Regina Mikheeva

Background. The filaggrin protein, encoded by the FLG gene, is key to maintaining a healthy skin barrier and normal immune system function. Filaggrin loss-of-function mutations are the primary cause of ichthyosis vulgaris, a common dermatological disorder. At the same time, high variability in individual variant frequencies between the studied populations and a large number of repeats in the third exon significantly complicate gene analysis using standard PCR methods. Objective. To examine the spectrum of genetic variants in the FLG gene associated with ichthyosis vulgaris in a cohort of Russian patients using whole-exome sequencing and analysis of publicly available data on this gene’s polymorphism. Material and methods. Clinical examination and molecular genetic testing were performed on 77 patients diagnosed with ichthyosis vulgaris (ICD-10: Q80.0) at the Moscow Scientific and Practical Center of Dermatovenereology and Cosmetology (MSPCDC). Whole-exome sequencing (WES) was done to analyze the coding region of the FLG gene in DNA samples from peripheral blood leukocytes. To analyze the spectrum of genetic variants across the Russian Federation (RF), a database of population frequencies of the RF was used. Results and conclusion. Among 120 762 individuals whose FLG gene variant data were evaluated in the RF population frequency database, 292 different pathogenic and likely pathogenic variants were identified, of which only 78 were registered as pathogenic in the ClinVar database. All detected variants were mapped across the amino acid sequence of the protein with a relative enrichment in the region of filaggrin repeats. The overall carrier frequency of pathogenic variants was 8.0%, of which the 3 most common pathogenic variants (p.Ser761fs, p.Arg2447* and p.Arg501*) accounted for 5.6%. Furthermore, individual allele frequency ratios relative to the gnomAD database varied from 0.06× to 26×, which indicates significant differences in the spectrum of pathogenic variants compared to global statistics. Obtaining whole-exome data for patients with ichthyosis vulgaris allowed us not only to confirm the pathogenicity of the p.Ser761fs variant, identified in 32 of 77 patients, but also to reveal the previously undescribed p.Ser1515* variant. The large number of variants of uncertain significance (2475), most of which are missense substitutions, suggests the need for in vitro functional testing, segregation studies, and comparison of their frequencies among patients with the disease and in the general population from Russia. In addition, there are ongoing doubts in the scientific community regarding the pathogenicity of many nonsense and frameshift mutations. Accordingly, the obtained overall frequency of pathogenic variants may be revised in the future. Thus, in this study, the spectrum of pathogenic variants in the FLG gene was determined for the first time among patients with ichthyosis vulgaris and within a general population sample from Russia.

HCT116 is a colorectal cancer cell line frequently used in anti-tumor drug development experiments as well as in studies of the molecular machinery of eukaryotic cells. It is well characterized by the presence of several single-nucleotide and short mutations in multiple oncogenes and tumor suppressor genes, including KRAS, PIK3CA, MLH1, CTNNB1, CDKN2A, TGFBR2, and BRCA2. However, its landscape of large genomic rearrangements (LGRs) and copy number variants (CNVs) is still far from being fully understood. Therefore, the aim of this study was to identify LGRs and CNVs in several HCT116 cell line samples using Oxford Nanopore sequencing technology, including three samples from the SRA NCBI database, and to compare common and unique variants across all samples. Using the recently developed eLaRodON tool, we identified 22,666 common LGRs, among which more than 70% of tandem duplications and deletions larger than 80 kb were confirmed by CNV analysis. Among LGRs affecting protein-coding sequences, two in-frame rearrangements were identified: a deletion of exons 4–6 and a duplication of exon 10 in the CCSER1 gene, which encodes a cell division regulator protein. Given its high rearrangement rate in various tumors and the clinical significance of its overexpression, this finding may be potentially useful in future research on this cell line. Regarding differences between samples, we found that LGRs in the laboratory sample and in one of the three SRA NCBI samples occurred more frequently via ALR/Alpha repeats than via Alu repeats, in contrast to common LGRs and those unique to the other samples, a finding that may indicate the presence of unique mechanisms of genomic instability. Thus, this study reveals a broad spectrum of large genomic rearrangements and copy number variants that can be identified in the HCT116 cell line using Oxford Nanopore sequencing, including rearrangements specific to distinct cell line samples.

HCT116 is a colorectal cancer cell line frequently used in anti-tumor drug development experiments as well as in studies of the molecular machinery of eukaryotic cells. It is well characterized by the presence of several single-nucleotide and short mutations in multiple oncogenes and tumor suppressor genes, including KRAS, PIK3CA, MLH1, CTNNB1, CDKN2A, TGFBR2 , and BRCA2 . However, its landscape of large genomic rearrangements (LGRs) and copy number variants (CNVs) is still far from being fully understood. Therefore, the aim of this study was to identify LGRs and CNVs in several HCT116 cell line samples using Oxford Nanopore sequencing technology, including three samples from the SRA NCBI database, and to compare common and unique variants across all samples. Using the recently developed eLaRodON tool, we identified 22,666 common LGRs, among which more than 70% of tandem duplications and deletions larger than 80 kb were confirmed by CNV analysis. Among LGRs affecting protein-coding sequences, two in-frame rearrangements were identified: a deletion of exons 4–6 and a duplication of exon 10 in the CCSER1 gene, which encodes a cell division regulator protein. Given its high rearrangement rate in various tumors and the clinical significance of its overexpression, this finding may be potentially useful in future research on this cell line. Regarding differences between samples, we found that LGRs in the laboratory sample and in one of the three SRA NCBI samples occurred more frequently via ALR/Alpha repeats than via Alu repeats, in contrast to common LGRs and those unique to the other samples, a finding that may indicate the presence of unique mechanisms of genomic instability. Thus, this study reveals a broad spectrum of large genomic rearrangements and copy number variants that can be identified in the HCT116 cell line using Oxford Nanopore sequencing, including rearrangements specific to distinct cell line samples.