of factors including age and developmental level at HSCT. We hypothesized that attenuated patients would have differential problems with memory and visual spatial ability, normal IQ, reading, and recognition memory but below average encoding and visual spatial ability and Hurler syndrome children matched for Full Scale IQ would have normal performance in all of the above domains. Results: 6 patients with attenuated MPS I after ERT were no different than 6 patients who had Hurler syndrome who underwent HSCT (at age two or earlier) on IQ measures, reading, vocabulary and recognition memory. Significant differences were found on memory encoding using Mann– Whitney U (Z = 2.04, pd.05), and on visual spatial ability (Z = 2.68, pd.004). We conclude that attenuated MPS patients show differential memory and visual spatial impairment, compared to IQ matched severe Hurler patients who had HSCT. Hippocampal damage, hypothesized to underlie these deficits, may be averted by early transplant in Hurler syndrome but may continue or intensify in attenuated MPS I. doi:10.1016/j.ymgme.2007.08.036
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activity-attenuating polymorphisms and disease-causing mutations. PCRagarose gel electrophoresis is used to detect the common 30 kb deletion and a less common 7.4 kb deletion in the GALC gene. Lastly, a semi-automated PCR-sequence analysis assay was developed to sequence all 17 exons, the intron–exon boundaries, and the promoter region of the GALC gene. Thus far, 71,721 newborns have been tested with the MS/MS method (mean activity = 4.24 mol/L h). DNA from those newborns with activities <10% of the daily mean activity were evaluated for sequence alterations. Of the newborns screened to date, fourteen (with activity 610%) have been tested for DNA variants, eight have been referred for follow-up diagnostic testing, and six have been found only to have non-disease causing polymorphisms. A summary of the screening results to date will be presented. Reference [1] Y. Li, C.R. Scott, N.A. Chamoles, A. Ghavami, B.M. Pinto, F. Turecek, M.H. Gelb, Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening, Clinical Chem. 50 (17) (2004) 85–96. doi:10.1016/j.ymgme.2007.08.039
33 Screening of newborns for lysosomal storage diseases C. Ronald Scott a, Frantisek Turecek b, Michael Gelb b, a University of Washington, Seattle, WA, USA, b Department of Pediatrics and Chemistry, University of Washington, Seattle, WA, USA Our group at the University of Washington has been developing direct, enzymatic assays that involve the use of a tandem mass spectrometer (MS/ MS) as a platform for the assay of lysosomal enzymes. Natural or artificial substrates are synthesized for each of the lysosomal enzymes of interest. The substrates and products each have a different mass (m/z) that can be uniquely identified by the mass spectrometer. We have demonstrated that lysosomal enzymes are stable on filter paper blood samples submitted for newborn screening and that individuals with Gaucher, Fabry, Krabbe, Niemann-Pick, Pompe, MPS I, and MPS II can be distinguished from normal infants. The assays appear to be sensitive, specific, and can be multiplexed as a single injection in a mass spectrometer, such that a single 3 mm blood spot can be analyzed for multiple disorders. We are planning to instigate a field trial for this technology in a newborn screening laboratory. We have selected Fabry disease and Pompe disease as the original disorders for early detection. The program will require careful and rapid confirmation of suspected diagnoses by a combination of clinical, enzymatic, and molecular techniques. It is expected that there may exist unanticipated results on the basis of biological heterogeneity, methodological variation, or environmental factors. doi:10.1016/j.ymgme.2007.08.038
34 Newborn screening for Krabbe disease Joseph J. Orsini a, Mark M. Morrissey b, Laura Helton b, Carlos Saavedra b, Joan Keutzer c, Kate X. Zhang c, Michele Caggana b, a New York State Department of Health, Albany, NY, USA, b New York State Department of Health, Wadsworth Center, Albany, NY, USA, c Genzyme Corporation, 1 Mountain Road, Framingham, MA, USA The application of tandem mass spectrometry (MS/MS) in screening newborns for inherited disorders is common in newborn screening programs. Li et al. [1] extended the application of the MS/MS methodology to screen for the enzyme deficiency in five lysosomal storage disorders. We evaluated and modified this method with a focus on Krabbe disease, and then performed a population study using the revised method. We tested over 100,000 unidentified newborns, 56 obligate carriers and 16 Krabbe patients. The galactocerebrosidase (GALC) activities were converted to percent of daily mean activities, and the results from diseased and non-diseased populations were used to establish cutoffs. In addition, we developed a rapid methodology using real-time PCR to detect the known common
35 Newborn screening for lysosomal storage diseases—A reality? Olaf Bodamer, A. Mu¨hl, Division of Biochemical Genetics, University Children’s Hospital, Vienna, Austria Neonatal screening programmes for inborn errors of metabolism are implemented throughout the world. Disorders include defects of fatty acid oxidation, defects of protein metabolism, galactosemia, hypothyroidism and others. Ideally, the natural history of such disorders is well understood while early diagnosis and treatment results in significant reduction of otherwise high morbidity and mortality. With the advent of novel treatment modalities in lysosomal storage diseases (LSD) such as bone marrow transplantation and/or enzyme replacement therapies, newborn screening for LSD has become a focus point. From a technological perspective highthroughput newborn screening for LSD may be feasible using different analytical approaches. Among these, screening by tandem-mass spectrometry using unique, specific substrates and internal standards seems to be the most promising method as enzyme activities can be readily measured in dry blood spots from neonatal filter cards. This technique may be incorporated into existing neonatal screening programmes using tandem mass spectrometry. Prior to implementation of neonatal screening programmes for LSD, pilot studies have to demonstrate its technical feasibility, sensitivity and specificity. In addition, strategies for confirmatory testing, treatment, follow-up care and scientific evaluation have to be defined and agreed upon at an international level. doi:10.1016/j.ymgme.2007.08.040
36 LSD enzyme assays in dried blood spots—From research to newborn screening Joan Keutzer a, Wuh-Liang Hwu b, Carole Elbin a, Wei-Lien Chuang a, X. Kate Zhang a, a Genzyme Corporation, Cambridge, MA, USA, b Department of Pediatrics and Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan Measuring lysosomal enzyme activity in dried blood spots (DBS) may permit the presymptomatic identification of patients with lysosomal storage disorders (LSD) through newborn screening. There are two strategies for measuring enzyme activity in DBS that differ in the substrate that is used. The first approach uses 4-MU-conjugates that are available commercially and a fluorometer to quantify enzyme activity. We proved the feasibility of this method in a Pompe disease pilot program in Taiwan. Three cases of Pompe disease were identified out of a total of 70,683 newborns screened. The second approach uses novel substrates and internal standard pairs that are in development for use in newborn screening and tandem mass spectrometry to quantify
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activity-attenuating polymorphisms and disease-causing mutations. PCRagarose gel electrophoresis is used to detect the common 30 kb deletion and a less common 7.4 kb deletion in the GALC gene. Lastly, a semi-automated PCR-sequence analysis assay was developed to sequence all 17 exons, the intron–exon boundaries, and the promoter region of the GALC gene. Thus far, 71,721 newborns have been tested with the MS/MS method (mean activity = 4.24 mol/L h). DNA from those newborns with activities <10% of the daily mean activity were evaluated for sequence alterations. Of the newborns screened to date, fourteen (with activity 610%) have been tested for DNA variants, eight have been referred for follow-up diagnostic testing, and six have been found only to have non-disease causing polymorphisms. A summary of the screening results to date will be presented. Reference [1] Y. Li, C.R. Scott, N.A. Chamoles, A. Ghavami, B.M. Pinto, F. Turecek, M.H. Gelb, Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening, Clinical Chem. 50 (17) (2004) 85–96. doi:10.1016/j.ymgme.2007.08.039
33 Screening of newborns for lysosomal storage diseases C. Ronald Scott a, Frantisek Turecek b, Michael Gelb b, a University of Washington, Seattle, WA, USA, b Department of Pediatrics and Chemistry, University of Washington, Seattle, WA, USA Our group at the University of Washington has been developing direct, enzymatic assays that involve the use of a tandem mass spectrometer (MS/ MS) as a platform for the assay of lysosomal enzymes. Natural or artificial substrates are synthesized for each of the lysosomal enzymes of interest. The substrates and products each have a different mass (m/z) that can be uniquely identified by the mass spectrometer. We have demonstrated that lysosomal enzymes are stable on filter paper blood samples submitted for newborn screening and that individuals with Gaucher, Fabry, Krabbe, Niemann-Pick, Pompe, MPS I, and MPS II can be distinguished from normal infants. The assays appear to be sensitive, specific, and can be multiplexed as a single injection in a mass spectrometer, such that a single 3 mm blood spot can be analyzed for multiple disorders. We are planning to instigate a field trial for this technology in a newborn screening laboratory. We have selected Fabry disease and Pompe disease as the original disorders for early detection. The program will require careful and rapid confirmation of suspected diagnoses by a combination of clinical, enzymatic, and molecular techniques. It is expected that there may exist unanticipated results on the basis of biological heterogeneity, methodological variation, or environmental factors. doi:10.1016/j.ymgme.2007.08.038
34 Newborn screening for Krabbe disease Joseph J. Orsini a, Mark M. Morrissey b, Laura Helton b, Carlos Saavedra b, Joan Keutzer c, Kate X. Zhang c, Michele Caggana b, a New York State Department of Health, Albany, NY, USA, b New York State Department of Health, Wadsworth Center, Albany, NY, USA, c Genzyme Corporation, 1 Mountain Road, Framingham, MA, USA The application of tandem mass spectrometry (MS/MS) in screening newborns for inherited disorders is common in newborn screening programs. Li et al. [1] extended the application of the MS/MS methodology to screen for the enzyme deficiency in five lysosomal storage disorders. We evaluated and modified this method with a focus on Krabbe disease, and then performed a population study using the revised method. We tested over 100,000 unidentified newborns, 56 obligate carriers and 16 Krabbe patients. The galactocerebrosidase (GALC) activities were converted to percent of daily mean activities, and the results from diseased and non-diseased populations were used to establish cutoffs. In addition, we developed a rapid methodology using real-time PCR to detect the known common
35 Newborn screening for lysosomal storage diseases—A reality? Olaf Bodamer, A. Mu¨hl, Division of Biochemical Genetics, University Children’s Hospital, Vienna, Austria Neonatal screening programmes for inborn errors of metabolism are implemented throughout the world. Disorders include defects of fatty acid oxidation, defects of protein metabolism, galactosemia, hypothyroidism and others. Ideally, the natural history of such disorders is well understood while early diagnosis and treatment results in significant reduction of otherwise high morbidity and mortality. With the advent of novel treatment modalities in lysosomal storage diseases (LSD) such as bone marrow transplantation and/or enzyme replacement therapies, newborn screening for LSD has become a focus point. From a technological perspective highthroughput newborn screening for LSD may be feasible using different analytical approaches. Among these, screening by tandem-mass spectrometry using unique, specific substrates and internal standards seems to be the most promising method as enzyme activities can be readily measured in dry blood spots from neonatal filter cards. This technique may be incorporated into existing neonatal screening programmes using tandem mass spectrometry. Prior to implementation of neonatal screening programmes for LSD, pilot studies have to demonstrate its technical feasibility, sensitivity and specificity. In addition, strategies for confirmatory testing, treatment, follow-up care and scientific evaluation have to be defined and agreed upon at an international level. doi:10.1016/j.ymgme.2007.08.040
36 LSD enzyme assays in dried blood spots—From research to newborn screening Joan Keutzer a, Wuh-Liang Hwu b, Carole Elbin a, Wei-Lien Chuang a, X. Kate Zhang a, a Genzyme Corporation, Cambridge, MA, USA, b Department of Pediatrics and Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan Measuring lysosomal enzyme activity in dried blood spots (DBS) may permit the presymptomatic identification of patients with lysosomal storage disorders (LSD) through newborn screening. There are two strategies for measuring enzyme activity in DBS that differ in the substrate that is used. The first approach uses 4-MU-conjugates that are available commercially and a fluorometer to quantify enzyme activity. We proved the feasibility of this method in a Pompe disease pilot program in Taiwan. Three cases of Pompe disease were identified out of a total of 70,683 newborns screened. The second approach uses novel substrates and internal standard pairs that are in development for use in newborn screening and tandem mass spectrometry to quantify