Determination of frequencies of alleles , associateD with the pseuDoDeficiency of lysosomal hyDrolases , in population of ukraine

The pseudodeficiency of lysosomal hydrolases described as a significant reduction in enzyme activi ty in vitro in clinically healthy individuals, can lead to diagnostic errors in the process of biochemical analysis of lysosomal storage disease in case of its combination with pathology of another origin. Pseudodeficiency is mostly caused by some non-pathogenic changes in the corresponding gene. These changes lead to the in vitro lability of the enzyme molecule, whereas in vivo the enzyme retains its functional activity. To assess the prevalence of the most common lysosomal hydrolases pseudodeficiency alleles in Ukraine, we have determined the frequency of alleles c.1055A>G and c.* 96A>G in the ARSA gene, substitutions c.739C>T (R247W) and c.745C>T (R249W) in the HEXA gene, c.1726G>A (G576S) and c.2065G>A (E689K) in the GAA gene, c.937G>T (D313Y) in the GLA1 gene and c.898G>A (A300T) in the IDUA gene in a group of 117 healthy individuals from different regions of the country and 14 heterozygous carriers of pathogenic mutations in the HEXA gene (parents of children with confirmed diagnosis of Tay-Sachs disease). The total frequency of haplotypes, associated with arylsulfatase A pseudodeficiency, in healthy people in Ukraine (c.1055G/c.*96G and c.1055G/c.*96A haplotypes) was 10.3%. The frequency of c.739C>T (R247W) allele, associated with hexosaminidase A pseudodeficiency, among Tay-Sachs carriers from Ukraine was 7.1%. The total frequency of α-glucosidase pseudodeficiency haplotypes in healthy individuals in Ukraine (c.1726A/c.2065A and c.1726G/ c.2065A haplotypes) was 2.6%. No person among examined individuals with the substitution c.937G>T (D313Y) in the GLA1 gene and c.898G>A (A300T) in the IDUA gene was found. The differential diagnostics of lysosomal storage diseases requires obligatory determination of the presence of the pseudodeficiency alleles, particularly the ones with high incidence in the total population. Ignoring phenomenon of pseudodeficiency may lead to serious diagnostic errors.

The pseudodeficiency of lysosomal hydrolases described as a significant reduction in enzyme activi ty in vitro in clinically healthy individuals, can lead to diagnostic errors in the process of biochemical analysis of lysosomal storage disease in case of its combination with pathology of another origin.Pseudodeficiency is mostly caused by some non-pathogenic changes in the corresponding gene.These changes lead to the in vitro lability of the enzyme molecule, whereas in vivo the enzyme retains its functional activity.To assess the prevalence of the most common lysosomal hydrolases pseudodeficiency alleles in Ukraine, we have determined the frequency of alleles c.1055A>G and c.* 96A>G in the ARSA gene, substitutions c.739C>T (R247W) and c.745C>T (R249W) in the HEXA gene, c.1726G>A (G576S) and c.2065G>A (E689K) in the GAA gene, c.937G>T (D313Y) in the GLA1 gene and c.898G>A (A300T) in the IDUA gene in a group of 117 healthy individuals from different regions of the country and 14 heterozygous carriers of pathogenic mutations in the HEXA gene (parents of children with confirmed diagnosis of Tay-Sachs disease).The total frequency of haplotypes, associated with arylsulfatase A pseudodeficiency, in healthy people in Ukraine (c.1055G/c.*96Gand c.1055G/c.*96Ahaplotypes) was 10.3%.The frequency of c.739C>T (R247W) allele, associated with hexosaminidase A pseudodeficiency, among Tay-Sachs carriers from Ukraine was 7.1%.The total frequency of α-glucosidase pseudodeficiency haplotypes in healthy individuals in Ukraine (c.1726A/c.2065Aand c.1726G/ c.2065A haplotypes) was 2.6%.No person among examined individuals with the substitution c.937G>T (D313Y) in the GLA1 gene and c.898G>A (A300T) in the IDUA gene was found.The differential diagnostics of lysosomal storage diseases requires obligatory determination of the presence of the pseudodeficiency alleles, particularly the ones with high incidence in the total population.Ignoring phenomenon of pseudodeficiency may lead to serious diagnostic errors.

K e y w o r d s: lysosomal hydrolases, pseudodeficiency of the enzyme, allele frequency.
I n most cases, the hereditary deficiency of lysosomal hydrolases is associated with the develop ment of severe neurodegenerative diseaseslysosomal storage disorders [1].However, some individuals remain clinically healthy despite a significant decrease in the activity of a certain enzyme, determined by standard techniques.This phenome non is called "pseudodeficiency" of the enzyme [2].In practice, this situation usually refers not to the absolute deficiency of the enzyme activity but, rather, to a decrease to the level which is lower than that for heterozygous carriers, thus resulting in impossibili ty of distinguishing between such individuals and patients with lysosomal storage disorders.The pseudodeficiency phenomenon is described not solely for lysosomal hydrolases, however, it is more common for this group of enzymes.
It was established that in most cases, the pseudodeficiency of lysosomal hydrolases is caused by some polymorphic changes in the corresponding gene [2].Usually, these changes are nonpathoge nic and lead to in vitro lability of the enzyme mole cule, whereas in vivo the enzyme retains functional activi ty.Such changes may be inherited either independently, i.e. in the absence of any other changes in the corresponding gene, or in the combination with pathogenic mutations.In the first case, any person in the entire population, regardless of the aggravation with pathogenic mutations of the corresponding gene, may carry the pseudodeficiency allele and express the decreased enzyme activity at biochemical examination [3].As for the other case, related to the combination of inheritance and pathogenic mutations, pseudodeficiency of enzyme activity is mostly doi: https://doi.org/10.15407/ubj88.05.096 manifested in heterozygous carriers of pathogenic mutations [4].Both situations can lead to serious errors during biochemical diagnostics of lysosomal storage disorders when the enzyme pseudodeficiency and pathology of some other genesis are combined.Therefore, to avoid false diagnosis, if a decreased activity of the corresponding enzyme was determined in a patient, the biochemical test of lysosomal enzymes, for which pseudodeficiency had been established, should include the determination of pseudodeficiency alleles.It is also essential to assess the pathogenicity of the identified genetic changes at the interpretation of the proband's genetic analysis.
The most striking feature of the phenomenon of lysosomal enzymes pseudodeficiency is rather high incidence in the total population.For instance, popu lationbased studies in different countries showed that 5 to 17% of the ArSA gene variants in the population are represented by the variant associated with arylsulfatase A pseudodeficiency [511].At the same time, other pseudodeficiencies, such as hexasominidase A and B, βmannosidase or βgalactosidase pseudodeficiency, occur quite rarely; at present only single cases of such changes have been described [1214].Therefore, to elaborate the most efficient algorithms for laboratory analysis for differential diagnostics of lysosomal storage disorders in a specific population, the frequency of alleles of pseudodeficiency of lysosomal hydrolases in this population should be taken into consideration.
Our work was aimed at determining the incidences of the most widespread alleles in genes ArSA, HEXA, GAA, GlAI and IDUA, which cause the arylsulfatase A, βhexasominidase A, αgalactosidase, αLiduronidase and αglucosidase pseudodeficiency in the Ukrainian population.

materials and methods
The studies were conducted using blood samples from 117 unrelated volunteers with no signs of lysosomal storage disorders in their clinical history from all the regions of Ukraine and 14 heterozygous carriers of pathogenic mutations in the HEXA gene (parents of children with confirmed diagnosis of TaySachs disease).
All participants gave informed consent for the study prior to the procedure of obtaining their blood samples.
Genomic DNA was isolated from whole periphe ral blood with EDTA using Neogene commercial sets (Ukraine).The determination of c.1049А>G and c.*96 А>G variants of ArSA gene was performed by PCR method with subsequent RFLPanalysis [8].The design of primers and the conditions of RFLPanalysis are presented in Table 1.
Analysis of the products was performed by electrophoresis in 8% PAAG followed by staining with ethidium bromide solution.
Analysis of the products was performed by electrophoresis in 2% agarose gel followed by staining with ethidium bromide solution.
Arlequin 3.5 software was used to assess allele frequencies, their correspondence to the Hardy Weinberg equilibrium, value of linkage disequilib-T a b l e 1. Design of primers and conditions of RFLP-analysis of c. 1049А>G and c rium, and incidences of haplotypes.The significance level p = 0.05 was used to assess the statistical significance of the differences.
We studied the incidence of polymorphic alleles of genes, associated with the pseudodeficiency of five lysosomal enzymes, namely alleles c.1055А>G and c.*96А>G in ArSA gene and alleles c.739C>T and c.745C>T in HEXA gene, as alleles with the highest incidence rate in many populations, as well as alleles

Gene
Allele Primers Fragment size The allelespecific nucleotides are underlined.
c.1726G>A and c.2065G>A in GAA gene,c.937G>T in GlA1 gene and c.898G>A in IDUA gene.The latter three have been chosen due to their relevance to early and accurate diagnostics of the corresponding diseases, related to specific enzyme replacement therapy.
The alleles of the pseudodeficiency of other lysosomal enzymes, such as βgalactosidase, βmannosidase and βhexosaminidase А and B, were not studied in this work.
Arylsulfatase A pseudodeficiency.It is known that alleles с.1055А>G and с.*96А>G in ArSA gene are the most common cause of the arylsulfatase A pseudodeficiency in most populations [8].They have been discovered in individuals both with and without pathogenic mutations in this gene.Therefore, our assessment of the frequency of these alleles in

Lysosomal enzyme
Disease, caused by the enzymatic deficiency Gene Alleles, associated with enzyme pseudodeficiency Ref.
Arylsulfatase A (EC 3.1.The total incidence of alleles was found to be 5.56% for the substitution of с.1055А>G and 4.7% for the substitution of с.*96 А>G.The genotype distribution among the investigated individuals corresponded to HardyWeinberg equilibrium for both substitutions (P > 0.05).It is known that in the vast majority of cases these two alleles are inheri ted together.A significantly linkage disequilibrium of alleles с.1055А>G and с.*96 А>G (r 2 = 0.84, P < 0.05) was observed in the investigated individuals.The total incidence of the haplotype with two substitutions (с.1055G/с.*96Ghaplotype) was found to be 9.4%.One person was homozygous for these two alleles, and nine patients were heterozygous.The isolated substitution с.1055А>G was revealed in two heterozygous individuals.No isolated substitution Hexosaminidase A pseudodeficiency.Two substitutions, associated with hexosaminidase A pseudodeficiency, namely с.739C>T (R247W) and с.745C>T (R249W), were described for the HEXA gene [4].These substitutions were found in heterozygous carriers of pathogenic mutations in HEXA gene in all to date published cases.Therefore, to estimate the frequency of these alleles in Ukraine we exami_ ned 117 healthy donors and 14 heterozygous carriers of pathogenic mutations in HEXA gene (parents of children with the confirmed TaySachs disease) for the presence of substitutions с.739C>T (R247W) and с.745C>T (R249W) in HEXA gene.No individual with at least one of the mentioned substitutions was found among the healthy volunteers.Among the carriers of pathogenic mutations in HEXA gene, there was one person (the father of a sick child) who had substitution с.739C>T (R247W).Thus, the frequency of allele с.739C>T (R247W), associated with the hexosaminidase А pseudodeficiency, among the carriers of pathogenic mutations in HEXA gene in Ukraine was determined to be 7.1%.

A -allele с.*96А>G of ARSA gene (1 -PCR fragment before restriction; 2-4 -PCR fragments after treatment with restriction endonuclease Ddel: 2,3wild type allele, 4 -* allele 96A>G heterozygous); B -allele с.1055А>G of ARSA gene (PCR fragments after treatment with restriction endonuclease Bsrl: 1 -allele с.1055А>G heterozygous; 2 -wild type allele)
Acid α-glucosidase pseudodeficiency.At present, the acid αglucosidase pseudodeficiency is associated with the substitutions c.1726G>A (G576S) and c.2065G>A (E689K) in GAA gene [18].Similar to ary lsulfatase A pseudodeficiency alleles, these alleles occur regardless of pathogenic mutations in the mentioned gene.Therefore, a group of 117 healthy volunteers was examined for the presen ce of these substitutions.The total incidence of alleles, associated with the αglucosidase pseudodeficiency, in our population was 0.43% for the substitution of с.1726G>A and 1.28% for the substitution of с.2065G>A.The distribution of genotypes among the investigated individuals corresponded to HardyWeinberg equilibrium for both substitutions (P > 0.05).Linkage disequilibrium of alleles с.1726G>A and с.2065G>A was less pronounced than that for the arylsulfatase А pseudodeficiency alleles (r 2 = 0.33, p = 0).The total frequency of the haplotype with two substitutions (1726A/2065A haplotype) was 0.9% (Table 3).One person was heterozygous for these two alleles.No isolated substitution с.1726G>A was observed in any of the individuals, and isolated substitution с.2065G>A was found in two individuals, which is consistent with published data [21].Thus, the total frequency of haplotypes, which cause the αglucosidase pseudodeficiency, among the individuals from Ukraine (1726A/2065A and 1726G/2065A haplotypes) was determined to be 2.6%.
α-Galactosidase and α-iduronidase pseudodeficiency.The samples from 117 volunteers were investigated for the presence of the substitution c.937G>T (D313Y) in GlA1 gene (αgalactosidase pseudodeficiency) and the substitution c.898G>A (A300T) in IDUA gene (αiduronidase pseudodeficiency).No person with the mentioned substitutions was found among the examined individuals.
Arylsulfatase A is a lysosomal enzyme, a deficiency of which results in development of metachromatic leukodystrophy (MLD), a severe neurodegenerative disorder [1].It was shown that the functionally active enzyme is produced in the cells of individuals with the arylsulfatase A pseudodeficiency, but it differs somewhat structurally from the normal one due to the loss of one of three oligosaccharide residues [3].In most cases, the arylsulfatase A pseudodeficiency allele is a complex of two mutations in ArSA gene -c.1055А>G and c.*96А>G.The first one corresponds to the substitution N350S and substitutes asparagine for serine, impairing the glycosylation site.The second substitution A>G in the position *96 impairs the work of polyadenylation signal.
Analyzing the obtained results on the distribution of the incidence of alleles c.1055А>G and c.*96А>G in ArSA gene, it should be noted that the isolated substitution c.*96А>G does not practically occur in Ukraine, as in other European populations (Table 4) [8].The total incidence of haplotypes associated with the arylsulfatase A pseudodeficiency in Ukraine is close to incidences, obtained by Middle European researchers.
The lowest incidence is observed in Finland, which is known for its peculiarities of gene inciden ces due to the demographic specificity of this count ry -a sparseness of first "settlers", the isolation due to low settlement density and geographic location.The highest incidence is in Portugal and Great Britain , which is close to the incidence of the arylsulfatase A pseudodeficiency allele in America (total incidence of haplotypes is about 0.2) and on the African continents (total incidence of haplotypes is 0.26-0.33)[8,22].This may be related to the great navigation history of these countries and the con- The deficiency in hexosaminidase A activity, which occurs due to the mutations in HEXA gene, usually results in TaySachs disease [1].However, there were described healthy individuals with pronounced deficiency of hexosaminidase A activity in vitro [4].The vast majority of individuals with the pseudodeficiency of the mentioned enzyme had one of the polymorphic substitutions c.739C>T (R247W) or c.745C>T (R249W) in the compound with pathogenic mutation in HEXA gene.This combination led to a considerable decrease in the hexosaminidase A activity regarding the 4methylumbelliferylderived synthetic substrate (0-25% of the control value), whereas the ability of this enzyme to hydrolyze the natural substrate was not impaired.The studies of the incidence of hexosaminidase A pseudodeficiency alleles demonstrated that about 2% of carriers of pathogenic mutations in HEXA gene among Ashkenazi Jews and 35% carriers of nonJewish ethnici ty had one of the polymorphic substitutions in the compound with the pathogenic mutation [4].The incidence of the hexosaminidase A deficiency allele among the carriers of pathogenic mutations in HEXA gene from Ukraine was found to be 7.1%.It should be noted that all the families with TaySachs disease, examined by us, were of Ukrainian ethnici ty.Thus, the determined incidence of the hexo saminidase A pseudodeficiency allele is related to the data for nonJewish populations.Such high incidence of hexosaminidase A deficiency alleles among the carriers of pathogenic mutations in HEXA gene of nonJewish ethnicity may lead to falsepositive diagnosis of this disease.

T a b l e 4. The incidence of alleles of the arylsulfatase A pseudodeficiency in Ukrainian and other European populations
The deficiency of lysosomal acid αglucosidase causes intralysosomal accumulation of glycogen, primarily in muscle tissues, that results in a severe progressive neuromuscular pathology -Pompe disea se or type II glycogenesis [1].
In view of the introduction of enzyme replacement therapy of the disease into clinical practice, there is now urgency for early diagnostics for achievement maximized treatment efficiency.One of the approaches to early diagnostics of Pompe disea se is the conducting of neonatal screening with the assessment of the acid αglucosidase activity in dry blood spot [21].One of the difficulties in interpre ting results of such a study is the described phenomenon of acid αglucosidase pseudodeficiency associated with two polymorphic substitutions in the GAA gene -c.1726G>A(G576S) and c.2065G>A (E689K) [18].It was demonstrated that the substitution c.1726 G>A often occurs in the cisposition with the substitution c.2065 G>A, and leads to a considerable decrease in the acid αglucosidase activity, to practically pathological levels, in healthy individuals.In Asian population, the incidence of the acid αglucosidase pseudodeficiency allele is rather high -3.3-3.9% of the total population [18].The isolated substitution c.2065G>A occurs with approximately the same incidence, whereas the isolated substitution c.1726G>A was not found in any person.The information about the incidence of substitution, associated with acid αglucosidase pseudodeficiency in European populations is very limited (Table 5).There are only published data on the incidence of acid αglucosidase pseudodeficiency alleles in the Netherlands, which is very close to our results, in contrast to the very high incidence of these alleles among the population of Japan and China.
No substitution c.937G>T (D313Y) in GlA1 gene (αgalactosidase pseudodeficiency) or substitution c.898G>A (A300T) in IDUA gene (αiduronidase pseudodeficiency) were found in any of the tested individuals.This indicates that the frequency of such mutations in Ukraine is smaller than 0.004, however, they may still be found.Therefore, in the process of diagnostics of Fabry disease and type I mucopolysaccharidosis, it is worth remembering that the presen ce of the mentioned mutations in patients requires obligatory further study to detect other diseasecausing mutations.
Thus, taking into consideration the significant clinical polymorphism and genetic heterogeneity of lysosomal storage disorders, the differential diag nostics of this large group of hereditary diseases requires a wide application of different biochemical and moleculargenetic methods.At present, the assessment of the specific enzyme activity is a method of choice not only at the stage of confirming nosological diagnostics but also for examination of family members of the sick person with the purpose of medical and genetic consultation.Therefore, the results of the enzyme activity assessment should obligatorily be interpreted with consideration of the data about the presence or absence of the pseudodeficiency allele in the proband.According to our study, T a b l e 5.The incidence of acid α-glucosidase pseudodeficiency alleles in Ukraine (our study) and other populations [18]

Population
UDC 616-056.7-07Determination of frequencies of alleles, associateD with the pseuDoDeficiency of lysosomalhyDrolases, in population of ukraineN.V. OlkhOVych, N. G. GOrOVeNkOState Institute of Genetic and regenerative Medicine, NAMS of Ukraine, kyiv; e-mail: nolhovich@gmail.com . -the individual incidence of alleles and haplotypes was not determined N. V. Olkhovych, N. G. Gorovenko sequent migration of the population.The similarity between the incidences of the arylsulfatase A pseudodeficiency alleles in Ukraine, and those in Polish and Turkish populations may also be explained by the common centurieslong history and the population migration.