The phylodynamics of SARS-CoV-2 during 2020 in Finland.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of infections and fatalities globally since its emergence in late 2019. The virus was first detected in Finland in January 2020, after which it rapidly spread among the populace in spring. However, compared to other European nations, Finland has had a low incidence of SARS-CoV-2. To gain insight into the origins and turnover of SARS-CoV-2 lineages circulating in Finland in 2020, we investigated the phylogeographic and -dynamic history of the virus. Methods: The origins of SARS-CoV-2 introductions were inferred via Travel-aware Bayesian time-measured phylogeographic analyses. Sequences for the analyses included virus genomes belonging to the B.1 lineage and with the D614G mutation from countries of likely origin, which were determined utilizing Google mobility data. We collected all available sequences from spring and fall peaks to study lineage dynamics. Results: We observed rapid turnover among Finnish lineages during this period. Clade 20C became the most prevalent among sequenced cases and was replaced by other strains in fall 2020. Bayesian phylogeographic reconstructions suggested 42 independent introductions into Finland during spring 2020, mainly from Italy, Austria, and Spain. Conclusions: A single introduction from Spain might have seeded one-third of cases in Finland during spring in 2020. The investigations of the original introductions of SARS-CoV-2 to Finland during the early stages of the pandemic and of the subsequent lineage dynamics could be utilized to assess the role of transboundary movements and the effects of early intervention and public health measures.

Introduction and Rapid Spread of SARS-CoV-2 Omicron Variant and Dynamics of BA.1 and BA.1.1 Sublineages, Finland, December 2021.

Multiple introductions of SARS-COV-2 Omicron variant BA.1 and BA.1.1. lineages to Finland were detected in early December 2021. Within 3 weeks, Omicron overtook Delta as the most common variant in the capital region. Sequence analysis demonstrated the emergence and spread through community transmission of a large cluster of BA.1.1 virus.

Incidence Trends for SARS-CoV-2 Alpha and Beta Variants, Finland, Spring 2021.

Severe acute respiratory syndrome coronavirus 2 Alpha and Beta variants became dominant in Finland in spring 2021 but had diminished by summer. We used phylogenetic clustering to identify sources of spreading. We found that outbreaks were mostly seeded by a few introductions, highlighting the importance of surveillance and prevention policies.

Somatic mutations in lymphocytes in patients with immune-mediated aplastic anemia.

The prevalence and functional impact of somatic mutations in nonleukemic T cells is not well characterized, although clonal T-cell expansions are common. In immune-mediated aplastic anemia (AA), cytotoxic T-cell expansions are shown to participate in disease pathogenesis. We investigated the mutation profiles of T cells in AA by a custom panel of 2533 genes. We sequenced CD4+ and CD8+ T cells of 24 AA patients and compared the results to 20 healthy controls and whole-exome sequencing of 37 patients with AA. Somatic variants were common both in patients and healthy controls but enriched to AA patients' CD8+ T cells, which accumulated most mutations on JAK-STAT and MAPK pathways. Mutation burden was associated with CD8+ T-cell clonality, assessed by T-cell receptor beta sequencing. To understand the effect of mutations, we performed single-cell sequencing of AA patients carrying STAT3 or other mutations in CD8+ T cells. STAT3 mutated clone was cytotoxic, clearly distinguishable from other CD8+ T cells, and attenuated by successful immunosuppressive treatment. Our results suggest that somatic mutations in T cells are common, associate with clonality, and can alter T-cell phenotype, warranting further investigation of their role in the pathogenesis of AA.

Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease.

Graft versus host disease (GvHD) is the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). Here we report studies of a patient with chronic GvHD (cGvHD) carrying persistent CD4+ T cell clonal expansion harboring somatic mTOR, NFKB2, and TLR2 mutations. In the screening cohort (n = 134), we detect the mTOR P2229R kinase domain mutation in two additional cGvHD patients, but not in healthy or HSCT patients without cGvHD. Functional analyses of the mTOR mutation indicate a gain-of-function alteration and activation of both mTORC1 and mTORC2 signaling pathways, leading to increased cell proliferation and decreased apoptosis. Single-cell RNA sequencing and real-time impedance measurements support increased cytotoxicity of mutated CD4+ T cells. High throughput drug-sensitivity testing suggests that mutations induce resistance to mTOR inhibitors, but increase sensitivity for HSP90 inhibitors. Our findings imply that somatic mutations may contribute to aberrant T cell proliferations and persistent immune activation in cGvHD, thereby paving the way for targeted therapies.

Comparison of targeted next-generation sequencing (NGS) and real-time PCR in the detection of EGFR, KRAS, and BRAF mutations on formalin-fixed, paraffin-embedded tumor material of non-small cell lung carcinoma-superiority of NGS.

The development of tyrosine kinase inhibitor treatments has made it important to test cancer patients for clinically significant gene mutations that influence the benefit of treatment. Targeted next-generation sequencing (NGS) provides a promising method for diagnostic purposes by enabling the simultaneous detection of multiple mutations in various genes in a single test. The aim of our study was to screen EGFR, KRAS, and BRAF mutations by targeted NGS and commonly used real-time polymerase chain reaction (PCR) methods to evaluate the feasibility of targeted NGS for the detection of the mutations. Furthermore, we aimed to identify potential novel mutations by targeted NGS. We analyzed formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens from 81 non-small cell lung carcinoma patients. We observed a significant concordance (from 96.3 to 100%) of the EGFR, KRAS, and BRAF mutation detection results between targeted NGS and real-time PCR. Moreover, targeted NGS revealed seven nonsynonymous single-nucleotide variations and one insertion-deletion variation in EGFR not detectable by the real-time PCR methods. The potential clinical significance of these variants requires elucidation in future studies. Our results support the use of targeted NGS in the screening of EGFR, KRAS, and BRAF mutations in FFPE tissue material.

Comparison of solution-based exome capture methods for next generation sequencing.

BACKGROUND: Techniques enabling targeted re-sequencing of the protein coding sequences of the human genome on next generation sequencing instruments are of great interest. We conducted a systematic comparison of the solution-based exome capture kits provided by Agilent and Roche NimbleGen. A control DNA sample was captured with all four capture methods and prepared for Illumina GAII sequencing. Sequence data from additional samples prepared with the same protocols were also used in the comparison. RESULTS: We developed a bioinformatics pipeline for quality control, short read alignment, variant identification and annotation of the sequence data. In our analysis, a larger percentage of the high quality reads from the NimbleGen captures than from the Agilent captures aligned to the capture target regions. High GC content of the target sequence was associated with poor capture success in all exome enrichment methods. Comparison of mean allele balances for heterozygous variants indicated a tendency to have more reference bases than variant bases in the heterozygous variant positions within the target regions in all methods. There was virtually no difference in the genotype concordance compared to genotypes derived from SNP arrays. A minimum of 11× coverage was required to make a heterozygote genotype call with 99% accuracy when compared to common SNPs on genome-wide association arrays. CONCLUSIONS: Libraries captured with NimbleGen kits aligned more accurately to the target regions. The updated NimbleGen kit most efficiently covered the exome with a minimum coverage of 20×, yet none of the kits captured all the Consensus Coding Sequence annotated exons.

Thaumatin-like protein and baker's respiratory allergy.

BACKGROUND: Baker's asthma and rhinitis are among the most common occupational diseases. Inhaled cereal flours, such as wheat, especially cause this disease. OBJECTIVE: To identify and test in vivo clinically important wheat allergens in baker's respiratory allergy in a Finnish population. METHODS: Potential wheat allergens were purified using chromatographic methods from salt-soluble protein extracts of wheat flour and were used in skin prick tests with serial 10-fold dilutions (0.5-0.005 mg/mL). Twenty patients with baker's rhinitis, asthma, or both participated in this study. All the patients had positive skin prick test reactions and specific IgE antibodies to wheat flour. The control group consisted of 10 healthy individuals. Molecular identities of purified wheat allergens were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tandem mass spectrometry. RESULTS: Allergen concentrations of 0.3-0.5 mg/mL revealed that 12 patients reacted to a-amylase inhibitor (alpha-AI), 9 to peroxidase I (PI), 9 to thaumatin-like protein (TLP), and 6 to lipid transfer protein 2G (LTP2G). Conversely, with allergen concentrations of 0.05 mg/mL, 5 patients responded to alpha-AI, 3 to PI, 4 to LTP2G, and 6 to TLP. Of these, TLP and LTP2G are now observed to be new allergens associated with baker's asthma. CONCLUSIONS: In addition to the earlier-described alpha-AI and PI, TLP and LTP2G are important in vivo wheat allergens in baker's allergies in Finland. Further studies are needed to elucidate the role of these novel wheat allergens in respiratory disorders.