This study evaluates primary template-directed amplification (PTA) for whole exome sequencing (WES) of small fibroblast cell groups, which mimics the limited cell quantities typical of trophectoderm embryo biopsies. PTA's consistent amplification reduces allelic dropout (ADO) and improves uniform coverage, overcoming challenges associated with conventional methods such as multiple displacement amplification (MDA). Using fibroblast samples alongside well-characterized genomic references (E701, NA12878), we benchmarked PTA-WES, achieving 97.5% target region coverage at 10x, meeting American College of Medical Genetics and Genomics (ACMG) standards. The completed filtering and variant calling provide a foundation for further optimization and analysis aimed at evaluating the reliability of PTA for routine clinical use. Preliminary results from embryo biopsies sequenced with PTA-WES revealed a median coverage of 102x, significantly improving upon the variability and coverage gaps observed with MDA-WES. These findings support the potential of PTA to increase the clinical applicability of WES for preimplantation genetic testing for monogenic disorders (PGT-M), expanding its ability to detect inherited and de novo mutations in embryos.

IntroductionThe study of the cervicovaginal microbiome is a critical area of research in medical science. According to scientific data, microorganisms inhabiting the lower female genital tract may influence susceptibility to and persistence of human papillomavirus (HPV), as well as the development and progression of squamous intraepithelial lesions (SIL) toward cervical cancer (CC).MethodsThe study included 67 patients with a histological diagnosis of low-grade squamous intraepithelial lesion (LSIL) and 85 patients with high-grade squamous intraepithelial lesion (HSIL). The cervical canal microbiome of patients with LSIL (n = 31) and HSIL (n = 45) was examined using the 16S rRNA gene sequencing method, while the microbiocenosis of the remaining patients with LSIL (n = 36) and HSIL (n = 40) was analyzed using PCR-RT.ResultsThere are patients with HSIL on average older than patients with LSIL about 3 years. HPV 16 was found to be the most common virus type in patients with SIL. When determining of 16S rRNA genes of prokaryotic microorganisms in women of reproductive age with SIL by the new generation sequencing, it turned out that the cervical canal microbiome is inhabited by many atypical representatives (soil, aquatic and aerobacteria). In addition to the genera Lactobacillus, Streptococcus, Staphylococcus, Gardnerella, Ureaplasma, Prevotella, Sneathia, Fusobacterium, Veillonella, Megasphaera, Dialister, Enterococcus, Escherichia/Shigella, Fannyhessea, Peptococcus, Peptostreptococcus, Finegoldia, Porphyromonas, Corynebacterium, Alloscardovia, Mageeibacillus, Haemophilus, Duncaniella, Ralstonia, Sphingomonas, Pedobacter, Methylobacterium, Ruminococcus, Sediminibacterium, Pseudomonas, Aerococcus, Acinetobacter, Campylobacter, Stenotrophomonas, Sphingobacterium, Phyllobacterium and others may be included in the microbial composition of the cervical canal. Dysbiotic disorders were more common in patients with HSIL. Streptococcus spp. always accompanied aerobic vaginitis (AV), whereas bacterial vaginosis (BV) was associated with the genera Gardnerella, Fannyhessea, Prevotella, Dialister, Sneathia, Anaerococcus, Megasphaera, Finegoldia, Peptoniphilus, Porphyromonas, Parvimonas and Eubacterium spp. When comparing the two methods, the genera Peptoniphilus, Methylobacterium, Ralstonia spp, Sphingomonas, Phyllobacterium, Parvimonas, Anaerococcus and Ruminococcus may be included in the microbial biomass in a significant proportion. Eubacterium spp. did not occur in the 16S sequencing method. Some representatives are detected together with each other in the RT-qPCR method (Femoflor - 16), e.g., Sneathia spp. + Leptotrichia spp. + Fusobacterium spp.; Megasphaera spp. + Veillonella spp. + Dialister spp.; Mobiluncus spp. + Corynebacterium spp., however, Corynebacterium spp., Veillonella spp., Mobiluncus spp., Fusobacterium spp., Leptotrichia spp. and Peptostreptococcus spp. were practically absent (or in a low percentage) in the microbiome of women with SIL.

The first Russian human genome standard E701 was developed through a collaborative research involving four laboratories: Pirogov Russian National Research Medical University, National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I.Kulakov, National Research Center Kurchatov Institute, and National Medical Research Radiological Centre. Whole-genome sequencing of short reads on various platforms (MGI Tech, Illumina) and alignment to the reference human genome GRCh38.p14 were performed for the E701 sample. Subsequently, 3842 877 genomic variants were identified, which can be used as a standard for calculating statistical quality metrics while analyzing sequencing data. This variant set was additionally filtered by the GIAB high-confidence regions version 4.2.1, yielding 3262 154 variants. Moreover, E701 sample has undergone multiple whole-exome sequencing using five different enrichment panels for exome-genome concordance analysis. Furthermore, 9096 bi-allelic variants were identified on the autosomes and the X chromosome, with a minor allele frequency exceeding 0.4. Additionally, mitochondrial DNA sequencing was performed with the breadth of coverage over 99.9% at 1000×.

ABSTRACTThe new HLA‐C*12:02:55 allele showed one synonymous nucleotide difference compared to the HLA‐С*12:02:02:01 allele in codon 134.

The new HLA-B*52:130 allele showed one nonsynonymous nucleotide difference compared to the HLA-B*52:01:01:01 allele in codon 170.

Introduction: Langerhans cell histiocytosis (LCH) is a rare clonal myeloid disorder characterized by tissue infiltration with pathological antigen-presenting cells morphologically and phenotypically similar to Langerhans cells. Activating mutations in the MAPK/ERK signaling pathway play a key role in the pathogenesis of LCH. The aim of this study was to characterize the morphological and molecular features of the neoplastic process. Materials and Methods: This study presents a clinical case of a 34-year-old patient with a rare mixed form of LCH combined with Erdheim-Chester disease (ECD), involving multiple organs including kidneys, skin, bones, ENT organs, eyes, and the central nervous system. The patient also had diabetes insipidus, chronic external otitis, and presumptive hereditary forms of anemia and hemochromatosis. A comprehensive histopathological evaluation was performed, including histochemical, immunohistochemical, and immunofluorescent analyses, alongside molecular genetic testing using whole exome sequencing (WES) of blood and skin samples. The patient demonstrated a partial clinical and laboratory response to targeted therapy with trametinib and desmopressin. Results: Comparison of WES data from blood and skin revealed 71,953 shared variants consistent with likely germline changes, as well as 3,081 unique somatic variants in skin and 2,633 unique variants in blood. Low-level mosaic BRAF V600E mutation (VAF = 1%) was detected in the skin, alongside pathogenic germline variants in the STEAP3 (c.523-2A>T, NM_182915.3) and SLC40A1 (c.332T>A, p.Met111Lys, NM_014585.6) genes. Immunofluorescence analysis of the trephine biopsy revealed diverse cellular populations, including sparse tryptase-positive mast cells, which showed minimal contact with each other or other labeled cells. The populations of Vimentin+CD11b–CD34+ and Vimentin+CD11b+CD34– cells (excluding endothelial cells) accounted for median values of 0.68% and 7.78%, respectively, of the total Vimentin+ cells. Regarding CD68 and S100 expression, CD68+ cells predominated (median = 55.61%), S100+ cells constituted a median of 43.02%, whereas CD68+S100+ cells were the rarest (median = 2.174%), reflecting the transition of Langerhans cells to LCH cells, encompassing up to 4.81% of the cellular population. This hybrid microenvironment paralleled the patient’s systemic inflammatory activity (elevated acute-phase reactants), multi-organ involvement, and renal interstitial fibrosis/tubular atrophy with reduced kidney function, without serological or histological evidence of vasculitis. Conclusion: This case illustrates the histological heterogeneity and hybrid pathology of mixed histiocytosis, where canonical dendritic LCH cells coexist with macrophage-rich ECD-like stroma. An integrated approach involving a multidisciplinary clinical team, thorough molecular genetic testing, and the application of targeted therapy is essential for improving prognosis and quality of life in young patients.

The new HLA-B*38:01:26 allele showed one synonymous nucleotide difference compared to the HLA-B*38:01:01:01 allele in codon 101.

Whole exome sequencing (WES) is essential for identifying genetic variants linked to diseases. This study compares available to date four exome enrichment kits: Agilent SureSelect Human All Exon v8, Roche KAPA HyperExome, Vazyme VAHTS Target Capture Core Exome Panel, and Nanodigmbio NEXome Plus Panel v1. We evaluated target design, coverage statistics, and variant calling accuracy across these four different exome capture products. All kits showed high target coverage, with 10x coverage exceeding 97.5% and 20x coverage above 95%. Roche exhibited the most uniform coverage, indicated by the lowest fold-80 scores, while Nanodigmbio had more on-target reads due to fewer off-target reads. Variant calling performance, evaluated using in-lab standard E701 DNA sample, showed high recall rates for all kits, especially Agilent v8. All kits achieved an F-measure above 95.87%. Nanodigmbio had the highest precision with the fewest false positives but a slightly lower F-measure than other kits. This study also highlights the performance of new solutions from Vazyme (China) and Nanodigmbio (China), which were comparable to Agilent v8 and Roche KAPA kits. These findings assist researchers and clinicians in selecting appropriate exome capture solutions.

The latest scientific research development by Russian scientists – the certified standard sample of the human full-genomic DNA composition E701 – CRM 12688-2024 – was able to give people confidence in their health and the opportunity to live at a new qualitative level. The CRM, developed by specialists from the Research Center for Applied Metrology - Rostest (formerly VNIIMS) – the national metrology institute of the Russian Federation, and the Russian Research Medical University named after N.I. Pirogov of the Ministry of Health of the Russian Federation, is a means of metrological support for measurements performed in the study of hereditary genetic diseases, establishing kinship, identifying mutations in the human genome, and determining oncological diseases. For the first time, the use of a CRM of the full human genome allows calibrating measuring instruments intended for the amplification and sequencing of DNA and RNA nucleotide sequences, and human genome analysis. This became possible, among other things, due to the use of specially selected primers and probes for a specific short fragment of full-genomic DNA. The range of ruGIAB application is very wide, it can be used in the field of metrological support not only in healthcare, but also in pharmaceuticals, in the field of ecology, in the implementation of agricultural and industrial biotechnological processes. ruGIAB was created using four generations of sequencing platforms through interlaboratory and intra-laboratory studies. Calibrated measuring instruments and CRM were used to determine its metrological characteristics. The values of the mass concentration of the CRM were obtained by spectrophotometry and real-time PCR. The values of the mass fractions of each nucleotide were obtained by next-generation sequencing (NGS) methods using DNBSEQ-G400 sequencers, and Illumina high-throughput sequencing methods using the NovaSeq sequencer. The total relative uncertainty of the certified value of the mass fractions of CRM nucleotides was no more than 1.5%, the total relative uncertainty of the certified value of the mass concentration of human genomic DNA was no more than 8% at a probability of 0.95 with a coverage factor k = 2. The CRM material is a preparation of human genomic DNA (Caucasian males with 46XY karyotype without hereditary pathologies in the anamnesis) in a buffer solution. The conducted studies have shown the comparability of the results of measurements of the metrological characteristics of the CRM, obtained by various methods and on various sequencing platforms, obtained under conditions of the greatest spread of readings under reproducibility conditions. The certified characteristics of the CRM are metrologically traceable to the state primary standards of the Russian Federation. Practical application of CRM in 18 organizations – laboratories of genetic research, research institutes demonstrated the comparability of results at the level of 8% of the total uncertainty in measuring the mass concentration, 1.5% of the total uncertainty in determining the nucleotide sequence of the CRM. This confirms the successful solution of the set task of metrological support of measurements performed in studies of hereditary genetic diseases of humans, on the vast territory of the European part of the Russian Federation.

AbstractBackgroundAuditory Neuropathy Spectrum Disorder (ANSD) is often missed by common hearing screening tests, still accounting for up to 10% of hearing impairments. ANSD has an underlying genetic factor being mostly caused by pathogenic variants in 13 genes.MethodsWe examined 122 children with impaired hearing, including 102 pediatric patients (mean age was 3.7±4.1 years) with sensorineural hearing loss (SNHL) of varying severity and 20 children with a clinically confirmed ANSD (mean age was 5.65±4.63 years). For children with SNHL, we genotyped the most frequent variants (c.35delG, c.167delT, c.235delC, c.313-326del14 and c.358-360delGAG) in theGJB2gene using quantitative PCR. For children with a clinically confirmed ANSD, we performed the whole exome sequencing and studied the obtained variants in a custom panel of 248 genes.ResultsOur findings show that fifty-six (54.9%) SNHL patients carried homozygous variants inGJB2gene. In 12 (60%) ANSD patients, we detected variants in nucleotide sequences of theOTOF(25%),CDH23, TMC1, COL11A1, PRPS1andHOMER2genes (8 of which had not been previously described). Transient Evoked Otoacoustic Emissions testing revealed differences at 500 Hz (AS, p = 0.04181) and 4000 Hz (AD, p = 0.00126) between the ANSD and SNHL patient groups. The Auditory Steady-State Response (ASSR) test demonstrated significant differences at all frequencies (p < 0.01). When comparing the results obtained from the pure-tone audiometry and ASSR tests in ANSD patients revealed statistically significant differences at 500 (AD, AS) and 1000 (AD) Hz.ConclusionsThese findings indicate that audiologists and otorhinolaryngologists should use the ASSR test in clinical practice for differential diagnosis of auditory neuropathies. According to the survey data from the parents of hearing-impaired children, rehabilitation was more successful in SNHL patients compared to ANSD patients.

Auditory neuropathy spectrum disorder (ANSD) is often missed by standard hearing tests, accounting for up to 10% of hearing impairments (HI) and commonly linked to variants in 23 genes. We assessed 122 children with HI, including 102 with sensorineural hearing loss (SNHL) and 20 with ANSD. SNHL patients were genotyped for common GJB2 variants using qPCR, while ANSD patients underwent whole exome sequencing, with variants analyzed across 249 genes. Homozygous GJB2 variants were found in 54.9% of SNHL patients. In 60% of ANSD patients, variants were detected in OTOF (25%), CDH23, TMC1, COL11A1, PRPS1, TWNK, and HOMER2 genes, including eight novel variants. Transient evoked otoacoustic emissions testing revealed differences at 4000 Hz (p = 0.0084) between the ANSD and SNHL groups. The auditory steady-state response (ASSR) test showed significant differences at 500 Hz (p = 2.69 x 10-4) and 1000 Hz (p = 0.0255) compared to pure-tone audiometry (PTA) in ANSD patients. Our questionnaire shows that the parents of children with SNHL often report an improved quality of life with hearing aids or cochlear implants, while parents of children with ANSD frequently experience uncertainty about outcomes (p = 0.0026), leading to lower satisfaction.

The new HLA-B*44:418 allele showed one non-synonymous nucleotide difference compared with the HLA-B*44:05:01:01 allele in codon 12.

Autoimmune adrenal insufficiency (AAI) is a rare disease. This research evaluates three patients with AAI, including autoimmune polyglandular syndrome (APS) type 2. Two patients had APS or AAI during childhood, and one had a history of endocrine autoimmune disease, indicating a possible hereditary basis of the condition. Trio-based exome sequencing and high-resolution HLA typing were employed to analyze patients and their parents. Benign or likely benign variants of the AIRE gene were identified in all participants of the study. These variants, coupled with clinical data and the results of antibody studies to type I interferons, helped to exclude APS-1. Patients with APS-2, in contrast to patient with AAI, inherited distinct variants of unknown significance in the CLEC16A gene, which is associated with autoimmune diseases, including AAI. Various risk alleles in other genes associated with autoimmunity were identified in all patients. HLA typing of class II loci revealed alleles related to APS. Nevertheless, the frequencies of the haplotypes identified are substantial in the healthy Russian population. Immunological tests can detect antibody carriers and assess the risk of autoimmune disease development. In the future, to identify genetic predictors of autoimmune endocrinopathies, it is recommended to analyze the whole genome of patients and their relatives, examining clinically relevant variants in non-coding regions.

Modern medical scientific sources offer descriptions of cases of hereditary bone dysplasias occurring under the guise of rheumatoid arthritis (RA). These include the progressive pseudorheumatoid dysplasia (PPRD), an autosomal recessive disease caused by defects in the CCN6 gene, as well as autosomal dominant forms of pseudorheumatoid dysplasia associated with variants in the COL2A1 gene. Though there are no references to pseudorheumatoid dysplasia case observations in available domestic sources. This Article represents a discussion over the bibliographic sources on PPRD and a clinical case observation of a patient initially observed for RA but diagnosed subsequently with an autosomal dominant form of bone dysplasia caused by a de novo variant in the COL2A1 gene.

An analysis of 1300+ existing ACE mutations revealed that 400+ are damaging and led us to hy-pothesize that carriers of heterozygous loss-of-function (LoF) ACE mutations (which result in low ACE levels) could be at risk for the development of late-onset Alzheimer’s disease (AD) [Danilov, 2024]. Here we quantified blood ACE levels in EDTA-plasma from 41patients with 10 different heterozygous ACE mutations, as well as 33 controls, and estimated the effect of these mutations on ACE phenotype using a set of mAbs to ACE and two ACE substrates. We found that relatively frequent (~1%) AD-associated ACE mutations in the N domain of ACE, Y215C and G325R are truly damaging and, likely, transport-deficient, with ACE levels in plasma only ~50% of controls. Another AD-associated ACE mutation, R1250Q, in the cytoplasmic tail, did not cause a decrease in ACE and, likely, did not affect surface ACE expression. We have also developed a method to identify patients with anti-catalytic mutations in the N domain. These mutations may result in reduced degradation of amyloid beta peptide Aβ42, an important component for amyloid deposition. Consequently, these could pose a risk factor for the development of AD. Therefore, a systematic analysis of blood ACE levels in patients with all ACE mutations has potential to identify individuals at an increased risk of late-onset AD. These individuals may benefit from future preventive or therapeutic interventions involving a combination of chemical and pharmacological chaperones, as well as proteasome inhibitors, aiming to enhance ACE protein traffic. This approach has been previously demonstrated in a cell model of the transport-deficient ACE mutation, Q1069R [Danilov, 2010].

The MGI (MGI Tech Co. Ltd., China) next-generation sequencing platform, including the DNBSEQ-G50, -G400, and -T7 sequencers, is being actively adopted in research. Despite its widespread adoption, challenges persist in the form of limitations associated with the manufacturer's provided barcode set for library preparation. These limitations include constraints on the number of samples that can be concurrently sequenced, compatibility issues with barcodes from diverse or incomplete sets, and restrictions on the sample ratio. Purpose: to develop a universal method that allows sequencing of up to 252 samples simultaneously on a single sequencer lane, while eliminating barcode-related limitations. We proposed a “quad method” that provides 4 or 4n+2 equilibration of barcodes. This paper also delves into its comprehensive analysis, verification procedures, seamless integration into the sequencing process and validation of the method on the DNBSEQ G-400 platform. The quad method showed efficiency and reliability, allowing sequencing of up to 252 samples simultaneously without compromising data quality. The proposed method optimizes library preparation and improves the flexibility of sequencing on the MGI platform.

Pseudomonas aeruginosa is characterized by a high adaptive potential, developing resistance in response to antimicrobial pressure. We employed a spatiotemporal evolution model to disclose the pathways of adaptation to colistin, a last-resort polymyxin antimicrobial, among three unrelated P. aeruginosa lineages. The P. aeruginosa ATCC-27833 reference strain (Pa_ATCC), an environmental P. aeruginosa isolate (Pa_Environment), and a clinical isolate with multiple drug resistance (Pa_MDR) were grown over an increasing 5-step colistin concentration gradient from 0 to 400 mg/L. Pa_Environment demonstrated the highest growth pace, achieving the 400 mg/L band in 15 days, whereas it took 37 and 60 days for Pa_MDR and Pa_ATCC, respectively. To identify the genome changes that occurred during adaptation to colistin, the isolates selected during the growth of the bacteria (n = 185) were subjected to whole genome sequencing. In total, 17 mutation variants in eight lipopolysaccharide-synthesis-associated genes were detected. phoQ and lpxL/PA0011 were affected in all three lineages, whereas changes in pmrB were found in Pa_Environment and Pa_MDR but not in Pa_ATCC. In addition, mutations were detected in 34 general metabolism genes, and each lineage developed mutations in a unique set of such genes. Thus, the three examined distinct P. aeruginosa strains demonstrated different capabilities and genetic pathways of colistin adaptation.

About 7% of all cancer deaths are caused by pancreatic cancer (PCa). PCa is known for its lowest survival rates among all oncological diseases and heterogenic molecular profile. Enormous amount of genetic changes, including somatic mutations, exceeds the limits of routine clinical genetic laboratory tests and further stagnates the development of personalized treatments. We aimed to build a mutational landscape of PCa in the Russian population based on full exome next-generation sequencing (NGS) of the limited group of patients. Applying a machine learning model on full exome individual data we received personalized recommendations for targeted treatment options for each clinical case and summarized them in the unique therapeutic landscape.