Erythrocytosis is a pathological condition characterized by a persistent increase in red blood cell mass in the circulation, reflected by elevated hemoglobin and hematocrit levels. According to the classical classification, erythrocytosis is divided into primary and secondary forms based on the site of origin, and into acquired and congenital forms according to its etiology. Although most cases are associated with external factors or underlying chronic diseases, the condition may also result from genetic alterations. Congenital forms, known as familial erythrocytosis (ECYT), are diagnosed in clinical practice through genetic testing of nine genes involved in the regulation of oxygen sensing (VHL, EGLN1, EPAS1), hematopoietic cell maturation signaling (EPOR and EPO), or oxygen transport (HBB, HBA1, HBA2, BPGM). The cause of the disease remains unexplained in more than 70% of patients, suggesting the existence of additional, as yet unidentified genes and molecular mechanisms. Among the more recently proposed candidate genes, PIEZO1 has attracted considerable attention. It encodes a mechanosensitive ion channel involved in mechanotransduction and in maintaining red blood cell hydration, volume, and deformability.
The initial part of the thesis included a review of variants in the literature and a bioinformatic analysis of their location and impact on protein function. Based on the obtained data and previous next-generation sequencing results from three patients with idiopathic erythrocytosis, three target regions of the PIEZO1 gene were selected, encompassing exon 18, exon 37, and exons 42–44. The conditions for their amplification by polymerase chain reaction were optimized. The efficiency and specificity of amplification were assessed by agarose gel electrophoresis, and the purified products were subsequently analyzed by Sanger sequencing.
In all three patients, Sanger sequencing confirmed four variants previously identified by next-generation sequencing: NM_001142864.4 (PIEZO1):c.2344G>A (p.Gly782Ser) and c.2423G>A (p.Arg808Gln) in exon 18, c.5195C>T (p.Thr1732Met) in exon 37, and c.6380C>T (p.Thr2127Met) in exon 44. In addition to the previously identified variants, a novel synonymous variant c.6390G>A (p.Thr2130=) was identified in exon 44 in one patient and was classified as benign. The confirmed variants are located within structurally and functionally important regions of the PIEZO1 protein, including transmembrane units and the anchor region, which are involved in mechanical activation, stabilization, and the transmission of mechanical forces.
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