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Družinska eritrocitoza je prirojeno bolezensko stanje, za katerega je značilna povečana masa eritrocitov, ki se odraža v povečanih ravneh hemoglobina in hematokrita. Eritrocitoza lahko obolelim zaradi hiperviskoznosti krvi povzroča različne zdravstvene težave, v najslabšem primeru tudi trombotične zaplete. Bisfosfoglicerat mutaza (BPGM) je gen, povezan z družinsko eritrocitozo tipa 8 (ECYT8). Gen BPGM kodira zapis za protein bisfosfoglicerat mutazo (BPGM), ki katalizira pretvorbo 1,3-bisfosfoglicerata (1,3-BPG) v 2,3-bisfosfoglicerat (2,3-BPG), alosterični efektor hemoglobina. Mutacije gena BPGM lahko povzročijo spremenjeno strukturo in zmanjšano delovanje proteina BPGM in zato nižje koncentracije 2,3-BPG v krvi, kar povzroči povečano afiniteto hemoglobina do kisika. Kisik se posledično v manjši meri sprošča v okoliška tkiva, kar vodi v kronično hipoksijo tkiv in nastanek ECYT8. Magistrsko nalogo smo izvedli v sodelovanju s Specializiranim hematološkim laboratorijem Kliničnega oddelka za hematologijo Univerzitetnega kliničnega centra v Ljubljani. S pomočjo bioinformacijske analize gena BPGM smo opredelili regije, vključene v genetsko analizo ter nato z optimizacijo metode PCR in sekvenciranjem po Sangerju uspešno vzpostavili genetski test za preverjanje prisotnosti različic v regijah prvega in drugega eksona gena BPGM. Delovanje vzpostavljenega genetskega testa smo nato potrdili pri dveh slovenskih bolnikih s sumom na družinsko eritrocitozo. Glede na analizo pridobljenih nukleotidnih zaporedij bolnika nista imela različic v testiranih regijah gena BPGM, s čimer je bila izključena možnost ECYT8. Vzpostavljen genetski test se bo v kombinaciji z drugimi testi v prihodnosti uporabljal za rutinsko analizo gena BPGM pri diagnostiki družinske eritrocitoze v Sloveniji. Familial erythrocytosis is a hereditary disease characterized by an increased mass of erythrocytes and increased levels of hemoglobin and hematocrit. Due to hyperviscosity of the blood, erythrocytosis can cause health problems such as thrombotic complications. Bisphosphoglycerate mutase (BPGM) is a gene associated with familial erythrocytosis type 8 (ECYT8). The BPGM gene encodes a transcript for bisphosphoglycerate mutase (BPGM), which is a protein that catalyzes the conversion of 1,3-bisphosphoglycerate (1,3-BPG) to 2,3-bisphosphoglycerate (2,3-BPG), the allosteric effector of hemoglobin. Mutations in the BPGM gene can cause altered structure and decreased function of BPGM protein and therefore lower concentrations of 2,3-BPG in the blood, causing an increased affinity of hemoglobin for oxygen. Hence, the oxygen delivery to the tissues decreases, causing chronic tissue hypoxia and consequent ECYT8. The master's thesis was performed in cooperation with the Specialized Hematology Laboratory of the Clinical Department of Hematology of the University Medical Center in Ljubljana. Using bioinformatical analysis, we first identified regions for genetic analysis. Then, we successfully established a genetic test to verify the presence of variants in the regions of the first and second exons of the BPGM gene, by PCR optimization and Sanger sequencing. The established genetic test was then confirmed in two Slovenian patients with suspected familial erythrocytosis. According to the analysis of the acquired nucleotide sequences, the patients had no variations in the tested regions of the BPGM gene, thus excluding the possibility of ECYT8. In the future, the established genetic test will be used in combination with other tests, for routine analysis of the BPGM gene in the diagnosis of familial erythrocytosis in Slovenia. |
