RESUMO
BACKGROUND: Alpha-mannosidosis is a rare lysosomal storage disorder, caused by decreased activity of α-D-mannosidase. This enzyme is involved in the hydrolysis of mannosidic linkages in N-linked oligosaccharides. Due to the mannosidase defect, undigested mannose-rich oligosaccharides (Man2GlcNAc - Man9GlcNAc) accumulating in cells are excreted in large quantities in urine. METHODS: In this work, we determined the levels of urinary mannose-rich oligosaccharides in a patient subjected to novel enzyme replacement therapy. Urinary oligosaccharides were extracted using solid phase extraction (SPE), labeled by fluorescent tag 2-aminobenzamide, and quantified by high-performance liquid chromatography (HPLC) with fluorescence detector (FLD). The identity of peaks was determined by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. In addition, the levels of urinary mannose-rich oligosaccharides were also quantified by 1H nuclear magnetic resonance (NMR) spectroscopy. The data were analyzed using one-tailed paired t-test and Pearson's correlation tests. RESULTS: Compared to levels before the administration of therapy, an approximately two-folds decrease in total mannose-rich oligosaccharides after one month of treatment was observed by NMR and HPLC. After four months, an approximately ten-folds significant decrease in total urinary mannose-rich oligosaccharides was detected, suggesting therapy effectiveness. A significant decrease in the levels of oligosaccharides with 7-9 mannose units was detected by HPLC. CONCLUSIONS: The application of both HPLC-FLD and NMR in quantification of oligosaccharide biomarkers is a suitable approach for monitoring of therapy efficacy in alpha-mannosidosis patients.
Assuntos
alfa-Manosidose , Humanos , Cromatografia Líquida de Alta Pressão , alfa-Manosidose/tratamento farmacológico , Manose , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância MagnéticaRESUMO
Abstract Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disorder (LSD) caused by deficiency of lysosomal N-sulphoglucosamine sulphohydrolase, which is one of four enzymes involved in heparan sulfate degradation. Traditional methods used for MPS IIIA diagnostics usually constitute of selective screening, based on the analysis of urinary glycosaminoglycans, further enzymatic assays in leukocytes, and mutation analysis. Nowadays, some LSDs, including mucopolysaccharidoses, can be precisely diagnosed by mass spectrometry-based techniques. Up to this date, there are no comprehensive studies of MPS IIIA diagnostics by MALDI-TOF analysis of free oligosaccharides in urine published. In the presented work, MALDI-TOF/TOF analysis of permethylated oligosaccharides was performed to obtain the set of glyco-biomarkers that together form the specific fingerprint of this disease. Early and accurate diagnostics of MPS IIIA is crucial to stabilize the progressive cellular damage and improve the overall well-being of patients.
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Fabry disease (FD, OMIM#301500) is a rare inborn error of the lysosomal enzyme α-galactosidase (α-Gal A, EC 3.2.1.22) and results in progressive substrate accumulation in tissues with a wide range of clinical presentations. Despite the X-linked inheritance, heterozygous females may also be affected. Hemizygous males are usually affected more severely, with an earlier manifestation of the symptoms. Rising awareness among health care professionals and more accessible diagnostics have positioned FD among the most-common inherited metabolic diseases in adults. An early and correct diagnosis of FD is crucial with a focus on personalised therapy. Preventing irreversible destruction of vital organs is the main goal of modern medicine. The aim of this study was to offer a complex report mapping the situation surrounding FD patients in Slovakia. A total of 48 patients (21 males, 27 females) with FD are registered in the Centre for Inborn Errors of Metabolism in Bratislava, Slovakia. In our cohort, we have identified three novel pathogenic variants in five patients. Three patients presented with the frameshift mutation c.736delA, and two others presented with the missense mutations c.203T>C, c.157A>C. Moreover, we present a new clinical picture of the pathogenic variant c.801+1G>A, which was previously described and associated with the renal phenotype.
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BACKGROUND: Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by defects in genes coding for different lysosomal enzymes which degrade glycosaminoglycans. Impaired lysosomal degradation causes cell dysfunction leading to progressive multiorgan involvement, disabling consequences and poor life expectancy. Enzyme replacement therapy (ERT) is now available for most MPS types, offering beneficial effects on disease progression and improving quality of life of patients. The landscape of MPS in Europe is not completely described and studies on availability of treatment show that ERT is not adequately implemented, particularly in Southern and Eastern Europe. In this study we performed a survey analysis in main specialist centers in Southern and Eastern European countries, to outline the picture of disease management in the region and understand ERT implementation. Since the considerable number of MPS IVA patients in the region, particularly adults, the study mainly focused on MPS IVA management and treatment. RESULTS: 19 experts from 14 Southern and Eastern European countries in total responded to the survey. Results outlined a picture of MPS management in the region, with a high number of MPS patients managed in the centers and a high level of care. MPS II was the most prevalent followed by MPS IVA, with a particular high number of adult patients. The study particularly focused on management and treatment of MPS IVA patients. Adherence to current European Guidelines for follow-up of MPS IVA patients is generally adequate, although some important assessments are reported as difficult due to the lack of MPS skilled specialists. Availability of ERT in Southern and Eastern European countries is generally in line with other European regions, even though regulatory, organizational and reimbursement constrains are demanding. CONCLUSIONS: The landscape of MPS in Southern and Eastern European countries is generally comparable to that of other European regions, regarding epidemiology, treatment accessibility and follow up difficulties. However, issues limiting ERT availability and reimbursement should be simplified, to start treatment as early as possible and make it available for more patients. Besides, educational programs dedicated to specialists should be implemented, particularly for pediatricians, clinical geneticists, surgeons, anesthesiologists and neurologists.
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Mucopolissacaridoses , Mucopolissacaridose II , Mucopolissacaridose IV , Adulto , Terapia de Reposição de Enzimas/métodos , Humanos , Mucopolissacaridoses/tratamento farmacológico , Mucopolissacaridoses/terapia , Mucopolissacaridose II/tratamento farmacológico , Mucopolissacaridose IV/tratamento farmacológico , Qualidade de VidaRESUMO
Congenital disorder of glycosylation type Ig (ALG12-CDG) is a rare inherited metabolic disease caused by a defect in alpha-mannosyltransferase 8, encoded by the ALG12 gene (22q13.33). To date, only 15 patients have been diagnosed with ALG12-CDG globally. Due to a newborn Slovak patient's clinical and biochemical abnormalities, the isoelectric focusing of transferrin was performed with observed significant hypoglycosylation typical of CDG I. Furthermore, analysis of neutral serum N-glycans by mass spectrometry revealed the accumulation of GlcNAc2Man5-7 and decreased levels of GlcNAc2Man8-9, which indicated impaired ALG12 enzymatic activity. Genetic analysis of the coding regions of the ALG12 gene of the patient revealed a novel homozygous substitution mutation c.1439T>C p.(Leu480Pro) within Exon 10. Furthermore, both of the patient's parents and his twin sister were asymptomatic heterozygous carriers of the variant. This comprehensive genomic and glycomic approach led to the confirmation of the ALG12 pathogenic variant responsible for the clinical manifestation of the disorder in the patient described.
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Defeitos Congênitos da Glicosilação/genética , Predisposição Genética para Doença , Manosiltransferases/genética , Polissacarídeos/genética , Defeitos Congênitos da Glicosilação/epidemiologia , Defeitos Congênitos da Glicosilação/patologia , Feminino , Testes Genéticos , Glicosilação , Homozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Mutação de Sentido Incorreto/genética , Fenótipo , Polissacarídeos/metabolismo , Eslováquia/epidemiologia , Transferrina/genéticaRESUMO
OBJECTIVE: Despite profound neurological symptomatology there are only few MRI studies focused on the brain abnormalities in alpha-mannosidosis (AM). Our aim was to characterize brain MRI findings in a large cohort of AM patients along with clinical manifestations. METHODS: Twenty-two brain MRIs acquired in 13 untreated AM patients (8 M/5F; median age 17 years) were independently assessed by three experienced readers and compared to 16 controls. RESULTS: Focal and/or diffuse hyperintense signals in the cerebral white matter were present in most (85%) patients. Cerebellar atrophy was common (62%), present from the age of 5 years. Progression was observed in two out of 6 patients with follow-up scans. Cortical atrophy (62%) and corpus callosum thinning (23%) were already present in a 13-month-old child. The presence of low T2 signal intensity in basal ganglia and thalami was excluded by the normalized signal intensity profiling. The enlargement of perivascular spaces in white matter (38%), widening of perioptic CSF spaces (62%), and enlargement of cisterna magna (85%) were also observed. Diploic space thickening (100%), mucosal thickening (69%) and sinus hypoplasia (54%) were the most frequent non-CNS abnormalities. CONCLUSION: White matter changes and cerebellar atrophy are proposed to be the characteristic brain MRI features of AM. The previously reported decreased T2 signal intensity in basal ganglia and thalami was not detected in this quantitative study. Rather, this relative MR appearance seems to be related to the diffuse high T2 signal in the adjacent white matter and not the gray matter iron deposition that has been hypothesized.
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Atrofia/diagnóstico , Cerebelo/diagnóstico por imagem , Substância Branca/diagnóstico por imagem , alfa-Manosidose/diagnóstico , Adolescente , Adulto , Atrofia/diagnóstico por imagem , Atrofia/patologia , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Cerebelo/patologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Imageamento por Ressonância Magnética/métodos , Masculino , Malformações do Sistema Nervoso/diagnóstico , Malformações do Sistema Nervoso/diagnóstico por imagem , Malformações do Sistema Nervoso/patologia , Neuroimagem/métodos , Substância Branca/patologia , Adulto Jovem , alfa-Manosidose/diagnóstico por imagem , alfa-Manosidose/patologiaRESUMO
BACKGROUND: Enzyme replacement therapy (ERT) with laronidase (recombinant human α-L-iduronidase, Aldurazyme®) is indicated for non-neurological signs and symptoms of mucopolysaccharidosis type I (MPS I). The approved laronidase dose regimen is weekly infusions of 0.58mg/kg, however, patients and caregivers may have difficulty complying with the weekly regimen. We examined clinical outcomes, tolerability, compliance, and satisfaction in a series of patients who switched to every other week infusions. METHODS: This multinational, retrospective, chart review case series analyzed data from 20 patients who had undergone ERT with laronidase 0.58mg/kg weekly for more than one year, and who then switched to 1.2mg/kg every other week. RESULTS: The majority of patients had attenuated MPS I phenotypes (9 with Hurler-Scheie and 8 with Scheie syndromes) and 3 patients had severe MPS I (Hurler syndrome). Most patients presented with organomegaly (17/20), umbilical and/or inguinal hernia (16/20), cardiac abnormalities (17/20), musculoskeletal abnormalities (19/20), and neurological and/or developmental deficits (15/20). Following laronidase treatment, signs stabilized or improved. No deterioration or reversal of clinical outcome was noted in any patient who switched from the weekly dose of 0.58mg.kg to 1.2mg/kg every other week. There were no safety issues during the duration of every other week dosing. Patient compliance and satisfaction with the dosing regimen were greater with every other week dosing than weekly dosing. CONCLUSIONS: An alternative dose regimen of 1.2mg/kg laronidase every other week was well tolerated and clinically similar to the standard dose for patients who were stabilized with weekly 0.58 mg/kg for one year or more. When an individualized approach to laronidase therapy is necessary, every other week dosing may be an alternative for patients with difficulty receiving weekly infusions.
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Iduronidase/administração & dosagem , Iduronidase/uso terapêutico , Mucopolissacaridose I/tratamento farmacológico , Adolescente , Criança , Pré-Escolar , Relação Dose-Resposta a Droga , Humanos , Lactente , Estudos Retrospectivos , Adulto JovemRESUMO
Melanocortin-4 receptor (MC4R) deficiency is the most frequent monogenic form of obesity. The contribution of MC4R mutations to the Slovak population has not been investigated as yet. We screened the coding sequence of the MC4R gene in a cohort of 210 Slovak obese children and adolescents. We identified four different mutations in four patients, giving a mutation detection rate of 0.95%. Of these, three were missense mutations previously identified and characterized by other research groups (p.R7C, p.S127L and p. R305W, respectively). One was a novel nonsense mutation p.W174* detected in a severely obese 7-year-old boy. This mutation was further analyzed in family segregation analysis and exhibited variable penetrance. Two known amino acid polymorphisms (p.V103I and p.I251L) were also identified in seven subjects of our cohort group. We also performed multifactorial statistical analysis to determine the influence of genotypes on standard biochemical blood markers. No significant influence was observed in carriers of DNA variants on tested parameters. We conclude that rare heterozygous MC4R mutations contribute to the onset of obesity only in a few cases in the Slovak population.
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Mutação/genética , Obesidade Mórbida/epidemiologia , Obesidade Mórbida/genética , Receptor Tipo 4 de Melanocortina/genética , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Linhagem , Prevalência , Eslováquia/epidemiologia , Adulto JovemRESUMO
BACKGROUND: Gaucher disease is the most common lysosomal storage disorder and is caused by a deficiency of the enzyme glucocerebrosidase. Enzyme deficiency leads to the accumulation of undegraded substrates, mainly in cells of the monocyte/ macrophage lineage, which is responsible for the clinical manifestations of the disease. To date, no study has attempted to identify the mutation spectrum of the glucocerebrosidase gene (GBA) in Slovak patients OBJECTIVES: To identify mutations in 14 Slovak patients with confirmed glucocerebrosidase deficiency. METHODS: Using molecular genetics methods PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) and direct sequencing of coding region GBA we identified the spectrum of mutations in our patients. RESULTS: Five mutations (N370S, L444P, G377S, D409H and RecNciI) accounted for 75% of the mutant alleles. The remaining 25% were rare and probably individual mutations. CONCLUSIONS: The mutational spectrum in our patients is similar to that observed in other European countries and corresponds to a Caucasian population, with N370S, L444P, RecNciI being the most common. Interestingly, mutation G377S was more frequent in our patients as compared to other published data. The C4W, L96P, H311N, 745delG and 1127_1128delTT mutations are described here for the first time in Gaucher disease, contributing to the panel of published GBA mutations.
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Doença de Gaucher , Glucosilceramidase/genética , Feminino , Doença de Gaucher/epidemiologia , Doença de Gaucher/genética , Estudos de Associação Genética , Humanos , Masculino , Mutação , Eslováquia/epidemiologiaRESUMO
In humans, uric acid represents a biologically active end-product of purine nucleotides. Several studies in children and adolescents documented an association between hyperuricaemia and the components metabolic syndrome. High intake of fructose-sweetened beverages might increase uricaemia, since fructose is the only saccharide which metabolism results in the formation of uric acid. Current knowledge does not allow distinguishing whether hyperuricaemia is causally related to the components of metabolic syndrome, or rather represents a marker of an enhanced risk, and poor outcome. No guidelines exist whether or not to treat asymptomatic hyperuricaemia in the adolescents. Randomized controlled prospective clinical studies are needed to elucidate whether uric acid-lowering management would beneficially affect the prevalence of metabolic syndrome, and the incidence of cardiovascular disease.
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Doenças Assintomáticas , Hiperuricemia/complicações , Síndrome Metabólica/complicações , Adolescente , Bebidas/efeitos adversos , Criança , Frutose/efeitos adversos , Humanos , Hiperuricemia/induzido quimicamente , Estudos Prospectivos , Fatores de Risco , Edulcorantes/efeitos adversosRESUMO
AIM: Obesity is the major determinant of metabolic syndrome. Being born small for gestational age (SGA) may be co-responsible. We aimed at evaluating the association between 1. obesity and 2. being born SGA and the presence of endocrine-metabolic abnormalities in prepubertal Slovak children. METHODS: The study included 98 children, aged 3-10.9 years: 36 AGA-born obese children (OB), 31 SGA-born children (SGA) and 31 appropriate for gestational age born non-obese children (AGA). Fasting serum levels of glucose, total cholesterol, LDL, HDL, triglycerides, fT4, TSH, cortisol and insulin were determined. HOMA-IR was calculated. Personal data about birth weight and length and family history were collected. Actual anthropometric measurement was done. RESULTS: In every group, high prevalence of positive family history of metabolic disorder was found. In comparison with AGA children, OB children were taller (p<0.01) with higher body mass index (BMI) (p<0.001), and had increased insulin levels and homeostasis model assessment for insulin resistance (HOMA-IR) (p<0.001), decreased high-density lipoprotein (HDL) (p<0.001), and a trend to higher cortisol levels (p=0.069) was noted. SGA-born children were shorter (p<0.001), with BMI comparable to the AGA group. They had higher glucose levels (p<0.001), a trend to decreased HDL levels (p=0.085) and increased fT4 levels (p<0.001). A three-fold higher occurrence of metabolic abnormalities was present in obese children and twice more metabolic abnormalities were present in SGA-born children in comparison with AGA-born children. CONCLUSIONS: SGA-born children are more prone to developing endocrine-metabolic abnormalities than non-obese children born AGA, but they are at less risk than obese AGA-born children. We should provide specialized care for obese children already in prepubertal age and pay attention to SGA-born children.
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Obesidade Infantil/sangue , Glicemia/metabolismo , Criança , Pré-Escolar , Saúde da Família , Feminino , Idade Gestacional , Humanos , Hidrocortisona/sangue , Recém-Nascido Pequeno para a Idade Gestacional/sangue , Insulina/sangue , Resistência à Insulina , Metabolismo dos Lipídeos , Masculino , Eslováquia , Hormônios Tireóideos/sangueRESUMO
We performed a complex analysis of the neurofibromatosis type 1 (NF1) gene in Slovakia based on direct cDNA sequencing supplemented by multiple ligation dependent probe amplification (MLPA) analysis. All 108 patients had café-au-lait spots, 85% had axilary and/or inguinal freckling, 61% neurofibromas, 36% Lisch nodules of the iris and 31% optic pathway glioma, 5% suffered from typical skeletal disorders, and 51% of patients had family members with NF1. In 78 of the 86 (90.7%) index patients our analysis revealed the presence of NF1 mutations, 68 of which were small changes (87.2%), including 39 (50%) novel. Among the identified mutations the most prevalent were small deletions and insertions causing frameshift (42.3%), followed by nonsense (14.1%), missense (12.8%), and typical splicing (11.5%) mutations. Type 1 NF1 deletions and intragenic deletions/duplication were identified in five cases each (6.4%). Interestingly, in five other cases nontypical splicing variants were found, whose real effect on NF1 transcript would have remained undetected if using a DNA-based method alone, thus underlying the advantage of using the cDNA-based sequencing. We show that Slovak NF1 patients have a similar repertoire of NF1 germline mutations compared to other populations, with some prevalence of small deletions/insertions and a decreased proportion of nonsense mutations.
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Genes da Neurofibromatose 1 , Mutação , Neurofibromatose 1/genética , População Branca/genética , Adolescente , Adulto , Idoso , Substituição de Aminoácidos , Criança , Pré-Escolar , Análise Mutacional de DNA , Éxons , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neurofibromatose 1/diagnóstico , Eslováquia , Adulto JovemRESUMO
AIM OF THE STUDY: In 2006-2008 a survey analyzing food patterns, intake of main food items and leisure time activities of 5,410 schoolchildren was carried out. METHODS: The study was performed in 13 randomly selected regions of Slovakia. 5,410 elementary school children (2,848 girls and 2,562 boys) aged from 6.3 to 15.9 years, mean age was 11 +/- 2.6 years, were included. The data collected by questionnaire concerned nutrition and leisure time activities. RESULTS: Noteworthy results are that only 63% of participants eat breakfast regularly. Almost all of the children eat lunch during workdays regularly and 60.9% prefer a cooked (hot) dish for supper. Although dairy products are a substantial part of child nutrition, in general their consumption in our sample was low. Only 50.1% of children consume these daily and 62% of children drink milk daily, more often boys than girls. A striking observation is that only 65.5% of interviewed pupils eat fruit every day and 30.9% of them eat vegetables daily. The frequency of consumption of poultry and pork in our sample was almost the same, however, the analysis showed that only 14% of children consume fish once per week. Moreover, only 12.6% of subjects prefer wholegrain bread. In our sample 56.8% of children eat sweets daily, 32% prefer a salty snack almost 2 times per week. Within the group of pupils 35.8% do not attend physical trainings even once a week. Almost every child is used to watch TV and 64% of them play PC games daily. While both girls and boys watched TV on average over 2 hours, boys spend more time on PC per day than girls (girls 0.72 hours per day vs. boys 1.13 hours per day, p < 0.001). The food patterns and leisure time activities of children older than 11 years and rural pupils were less favourable. CONCLUSIONS: According to results of our analysis we recommend to increase the consumption of dairy products, fresh fruits and vegetables in Slovak schoolchildren and spare no effort in making children to take breakfast regularly. It is necessary to promote daily moderate physical activity. Nutritional and lifestyle education should begin already in childhood.
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Dieta/estatística & dados numéricos , Exercício Físico , Atividades de Lazer , Obesidade/epidemiologia , Adolescente , Distribuição por Idade , Índice de Massa Corporal , Criança , Feminino , Preferências Alimentares , Humanos , Masculino , Sobrepeso/epidemiologia , Prevalência , Eslováquia/epidemiologiaRESUMO
BACKGROUND: The periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA) syndrome appears to be more common than generally appreciated and should be differentiated from hereditary periodic fever syndromes, particularly from mevalonate kinase deficiency (MKD). PATIENTS AND METHODS: 14 unrelated patients (7 males, 7 females) met clinical criteria for both the PFAPA syndrome and MKD. Immunoglobulin D (IgD) levels, mevalonic aciduria and mevalonate kinase (MVK) genotype was determined in all patients. RESULTS: Children experienced their first febrile episode at the age of 24.5±5.9 months (mean±SD), the clinical diagnosis of PFAPA syndrome was established with delay at 42.7±11.7 months. The duration of febrile episodes was 3.4±0.2 days, the asymptomatic interval between them lasted 5.4±0.9 weeks. Accompanying symptoms included pharyngitis (92.8%), cervical lymphadenitis (85.7%), aphthous stomatitis (21.4%), arthralgia (14.3%) and skin erythema (35.7%). Neither mevalonic aciduria nor MVK gene mutations were found in any of the subjects, however, unexpectedly, increased plasma IgD (322.2±29.2 U/l) levels were detected in all patients. CONCLUSION: Raised IgD levels may represent a non-specific epiphenomenon, which frequently accompanies PFAPA syndrome as well as MKD. Because of the overlapping clinical and laboratory features, genetic testing of the MVK gene is indicated to differentiate these two conditions, if clinical criteria for both are fulfilled.
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Febre/imunologia , Imunoglobulina D/sangue , Linfadenite/diagnóstico , Faringite/diagnóstico , Estomatite Aftosa/diagnóstico , Artralgia/diagnóstico , Artralgia/imunologia , Pré-Escolar , Estudos de Coortes , Diagnóstico Diferencial , Eritema/diagnóstico , Eritema/imunologia , Exantema/diagnóstico , Exantema/imunologia , Feminino , Febre/sangue , Febre/genética , Genótipo , Humanos , Lactente , Linfadenite/imunologia , Masculino , Deficiência de Mevalonato Quinase/diagnóstico , Deficiência de Mevalonato Quinase/genética , Ácido Mevalônico/urina , Faringite/imunologia , Estomatite Aftosa/imunologia , Síndrome , Fatores de TempoRESUMO
Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder that is caused by a deficiency of the enzyme alpha-L-iduronidase (IDUA). Of the 21 Czech and Slovak patients who have been diagnosed with MPS I in the last 30 years, 16 have a severe clinical presentation (Hurler syndrome), 2 less severe manifestations (Scheie syndrome), and 3 an intermediate severity (Hurler/Scheie phenotype). Mutation analysis was performed in 20 MPS I patients and 39 mutant alleles were identified. There was a high prevalence of the null mutations p.W402X (12 alleles) and p.Q70X (7 alleles) in this cohort. Four of the 13 different mutations were novel: p.V620F (3 alleles), p.W626X (1 allele), c.1727 + 2T > G (1 allele) and c.1918_1927del (2 alleles). The pathogenicity of the novel mutations was verified by transient expression studies in Chinese hamster ovary cells. Seven haplotypes were observed in the patient alleles using 13 intragenic polymorphisms. One of the two haplotypes associated with the mutation p.Q70X was not found in any of the controls. Haplotype analysis showed, that mutations p.Q70X, p.V620F, and p.D315Y probably have more than one ancestor. Missense mutations localized predominantly in the hydrophobic core of the enzyme are associated with the severe phenotype, whereas missense mutations localized to the surface of the enzyme are usually associated with the attenuated phenotypes. Mutations in the 130 C-terminal amino acids lead to clinical manifestations, which indicates a functional importance of the C-terminus of the IDUA protein.