engleski

Congenital disorders of glycosyltion ic (CDG-Ic): novel polymorphisms / mutations in alg6 gene

Congenital disorders of glycosylation (CDGs) are a growing group of autosomal recessive diseases caused by deficient assembly or processing of glycoproteins. The primary defects either impair the formation of the lipid-linked oligosaccharide (LLO) precursor of N-linked glycosylation (CDGs type I – 13 types ; Ia-m) or affect processing of the protein-bound sugar chains (CDGs type II – 8 types ; IIa-h). Although only  1000 CDG patients were diagnosed so far, the true incidence was estimated to be much higher (based on frequency analysis for certain common mutations). The clinical spectrum of the different types of CDGs discovered so far is highly variable, ranging from disorders restricted to specific organs to severe multisystemic disorders. Because of the resemblance of CDG symptoms to some other diseases, especially neurological ones, CDGs are often under- or misdiagnosed. The second most frequent type of CDGs, CDG-Ic is caused by mutations in ALG6 gene that encode Man9GlcNAc2-PP-Dol alpha1, 3-glucosyltransferase. The frequencies of heterozygotes for specific mutations, as well as CDG patients were shown to be specific for every population. Until now no patient with CDG syndrome was detected in Croatia. The aim of this study was to determine the frequencies of various mutations/polymorphisms in ALG6 gene in the population of Croatia. The blood samples of 600 healthy volunteers were collected on Blood Stain Cards® ; ; . For determination of the frequency of A333V mutation, single strand conformation polymorphism (SSSP) analysis was optimized, and for the first time employed for this purpose. No heterozygote for A333V mutation was found in 600 analyzed samples representing population of Croatia. Assuming that these results are valid for the Croatian population, the incidence of homozygous A333V (CDG-Ic patients) would be below 1 in 1.4x106. For determination of the frequencies of F304S polymorphism (998T>C in exon 10) and Y131H mutation (391T>C in exon 5) real-time PCR (TaqMan SNP Genotyping Assay) was used. The frequency of F304S polymorphism was shown to be similar to the frequencies found in other tested populations (29%). Surprisingly, eight homozygotes for Y131H were detected, but since the samples were obtained from healthy persons, direct sequencing was employed for checking these weird results. It revealed 3 novel polymorphisms in exon 5 of ALG6 gene (383T C, 390G>A and 429G C) and in 2 novel polymorphisms (IVS5+17C>T and IVS5+34G>A) in downstream intervening sequence. It seems that presence of these until now unknown mutation caused incorrect/inappropriate binding of the designed probes and sondes thus yielding false positive results. Tthe base substitution T C on the position 383 does not cause amino acid substitution, but 390G>A and 429G C cause substitution of V128A and K143N, respectively. Finally, the frequency of heterozygotes for Y131H was found to be 6.53% that is 3 times higher than in the United States. If the obtained results are valid for Croatian population (1/15) the estimated incidence of homozygotes should be 1 in 1000, suggesting  4500 CDG-Ic patients should be in Croatia. How come none of them is detected so far? Do they exist at all or they stayed under- or misdiagnosed by now? These questions wait to be answered soon! To achieve that task, the awareness of the physicians on CDGs should be significantly improved, especially since the primary diagnostic screening could be performed by simple isoelectric focusing of serum transferrin.

congenital disorders of glycosylation; polymorphysms; mutations

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