Molecular-genetic characterization of Ukrainian patients with mucopolysaccharidosis I : identification of three new mutations in α-L-iduronidase gene

N. S. Trofimova, N. V. Olkhovich © 2016 N. S. Trofimova et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 616-056.7-07


Introduction
Mucopolysaccharidosis I (MPS I) is a rare hereditary autosomal-recessive metabolic disorder, which occurs due to the deficiency of the lysosomal enzyme α-L-iduronidase (IDUA; EC 3.2.1.76),involved in the multistage catabolism of dermatan sulphate (DS) and heparan sulphate (HS).The deficiency in α-L-iduronidase leads to the accumulation of non-degraded GAG substrate in the lysosomes of cells of different organs and tissues [1,2].
Although the clinical phenotype of MPS I is a continuous spectrum from severe forms to mild ones, generally three clinical forms are distinguished: 1) severe form (Hurler syndrome, MPS I H; MIM # 607014, ORPHA 93473), with the manifestation of symptoms by the age of 12 months, life span of no more than 10 years and mental retardation, which is manifested at the age under three; 2) intermediate form (Hurler-Scheie syndrome, MPS I H/S; MIM # 607015, ORPHA 93476), with the manifestation of symptoms at the age from 1 to 6 years, longer life span and no or mild mental retardation, which is never manifested under 3; 3) mild form (Scheie syndrome, MPS I S; MIM # 607016, ORPHA 93474), with the manifestation of the first symptoms of the disease af-Biomedicine ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2016. Vol. 32.N 6. P 442-449 doi: http://dx.doi.org/10.7124/bc.00093B ter the age of 5, unchanged life span and complete absence of mental retardation [1].
The gene, encoding α-L-iduronidase (IDUA; MIM # 252800), was mapped in locus 4p16.3; it consists of 14 exons, separated by 13 introns.An open reading frame of cDNA (ORF) is ~ 2 kilobases long and encodes the polypeptide of 653 amino acids [3].At present there are over 220 mutations, identified and characterized in the IDUA gene, including missence/nonsense mutations, small deletions and insertions, four gross-deletions, one grossinsertion, 36 splicing mutations and three complex mutations (Human Gene Mutation Database-HGMD, http://www.hgmd.cf.ac.uk/ac/index.php).In general most mutations occur less than in 3 % of cases or are single.
The missence mutations p.W402*, p.Q70*, p.P533R and p.G51D are the most common for the gene IDUA.Mutation p.W402* has about 50 % distribution frequency in Northern Europe, Great Britain, North America, and Spain, whereas its frequency in the Russian Federation, Italy, and Brazil is 4 %, 11 %, and 20 % respectively, according to the estimates of different researchers [4,5].On the contrary, mutation p.Q70* occurs in Scandinavia and the Russian Federation much more frequently (up to 50 %) than in other countries [6].The missence mutation p.P533R of probable North African origin is wide-spread in the Mediterranean region, and has 13 % and 10 % of mutant alleles in the IDUA gene in Italy and Spain, respectively.According to the data of Italian researchers, the missence mutation p.G51D is major for Italy only, and is registered with the frequency of 13 % for mutant alleles in the IDUA gene among Italian patients with mucopolysaccharidosis I [4].The frequency of mutations in the IDUA gene for Ukraine has not been defined yet.
The aim of our work was to identify the whole spectrum of mutations in the IDUA gene in Ukrainian patients with MPS I.

Materials and Methods
The material of the research was the blood of 17 patients with MPS I (7 boys and 10 girls), whose diagnosis was confirmed in the Clinical Genetics Laboratory, NCSH OKHMATDYT of the Ministry of Health of Ukraine, using biochemical methods with the determination of the activity of α-L-iduronidase enzyme in lysosomes, and the blood of their 37 relatives (siblings and parents) from different regions of Ukraine [7].The age of the patients at the moment of the diagnosis determination was 2 ± 1 years for Hurler syndrome, 14 ± 1 years for Hurler-Scheie syndrome and Scheie syndrome.One proband had a sibling with the identical genotype, who was not taken into consideration while identifying the mutation frequency.For control of the pathogenicity of new mutations, the blood samples of 100 volunteers aged 18 to 60 years without any clinical signs of lysosomal pathology were used.The parents of all the patients and volunteers gave their informed consent for the studies.The bioethics committee approved the research.
DNAs were extracted from the whole blood using the commercial NeoSorb kits (Neogen, Ukraine).The identification of major mutations p.Q70* and p.W402* was conducted using the RFLP-analysis as described in our previous publication [7].
The identification of rare mutations in the IDUA gene involved the method of direct automated sequencing by the Sanger's method using ABI 3130 analyzer (Applied Biosystems) according to the manufacturer's protocol.The sequences of primers for each of fourteen exons of the IDUA gene were selected using Primer3 program (http://simgene.com/Primer3).The analysis of sequencing results was made using programs Sequencing Analysis, v.1.1/3.1 (Applied Biosystems, Life Technologies Corporation, USA), Chromas and Blast (http://www.ncbi.nlm.nih.gov/blast).The analysis of pathogenicity of new mutations was made using programs PolyPhen2 and Provean (http://genetics.bwh.harvard.edu/pph2/,http://provean.jcvi.org/index.php).

Results and Discussion
The studies, conducted by us, allowed identifying 100 % (34/34) mutant alleles of the IDUA gene in Ukrainian patients with MPS I (Table 1).
The most common mutation for the investigated Ukrainian patients with MPS I was nonsense -muta-tion p.Q70* -11 alleles out of 34.The frequency of this mutation is 32.4 % (11/34) which is close to the indices of the distribution of this mutation in Europe [6].Two patients had this mutation in the homozygous state, seven patients -in the heterozygous state.
The other major mutation p.W402* in the IDUA gene was found in five patients with MPS I in the heterozygous state, which is 14.7 % (5/34), i.e. its distribution in Ukraine is close to the indices of Italy [4].Two compound heterozygotes p.Q70*/p.W402* were revealed.
In addition to major mutations, the previously described deletion с.1398delC was identified with high frequency of 8.8% (3/34) in Ukraine, in homo-and heterozygous state in the patients with MPS I.
Four previously revealed mutations were described among the missence mutations, identified during the analysis, in addition to major p.Q70* and p.W402*: in exon 2 -p.A75T, in exon 8 -p.А327P, in exon 11 -p.P533L, and in exon 14 -p.S633I.The previously described mutation in the splicing zone for exon 11 IVS11ds+5G-A was identified for one patient in one allele.A previously known insertion of 12 basic pairs с.889_899_ins_12 was revealed in exon 7 in four patients, two of them were siblings.A previously described deletion of 12 bp in exon 1 c.46_57del_12 was found in one patient.
Additionally, three mutations in the IDUA gene, which have been neither previously described nor included into HGMD database, were identified by us, namely, three missence mutations: p.N372S in exon 8 (in three alleles in two patients), p.Q563P in exon 12 (in one allele), and nonsense-mutation p.S633* in exon 14 (in two alleles in one patient) (Fig. 1).
No changes in the sequence of nucleotides of the IDUA gene were identified by us in the individuals of the control group, i.e. they may be estimated as mutations, and not as a polymorphic variant.
The detailed analysis of these mutations using programs PolyPhen2 and Provean confirmed their pathological character (Fig. 2).
A new missence mutation p.N372S, which was identified in exon 8, leads to the replacement of asparagine for serine, due to which the length of protein does not change, but there is a disorder in the functioning of the protein, which causes the change in the splicing site.A new missence mutation p. Q563P, which was identified in exon 12, results in the replacement of glutamine for proline, which does not have any significant impact on the protein func- tion, leading only to the point replacement of the aminoacid in its structure.A new nonsense-mutation p.S633* in exon 14 forms a stop-codon and shortens the protein by 21 aminoacids (~10 % from the total length of the protein).Therefore, the spectrum of mutations in the IDUA gene in Ukrainian patients with MPS I at presen is represented by 9 missence mutations, 2 deletions of 1 and 12 bp, 1 insertion of 12 bp, and 1 mutation in the splicing zone (Fig. 3).
Similar to many other European populations, two mutations may be considered to be major for Ukrainian patients with MPS I: p.Q70* (32.35 %) and p.W402* (14.7 %).As for rare mutations, the most common ones were found to be deletion с.1398delC (8.8 %) and a new missence mutation p.N372S (8.8 %), which were revealed in the homo- and heterozygous state among Ukrainian patients with MPS I.The insertion с.889_899_ins_12 (8.8 %), identified in four patients in the heterozygous state, two of whom were siblings, was also found to be quite common.A new nonsense mutation p.S633* (5.9 %), which was identified in one patient, may also potentially be more common for Ukraine, as it was identified in the homozygous state.However, this may also be a result of the intrafamilial accumulation of the pathological mutation after the marriage of close relatives, who are parents of this patient.Other mutations were represented by single cases.Probably, these frequencies may change with further identification of new patients.The most rare mutations, identified by us in Ukrainian patients with MPS I, were localized in exons 2, 7, 8, 9, and 14, and one in each -in exons 1, 11, and 12 of the IDUA gene.
The complete characterization of the patients with MPS I with the consideration of the clinical form of the disease is presented in Table 2.
The results of molecular-genetic analysis of Ukrainian patients with MPS I, obtained by us, demonstrated that all the patients with major mutation p.Q70* in the homozygous state, and two patients, who were found to be compound heterozygotes of p.Q70X*/ p.W402*, have a severe form of the disease -Hurler syndrome, which is in good agreement with the data of other researchers about the associa-tion of major nonsense -mutations p.Q70* and p.W402* with the severe disease progression [6,8].The severe progression of MPS I was also noted for patients with the major mutation p.W402* in one allele and the missence mutation p.A75T (1 person) [15].A previously known single-nucleotide deletion с.1398delC, identified in two patients in both the homozygous state and the compound with the major mutation p.W402*, also characterized the presence of the severe clinical form of MPS I, Hurler syndrome, in patients [4].
Two patients, who had a new, previously not described missence mutation p.N372S in the homozygous state and in the compound with the major mutation p. Q70*, were characterized by early onset of the disease and a severe phenotype.Three patients with the compounds combination of major mutations and rare mutations (genotypes p.Q70*/p.P533L, p.Q70*/c.46_57_ del_12 and p.W402*/p.S633I) had a severe form of the disease -Hurler syndrome with early onset of the disease.
Scheie syndrome was diagnosed in four patients, two of whom were siblings, with genotypes с.889_899_ins_12/p.Q70* -in three patients, and the missence mutation p.А327P in the Therefore, our analysis of the molecular-genetic characterization of Ukrainian patients with MPS I allowed identifying 100 % mutant alleles.High frequency of mutations p.Q70* (32.35 %) and p.W402* (14.7 %) among Ukrainian patients substantiates RFLP-screening for the presence of these mutations at the first stage of the molecular-genetic analysis.It would be reasonable to start the analysis during the identification of rare mutations, which have led to the development of the disease in the proband, with "hot" regions of the IDUA gene -exons 2, 7, 8, 9, and 14, where most pathogenic mutations were localized in the patients, studied by us.During the planning of the analysis, this order would allow optimizing time and resources for the analysis and ensuring the maximal efficiency of the molecular genetic diagnostics of MPS I in Ukraine.

Conclusions
The molecular genetic analysis of pathogenic mutations in the IDUA gene in 18 Ukrainian patients with MPS I from 17 families (34 alleles) revealed 100 % mutant alleles.
The share of the alleles with the major mutation p.Q70* was the largest -32.35 % (11/34).The other major mutation for European populations, p.W402*, was found in five patients (14.7 %) in one allele, so it is also major for Ukraine.
Most rare mutations, identified by us in Ukrainian patients with MPS I, were localized in exons 2, 7, 8, 9, and 14 of the IDUA gene, and one in each -in exons 1, 11, and 12.
Three previously not described missence mutations in the IDUA gene -p.N372S, p.Q563P and p.S633* -were also revealed.
The data of the conducted molecular genetic analysis of mutations in the IDUA gene may be used

Fig. 1 .BFig. 2 .
Fig. 1.The chromatograms of the DNA sequence of new mutations in the IDUA gene: А -missence mutation p.N372S in the homozygous state in exon 8; B -missence mutation p.N372S in the heterozygous state in exon 8; C -missence mutation p.Q563P in the heterozygous state in exon 12; D -missence mutation p.S633* in the homozygous state in exon 14.

Fig. 3 .
Fig. 3.The localization of mutations in separate exons of the IDUA gene in Ukrainian patients with MPS I