XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta
XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta
XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta
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Lectures<br />
INTrONIC LINE-1 INSErTION IN THE β-gloBIn GENE caUSES<br />
β-THalaSSEMIa DUE TO aBErraNT SPLICING, NONSENSE-MEDIaTED<br />
DECay aND DECreaSED raTE of β-gloBIn L1<br />
aLLELE traNSCrIPTION<br />
Lucie Piterková 1 , Jana Kučerová 1 , Karel Indrák 1,2 and Vladimír Divoký 1,2<br />
1<br />
Department of Biology, Faculty of Medicine, UP Olomouc<br />
2<br />
Department of Hemato-Oncology, University Hospital Olomouc<br />
ß-thalassemia is a common hereditary hemoglobin disorder characterized by quantitative<br />
reduction of functional ß globin chains. The patients, mother and daughter of Ukrainian<br />
descent exhibited typical laboratory features of ß -thalassemia trait. Molecular analyses<br />
revealed that the full-length (6 kb) retrotransposon L1 was inserted in the antisense<br />
orientation into the intron-2 of the ß-globin gene (collaboration with Dr. J. Prchal, Salt<br />
Lake City, UT). The total level of expression of the affected ß-globin gene transcript was<br />
reduced to 10-15% of the total ß-globin mRNA and thus leading to ß + -thalassemia. Based<br />
on recently published data we hypothesize that RNA production of mutated gene was<br />
affected by the combination of several events. We demonstrated that the observed<br />
reduction in steady-state level of ß-globin mRNA is partially caused by aberrant splicing<br />
followed by activation of nonsense-mediated decay (NMD) pathway, leading to increased<br />
degradation of aberrant ß-globin mRNA variants. Reduction in expression of ß-globin<br />
mRNA from ß -globin L1<br />
allele comes also from altered rate of transcription. We performed<br />
PCR-based nuclear run-on assay and forty minutes of in vitro transcription revealed 30%<br />
decrease in ß-globin L1<br />
allele transcription rate compared to wild-type ß-globin allele.<br />
We also observed the ß-globin L1<br />
3’ enhancer sequence was fully methylated. However,<br />
treatment with a demethylating agent did not increase the expression of the ß-globin<br />
transcript of the ß-globin L1<br />
gene. Therefore the methylation of the ß-globin L1<br />
3’ enhancer<br />
sequence is only a secondary event probably associated with enhancer displacement by<br />
L1 insertion. The other known mechanisms of intronic L1-mediated gene disruption as<br />
premature polyadenylation and gene breaking were not detected in our case.<br />
88 <strong>XXII</strong>. Biochemistry Congress, Martin
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