Single-cell DNA-seq depicts clonal evolution of multiple driver alterations in osimertinib-resistant patients.

BACKGROUND: The development of targeted agents, such as osimertinib for EGFR-mutated non-small-cell lung cancer (NSCLC), has drastically improved patient outcome, but tumor resistance eventually always occurs. In osimertinib-resistant NSCLC, the emergence of a second molecular driver alteration (such as ALK, RET, FGFR3 fusions or BRAF, KRAS mutations) has been described. Whether those alterations and the activating EGFR mutations occur within a single cancer cell or in distinct cell populations is largely debated. PATIENTS AND METHODS: Tumor sequencing was used to identify the acquired resistance mechanisms to osimertinib in the MATCH-R trial (NCT0251782). We implemented single-cell next-generation sequencing to investigate tumor heterogeneity on patient's frozen tissues in which multiple alterations have been identified. Patient-derived models, cell lines, and patient-derived xenografts were exposed to specific inhibitors to investigate combination treatment strategies. RESULTS: Among the 45 patients included in MATCH-R who progressed on osimertinib, 9 developed a second targetable alteration (n = 2 FGFR3-TACC3, n = 1 KIF5B-RET, n = 1 STRN-ALK fusions; n = 2 BRAFV600E, n = 1 KRASG12V, n = 1 KRASG12R, n = 1 KRASG12D mutations). Single-cell analysis revealed that the two driver alterations coexist within one single cancer cell in the four patients whose frozen samples were fully contributive. A high degree of heterogeneity within samples and sequential acquisitions of molecular events were highlighted. A combination treatment concomitantly targeting the two driver alterations was required on the corresponding patient-derived models to restore cell sensitivity, which was consistent with clinical data showing efficacy of brigatinib in the patient with ALK fusion after progression to osimertinib and crizotinib administered sequentially. CONCLUSIONS: Distinct molecular driver alterations at osimertinib resistance coexist with initial EGFR mutations in single cancer cells. The clonal evolution of cancer cell populations emphasized their heterogeneity leading to osimertinib relapse. Combining two targeted treatments is effective to achieve clinical benefit.

Copy number variants in genomes of local sheep breeds from Russia.

Copy number variants (CNVs) are genomic structural variations that contribute to many adaptive and economically important traits in livestock. In this study, we detected CNVs in 354 animals from 16 Russian indigenous sheep breeds and analysed their possible functional roles. Our analysis of the entire sample set resulted in 4527 CNVs forming 1450 CNV regions (CNVRs). When constructing CNVRs for individual breeds, a total of 2715 regions ranging from 88 in Groznensk to 337 in Osetin breeds were identified. To make interbreed CNVR frequency comparison possible, we also identified core CNVRs using CNVs with overlapping chromosomal locations found in different breeds. This resulted in 137 interbreed CNVRs with frequency >15% in at least one breed. Functional enrichment analysis of genes affected by CNVRs in individual breeds revealed 12 breeds with significant enrichments in olfactory perception, PRAME family proteins, and immune response. Function of genes affected by interbreed and breed-specific CNVRs revealed candidates related to domestication, adaptation to high altitudes and cold climates, reproduction, parasite resistance, milk and meat qualities, wool traits, fat storage, and fat metabolism. Our work is the first attempt to uncover and characterise the CNV makeup of Russian indigenous sheep breeds. Further experimental and functional validation of CNVRs would help in developing new and improving existing sheep breeds.

[Signatures of selection and candidate genes for adaptation to extreme environmental factors in the genomes of Turano-Mongolian cattle breeds]. / Следы отбора и гены-кандидаты адаптации к экстремальным факторам среды в Ð³ÐµÐ½Ð¾Ð¼Ð°Ñ Ñ‚ÑƒÑ€Ð°Ð½Ð¾-Ð¼Ð¾Ð½Ð³Ð¾Ð»ÑŒÑÐºÐ¸Ñ Ð¿Ð¾Ñ€Ð¾Ð´ крупного рогатого скота.

Changes in the environment force populations of organisms to adapt to new conditions, either through phenotypic plasticity or through genetic or epigenetic changes. Signatures of selection, such as specific changes in the frequency of alleles and haplotypes, as well as the reduction or increase in genetic diversity, help to identify changes in the cattle genome in response to natural and artificial selection, as well as loci and genetic variants directly affecting adaptive and economically important traits. Advances in genetics and biotechnology enable a rapid transfer of unique genetic variants that have originated in local cattle breeds in the process of adaptation to local environments into the genomes of cosmopolitan high-performance breeds, in order to preserve their outstanding performance in new environments. It is also possible to use genomic selection approach to increase the frequency of already present adaptive alleles in cosmopolitan breeds. The review examines recent work on the origin and evolution of Turano-Mongolian cattle breeds, adaptation of Turano-Mongolian cattle to extreme environments, and summarizes available information on potential candidate genes for climate adaptation of Turano-Mongolian breeds, including cold resistance genes, immune response genes, and high-altitude adaptation genes. The authors conclude that the current literature data do not provide preference to one of the two possible scenarios of Turano-Mongolian breed origins: as a result of the domestication of a wild aurochs at East Asia or as a result of the migration of taurine proto-population from the Middle East. Turano-Mongolian breeds show a high degree of adaptation to extreme climatic conditions (cold, heat, lack of oxygen in the highlands) and parasites (mosquitoes, ticks, bacterial and viral infections). As a result of high-density genotyping and sequencing of genomes and transcriptomes, prospective candidate genes and genetic variants involved in adaptation to environmental factors have recently been identified.

Genomic profiling of late-onset basal cell carcinomas from two brothers with nevoid basal cell carcinoma syndrome.

BACKGROUND: Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant genetic disorder. It is commonly caused by mutations in PTCH1 and chiefly characterized by multiple basal cell carcinomas (BCCs) developing prior to the age of 30 years. In rare cases, NBCCS presents with a late onset of BCC development. OBJECTIVE: To investigate BCC tumorigenesis in two brothers, who showed characteristic features of NBCCS but developed their first BCCs only after the age of 40 years. Two other siblings did not show signs of NBCCS. RESULTS: We obtained blood samples from four siblings and nine BCCs from the two brothers with NBCCS. Whole exome sequencing and RNA sequencing revealed loss of heterozygosity (LOH) of PTCH1 in eight out of nine tumours that consistently involved the same haplotype on chromosome 9. This haplotype contained a germinal splice site mutation in PTCH1 (NM_001083605:exon9:c.763-6C>A). Analysis of germline DNA confirmed segregation of this mutation with the disease. All BCCs harboured additional somatic loss-of-function (LoF) mutations in the remaining PTCH1 allele which are not typically seen in other cases of NBCCS. This suggests a hypomorphic nature of the germinal PTCH1 mutation in this family. Furthermore, all BCCs had a similar tumour mutational burden compared to BCCs of unrelated NBCCS patients while harbouring a higher number of damaging PTCH1 mutations. CONCLUSIONS: Our data suggest that a sequence of three genetic hits leads to the late development of BCCs in two brothers with NBCCS: a hypomorphic germline mutation, followed by somatic LOH and additional mutations that complete PTCH1 inactivation. These genetic events are in line with the late occurrence of the first BCC and with the higher number of damaging PTCH1 mutations compared to usual cases of NBCCS.

Resequencing and signatures of selection scan in two Siberian native sheep breeds point to candidate genetic variants for adaptation and economically important traits.

Russian sheep breeds represent an important economic asset by providing meat and wool, whilst being adapted to extreme climates. By resequencing two Russian breeds from Siberia: Tuva (n = 20) and Baikal (n = 20); and comparing them with a European (UK) sheep outgroup (n = 14), 41 million variants were called, and signatures of selection were identified. High-frequency missense mutations on top of selection peaks were found in genes related to immunity (LOC101109746) in the Baikal breed and wool traits (IDUA), cell differentiation (GLIS1) and fat deposition (AADACL3) in the Tuva breed. In addition, genes found under selection owing to haplotype frequency changes were related to wool traits (DSC2), parasite resistance (CLCA1), insulin receptor pathway (SOCS6) and DNA repair (DDB2) in the Baikal breed, and vision (GPR179) in the Tuva breed. Our results present candidate genes and SNPs for future selection programmes, which are necessary to maintain and increase socioeconomic gain from Siberian breeds.

The Iberian legacy into a young genetic xeroderma pigmentosum cluster in central Brazil.

In central Brazil, in the municipality of Faina (state of Goiás), the small and isolated village of Araras comprises a genetic cluster of xeroderma pigmentosum (XP) patients. The high level of consanguinity and the geographical isolation gave rise to a high frequency of XP patients. Recently, two founder events were identified affecting that community, with two independent mutations at the POLH gene, c.764 + 1 G > A (intron 6) and c.907 C > T; p.Arg303* (exon 8). These deleterious mutations lead to the xeroderma pigmentosum variant syndrome (XP-V). Previous reports identified both mutations in other countries: the intron 6 mutation in six patients (four families) from Northern Spain (Basque Country and Cantabria) and the exon 8 mutation in two patients from different families in Europe, one of them from Kosovo. In order to investigate the ancestry of the XP patients and the age for these mutations at Araras, we generated genotyping information for 22 XP-V patients from Brazil (16), Spain (6) and Kosovo (1). The local genomic ancestry and the shared haplotype segments among the patients showed that the intron 6 mutation at Araras is associated with an Iberian genetic legacy. All patients from Goiás, homozygotes for intron 6 mutation, share with the Spanish patients identical-by-descent (IBD) genomic segments comprising the mutation. The entrance date for the Iberian haplotype at the village was calculated to be approximately 200 years old. This result is in agreement with the historical arrival of Iberian individuals at the Goiás state (BR). Patients from Goiás and the three families from Spain share 1.8 cM (family 14), 1.7 cM (family 15), and a more significant segment of 4.7 cM within family 13. On the other hand, the patients carrying the exon 8 mutation do not share any specific genetic segment, indicating an old genetic distance between them or even no common ancestry.

A rare splice site mutation in the gene encoding glucokinase/hexokinase 4 in a patient with MODY type 2.

The article presents a variant of maturity onset diabetes of the young type 2, caused by a rare mutation in the GCK gene. Maturity onset diabetes of the young (MODY) is a hereditary form of diabetes with an autosomal dominant type of inheritance, an onset at a young age, and a primary defect in pancreatic ß-cell function. This type of diabetes is different from classical types of diabetes mellitus (DM1 and DM2) in its clinical course, treatment strategies, and prognosis. Clinical manifestations of MODY are heterogeneous and may vary even among members of the same family, i. e., carriers of identical mutations. This phenotypic variation is due to the interaction of mutations with different genetic backgrounds and the influence of environmental factors (e. g., lifestyle). Using next-generation sequencing technology, the c.580-1G>A substitution (IVS5 -1G>A, rs1554335421) located in an acceptor splice site of intron 5 of the GCK gene was found in a proband. The identified variant cosegregated with a pathological phenotype in the examined family members. The GCK gene encodes glucokinase (hexokinase 4), which catalyzes the first step in a large number of glucose metabolic pathways such as glycolysis. Mutations in this gene are the cause of MODY2. The illness is characterized by an insignificant increase in the fasting glucose level, is a well-controlled disease without medication, and has a low prevalence of micro- and macrovascular complications of diabetes. The presented case of MODY2 reveals the clinical significance of a mutation in the splice site of the GCK gene. When nonclassical diabetes mellitus is being diagnosed in young people and pregnant women, genetic testing is needed to verify the diagnosis and to select the optimal treatment method. Key words: human; maturity onset diabetes of the young; MODY2; glucokinase gene; next-generation sequencing; genetic analysis; bioinformatics.

Cloning and Functional Analysis of SaCLCc1, a Gene Belonging to the Chloride Channel Family (CLC), from the Halophyte Suaeda altissima (L.) Pall.

One of the genes of the CLC (Chloride Channel) family, SaCLCc1, from the halophyte Suaeda altissima (L.) Pall. was cloned. To investigate the function of SaCLCc1, it was expressed in the S. cerevisiae deletion mutant Δgef1::LEU2 for the only gene of the CLC family in this organism. The growth of the transformed SaCLCc1-expressing mutant Δgef1 was restored when cells were grown in Fe2+-deficient YPEG medium, in minimal synthetic media SD and SR (pH 7.0), and in rich YPD medium containing Mn2+. The complementation of the Δgef1 mutant phenotype with the SaClCc1 gene indicates the involvement of the SaClCc1 protein in the transport of Cl- ions.

[In silico Analyses of Transcriptomes of the Marine Green Microalga Dunaliella tertiolecta: Identification of Sequences Encoding P-type ATPases].

De novo assembled transcriptomes of the marine microalga Dunaliella tertiolecta (Chlorophyta) were analyzed. Transcriptome assemblies were performed using short-read RNA-seq data deposited in the SRA database (DNA and RNA Sequence Read Archive, NCBI). A merged transcriptome was assembled using a pooled RNA-seq data set. The goal of the study was in silico identification of nucleotide sequences encoding P-type ATPases in D. tertiolecta transcriptomes. P-type ATPases play a considerable role in the adaptation of an organism to a variable environment, and this problem is particularly significant for microalgae inhabiting an environment with an unstable ionic composition. Particular emphasis was given to searching for a sequence coding Na^(+)-ATPase. This enzyme is expected to function in the plasma membrane of D. tertiolecta like in some marine algae, in particular, in the closely related alga Dunaliella maritima. An ensemble of 12 P-type ATPases consisting of members belonging to the five main subfamilies of the P-type ATPase family was revealed in the assembled transcriptomes. The genes of the following P-type ATPases were found: (1) heavy metal ATPases (subfamily PIB); (2) Ca^(2+)-ATPases of SERCA type (subfamily P2A); (3) H^(+)-ATPases (subfamily P3); (4) phospholipid-transporting ATPases (flippases) (subfamily P4); (5) cation-transporting ATPases of uncertain specificities (subfamily P5). The presence of functional Na^(+)-ATPases in marine algae is presently undoubted. However, contrary to expectations, we failed to find a nucleotide sequence encoding a protein that could unequivocally be considered a Na^(+)-ATPase. Further study is necessary to elucidate the roles of in silico revealed D. tertiolecta ATPases in Na^(+) transport.

Comparative enzyme immunoassay of matrix metalloproteinases-2, -7, -9 and their tissue inhibitor-2 in tumors and plasma of patients with gastric cancer.

The content of matrix metalloproteinases (MMP) -2, -7, and -9 was significantly higher in tumors in comparison with the adjacent histologically intact gastric mucosa in 80, 70, and 72% patients with gastric cancer, respectively, the increase in the level of MMP tissue inhibitor-2 (TIMP-2) detected in 61% tumors was insignificant. Only plasma level of MMP-7 was elevated in primary patients in comparison with the control and positively correlated with the expression of this protein in the tumor. The concentration of MMP-7 was maximum in the blood of patients with tumor invasion in lymph vessels. These data suggest MMP-7 as a possible serological marker of gastric cancer.

Analysis of NM23 protein and components of plasminogen activation system in tumors of patients with stomach cancer with consideration for disease clinical picture and morphology.

Immunohistochemical analysis of the expression and distribution of nm23 protein and biochemical analysis of the main components of plasminogen activation system in tumors were carried out in stomach cancer patients. The data indicate that the expression of nm23 protein in malignant epithelial gastric tumors is heterogeneous, characterized by cytoplasmic and nuclear immunoreactivity. Reduced expression of the marker is more typical of poorly or undifferentiated tumors. The expression of nm23 protein positively correlated with tPA level (r(s)=0.4; p=0.01) and did not correlate with the content of uPA and PAI-1 in tumors. High PAI-1 values in tumors (>0.5 ng/mg protein) significantly correlated with lower 3-year overall survival of stomach cancer patients. These data confirm the role of the studied proteins in invasion and metastases of malignant tumors and suggest a relationship between changes in the expression of nm23 protein and mechanisms of stomach cancer progress.

Relationship between expression of NM23 protein and clinical morphological factors and content of plasminogen activation system components in tumors of patients with gastric cancer.

Immunohistochemical studies revealed a relationship between the expression of nm23 protein in gastric cancer cells and some parameters characterizing plasminogen activation system and clinical morphological factors. Expression of nm23 in the cytoplasm was detected almost 3-fold more often than in the nuclei; cytoplasmic expression more strictly correlated with patient's age, tumor location, and its histological structure than nuclear expression.