Background: The mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease resulting from the defective activity of the enzyme α-L-iduronidase (IDUA). The disease has three major clinical subtypes (severe Hurler syndrome, intermediate Hurler–Scheie syndrome and attenuated Scheie syndrome). We aim to identify the genetic variants in MPS I patients and to investigate the effect of the novel splice site mutation on splicing of IDUA- mRNA variability using bioinformatics tools. Methods: The IDUA mutations were determined in four MPS I patients from four families from Northern Tunisia, by amplifying and sequencing each of the IDUA exons and intron–exon junctions. Results: One novel splice site IDUA mutation, c.1650 + 1G > T in intron 11 and two previously reported mutations, p.A75T and p.R555H, were detected. The patients in families 1 and 2 who have the Hurler phenotype were homozygotes for the novel splice site mutation c.1650 + 1G > T. The patient in family 3, who also had the Hurler phenotype, was a compound heterozygote for the novel splice site mutation c.1650 + 1G > T and for the previously reported missense mutation p.A75T. The patient in family 4 who had the Hurler–Scheie phenotype was a compound heterozygote for the novel splice site mutation c.1650 + 1G > T and for the previously reported missense mutation p.R555H. In addition, four known IDUA polymorphisms were identified. Bioinformatics tools allowed us to associate the variant c.1650 + 1G > T with the severe clinical phenotype of MPS I. This variant affects the essential nucleotide + 1 (G to T) of the donor splice site of IDUA intron 11. The G > T in intron 11 leads to wild type donor site broken with minus 19.97% value compared to normal value with 0%, hence the new splice site acceptor has plus 5.59%. Conclusions: The present findings indicate that the identified mutations facilitate the accurate carrier detection (genetic counseling of at-risk relatives) and the molecular prenatal diagnosis in Tunisia. Keywords: Mucopolysaccharidosis type I, α-L-iduronidase, Splice site mutation, Homozygous, Compound heterozygote Background characterized by infantile onset, severe organomegaly and Mucopolysaccharidosis type I (MPS I) is an autosomal re- bone involvement, and mental retardation. The intermedi- cessive lysosomal storage disorder caused by the deficient ate Hurler–Scheie syndrome (MPS IH/S; 607015) is char- activity of the enzyme α-L-iduronidase (IDUA, EC 220.127.116.11). acterized by onset in childhood, severe organomegaly and This glycosidase is required for the hydrolysis of α- bone involvement, and usually limited, if any, neurological L-iduronide residues of dermatan sulphate and heparan involvement. The attenuated Scheie syndrome (MPS IS; sulphate . 607016) is characterized by later onset, visceral and bone MPS I has three major clinical subtypes among which disease, and neurological developmental delay . the severe Hurler syndrome (MPS IH; 607014). It is The IDUA gene has 19 kb in length, containing 14 exons and 13 introns. It is mapped on the short arm * Correspondence: email@example.com of chromosome 4 at region p16.3 being tran- Faculty of pharmacy, University of Monastir, 5000 Monastir, Tunisia scribed into a 2.3 kb cDNA, which encodes a Faculty of pharmacy of Monastir, University of Monastir, Avenue Avicenne, 653-residue glycopeptide . 5019 Monastir, Tunisia Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chkioua et al. Diagnostic Pathology (2018) 13:35 Page 2 of 8 To date, more than 201 mutations and 32 polymorphisms Biochemical diagnosis have been identified [Human Gene Mutation Database] The diagnosis of these diseases was based on the follow- (http://www.hgmd.org;2017).Previousmutations include ing approach after a clinical and paraclinical suspicion. 113 missense/nonsense, 33 splicing, 31 small deletions, 15 small insertions, 4 gross deletions and 3 complex rearrange- Quantitative and qualitative analysis of total urinary ments. Genetic testing MPS I patients is useful for the glycosaminoglycans identification of specific genotypes, genotype-phenotype Study of urinary glycosaminoglycans was performed first. correlations and also for prenatal diagnosis. Urinary GAGs were quantified using a dimethylmethylene The splice site mutations are DNA sequence changes blue test (DMB) . The quantity of DMB bound to sul- that alter or abolish correct mRNA splicing during the fated glycosaminoglycans was measured via spectropho- process of precursor mRNA maturation. The modification tometry at wavelength of 656 nm. Electrophoresis on in the consensus sequence, known as splice-donor and cellulose acetate plate was performed to identify which splice-acceptor sequences, which surround each exon may type of GAGs is present in excess (e.g., dermatan sulphate, lead to: exon skipping, cryptic splice site activation, cre- heparan sulphate, keratan sulphate). Discontinuous elec- ation of a pseudo-exon within an intron and intron reten- trophoresis on cellulose acetate plate separated the differ- tion . Hence some splice site mutations do not abolish ent GAGs based on their charge and differential solubility completely the wild-type transcript expression, which may in ethanol, and the mucopolysaccharides were visualized lead to less severe phenotypes . In our study, we have by staining with alcian blue . analyzed the novel splice donor site c.1650 + 1G > T in in- tron 11 using bioinformatics tools to determine the im- Enzyme analysis pact of this variant in MPS I phenotypic expression. Enzyme analysis for α-L-iduronidase (MPSI, EC 18.104.22.168) was performed in sonicated leukocytes pellets as described Methods using the 4-methylumbelliferyl-α-L-iduronide . Ethics statements Written informed consent was obtained and signed by Molecular analysis and DNA sequencing analysis all studied families after a full explanation of this study, We analyzed the IDUA gene of 4 MPS I patients from which was approved by the local ethic committees for Northern Tunisia using PCR, PCR-based restriction scientific research of the La Rabta Hospital Tunis, fragment length polymorphism (RFLP) and direct se- Tunisia. Additional informed consent was obtained from quencing methods. all patients for whom identifying information is included Genomic DNA was isolated from venous blood by the in this study. All procedures were in accordance with phenol/chloroform procedure according to standard the ethical standards of the responsible committee on protocols as described previously . All the exons and human experimentation (institutional and national) and flanking intron/exon junctions of the IDUA gene were with the Helsinki Declaration. amplified and sequenced. For patients with a family his- tory of known or suspected pathogenic mutations or for Study populations the indexed cases of parents, the targeted DNA locus This is a series of four patients (P1, P2, P3 and P4) with was analyzed. MPS I disease aged 2–5 years who were recruited in the PCR reaction consisted of 50 ng of DNA,1 X HotStar- pediatric department of La Rabta Hospital in Tunisia. Taq buffer (Qiagen, Paris, France), 2 mM MgCl2, Among the four explored MPS I families, three (family 1, 200 μM of each dNTP, 10 pmol of each primer, 2.5 U of family 2 and family 4) are related as second cousins. The HotStarTaq (Qiagen, Paris, France) and 1 X Q solution. MPS I patients had a clinical diagnosis of Hurler syndrome The final reaction volume was 18 μl. Thermal PCR pro- which was further confirmed with biological analysis by file consisted of an initial denaturation at 95 °C for demonstrating a high excretion of GAGs in the urine and a 15 min, 35 cycles of denaturation at 94 °C for 30s, an- deficiency in α-L-iduronidase activity in leukocytes. The nealing at 68 °C for 30s and extension at 72 °C for 30s parents and other family members of each studied family followed by a final extension step at 72 °C for 10 min. were investigated in order to create a clearer profile of the PCR products were resolved in 2% agarose gel and were disease’s transmission to facilitate prenatal diagnosis and visualized under UV light. counseling for families at risk in Tunisia. In this study based on clinical manifestations of MPS I In silico predictions patients, an analysis of urine glycosaminoglycans The splice site mutation was located in the defined (GAGs) was done in first intention but this screening re- splice site consensus sequences which were (C/ quires a differential diagnosis with the Hunter syndrome A)AG|gt(a/g)agt and cag|G, for the donor splice site and for which we obtained the same GAG profile. the acceptor splice site, respectively . Chkioua et al. Diagnostic Pathology (2018) 13:35 Page 3 of 8 We have analyzed the splice site mutation in intron 11 to a Histidine missense mutation (p.R555H) and to the of IDUA gene by using the bioinformatics tools: Human novel splice site mutation c.1650 + 1G > T. Splicing Finder (HSF) version 2.4 (http:// In addition, four previously reported polymorphisms www.umd.be/HSF/) which includes several matrices to in exon 11 of IDUA gene were identified in the IDUA analyze splice site and Exonic Splicing Enhancer (ESE) patients and their parents: rs141046991, rs773947412, finder predictions (http://krainer01.cshl.edu/cgi-bin/ rs759123051, rs773184536 (Table 1). tools/ESE3/esefinder.cgi?process=home) to examine the conservation of ESE motifs. Splice site mutation analysis The relative strength of the splicing sites obtained from the bioinformatics tool is given as a consensual value Results (CVs), which varies from 0 to 100. Therefore, the effect Clinical features and biochemical analysis of a splice site mutation depends on the CVs value. The clinical features of each patient are presented in Splice sites with CVs over 80 are solid splicing sites, but Table 1. splicing sites with CVs ranging from 65 to 70 are weak Phenotypic analysis confirmed the diagnosis of all the sites because only a few of these sites are active . MPS IH studied patients. Indeed, for all the studied pa- The splice site mutation leads to the use of cryptic tients, the electrophoresis on cellulose acetate plate of sites thus the most cryptic splice sites are to be located GAGs showed the presence of heparan sulphate (HS) +/− 100 bp on each side of the exon-intron boundary. and dermatan sulphate (DS), an abnormal band, com- We have analyzed this region for the presence of poten- pared to the control case, in addition to an abnormal tial splice sites using HSF version 2.4 (http:// band of HS in MPSIII patient (Fig. 1). www.umd.be/HSF/) in the case of c.1650 + 1G > T muta- IDUA activity in MPS IH patients ranged from 0.00 to tion and we have found that the abolition of the wild 0.044 μKat/Kg protein. type donor splice site, with minus 19.97% value, and its substitution by a new splice acceptor site, with plus IDUA mutation analysis 5.59% value, leads to three possible conclusions (Fig. 3): Clinical and identified genotypes of studied patients are firstly, the loss of exon 11; secondly, the retention of the summarized in Table 1. part of the intron 11 sequence; and finally, the activation As a result of DNA sequencing analysis and of an alternative cryptic splicing site in exon 11 giving a RFLP-PCR, one novel and two previously reported mu- new size of exon 11 of about 135 bp compared to the tations were identified in this study including: two mis- normal length of 304 bp (Table 2). sense mutations p.A75T and p.R555H, and one novel The results using the Human Splicing Finder, showed splice site mutation c.1650 + 1G > T (Table 1). that the wild phenotype has a donor splice site CVs close Patients 1 and 2 from families 1, and 2 (Fig. 2) were all to 100, thus this new splice site could be strong and homozygous for a novel G to T transition in the con- functional enough to justify the mutant phenotype which served 5′ splice donor site of IDUA intron 11 (CAGGc> presented a new splice acceptor site with a CV at about CAGTc) (Table 2). The splice site mutation obliterated a 80 which would justify the new isoform of mRNA in pa- Cac8I restriction enzyme site. The amplicon of exon 11 tients (Table 2). from genomic DNA and its digestion with Cac8I re- sulted in three fragments (66, 101 and 378 bp) in the pa- Discussion tients with the splice site mutation, instead of the four This work was conducted as a straight continuation of fragments (23, 66, 101 and 356 bp) observed in normal studies carried out in other Tunisian MPS I patients and individuals (data not shown). their families [12–15]. In this cohort we were interested Patient 3 was a compound heterozygote for a G to A in patients presenting a severe phenotype of MPS I. All transition in exon 2 (Fig. 2) predicting an Alanine to a MPS IH patients have the splice site c.1650 + 1G > T Threonine missense mutation (p.A75T) and to a novel mutant allele in homozygous and/or heterozygous splice site mutation c.1650 + 1G > T. The p.A75T mis- forms. sense mutation created a MSLI restriction enzyme site. Our study showed that all studied patients were from The MSLI digestion of the amplicon of exon 2 from gen- different regions within the Northern part of Tunisia: omic DNA resulted in two fragments (166 and 139 bp) Tunis (Oued Ellil) and Nabeul (Korba and Elmaamoura) in the patient with the missense mutation, instead of the that are over 60 km away from each other; however, one fragment (305 bp) observed in normal individuals three families (P1, P2 and P4) were known to be related. (data not shown). Patients 1 and 2 (P1 and P2) who developed a severe Patient 4 was also a compound heterozygote for a G form of MPS I were homozygotes for the novel splice to A transition in exon 11 (Fig. 2) predicting an Arginine site mutation c.1650 + 1G > T. Chkioua et al. Diagnostic Pathology (2018) 13:35 Page 4 of 8 Table 1 Characteristics of the Tunisian MPS IH families Family Family 1 Family 2 Family 3 Family 4 Mother Father Patient 1 Mother Father Patient 2 Mother Father Patient 3 Mother Father Patient 4 Consanguinity 2nd cousins Unrelated 1st cousins 2nd cousins Tunisian Origin Nabeul Elmaamoura Korba Oued Ellil Sex/age (Years) 36 46 M/3 32 40 F/5 (died) 37 45 M /3 (died) 34 43 M/2 Leukocyte IDUA Activity μKat/Kg Unrealized Unrealized 0.00 Unrealized Unrealized 0.00 Unrealized Unrealized 0.044 Unrealized Unrealized 0.00 Protein Infantile onset –– + –– + –– + – + Organomegaly –– Severe –– Severe –– Severe –– Severe Bone involvement –– + –– + –– + –– + Mental retardation –– Severe –– Severe –– Severe –– Non marked Growth retardation –– Marked –– Marked –– Non marked–– Non marked Allele 1 c.1650 + c.1650 + c.1650 + c.1650 + c.1650 + c.1650 + c.1650 + p.A75T c.1650 + c.1650 + p.R555H c.1650 + 1G > T 1G > T 1G > T 1G > T 1G > T 1G > T 1G > T 1G > T 1G > T 1G > T Position Intron 11 Intron 11 Intron 11 Intron 11 Intron 11 Intron 11 Intron 11 Exon 2 Intron 11 Intron 11 Exon 11 Intron 11 Allele 2 NL NL c.1650 + NL NL c.1650 + NL NL p.A75T NL NL p.R555H 1G > T 1G > T Position Intron 11 Intron 11 Exon 2 Exon 11 Polymorphism None None None rs141046991 rs141046991 rs773947412 NL rs773947412 rs759123051 NL rs759123051 rs773184536 NL rs773184536 NL normal, F female, M Male Chkioua et al. Diagnostic Pathology (2018) 13:35 Page 5 of 8 legitimate splice site. The G > T mutation at the 5′-donor splice site of intron 11 presumably causes exon skipping, the loss of exon 11, and subsequently an aberrant poly- peptide that is misfolded (http://rulai.cshl.edu/tools/ESE3) which should explain the severe phenotype of MPS I. In further studies, we may suggest to perform in vitro ana- lysis in order to confirm the in silico predictions and therefore i) to study the impact of the splice site mutation during the process of precursor mRNA maturation, ii) to analyze the phenotypic expression of this disease. Fig. 1 MPS I electrophoresis profile on a cellulose acetate plate of Prenatal diagnosis was performed in family 1. The the urinary GAGs. 1 and 5: MPS III controls; 2, 3 and 4: MPS I or MPS fetus was found homozygous for the splice site mutation II patients; 6 and 7: Normal controls. CS: chondroitin sulphate; DS: dermatan sulphate; HS: heparan sulphate however the parents refused the interruption of preg- nancy. Then, the patient 1 was included in the register Our results about P1 and P2 using in silico predictions of patients wishing to perform a bone marrow transplant showed that the wild donor splice site and the mutant but neither matching relative nor an unrelated matched splice site have higher CVs and below 80 respectively. donor has been found so far. A splice site mutation may cause activation of an alter- The P3 of family 3 does not present any relationship with native cryptic splice site, preferred to the use of the the other investigated families but he lives in the same Fig. 2 Sequence electropherograms of the IDUA mutations identified in the patients with Hurler syndrome: a and e showing the homozygous c.1650 + 1G > T splice site mutation (P1 and P2). b and d showing the heterozygous c.1650 + 1G > T splice site mutation (P3 and P4). c showing the heterozygous p.R555H missense mutation (P4) Chkioua et al. Diagnostic Pathology (2018) 13:35 Page 6 of 8 Table 2 Data findings: CVs and percentage variation of wild type and mutant sequences in intron 11 of IDUA gene (http:// www.umd.be/HSF/) Splice site type Motif New splice site Wild type (WT) Mutant CV If cryptic site use, exon Variation (%) CV (0–100) (0–100) length variation (bp) Acceptor CCGCCCGGGCAGgc ccgcccgggcagTC 84.05 79.76 NA −5.1 Acceptor CGGGCAGgcaagtg ccgggcagtcaagTG 64.94 68.57 NA New site + 5.59 Donor CAGgcaagt CAGtcaagt 100 80.03 135 WT site broken −19.97 Acceptor Ggcaagtggcagtc gtcaagtggcagTC 73.99 77.65 NA + 4.95 region of Tunisia. He was found to be a compound hetero- phenotype observed in this patient, results from the associ- zygote forthe novelsplicesitemutationand thepreviously ation of both mutated alleles: c.1650 + 1G > T and p.A75T. reported missense mutation p.A75T. He developed severe Thus, this new splice site appears to be strong and func- features at an early age (9 months); this finding is in agree- tional enough to justify the mutant phenotype observed in ment with those reported in the literature . The severe P3 which presents a new splice acceptor site with a CV at Fig. 3 Effects of IDUA splice site mutation. a Mutation c.1650 + 1G > T, which abolies a splice donor (GT) within intron 11, resulting in skipping of the exon 11. b Mutation c.1650 + 1G > T, which creates an alternate cryptic splicing site, resulting in the deletion of 169 nucleotides at the end of the exon 11 giving a new size of exon 11 about 135 bp. c Mutation c.1650 + 1G > T which retains the part of the intron 11 Chkioua et al. Diagnostic Pathology (2018) 13:35 Page 7 of 8 about 80 and which would justify the new isoform of Abbreviations DMB: Dimethylmethylene blue test; GAGs: Glycosaminoglycans; HSF: Human mRNA in this patient. The p.A75T mutation has also an Splicing Finder; IDUA: α-L-iduronidase; MPS IH: Mucopolysaccharidosis type impact on phenotype manifestation. This genetic lesion was IH; MPSI: Mucopolysaccharidosis type I; WT: Wild type a non conservative mutation resulting from a change of a Acknowledgements non polar Alanine to a polar Threonine at position 75 of We thank all the families with MPS IH for their participation in this study and IDUA protein. The p.A75T mutation was described for the the clinicians for their fruitful involvement in this work. first time in a patient with a severe MPS I in North Amer- Availability of data and materials ica . Patient 3 died at the age of three and his brother, Novel genetic data from this study has been deposited in the European who developed the similar severe clinical phenotype died Nucleotide Archive [Accession number: LT960372] (https://www.ebi.ac.uk/ before our molecular investigation; he probably had the ena/data/view/LT960372). same genetic lesion as patient P3. Authors’ contributions Family 4 appears to present a relationship with the other LC carried out all the experiments, the data analyses, and wrote the studied families (1 and 2), as second cousins. P4 developed manuscript. HB and IJ supported the analysis and interpretation of the data. early clinical manifestations at 9 months of age, e.g. dys- OG, HB, NT and SL revised the manuscript. All authors participated in writing the manuscript and approved the final version. morphic facial appearance and hepatosplenomegaly, hence these features were presented in moderate form at this Ethics approval and consent to participate age. Patient 4 was a compound heterozygote for the novel The study was approved by the ethics committees for scientific research of the La Rabta Hospital Tunis, Tunisia; no reference number was issued. splice site mutation and the previously reported missense mutation p.R555H. The molecular analysis of genomic Consent for publication DNA of parents confirmed the segregation of the mutants’ Written informed consent for publication of their clinical details and/or alleles. His parents were heterozygous for the splice site clinical images was obtained from the parents of each patient. Copies of the consent forms are available for review by the Editor of this journal. mutation c.1650 + 1G > T and for the missense mutation p.R555H, respectively. Competing interests The p.R555H missense mutation in exon 11 occurred The authors declare that they have no competing interests. at a CpG dinucleotide, a hotspot for mutation , and resulted in a nonconservative transition of a basic Argin- Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in ine to a neutral Histidine. Homozygous patients for the published maps and institutional affiliations. p.R555H mutation have been reported from European countries in patients with the severe phenotype. Author details 1 2 Faculty of pharmacy, University of Monastir, 5000 Monastir, Tunisia. La The investigation of Tunisian patients suffering from Rabta Hospital, 1007 Tunis, Tunisia. The Auvergne-Rhône-Alpes Regional Hurler disease allowed us to define a geographical distribu- Branch of the French National Blood System EFS/GIMAP-EA-3064, 42023 tion of the IDUA mutations. Thus, the missense mutation Saint Etienne, France. Faculty of pharmacy of Monastir, University of Monastir, Avenue Avicenne, 5019 Monastir, Tunisia. p.P533R was recurrent in MPS I patients of Southern Tunisia whereas thesplicesitemutationc.1650+1G> Received: 11 September 2017 Accepted: 13 May 2018 T seems to be frequent in Northern Tunisia. Neither of these mutations have been identified in a different territory References from those in their regions of origin. It is noteworthy that 1. Neufeld EF. The mucopolysaccharidoses. The metabolic and molecular in the Maghreb (Morocco, Tunisia) only the p.P533R-MPS bases of inherited disease; 2001. p. 3421–52. I missense mutation has been identified so far [10, 11]. 2. Scott HS, Bunge S, Gal A, Clarke LA, Morris CP, Hopwood JJ. Molecular genetics of mucopolysaccharidosis type I: diagnostic, clinical, and biological Unfortunately, some MPS I patients die before their implications. Hum Mutat. 1995;6:288–302. investigations. This can be explained by the clinical diffi- 3. Scott H, Anson D, Orsborn A, Nelson P, Clements P, Morris C, Hopwood J. culty and thus the delay of diagnosis of the MPS I inher- Human alpha-L-iduronidase. cDNA isolation and expression. Proc Natl Acad Sci. 1991;88:9695–9. ited disease. The adverse socioeconomic conditions of 4. Berget SM. Exon recognition in vertebrate splicing. J Biol Chem. 1995;270: those patients make the situation even more difficult. 2411–4. 5. Varon R, Dutrannoy V, Weikert G, Tanzarella C, Antoccia A, Stöckl L, Spadoni E, Krüger LA, di Masi A, Sperling K. Mild Nijmegen breakage syndrome Conclusion phenotype due to alternative splicing. Hum Mol Genet. 2006;15:679–89. The novel mutation expands the IDUA gene mutation 6. Stone J. Urine analysis in the diagnosis of mucopolysaccharide disorders. spectrum and contributes to the recognition of its im- Ann Clin Biochem. 1998;35:207–25. 7. Hopwood J, Muller V. Biochemical discrimination of Hurler and Scheie pact on phenotypic expression in MPS I patients. 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Diagnostic Pathology – Springer Journals
Published: May 29, 2018
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