none16noopenGabrielli, Orazio; Zampini, Lucia; Monachesi, Chiara; Marchesiello, Rita Lucia; Padella, Lucia; Santoro, Lucia; Volpi, Nicola; Concolino, Daniela; Fiumara, Agata; Rigon, Laura; Mazzoli, Milena; Carnielli, Virgilio Paolo; Giovagnoni, Andrea; Catassi, Carlo; Galeazzi, Tiziana; Coppa, Giovanni ValentinoGabrielli, Orazio; Zampini, Lucia; Monachesi, Chiara; Marchesiello, Rita Lucia; Padella, Lucia; Santoro, Lucia; Volpi, Nicola; Concolino, Daniela; Fiumara, Agata; Rigon, Laura; Mazzoli, Milena; Carnielli, Virgilio Paolo; Giovagnoni, Andrea; Catassi, Carlo; Galeazzi, Tiziana; Coppa, Giovanni Valentin

Carnielli, Vp

Catassi, C

Concolino, D

Coppa, Gv

Fiumara, A

Gabrielli, O

Galeazzi, T

Giovagnoni, A

Marchesiello, Rl

Mazzoli, M

Monachesi, C

Padella, L

Rigon, L

Santoro, L

Volpi, Nicola

Zampini, L

English

Gabrielli, Orazio

ZAMPINI, LUCIA

MONACHESI, Chiara

Marchesiello, Rita Lucia

PADELLA, LUCIA

SANTORO, LUCIA

Concolino, Daniela

FIUMARA, AGATA

RIGON, LAURA

Mazzoli, Milena

Carnielli, Virgilio Paolo

Giovagnoni, Andrea

Catassi, Carlo

Galeazzi, Tiziana

Coppa, Giovanni Valentino

IRIS UniversitÃ  Politecnica delle Marche

Accepted ManuscriptEarly diagnosis of mucopolysaccharidoses in developingcountries: A low cost and easy execution approachOrazio Gabrielli, Lucia Zampini, Chiara Monachesi, Rita LuciaMarchesiello, Lucia Padella, Lucia Santoro, Nicola Volpi, DanielaConcolino, Agata Fiumara, Laura Rigon, Milena Mazzoli,Virgilio Paolo Carnielli, Andrea Giovagnoni, Carlo Catassi,Tiziana Galeazzi, Giovanni Valentino CoppaPII: S0009-8981(17)30072-4DOI: doi: 10.1016/j.cca.2017.02.020Reference: CCA 14668To appear in: Clinica Chimica ActaReceived date: 16 February 2017Revised date: 27 February 2017Accepted date: 27 February 2017Please cite this article as: Orazio Gabrielli, Lucia Zampini, Chiara Monachesi, Rita LuciaMarchesiello, Lucia Padella, Lucia Santoro, Nicola Volpi, Daniela Concolino, AgataFiumara, Laura Rigon, Milena Mazzoli, Virgilio Paolo Carnielli, Andrea Giovagnoni,Carlo Catassi, Tiziana Galeazzi, Giovanni Valentino Coppa , Early diagnosis ofmucopolysaccharidoses in developing countries: A low cost and easy execution approach.The address for the corresponding author was captured as affiliation for all authors. Pleasecheck if appropriate. Cca(2016), doi: 10.1016/j.cca.2017.02.020This is a PDF file of an unedited manuscript that has been accepted for publication. Asa service to our customers we are providing this early version of the manuscript. Themanuscript will undergo copyediting, typesetting, and review of the resulting proof beforeit is published in its final form. Please note that during the production process errors maybe discovered which could affect the content, and all legal disclaimers that apply to thejournal pertain.ACCEPTED MANUSCRIPT 1 Early diagnosis of Mucopolysaccharidoses in developing countries: a low cost and easy execution approach.  Orazio Gabrielli1, Lucia Zampini1, Chiara Monachesi1, Rita Lucia Marchesiello1, Lucia Padella1, Lucia Santoro1, Nicola Volpi3*, Daniela Concolino4, Agata Fiumara5, Laura Rigon6, Milena Mazzoli7, Virgilio Paolo Carnielli7, Andrea Giovagnoni2, Carlo Catassi1, Tiziana Galeazzi1, Giovanni Valentino Coppa1.  Corresponding Author: *Prof. Nicola Volpi Department of Life Sciences University of Modena and Reggio Emilia Via Campi 213/D 41100 Modena, Italy e-mail: volpi@unimo.it fax number: 0039 59 2055548 tel number: 0039 59 2055543  Author affiliations: 1Division of Pediatric and 2Radiological Sciences, Department of Clinical Sciences, Università Politecnica delle Marche, Ospedali Riuniti, Ancona, Italy   3 Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy 4 Department of Paediatrics, University of Catanzaro, Catanzaro, Italy 5 Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy 6Laboratory of Diagnosis and Therapy of Lysosomal Disorders Dept. of Women’s and Children’s Health, University of Padova, Italy   7 Division of Neonatology, Università Politecnica delle Marche, Ospedali Riuniti, Presidio Salesi, Ancona, Italy      Keywords: Newborns, Glycosaminoglycans, Mucopolysaccharidoses, DMB and electrophoresis.    ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT 2 The guidelines for an accurate diagnosis of Mucopolysaccharidoses (MPS) recommend first line screening methods on urine sample to determine quantitative and qualitative glycosaminoglycans (GAG) and successively the identification of specific enzyme deficiency and genetic analysis [1]. Actually several methods, requiring sophisticated equipments, are available [2-6]; nevertheless, the great majority of world population cannot benefit of the above approaches due to their high cost.  The aim of the present study was to apply a combined method based on the quantitative evaluation and qualitative characterization of the urinary GAGs for the diagnosis of MPS.                  The GAG evaluation [7] has been performed on 1 ml of spot urine samples collected from 625 newborns (356 males and 269 females) at the 2nd and 3rd day of life. As regard as the total amount of urinary GAGs, performed by 1,9-dimethylmethylene blue (DMB) method, the higher values, up to 745 µg/mg Creatinine (Cr), were found in newborns with Cr lower than 40 mg/dl; on the contrary, in newborns with Cr levels higher than 40 mg/dl the range of GAG concentration was between 63.9 and 384.0 µg/mg Cr (Tab.1). No differences between males and females were observed. A significant difference of GAG concentration was found in urine samples with Cr levels < 40 mg/dl compared to urine samples with Cr levels > 40 mg/dl (p<0.0001). These results call the attention of the role of Cr levels to obtain reference values of GAGs in normal term newborns.  As regards as the qualitative characterization, the electrophoretic GAG pattern shows in all samples a major fast band of chondroitin A sulfate  while a slower faint band, corresponding to heparan-sulfate, was observed in about 60% of urine samples.  Our results demonstrate that the combined method is the first step for the identification of all types of MPS; in fact, performing a qualitative and quantitative analyses simultaneously, a 100% of sensitivity and specificity are obtained [7]. On the contrary, DMB test alone can give false negative results, especially concerning the identification of MPS III and IV patients [6, 8]. Moreover, our combined method can be applied not only in the neonatal period, but also in the first months of life, such as in the occasion of vaccines administration or of a hospitalization for any health problems. It is important to underline that in the above reported situations, it is possible to identify the MPS patients in pre-symptomatic phase, using the reference values of urinary GAGs of the first year of life, recently published by our group [7].   We are aware that in developing countries there are situations of serious deficiencies in welfare state, even for health care system and the treatment of very common diseases; nevertheless, it is our opinion that to have available a simple method for diagnosis of these rare metabolic diseases could be helpful for the importance of genetic counseling and pre-symptomatic early treatment. Moreover, the use of the combined method could be applied in the poor countries where clusters of these diseases or when a high percentage of consanguineous marriages are present. In conclusion, this combined test represents an ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT 3 important tool for its simplicity, short working time, low cost, and it does not require any sophisticated equipments.   Table 1. Urinary GAG concentration (µg GAG/mg Creatinine) according to Creatinine (CR) values < 40 mg/dl a) and >40 mg/dl b) in healthy newborns.                                   Creatinine (CR) mg/dl DMB µg GAG/mg CR a) range n mean ± SD MIN-MAX     1-10 12  492.0 ± 158.7 191.7 - 745.1 11-20 53 296.9 ± 77.4 162.7 - 568.6 21-30 61 262.2 ± 80.3 163.4 - 511.2 31-40 63 246.4 ± 66.8 142.7 - 429.1  n tot   1-40 189 281.5 ± 100.7 142.7 - 745.1 b) range n mean ± SD MIN-MAX 41-50 51 203.0 ± 51.1 117.5 - 361.1 51-60 44 220.9 ± 56.4 132.2 - 384.0 61-70 47 198.4 ± 60.6 99.4 - 360.7 71-80 44 192.6 ± 61.8 84.7 - 364.8 81-90 28 187.8 ± 61.8 95.4 - 355.1 91-100 39 172.3 ± 45.7 102.1 - 306.5 101-110 26 178.0 ± 43.3 94.8 - 262.7 111-120 39 178.7 ± 51.6 100.9 - 347.8 121-130 30 158.9 ± 32.3 82.7 - 228.8 131-140 24 152.7 ± 53.5 63.9 - 291.9 141-150 16 182.5 ± 61.7 83.2 - 316.7 151-160 21 177.8 ± 68.3 94.6 - 313 161-170 10 153.2 ± 62.3 71.3 - 281.7 171-200 20 165.5 ± 49.0 91.6 - 304.1  n tot   41-200 436 185.6 ± 56.9 63.9 - 384 ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT 4 Contributors OG, AG, LS, CC, and GVC designed the study and prepared the last draft of the manuscript; LZ, LP, TG, RTM, CM, LR and NV performed the experimental procedures and analyses; DC, AF, MM, VPC recruited the newborn urinary samples; OG and GVC performed the statistical analysis and wrote the first draft. All authors read and approved the final manuscript.   Declaration of interest  The authors declare that they have no competing interests.   Acknowledgments This research has been co-funded by PRIN 2012 National Research Program and Mancini Foundation.   Funding.  Supported by MIUR, Ministero dell'Istruzione, dell' Università e della Ricerca, for the project PRIN 2012 National Research Program, Prot. 20122EK9SZ_002, entitled "Comprehensive approach to mucopolysaccharidoses: application of highly specific methods for neonatal diagnosis and assessment of therapeutic efficacy in patients and in experimental animals".This research has been supported also by Mancini Foundation.         ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT 5 References [1] T.J.A. Lehman, N. Miller, B. Norquist, L. Underhill, J. Keutzer, Diagnosis of the mucopolysaccharidoses, Rheumatology 50 (2011)  41-48.  [2] J. de Ruijter, M.H. de Ru, T. Wagemans, L. Ijlst, A.M. Lund, et al., Heparan sulfate and dermatan sulfate derived disaccharides are sensitive markers for newborn screening for mucopolysaccharidoses types I, II and III, Mol. Genet. Metab. 107 (2012) 705-710.   [3] C. Auray-Blais, P. Bherer, R. Gagnon, et al. Efficient analysis of urinary glycosaminoglycans by LC-MS/MS in mucopolysaccharidoses type I, II and VI. Mol Genet Metab 102 (2011) 49-56.  [4] C.K. Chung, H.Y. Lin, T.J. Wang, C.C. Tsai, K.L. Liu, S.P. Lin, A modified liquid chromatography/tandem mass spectrometry method for predominant disaccharide units of urinary glycosaminoglycans in patients with mucopolysaccharidoses, Orphanet J Rare Dis 9 (2014) 135-145.  [5] H. Zhang, T. Wood, S.P. Young, D.S. Millington, A straightforward, quantitative ultra-performance liquid chromatography-tandem mass spectrometric method for heparin sulfate, dermatan sulfate and chondroitin sulfate in urine: an improved clinical screening test for the mucopolysaccharidoses. Mol Genet Metab 114 (2015) 123-128.  [6] C. Auray-Blais, P. Lavoie, S. Tomatsu, et al. UPLC-MS/MS detection of disaccharides derived from glycosaminoglycans as biomarkers of mucopolysaccharidoses. Anal Chim Acta 936 (2016) 139-148.  [7] L. Zampini, L. Padella, R.L. Marchesiello, et al. Importance of the combined urinary procedure for the diagnosis of mucopolysaccharidoses. Clin Chim Acta 464 (2017) 165-169.  [8] J. Zschocke, G.F. Hoffmann Special metabolic investigations, In: Vademecum Metabolicum Diagnosis and Treatment of inborn errors of metabolism., eds. Milupa Metabolics GmbH&Co. KG, Friedrichsdorf, Germany, 2011, p.36 ACCEPTED MANUSCRIPT

Early diagnosis of mucopolysaccharidoses in developing countries: A low cost and easy execution approach

Clin Chim Acta. 2017 Feb 28;468:150-151. doi: 10.1016/j.cca.2017.02.020. [Epub ahead of print]

Early diagnosis of mucopolysaccharidoses in developing countries: A low cost and easy execution approach.

Gabrielli O1, Zampini L1, Monachesi C1, Marchesiello RL1, Padella L1, Santoro L1, Volpi N2, Concolino D3, Fiumara A4, Rigon L5, Mazzoli M6, Carnielli VP6, Giovagnoni A7, Catassi C1, Galeazzi T1, Coppa GV1.

KEYWORDS: 
DMB and electrophoresis; Glycosaminoglycans; Mucopolysaccharidoses; Newborn

VOLPI, Nicola

Archivio istituzionale della ricerca - Università di Modena e Reggio Emilia

Early diagnosis of mucopolysaccharidoses in developing countries: A low cost and easy execution approach.

https://iris.univpm.it/bitstream/11566/245859/1/CCA%202017.pdf

Early diagnosis of mucopolysaccharidoses in developing countries: A low cost and easy execution approach

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IRIS UniversitÃ Politecnica delle Marche

Archivio istituzionale della ricerca - Università di Modena e Reggio Emilia