Mutation analysis and clinical characterization of Iranian patients with mucopolysaccharidosis type I

Abstract Background Mucopolysaccharidosis type I (MPSI) is a rare autosomal recessive disorder caused by a deficiency of α‐l‐iduronidase (IDUA) encoded by the IDUA gene. We examined the mutation spectrum of the IDUA gene to explain the clinical, biochemical, and molecular features in 21 Iranian patients with MPSI. Methods Sanger sequencing was used to measure the IDUA gene sequence in the coding region and exon‐intron boundaries. We recorded the clinical findings of studied patients at the first diagnosis of disease and then during the treatment and follow‐up. Results Five different missense disease‐causing mutations were determined in our patient groups, indicating 90.48% of detection rate. The most widespread mutation was the p.Y109H, occurring in 15.625% of all alleles, which was reported for the first time in our study. Other frequent mutations were as follows: p.Ser157Pro (12.5%), p.Gly84Arg (12.5%), p.Asp257His (9.375%), and p.Asp301Glu (9.375%). Three ones of them were new missense mutations: p.Ser157Pro, p.Asp257His, and p.Asp301Glu. Discussion The results of this study explain the different spectrum of IDUA gene mutations in our patients with MPSI. We introduced here 32 different variants including four new variants: p.Y109H (15.625%), p.S157P (12.5%), p.D257H (9.375%), and p.D301E (9.375%). In this series, there was no relationship between the happening of clinical features and genotype variations and biochemical findings.


| INTRODUC TI ON
Mucopolysaccharidosis type I (MPSI; MIM 252800) is an autosomal recessive disorder caused by the deficiency of α-l-iduronidase (IDUA; EC 3.2.1.76). The main role of IDUA is to catalyze the lysosomal degradation of two main glycosaminoglycans (GAGs) including heparan sulfate (HS) and dermatan sulfate (DS). The defect of IDUA enzyme is caused by mutations in the IDUA gene (NCBI Reference Sequence: NG_008103.1). Mucopolysaccharidosis type I is provided with different phenotypes and classified according to the severity of symptoms ranging from severe Hurler syndrome to restively mild Scheie syndrome. 1 The progressive accumulation of DS and HS in lysosomes results in the severe decline of cells, tissues, and organs. 1 Among the other approaches, enzyme replacement therapy with Laronidase (recombinant human IDUA; Aldurazyme (Genzyme Corporation) is mainly used to treat MPSI in United States, Europe, and many other countries. 2 MPSI is considered to be one of the most commonly lysosomal storage disorders in different populations with a mean incidence of 1:100 000-1:150 000 newborns. 3,4 The IDUA gene is located on chromosome 4p16. 3  (rs121965019) and p.Pro533Arg (rs121965021), have been already reported to account for more than 50% of MPSI alleles in most populations. 7 The influence of mutations on IDUA enzyme activity has broad variability, and thus, IDUA is provided in different forms; however, there is not a significant relationship between the IDUA genotypes and various phenotypes that are observed in MPSI. In addition, there is no proved significance relationship in studies published elsewhere between residual l-iduronidase levels and MPSI phenotype. In the same way, there is also no clear genotype-phenotype relationship for this disease. Therefore, the similar residual enzyme activity values can be obtained for described three phenotypes. 8  It is seriously important to always determine and update the genotypic information of a population for a specific disease. In Iran, there is an insufficiency of studies based on a limited number of patients, and therefore, its application was confined to the needs at that time. Therefore, this study was aimed at measuring the mutation variety, compare it to other studies from neighbor regions and finally provide a study that indicates the current Iranian population.

| Biochemical analysis
Quantitative measurement of urinary GAGs: Urinary GAGs was measured calorimetrically according to the reaction of DMB with urinary mucopolysaccharides according to our previous study, and results were expressed in mg/g creatinine. 9 Fluorometric measurement of IDUA in dried blood spots (DBS): IDUA activity in DBS samples was measured according to our previously standardized method. 8 Results were expressed as µmol/spot incubation time, and the low range results were confirmed by IDUA leukocyte assay.

| Molecular study
Genomic DNA was extracted from venous blood using commercial kit of SinaClon Co. Multiple pairs of primers were used to reinforce 10 DNA fragments covering 14 exons and exon-intron boundaries of the IDUA gene. Primers used to amplify the genomic sequences were designed according to the sequence NG_008103. Table 1 shows the primers, PCR product fragments, and annealing temperature for each pairs of primer. Direct DNA fragments sequencing was performed with an ABI-3730XL capillary machine by BioNeer Inc according to Sanger sequencing technique. Patients' genomic sequences are performed compared with the reference sequence by the CLC software (CLCbio). Finally, we compared our results with the published reference sequence. We also sequenced thirty healthy subjects to confirm whether the novel DNA alterations are causative mutations.

| Model building
We have been used the three-dimensional structure of human IDUA (PDB code 3W81) as a model to conduct the mutational studies.

| RE SULTS
The studied population included 21 unrelated MPSI patients  There was the lowest activity in patient 14. There was the highest urinary GAG exertion in patient 5. There was no significant relationship between genotype and related phenotype in the studied subject (data not shown). Table 5 shows the mutation-caused disease and biochemical findings at the time of diagnosis in studied subjects.

| D ISCUSS I ON
In this study, the clinical features, biochemical, and molecular profil- Variation in glycine 84 of IDUA gene to arginine was previously described in some populations. This substitution occurs near to the active site of IDUA, and in addition to gain of charge, it causes loss of main chain flexibility and also solvent available surface. We identified this mutation in 12.5% of our studied subjects. This is a rare mutation and highly effects on solvent available residues. 18,19 The HumVar score for the new substitutions was close to 1 (except for p.Ser157Pro that is 0.772), thus predicted the severe pathological effect of this mutation (Table 3). Moreover, we sequenced  therefore, it explains that biochemical analysis was not significant to determine the clinical types of MPSI ( There were the following inconsistencies in genotype-phenotype correlations. In four cases, mutation p.Ser157Pro was involved.
One homozygote for the p.Ser157Pro mutation was classified as HS, and three homozygous patients were classified as S. However, here it should be noted that another mutation rather than 14 exons and exon-intron boundaries of the IDUA gene could be present, but undetected in our study. This mutation possibly might determine the metabolic phenotype.
Two patients with unknown genotype showed the most interesting contradiction in our study was observed. One patient with unknown genotypes was classified as mild Scheie form and one as sever hurler form. Examining the IDUA gene promoter and a powerful method to detect the duplications could indicate the molecular basis of the disease in these cases. Moreover, modifying factors may highly effect on metabolic phenotype. 21,22 In short, in this study, we extended information on the mutational spectrum of the IDUA gene by reporting data representative of the Iranian MPSI population. A spectrum of five mutations and a total of 32 different genotypes were identified. The detection rate was 90.48%. Our findings should be used as an effective tool for all clinical and diagnostic workplaces in Iran, and they also provide useful information for more clinical trials. In addition, results herein will help to complete relevant genetic information on the Middle East populations and provide an acceptable contribution to the largescale Asian and Middle East studies.

ACK N OWLED G M ENTS
The authors wish to thank all patients, their parents, and health stuffs who participated in this study. Financial support from Tarbiat Modares University is highly appreciated.

AUTH O R S ' CO NTR I B UTI O N S
All authors contributed equally in this work.

E TH I C A L A PPROVA L
All procedures performed in studies involving human participants were in accordance with the ethical standards of the ethics committee of Tarbiat Modares University and Pasteur Institute of Iran and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

I N FO R M E D CO N S E NT
Informed consent was obtained from all individual participants included in the study.

FI N A N CI A L D I SCLOS U R E
The author has no financial relationships relevant to this article to disclose.