Mucopolysaccharidosis type II (MPSII) is an X-linked lysosomal storage disorder caused by deficiency of the enzyme iduronate-2-sulfatase (IDS). The human IDS gene is located in chromosome Xq28. This is the first report of genotype and phenotype characterization of 49 Hunter patients from40 families of Argentina. Thirty different alleles have been identified, and 57%were novel. The frequency of de novomutationswas 10%. Overall, the percentage of private mutations in our series was 75%.Facultad de Ciencias Exacta

Amartino, H.

Arberas, C.

Bay, L.

Cabrera, A.

Ceci, Romina

Dipierri, J.

Gal, A.

Ilari, R.

Masllorens, F.

Rozenfeld, P.

Specola, N.

Molecular Genetics and Metabolism Reports

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Molecular Genetics and Metabolism Reports 1 (2014) 401–406Contents lists available at ScienceDirectMolecular Genetics andMetabolism Reportsj ou rna l homepage: h t tp : / /www. jou rna l s .e lsev ie r .com/molecu la r -genet ics -and-metabo l i sm- repor ts /Short CommunicationIdentiﬁcation of 17 novel mutations in 40Argentinean unrelated families withmucopolysaccharidosis type II (Hunter syndrome)H. Amartino a, R. Ceci b, F. Masllorens c, A. Gal d, C. Arberas e, L. Bay f, R. Ilari g,J. Dipierri h, N. Specola i, A. Cabrera j, P. Rozenfeld b,⁎a Hospital Universitario Austral, Pilar, Argentinab IIFP, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata y CONICET, Argentinac Hospital Posadas, Buenos Aires, Argentinad UKE, Hamburgo, Germanye Hospital de Niños Dr Ricardo Gutierrez, Buenos Aires, Argentinaf Hospital Garrahan, Buenos Aires, Argentinag Policlínico Bancario, Buenos Aires, Argentinah Universidad Nacional de Jujuy, Argentinai Hospital de Niños Sor María Ludovica, La Plata, Argentinaj Hospital de Niños VJ Vilela, Rosario, Argentinaa r t i c l e i n f o⁎ Corresponding author at: 47 y 115, La Plata (190E-mail address: paurozen@biol.unlp.edu.ar (P. Rohttp://dx.doi.org/10.1016/j.ymgmr.2014.08.0062214-4269/© 2014 The Authors. Published by Els(http://creativecommons.org/licenses/by-nc-nd/3.0a b s t r a c tArticle history:Received 30 July 2014Received in revised form 25 August 2014Accepted 26 August 2014Available online 18 September 2014Mucopolysaccharidosis type II (MPSII) is an X-linked lysosomal storagedisorder caused by deﬁciency of the enzyme iduronate-2-sulfatase(IDS). The human IDS gene is located in chromosome Xq28. This is theﬁrst report of genotype and phenotype characterization of 49 Hunterpatients from 40 families of Argentina. Thirty different alleles have beenidentiﬁed, and 57%were novel. The frequency of de novomutationswas10%. Overall, the percentage of private mutations in our series was 75%.© 2014 The Authors. Published by Elsevier Inc. This is an open accessarticle under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/3.0/).Keywords:Genetic testingGenotype–phenotype correlationHunter syndromeLysosomal storage disorderMucopolysaccharidosis type II1. IntroductionMucopolysaccharidosis type II (MPS II, Hunter disease, MIM 309900) is an X-linked lysosomal storagedisorder caused by deﬁciency of the enzyme iduronate-2-sulfatase (IDS). Clinical manifestations are coarse0), Argentina. Tel.: +54 221 4250497; fax: +54 221 4226947.zenfeld).evier Inc. This is an open access article under the CC BY-NC-ND license/).Table 1List of mutations from 40 families of Argentinean Hunter patients. NA: sample not available.Family Number ofpatientsType of mutation Genotype Protein alteration Exon Phenotype Presence of the mutation inthe motherReference1 1 Complete IDS deletion Severe NA2 1 Complete IDS deletion Severe NA3 1 Complete IDS deletion Severe NA4 1 Gene–pseudogenerecombinationSevere NA5 1 Gene–pseudogenerecombinationSevere NA6 1 Gene–pseudogenerecombinationSevere Y7 2 Missense c.683C N A p.Pro228Gln 5 Severe Y This study8 4 Missense c.1403G N A⁎ p.Arg468Gln 9 Severe Y This study, Whitleyet al. [7]9 1 Missense c.253G N A⁎ p.Ala85Thr 3 Mild NA This study, Li et al. [8]10 1 Missense c.1403G N A p.Arg468Gln 9 Severe N This study, Whitleyet al. [7]11 1 Missense c.1403G N A + c.1394A N T p.Arg468Gln + p.Gln465Leu 9 Severe Y This study12 1 Missense c.884A N T p.Lys295Ile 7 Severe Y This study13 1 Missense c.181 T N C p.Ser61Pro 2 Mild Y This study, Sohn et al.[9]14 1 Missense c.998C N T p.Ser333Leu 7 Severe Y This study, Flomenet al. [10]15 1 Missense c.592G N A p.Asp198Asn 5 Severe N This study16 1 Missense c.1033 T N A p.Trp345Arg 8 Severe NA This study17 1 Missense c.425C N A p.Ser142Tyr 4 Severe Y This study18 1 Missense c.401G N A⁎ p.Gly134Glu 3 Mild Y This study19 2 Missense c.359C N A p.Pro120His 3 Severe Y This study, Hopwoodet al. [11]20 1 Missense c.935G N A p.Gly312Asp 7 Mild Y This study402H.Amartinoetal./MolecularGeneticsandMetabolismReports1(2014)401–406FamilyNumber ofpatientsType of mutation Genotype Protein alteration Exon Phenotype Presence of the mutation inthe motherReference21 1 Missense c.641C N T p.Thr214Met 5 Severe NA This study22 1 Missense c.253G N A p.Ala85Thr 3 Mild NA This study, Li et al. [8]23 1 Missense c.1403G N A p.Arg468Gln 9 Severe NA This study, Whitleyet al. [7]24 1 Missense c.1016 T N C p.Leu339Pro 8 Severe Y This study, Guo (2006)25 1 Missense c.998C N T p.Ser333Leu 7 Severe Y This study, Flomenet al. [10]26 1 Missense c.469C N T p.Pro157Ser 4 Severe Y This study27 1 Missense c.1433A N G p.Asp478Gly 9 Mild Y This study, Schröderet al. [12]28 Missense c.1393C N T p.Gln465X 9 Severe NA This study, Li et al. [13]29 4 Nonsense c.820G N T p.Glu274X 6 Severe Y This study30 1 Nonsense c.1327C N T p.Arg443X 9 Severe Y This study, Bunge et al.[14]31 1 Short deletion c.908_909delCT Frameshift 7 Severe Y This study32 1 Short deletion c.411delT Frameshift 3 Severe Y This study33 1 Short deletion c.22_37del16pb Frameshift 1 Severe N This study34 2 Splicing c.508-1 g N a Partial exclusion exon 5 3′ deIVS4Mild Y This study35 1 Splicing c.1122C N T G374G. Loss of 20 aa 8 Mild Y This study, Rathmannet al. [15]36 1 Splicing c.241-5a N t Exclusion of exon 3 3′ deIVS2Severe Y This study37 1 Splicing c.708 + 1 g N a⁎ Exclusion exon 5 5′deIVS5Severe Y This study38 1 Splicing c.1181-1 g N a Exclusion exon 9 3′ deIVS8Severe Y This study, Popowskaet al. [16]39 1 Splicing c.1122C N T⁎ G374G. Loss of 20 aa 8 Mild Y This study, Rathmannet al. [15]40 2 Splicing c.1122C N T G374G. Loss of 20 aa 8 Mild Y This study, Rathmannet al. [15]⁎ The polymorphism c.438C N T was present.403H.Amartinoetal./MolecularGeneticsandMetabolismReports1(2014)401–406404 H. Amartino et al. / Molecular Genetics and Metabolism Reports 1 (2014) 401–406facial features, hepatosplenomegaly, dysostosis multiplex, heart disease, obstructive upper respiratorydisease, and frequent ear infections. Patients present a spectrum of phenotypes, ranging from severe toattenuated course [1].The human IDS gene is located in chromosome Xq28, spans approximately 24 kb and contains nine exons.A full length cDNA clone of the gene showed an open reading frame of 1650 bp long predicting a proteinwith550 aminoacids [2]. An IDS pseudogene (IDSP1) has been detected 20 kb distal to the telomeric side from thefunctional gene, whose sequences are highly homologous to exons 2 and 3 and introns 2,3 and 7 of the IDSgene [3]. To date, over 500 different mutations associated with MPS II have been identiﬁed [Human GeneMutation Database (HGMD); http://www.hgmd.org/].We report the results of genotype and phenotype characterization of 49 Hunter patients of 40 familiesfrom Argentina.2. Materials and methods2.1. PatientsForty nine Hunter patients and their mothers (only 28 of mother samples where available) from 40families were included in this study. Parents of the patients gave their informed consent previous to theparticipation in the study. MPS II diagnosis was conﬁrmed by clinical examination and deﬁcient IDS activity(activity of arilsulfatase B was normal in all cases, ruling out multiple sulfatase deﬁciency). Phenotype ofMPSII patients has been classiﬁed into attenuated (non-neuronopathic) or severe (neuronopathic) accordingto the clinical evidence of cognitive or developmental involvement.2.2. IDS genomic DNA analysisGenomic DNA was extracted from EDTA blood using a QIAamp DNA Blood Mini kit (Qiagen, Valencia,CA, USA). The 9 exons of the IDS gene including exon/intron boundaries were ampliﬁed by PCR usingspeciﬁc primers as described [3]. Sequencing was carried out in both directions in a MegaBASE 1000sequencer (Amersham Biosciences, Sunnyvale, CA, USA).Recombination between IDS and IDSP1 leading to any inversion mutation was analyzed by a PCRampliﬁcation assay as described [4].2.3. IDS cDNA analysisTotal cellular RNA was isolated from blood leukocytes by means of a total RNA isolation system(Macherey Nagel, Duren, Germany). The isolated RNA samples were reverse transcribed using a speciﬁcprimer, according to the method of Jonsson [5] and then ampliﬁed in 2 overlapping fragments andsequenced.3. Results and discussionUntil now, we have found the mutation in all the patients analyzed from Argentina. Thirty differentMPS II alleles have been identiﬁed in a cohort of 49 Argentinean patients from 40 families: 18 missense(60%), 5 splicing (17%), 3 short deletions (10%), 2 nonsense mutations (7%), 1 complex rearrangement(a gene–pseudogene recombination leading to inversion) (3%) aswell as 1 gross gene deletion (3%) (Table 1).We have not detected any insertions.Of the mutations in this cohort, 17 out of 30 different alleles (57%) were novel (missense: p.Pro228Gln,p.Lys295Ile, p.Asp198Asn, p.Trp345Arg, p.Ser142Tyr, p.Gly134Glu, p.Gly312Asp, p.Thr214Met, p.Gln465Leu,p.Pro157Ser; splicing: c.508-1G N A, c.241-5A N T, c.708+ 1 g N a; nonsense: p.Glu274X; and short deletions:c.908_909delCT, c.411delT, c.22_37del16pb).The effect of novel missense mutations was analyzed by different means. They are all predicted to beprobably damaging, as analyzed by the prediction tool PolyPhen, and are localized in conservedaminoacids between human and murine IDS but found in patients with both attenuated and severephenotypes. Glycines 134 and 312 are highly conserved among human sulfatases. Mutations found by us405H. Amartino et al. / Molecular Genetics and Metabolism Reports 1 (2014) 401–406at these positions introduce negative charged aminoacids. Although the change in the structure of thesehighly conserved aminoacids is high, both patients display an attenuated phenotype. The mutationsp.Pro228Gln and p.Pro157Ser in severe patients result from the replacement of a rigid nonpolar aminoacidby a polar one. The rest of the novel missense mutations are not localized in conserved aminoacids,however the effect in the phenotype is more devastating.Previously described mutations detected by us were p.Arg468Gln, p.Ala85Thr, p.Ser333Leu, p.Pro120His,p.Ser61Pro, p.Leu339Pro, p.Asp478Gly, p.Gln465X, p.Arg443X, the splice mutations c.1122C N T,c.1181-1 g N a, and the recombination gene–pseudogene.We found 3 novel splicing mutations. The mutation c.508-1 g N a was found in a patient with anattenuated phenotype. At the molecular level, the mutation produced a predominant alternative transcriptand a minor quantity of the correct transcript. The mutant transcript results from the disruption of the 3′original splice-site leading to the deletion of exon 5. The presence of the correct mRNA, although in minorquantity, would explain the less severe clinical manifestations observed in this patient. On the other side,the splicing mutations c.241-5a N t and c.708 + 1 g N a, each generate a unique mRNA product, whosesequence corresponds to the complete deletion of exons 3 and 5, respectively, and are associated with asevere phenotype.In one patient, two different “neighbor” mutations (p.Arg468Gln + p.Gln465Leu) were found. Themother was a carrier of both mutations, but not the grandmother and the 4 maternal aunts. Few otherreports described the presence of “neighbor” mutations [6]. The ﬁnding of these two close mutations inthe patient and his mother but not in the grandmother suggests that they are de novo mutations in amutation-prone region and reinforces the idea that they arose simultaneously.Three patients had a complete gene deletion. None of the ﬂanking markers tested were detected,suggesting that the deletion spans more than 20 kb upstream and 7 kb downstream of the IDS locus, andincludes the IDSP1 locus. No atypical symptoms of Hunter syndrome were found in these patients,indicating that the deletion did not compromise contiguous genes. The phenotype of these patients is thesevere form of the disease. The same phenotype was observed for the three of the patients who exhibited arecombination between gene and pseudogene.The absence of the probands' mutations in their mother suggests that in the three cases, themutations occurred de novo (10%). However, the possibility of germinal mosaicism could not be ruledout. The theoretical rate calculated by the Bayesian methods for this condition would be 33%, so thevalue found in our cohort is much less. In the cohort reviewed by Froissart et al., they found values ofde novo mutations close to the expected in any X chromosomal disease in which male reproductiveﬁtness is extremely low [2]. The low value found in our group could be because much of the patientsfrom Argentina were diagnosed because of a previous family history of MPS II, meaning there are stillmany undiagnosed patients in Argentina. We also found a low rate of the polymorphism in exon4 c.438C N T, only in 4 families (10%) as compared to the frequency reported in normal controls ofaround 30% [3].The percentage of private mutations in our series was 75%, a high proportion as usually seen in otherseries.Although genotype–phenotype correlation is difﬁcult to establish, it is worth mentioning thatboth missense (p.Ser61Pro, p.Ala85Thr, p.Gly134Glu, p.Gly312Asp, p.Asp478Gly) and splice mutations(c.1122C N T, c.508-1G N A) were associated with non-neuronopathic (“attenuated”) phenotype. From ourseries of 30 alleles, 7 (23%) were found associated with mild forms. Three of the mutations associated withthe non-neurological phenotype, are new (c.508-1 g N a, p.Gly134Glu, p.Gly312Asp).This is the ﬁrst report of mutations of Hunter patients from Argentina. Our results provide furtherevidence of mutational heterogeneity of the IDS gene observed in patients with MPS II.Role of the funding sourceThis work was ﬁnancially supported by a grant from Agencia Nacional de Promoción Cientíﬁca yTecnológica, Argentina (PICT 01070) to PR, however the funding source had no involvement in studydesign; in the collection, analysis and interpretation of data; in the writing of the report nor in the decisionto submit the article for publication.406 H. Amartino et al. / Molecular Genetics and Metabolism Reports 1 (2014) 401–406References[1] J.E. Wraith, M. Scarpa, M. Beck, et al., Mucopolysaccharidosis type II (Hunter syndrome): a clinical review andrecommendations for treatment in the era of enzyme replacement therapy, Eur. J. Pediatr. 167 (2008) 267–277.[2] R. Froissart, I. Da Silva, I. Maire, Mucopolysaccharidosis type II: an update on mutations spectrum, Acta Paediatr. 96 (2007) 71–77.[3] P. Li, A.B. Bellows, J.N. Thompson, Molecular basis of iduronate-2-sulphatase gene mutations in patients withmucopolysaccharidosis type II (Hunter syndrome), J. Med. Genet. 36 (1999) 21–27.[4] K. Lagerstedt, S.L. Karsten, B.M. Carlberg, et al., Double-strand breaks may initiate the inversion mutation causing the Huntersyndrome, Hum. Mol. Genet. 6 (1997) 627–633.[5] J.J. Jonsson, E.L. Aronovich, S.E. Braun, et al., Molecular diagnosis of mucopolysaccharidosis type II (Hunter syndrome) byautomated sequencing and computer-assisted interpretation: toward mutation mapping of the iduronate-2-sulfatase gene,Am. J. Hum. Genet. 56 (1995) 597–607.[6] I.V.D. Schwartz, L.C. Lima, K. Tylee, et al., Further cases of “neighbor”mutations inmucopolysaccharidosis type ii, Am. J. Med. Genet.A 140A (2006) 1684–1686.[7] C.B. Whitley, R.A. Anderson, E.L. 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Identification of 17 novel mutations in 40 Argentinean unrelated families with mucopolysaccharidosis type II (Hunter syndrome)

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Identification of 17 novel mutations in 40 Argentinean unrelated families with mucopolysaccharidosis type II (Hunter syndrome)

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