Fabry disease is an X-linked lysosomal storage disorder caused by the impairment of α-galactosidase A. Enzyme replacement therapy is available to treat patients, who often experience delayed diagnosis. A newborn screening for Fabry disease was performed to study the prevalence of the pathology and to evaluate the possibility to implement the test in systematic screenings. We collected 14,600 dried blood spot samples (7575 males and 7025 females) and carried out a diagnostic study by fluorometric measurement of α-galactosidase A enzymatic activity and GLA gene sequencing. We detected one patient with a mutation in GLA associated with classical Fabry Disease (M290I), ten subjects carrying genetic variants of uncertain diagnosis (S126G, R118C, A143T), and a girl with the non-characterized variant F18Y, which was not previously described. Additional 25 samples presented nucleotide substitutions described as polymorphisms (D313Y, rs2071225, and rs2071397). The estimated prevalence for Fabry disease in north-western Spanish males is of 0.013%. Conclusion: These results confirm that the prevalence of Fabry disease is underestimated and systematic screening is feasible; however, further characterization of variants of uncertain clinical significance is necessary to establish protocols of patients’ management. What is Known: • Fabry disease is a rare disease of delayed diagnosis, whose prevalence is underestimated. However, early diagnosis is important for better efficiency of the current available treatment. What is New: • This newborn screening for Fabry disease performed on Spanish population reveals a prevalence of genetic alterations in GLA of 0.1% in males (0.013% with classic Fabry disease) and also characterizes these modifications in order to discriminate between pathogenic mutations and genetic variants of unknown significance.

Introduction: Inborn errors of metabolism (IEM) represent an important public health problem due to current diagnosis and treatment limitations, poor life quality of affected patients, and consequent untimely child death. In contrast to classical methods, tandem mass spectrometry (MS/MS) has allowed simultaneous evaluation of multiple metabolites associated with IEM offering higher sensitivity, low false positive rates and high throughput. Aims: Determine concentration levels for amino acids and acylcarnitines in blood of newborns from Colombia, to establish reference values for further use in diagnosis of IEM. Methods: Implementation of a method to determine amino acids, acylcarnitines and succinylacetone in newborn dried blood spots using MS/MS, and its application in a cross-sectional study conducted in 891 healthy neonates from Cali and Quibdó cities is described. Results: fifty seven analytes that allow the diagnosis of 50 different pathologies were tested. The method showed to be linear, precise and accurate. Healthy neonates 2-18 days of age were included, 523 from Cali and 368 from Quibdó; 52% male and 48% female. Age-related differences on the concentration levels of amino acids and acylcarnitines were observed whereas no significant differences by gender were found. Conclusion: The study has contributed to reveal the usual concentration levels of amino acids, acylcarnitines and succinylacetone that could be used as reference for the establishment of a newborn metabolic screening program in Colombia.

Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) gene. Skeletal dysplasia and the related clinical features of MPS IVA are caused by disruption of the cartilage and its extracellular matrix, leading to a growth imbalance. Enzyme replacement therapy (ERT) with recombinant human GALNS has yielded positive results in activity of daily living and endurance tests. However, no data have demonstrated improvements in bone lesions and bone grow thin MPS IVA after ERT, and there is no correlation between therapeutic efficacy and urine levels of keratan sulfate, which accumulates in MPS IVA patients. Using qualitative and quantitative proteomics approaches, we analyzed leukocyte samples from healthy controls (n = 6) and from untreated (n = 5) and ERT-treated (n = 8, sampled before and after treatment) MPS IVA patients to identify potential biomarkers of disease. Out of 690 proteins identified in leukocytes, we selected a group of proteins that were dysregulated in MPS IVA patients with ERT. From these, we identified four potential protein biomarkers, all of which may influence bone and cartilage metabolism: lactotransferrin, coronin 1A, neutral alpha-glucosidase AB, and vitronectin. Further studies of cartilage and bone alterations in MPS IVA will be required to verify the validity of these proteins as potential biomarkers of MPS IVA.

Background: The fact that mucopolysaccharidoses (MPSes) are now treatable, and that the earlier treatment is initiated the better, is an indication for neonatal screening. The most efficient approach seems likely to be a multi‐tier procedure in which screening for urinary glycosaminoglycan (GAG) is followed by enzyme determinations in heelprick blood of newborns screening positive. Hitherto the method of choice for the determination of GAG has been the measurement of absorbance by a complex of GAG and 1,9‐dimethylmethylene blue (DMB). Method: We evaluated a DMB method in which absorbance by DMB is measured following its addition to the eluate obtained from paper‐borne newborn urine samples and is normalized relative to urinary creatinine. Calibration is performed with chondroitin‐6‐sulfate (Ch‐6‐S). Results: The limits of detection and quantification of GAG were 1.98 and 5.94 mg/dl, respectively. The within‐run coefficients of variation (CVs) of the GAG/creatinine ratio for 25, 31, and 70 mg/dl solutions of Ch‐6‐S in urine were 21.8, 16.4, and 10.5%, respectively, and the corresponding between‐run CVs were 25.0, 13.5, and 10.1%. Recovery from the urine spiked with 31 mg Ch‐6‐S/dl was 94.8%. Accuracy was also acceptable for all other GAGs except hyaluronic acid. For neonatal screening, the diagnostic threshold was tentatively established as 800 mg GAG/g creatinine, the 95th centile of samples from 903 infants aged 3–28 days, but the value of the GAG/creatinine ratio was negatively correlated with age. Application of the new method to samples from older individuals with and without MPS achieved 100% sensitivity and specificity when used with an age‐dependent threshold taken from the literature on the original DMB method. Conclusion: If used in the first tier of a multi‐tier screening protocol, the proposed method would allow the detection of abnormal levels of all GAGs except hyaluronic acid. J. Clin. Lab. Anal. 24:149–153, 2010.

Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal disease caused by mutations in the gene encoding the enzymeN-acetylgalactosamine-6-sulfate sulfatase (GALNS), and is characterized by systemic skeletal dysplasia due to excessive storage of keratan sulfate (KS) and chondroitin-6-sulfate in chondrocytes. Although improvements in the activity of daily living and endurance tests have been achieved with enzyme replacement therapy (ERT) with recombinant human GALNS, recovery of bone lesions and bone growth in MPS IVA has not been demonstrated to date. Moreover, no correlation has been described between therapeutic efficacy and urine levels of KS, which accumulates in MPS IVA patients. The objective of this study was to assess the validity of potential biomarkers proposed by other authors and to identify new biomarkers. To identify candidate biomarkers of this disease, we analyzed plasma samples from healthy controls (n=6) and from untreated (n=8) and ERT-treated (n=5, sampled before and after treatment) MPS IVA patients using both qualitative and quantitative proteomics analyses. The qualitative proteomics approach analyzed the proteomic profile of the different study groups. In the quantitative analysis, we identified/quantified 215 proteins after comparing healthy control untreated, ERT-treated MPSIVA patients. We selected a group of proteins that were dysregulated in MPS IVA patients. We identified four potential protein biomarkers, all of which may influence bone and cartilage metabolism: fetuin-A, vitronectin, alpha-1antitrypsin, and clusterin. Further studies of cartilage and bone samples from MPS IVA patients will be required to verify the validity of these proteins as potential biomarkers of MPS IVA.