The work is dedicated to the study of the spectrum and frequency of allelic variants of the ATP7B gene among people with suspected Wilson's disease from Ukraine, as well as to improving the detection of cases of Wilson's disease by developing and implementing an optimal algorithm for testing major mutations of candidate genes for hepatobiliary disorders in young people. For the differential diagnosis of idiopathic hepatobiliary disorders, genotyping of major mutations of the HFE and UGT1A1 genes was performed. Their frequency and contribution to the etiology of hepatobiliary disorders were also established. The first stage of the dissertation work was to establish the frequency of ATP7B, HFE, UGT1A1 gene mutations among practically healthy individuals and among individuals with idiopathic hepatobiliary disorders. Genotyping of the allelic variant c.3207C>A of the ATP7B gene was performed by the Bi-PASA PCR method, the low-functional allele A(TA)7TAA of the UGT1A1 gene by the heteroduplex analysis method, the allelic variants c.845G>A and c.187C>G of the HFE gene by the restriction analysis method. As a result of this stage of the work, the frequency of heterozygous carriers of the c.3207C>A mutation of the ATP7B gene was established (1:57, 1.75%) among practically healthy individuals, the frequency of heterozygous carriers of the c. 845G>C of the HFE gene in the total sample - 1:40 (2.5%). Every fourth person was a heterozygous carrier of the p.187C>G allelic variant of the HFE gene. The frequency of the homozygous genotype of the low-functioning allele (TA)7TAA of the UGT1A1 gene is 1:9 (11.7%). So, as a result of the first stage of work, the etiological factors of idiopathic hepatobiliary disorders were determined: mutations of the HFE gene (5.8%) and c.3207C>A mutation of the ATP7B gene (9.4%).The next stage of the work was the sequencing of the coding sequence of the ATP7B gene. Sequencing was performed in 23 DNA samples of individuals with clinical signs of Wilson's disease (three or more points according to the point scale of disease manifestations). Sequencing began with the 8th exon of the ATP7B gene. The same rearrangement was found in four patients - r.Glu770Argfs. In three of them, the mutation was in the compound heterozygous state with the major mutation p.3207C>A. In one patient, after sequencing the entire gene sequence and detection of large deletions and duplications (MLPA), only the c.2304dupC mutation was detected. One patient was found to have a c.2128G>A mutation within exon 8. The mutation is described in the HGMD database as pathogenic (rs137853285). According to the results of sequencing and comparison with the reference sequence, no rearrangements were detected in exon 9 of the gene in any of the DNA samples. Sequencing this region of the gene is not informative for the studied population. Within exon 13, a replacement of adenine for cytosine was found at position 3011 (c.3011A>C). In two individuals, an adenine to cytosine substitution was found within exon 13 at position 2973 cDNA (c.2973A>C). This variant does not lead to a change in amino acid sequences in the protein and is interpreted in databases as a single nucleotide polymorphism rs1801248. Also, two individuals with a substitution of guanine for adenine at position 3009 of the ATP7B gene (c.3009G>A) were found in the heterozygous state (rs1801247). Three allelic variants were found within eson 13. The c.3402delC mutation was detected in two individuals within exon 15. According to the results of the sequencing of exon 10 of the ATP7B gene, a missense substitution c was detected.2495A>G in the heterozygous state in 6 patients, and in two individuals in the homozygous state. In the results of sequencing from 16 to 21 exons of the ATP7B gene in the studied DNA samples of patients, no differences from the reference sequence of the gene were found. No differences from the reference sequence were found within exons 1, 2, 11 and 12 of the ATP7B gene. Within exon 3 of the ATP7B gene, a single-nucleotide polymorphism c.1366C>G (AG[C/T]GT) rs1801244 - replacement of the amino acid valine with leucine at position 456 in the heterozygous state, in three - in the homozygous state, was detected in four individuals. A variant in exon 4 c.1551C>T, which has not been described to date, was also detected. As a result of the work, no rearrangements were found in exons 5, 6 and 7. The obtained sequencing results indicate that, in addition to the predominant c.3207C>A mutation of the ATP7B gene, four other pathogenic variants were identified among this sample of individuals, which were detected in exons 8, 13, and 15. In a third of patients, in the absence of pathogenic alleles in the exonic sequences of the ATP7B gene one or more single-nucleotide polymorphisms of neutral value were detected.
