Molecular-genetic characterization of Ukrainian patients with mucopolysaccharidosis IIIA : identification of three new mutations in the heparan-N-sulfatase gene

N. S. Trofimova, N. V. Olkhovich, N. G. Gorovenko © 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

A decrease in the activity of any of these enzymes causes the accumulation of HS in the lysosomes of cells with subsequent excretion of the excess of this polysaccharide with biological liquids of the organism, thus resulting in the expressed degeneration of the central nervous system with progressing mental retardation and pathology of the connective tissue with some somatic and skeletal manifestations [1,2].
The human heparan-N-sulfatase gene (SGSH) is localized in locus 17q25.3; it is 11 thousand bp long, consists of 8 exons [8,9], and controls the synthesis of a protein, consisting of 502 aminoacids and containing 5 N-glycosylated sites.At present there are over 120 identified and characterized mutations in the SGSH gene, including missence\nonsense-mutations, small deletions and insertions, two large deletions and two splicing mutations (Human Gene Mutation Database-HGMD, http://www.hgmd.cf.ac.uk/ac/index.php).The missence-mutations R74C and R245H are the major for European populations, as their total frequency varies from 56 % in Germany, Poland, and the Netherlands to 33 % in Italy [10][11][12].According to the data of our previous studies, in Ukraine the total frequency of these major mutations in SGSH gene is 64.58 % [13], other kinds of mutations in this gene in patients with MPS ІІІ А have not been estimated.
The aim of our work was to identify the whole spectrum of mutations in the SGSH gene in Ukrainian patients with MPS ІІІ А.

Materials and Methods
The material of the research was the biological material (blood) of the patients with MPS ІІІ А (23 children from 21 family), whose diagnosis was confirmed by biochemical methods with the determination of the activity of heparan-N-sulfatase in lysosomes, as well as of their 47 relatives (siblings and parents) from different regions of Ukraine [13,14].The age of patients at the moment of the diagnosis determination was 5 ± 1 years; there were 15 boys and 8 girls.Two children had siblings with identical genotypes, thus, the genetic profiles of 21 patients with MPS III A were further analyzed.The parents of all the patients gave their informed consent for the research.The Bioethics Committee approved the decision about the possibility of conducting the research.

Results and Discussion
Our previous work highlighted the screening results of the major mutations R74C and R245H in the SGSH gene in Ukrainian patients with MPS ІІІ А [13], which demonstrated that the share of alleles, carrying the mutation R74C, was the highest -27/42 (64.3 %) (Table 1).Nine patients were homozygous carriers of this allele and nine -heterozygous carriers of this mutation.The mutation R245H in exon six of the SGSH gene was found in one patient only (2.4 %), who was defined as a compound heterozygote R74C/ R245H.According to the recent publications of molecular-genetic studies of the characteristics of patients with MPS III A in European populations, the highest amount of mutations in the SGSH gene is localized in exons 2, 4, 5, 7, and 8 [17].Our screening of all eight exons of the SGSH gene by SSCP methods in the patients, their parents, and in the control samples of healthy individuals was conducted to identify the abnormal conformation of DNA fragments during their refolding.The results obtained demonstrated that the highest number of abnormal DNA fragments of patients, which differed from the DNA fragments of healthy individuals, was found in exons 2, 7 and 8 of the SGSH gene, which is in good agreement with the published data about the highest number of mutations in these exons [17].
Our final identification of the mutations in the SGSH gene involved the method of direct automated sequencing of the exons, intron sites, and flanking non-coding regions of the SGSH gene.Firstly, the exons, marked as "hot" ones according to the primary screening results by SSCP method, were studied.Notably, the mutation R74C, major for both most European populations and Ukraine, is localized in the second exon of the SGSH gene, which conditioned a high number of abnormal DNA fragments in this exon (Fig. 1).
Overall, our studies allowed identifying 100 % (42/42) of mutant alleles of the SGSH gene.The complete molecular-genetic characteristic of the patients with MPS III A, with the consideration of the severity degree of the clinical course, is presented in Table 1.
Therefore, the spectrum of mutations in the SGSH gene in the Ukrainian patients with MPS ІІІ А is represented with 8 missence-mutations, 3 single nucleotide deletions, and a large deletion of 27 bp (Fig. 2).
Five previously described mutations in the exons 7 and 8 -T271M, E292K, S298P, E369K, N389K -were detected among missence-mutations, identified during the analysis in addition to major mutations R74C and R245H, the presence of which in Ukrainian patients with MPS III A was analyzed in the previous work [13] (Table 2).The described single nucleotide deletion с.1080delС in exon 7 was identified in one patient, in one allele.The deletion c.1135delG, previously described in exon 8, was identified in two patients.
Additionally, three mutations in the SGSH gene, not described previously, were found, including one missence-mutation G149R in exon 4, the deletion of one nucleotide in exon 2 с.216delC, and deletion 27 bp TCC^348CTCctgccggcgctggaggccgagcccctcT GGGCCACC in exon 8 (Fig. 3).The restriction fragment length polymorphism analysis of c.216delC deletion and analysis of electrophoretic mobility of the 8th exon of the SGSH gene in 100 alleles of healthy individuals did not reveal any similar rearrangements, thus, these new mutations may not be referred to polymorphisms.The detailed analysis of these mutations using PolyPhen2 and Provean programs also confirmed their pathological character (Fig. 3).
Therefore, two mutations may be considered as the major ones for the Ukrainian patients with MPS III A: R74C (64.3 %) and N389K (7.8 %).Other mu-  The studies of genotype-phenotype correlations for MPS III A, conducted in different countries, are based on the division of all the patients into two phenotypic groups: severe (with early onset and severe clinical course of the disease), and medium (with late onset and a milder phenotype) [23].
The results of the molecular-genetic analysis of the Ukrainian patients with MPS III A demonstrated that all the patients with the major mutation R74C in the homozygous state, and a patient, who was identi-  with the severe clinical course [18].The severe clinical course was also remarkable for patients with the major mutation R74C in one allele in single nucleotide deletions с.1080delС (1 person) and c.1135delG (2 siblings) and the missence-mutation E369K (1 person), which is in agreement with the previous data about the association of these mutations with early onset of the disease [10,18].This group also includes the patients with a single nucleotide deletion c.1135delG in the compound with the missencemutation T271M.It was demonstrated in several works that the missence-mutation S298P often has a mitigating effect on the phenotype, even with a "severe" mutation in another allele [24].Among the patients, studied by us, the mentioned mutation was identified in one person in the compound with the major mutation R74C, when it conditioned the development of a mild form of the disease with late onset.Some mitigating effect of the phenotype is also noted for the missence-mutations N389K and E292K, which conditioned medium-severe clinical course in the compound with "severe" R74C and E369K in three studied patients.
The patient with the compound combination of the major mutation R74C and the substitution G149R, previously described by us, as well as the patient with genotype c.1167C˃A /с.216delC were characterized with severe phenotype and early onset of the disease, while the patient with the mutation R74C in one allele and the deletion of 27 bp in another was remarkable for a milder phenotype and medium-severe clinical course.
Therefore, our analysis of molecular-genetic characteristics of Ukrainian patients with MPS III A identified 100 % mutant alleles.High incidence of the mutation R74C (64.3 %) among the patients from Ukraine substantiates the conducting of RFLP-screening at the first stage of the molecular-genetic analysis.The mutation R245H, very common in some European populations, was found only in one allele in the examined patients, thus RFLP-screening for it is not reasonable for our country.It would be feasible to start the identification of rare mutations, which conditioned the development of the disease in the proband, with "hot" fied as a compound heterozygote R74C/ R245H, had a severe form of the disease, which is in agreement with the data of other researchers about the association of major missence-mutations R74C and R245H regions of the SGSH gene -exons 2, 7 and 8, where the most pathogenic mutations were localized in the examined patients.This order of analysis planning would allow optimizing both time and resources for the analysis and ensuring the maximal efficiency of the molecular-genetic diagnostics of MPS III A in Ukraine.

Conclusions
100 % mutant alleles were identified while conducting the molecular-genetic analysis of pathogenic mutations in the SGSH gene in 23 patients with MPS ІІІ А from 21 families (42 alleles) in Ukraine.
The share of the alleles, carrying the major mutation R74C, was the highest -64.3 % (27/42).The second mutation, major for European populations, R245H, was identified only in one patient (2.4 %), in one allele, thus, it cannot be deemed major for Ukraine.
Most rare mutations, identified by us in Ukrainian patients with MPS III A, are localized in exons 7 and 8 of the SGSH gene and one for exons 2 and 4.
Three previously not described mutations were identified in the SGSH gene -c.216delC,c.446G˃A and c.1045_1070delCTGCCGGCGCTGGAGGCC GAGCCCCTC.
The data of the conducted molecular-genetic analysis of the mutations in the SGSH gene may be used to plan a more reasonable algorithm of the molecular-genetic analysis for Ukrainian patients with MPS III A in order to enhance the quality of medical services for the families, suffering from this pathology.

Fig. 2 .
Fig. 2. The localization of mutations in specific exons of the SGSH gene in Ukrainian patients with MPS ІІІ А

Table 1 . The results of molecular and genetic analysis of Ukrainian patients with MPS III A
The results of SSCP-analysis for fragments of exons 2 and 8, SGSH gene: