Molecular characteristics of patients with glycosaminoglycan storage disorders in Russia Dimitry A. Chistiakov a,b, ⁎, Kirill V. Savost'anov b , Lyudmila M. Kuzenkova c , Anait K. Gevorkyan d , Alexander A. Pushkov b , Alexey G. Nikitin b , Alexander V. Pakhomov b , Nato D. Vashakmadze c , Natalia V. Zhurkova b , Tatiana V. Podkletnova c , Nikolai A. Mayansky e , Leila S. Namazova-Baranova d , Alexander A. Baranov f a Department of Medical Nanobiotechnology, Pirogov Russian State Medical University, 117997 Moscow, Russia b Department of Molecular Genetic Diagnostics, Division of Laboratory Medicine, Institute of Pediatrics, Research Center for Children's Health, 119991 Moscow, Russia c Department of Psychoneurology and Psychosomatic Pathology, Institute of Pediatrics, Research Center for Children's Health, 119991 Moscow, Russia d Institute of Preventive Pediatrics and Rehabilitation, Research Center for Children's Health, 119991 Moscow, Russia e Department of Experimental Immunology and Virology, Division of Laboratory Medicine, Institute of Pediatrics, Research Center for Children's Health, 119991 Moscow, Russia f Research Center for Children's Health, 119991 Moscow, Russia abstract article info Article history: Received 3 May 2014 Received in revised form 16 May 2014 Accepted 18 May 2014 Available online 26 May 2014 Keywords: Mucopolysaccharidoses Glycosaminoglycans Mutation Enzyme activity Background: The mucopolysaccharidoses (MPSs) are rare genetic disorders caused by mutations in lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs). In this study, we analyzed a total of 48 patients including MPSI (n = 6), MPSII (n = 18), MPSIIIA (n = 11), MPSIVA (n = 3), and MPSVI (n = 10). Methods: In MPS patients, urinary GAGs were colorimetrically assayed. Enzyme activity was quantified by colorimetric and fluorimetric assays. To find mutations, all IDUA, IDS, SGSH, GALNS, and ARSB exons and intronic flanks were sequenced. New mutations were functionally assessed by reconstructing mutant alleles with site- directed mutagenesis followed with expression of wild-type and mutant genetic variants in CHO cells, measuring enzymatic activity, and Western blot analysis of protein expression of normal and mutated enzymes in cell lysates. Results: A total of five novel mutations were found including p.Asn348Lys (IDUA) in MPSI, p.Tyr240Cys (GALNS) in MPSIVA, and three ARSB mutations (p.Gln110*, p.Asn262Lysfs*14, and pArg315*) in MPSVI patients. In case of mutations p.Asn348Lys, p.Asn262Lysfs*14, and p.Gln110*, no mutant protein was detected while activity of the mutant protein was b 1% of that of the normal enzyme. For p.Tyr240Cys, a trace of mutant protein was observed with a remnant activity of 3.6% of the wild-type GALNS activity. For pArg315*, a truncated 30-kDa protein that had 7.9% of activity of the normal ARSB was detected. Conclusions: These data further enrich our knowledge of the genetic background of MPSs. © 2014 Elsevier B.V. All rights reserved. 1. Introduction The mucopolysaccharidoses (MPSs) comprise a group of lysosomal storage diseases caused by deficiency of enzymes catalyzing the hydroly- sis of glycosaminoglycans (GAGs) and characterized by intra-lysosomal deposits and increased excretion in urine of partially degraded GAGs that in turn results in ultimate dysfunction of organs and tissues across the whole body. GAGs (previously called mucopolysaccharides) are the products of lysosomal degradation of proteoglycans that are the compo- nents of the extracellular matrix. Depending on the catabolic pathway, four types of GAGs can be formed including chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate. The step-by-step degradation of GAGs involves a total of 10 enzymes four of which belong to the class of glycosidases, five are sulfatases, and finally one is a non- hydrolytic transferase [1]. Deficiencies of each one of these enzymes lead to seven different MPSs all of which share a series of clinical symptoms such as hepatosplenomegaly, facial dysmorphism, joints mobility, and frequent cardiovascular, visional, and neurological alterations [2]. The continued accumulation of non-degraded GAGs leads to the enlargement of lyso- somes. Due to increase in the number of lysosomes, cells expand in their size, an event that causes organomegaly. Usually, MPSs are recognized via analysis of urinary GAGs. However, it is not possible to distinguish between subtypes of MPSs that excrete similar types of GAGs. To perform a definitive analysis, an enzyme- specific assay should be done in patient's blood cells or skin fibroblasts Clinica Chimica Acta 436 (2014) 112–120 ⁎ Corresponding author at: Department of Medical Nanobiotechnology, Pirogov Russian State Medical University, Ulitsa Ostrovityanova 1, 117997 Moscow, Russia. Tel.: +7 495 434 13 01; fax: +7 495 434 14 22. E-mail address: dimitry.chistiakov@gmail.com (D.A. Chistiakov). http://dx.doi.org/10.1016/j.cca.2014.05.010 0009-8981/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim