Mutations in phosphomannomutase 2 gene that cause congenital disorder of glycosylation type Ia

Čačija, Maja (2008) Mutations in phosphomannomutase 2 gene that cause congenital disorder of glycosylation type Ia. Diploma thesis, Faculty of Science > Department of Biology.

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Congenital disorders of glycosylation (CDGs) are autosomal recessive disorders characterized by central nervous system dysfunction and multiorgan failure due to aberrant N-glycosylation of glycoproteins. CDG type I consist of defects in the assembly of lipid-linked oligosaccharide precursor and/or its transfer to the polypeptide chain. CDG-Ia, the most prevalent CDG-I, is caused by deficiency of phosphomannomutase 2 (PMM2), which is associated with mutations in PMM2 gene. PMM2 converts mannose 6-phosphate to mannose 1-phosphate, which then serves as GDP-mannose donor for the synthesis of lipid-linked oligosaccharide precursor. The R141H mutation (c.422G>A) is the most frequent mutation in exon 5 of PMM2 gene, accounting for low PMM2 activity. In this work, the allele frequencies of R141H mutation in exon 5 and single nucleotide polymorphisms (SNP) (IVS5+19T/C, IVS5+22T/A, IVS4-58-56delATG) in parts of introns related to exon 5 were studied on 60 samples of human genomic DNA (gDNA). The main goal was to determine the frequency of R141H heterozygotes in this sample of Croatian population, and also the frequency of polymorphisms in parts of intronic sequences IVS4 and IVS5 related to exon 5. Studies have shown that these polymorphisms are commonly related to specific mutations which cause CDG-Ia. After human genomic DNA was isolated from whole blood using Chelex® protocol, the target 291 bp DNA fragment (exon 5 of PMM2 gene and parts of its associated intronic sequences IVS4 and IVS5) was amplified using the PCR method and the resulting DNA bands were visualized by ethidium bromide on agarose gel. The mutation/polymorphism screening was preformed using the single-strand conformation polymorphism (SSCP) analyses; single-stranded DNA (ssDNA) bands were visualized by silver staining. Every ssDNA that showed different SSCP electrophoretic pattern compared to wild type ssDNA and control (known CDG-Ia) was sequenced and then analyzed using ABI Prism 310 Genetic Analyzer. The results of the analyses showed that in 60 analyzed human gDNA samples no R141H mutation in PMM2 gene was found, that means that no R141H heterozygotes were found in this sample of Croatian population. Seven samples showed polymorphisms in parts of intronic sequences (IVS4, IVS5) related to exon 5 of PMM2 gene: five IVS5+19T/C polymorphisms, one IVS4-58-56delATG and one IVS5+22T/A polymorphism were detected. However, we cannot make a general conclusion about the presence of R141H mutation in this population because of the limited number of samples. Nevertheless, the result complies with the fact that so far no CDG-Ia patients are found in Republic of Croatia. We can also make a conclusion that this sample of Croatian population is specific considering analyzed mutation/polymorphisms compared to some other populations, but to make a general conclusion it is necessary to analyze a larger set of samples from patients with disease having similar symptoms to those found in CDG-Ia patients.

Item Type: Thesis (Diploma thesis)
Keywords: glycosylation, CDG-Ia, PMM2 gene, PMM2, R141H mutation, intronic polymorphisms
Supervisor: Dumić Belamarić, Jerka
Co-supervisor: Pavlica, Mirjana
Date: 2008
Number of Pages: 83
Subjects: NATURAL SCIENCES > Biology
Divisions: Faculty of Science > Department of Biology
Depositing User: Silvana Šehić
Date Deposited: 15 Sep 2014 09:51
Last Modified: 15 Sep 2014 09:51

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