HLA genes play a major role for successful hematopoietic stem cell transplantation (HSCT). While the success of HSCT depends on a HLA compatibility between donor and patient, finding a suitable donor remains challenging because of the high polymorphic nature of HLA genes. In this study, HLA-A, -B, -C, -DRB1 and -DQB1 alleles were genotyped at the 3-fields resolution level using MiSeq Illumina of 3341 Russian volunteers from the Kirov bone marrow Registry. Full gene of HLA-A, -B and -C, exons 2-4 of HLA-DRB1 and exons 1-5 of HLA-DQB1 were amplified by multiplex long-range polymerase chain reaction (PCR) and each allele was determined by matching the targeted regions and the reference sequence consisting of the IPD-IMGT/HLA Database. A total of 79 alleles of HLA-A, 115 alleles of HLA-B, 67 alleles of HLA-C, 71 alleles of HLA-DRB1 and 34 alleles of HLA-DQB1 were identified. According to common, intermediate and well-documented catalogs, 38 alleles in HLA-A, 69 in HLA-B, 39 in HLA-C, 48 in HLA-DRB1 and 21 in HLA-DQB1 locus were common alleles, and 5, 7, 7, 7, 2 kinds, accordingly, to written above were well-documented alleles. A total of 12 novel alleles including 3 alleles in HLA-A, 3 alleles in HLA-B, 1 allele in HLA-C, 2 alleles in HLA-DRB1 and 3 alleles in HLA-DQB1 loci were found. Six haplotypes with a frequency of more than 1.0% accounted for 13.19% of the total haplotype frequencies. This information on rare and novel alleles found by HLA typing with NGS may be helpful for unrelated HSCT among Russians.

Evolution of SARS-CoV-2 in immunocompromised hosts may result in novel variants with changed properties. While escape from humoral immunity certainly contributes to intra-host evolution, escape from cellular immunity is poorly understood. Here, we report a case of long-term COVID-19 in an immunocompromised patient with non-Hodgkin’s lymphoma who received treatment with rituximab and lacked neutralizing antibodies. Over the 318 days of the disease, the SARS-CoV-2 genome gained a total of 40 changes, 34 of which were present by the end of the study period. Among the acquired mutations, 12 reduced or prevented the binding of known immunogenic SARS-CoV-2 HLA class I antigens. By experimentally assessing the effect of a subset of the escape mutations, we show that they resulted in a loss of as much as ~1% of effector CD8 T cell response. Our results indicate that CD8 T cell escape represents a major underappreciated contributor to SARS-CoV-2 evolution in humans.

HLA molecules are expressed in almost all nucleated cells of the body. These molecules are extremely variable and responsible for tissue specificity recognition. Precise HLA typing is crucial for tissue and organ transplantation. Usually, HLA-typing NGS methods are based on the assignment of reads to a certain previously reported HLA allele presented in the IPD-IMGT/HLA Database. But there is a limited number of tools able to identify novel alleles that have not yet been reported and thus absent in the database. Such alleles carry mismatches distinguishing them from all known alleles in the database. Therefore, manual evaluation of the identified HLA alleles in the genome browser is a compulsory step in the analysis, and one that is labor intensive and time consuming. We present the development and validation of a freely available web-application for the identification of novel HLA alleles in the most relevant HLA class I and II genes from NGS data. The tool can also be used for automated data quality assessment. The software was validated by analyzing 330 alleles. The results are concordant with orthogonal methods.

Наследственные болезни обмена веществ представляют собой обширный класс генетических заболеваний и вносят значительный вклад в детскую заболеваемость, при этом их диагностика с использованием биохимических методов зачастую вызывает затруднения. В СПбГКУЗ МГЦ были разработаны и внедрены три панели для секвенирования 88 генов, ответственных за развитие трех групп наследственных болезней обмена (НБО), и протестировано 84 ребенка, у которых данные заболевания были заподозрены по данным тандемной масс-спектрометрии (ТМС), либо по наличию клинических симптомов. У 6 детей методом NGS полностью установлена генетическая причина заболевания. Патогенные мутации выявлялись значительно чаще при повышении биохимических маркеров, демонстрируя ведущую роль предварительного биохимического скрининга в проведении NGS анализа. NGS значительно повышает результативность клинической диагностики НБО. Биохимическое тестирование и NGS играют взаимодополняющие роли, и их комплексное использование в алгоритме селективного скрининга позволяет повысить точность диагностики НБО. Inborn errors of metabolism are an extensive class of genetic diseases and contribute significantly to childhood morbidity, and their diagnosis using biochemical methods is often difficult. Three panels for sequencing of 88 genes responsible for the development of three groups of inborn errors of metabolism (IEM) were developed and introduced in St.Petersburg Medical and Genetic Center and 84 children were tested for which these diseases were suspected by tandem mass-spectrometry or by the presence of clinical symptoms. In 6 children, the NGS method fully established the genetic cause of the disease. Pathogenic mutations were detected significantly more frequently with increased biochemical markers, demonstrating the leading role of pre-biochemical screening in performing NGS analysis. NGS significantly improves the clinical diagnostic effectiveness of IEM. Biochemical testing and NGS play complementary roles and their complex use in selective screening algorithm allows to increase accuracy of IEM diagnostics.

The novel allele HLA-C*02:151 showed a single-nucleotide difference to C*02:02:02:01 at codon -23 (CGG/CCG).

The novel allele HLA‐C*02:151 showed a single‐nucleotide difference to C*02:02:02:01 at codon −23 (CGG/CCG).

The novel allele HLA-C*02:151 showed a single-nucleotide difference to C*02:02:02:01 at codon -23 (CGG/CCG).

The novel allele HLA-C*02:151 showed a single-nucleotide difference to C*02:02:02:01 at codon -23 (CGG/CCG).

Four novel alleles were identified by sequence based typing and next generation sequencing technology.

Four novel alleles were identified by sequence based typing and next generation sequencing technology.

The work describes the development of a reagent kit for high-performance HLA-typing using the Illumina MiSeq System platform. The developed reagent kit contains all the necessary components for target enrichment of five HLA genes, preparation of libraries, and software for automatic data analysis. The reagent kit verified on a 93 DNA samples with known genotypes. During the verification, the sensitivity and specificity of the reagent kit for each of the HLA loci were determined - for A and B they were 1.0 and 1.0, respectively, for the C-1.0 and 0.99 locus, for the DRB1 locus 0.98 and 0.99, for the DQB1 locus 0.98 and 0.93.

The mucoviscidosis is one of frequent monogenic diseases. In Russia, in case of mucoviscidosis carrying out of DNA-diagnostic is optional. However, its application permits shortening time of diagnosing, increasing efficiency of of therapeutic treatment and preventing secondary manifestation of disease in family. The DNA-diagnostic using panels on frequent mutations in gene CFTR is recommended in cases of uncertain clinical picture and under borderline values of specific laboratory indices. In Russia, application of such panels permit detecting up to 90% of pathological alleles in gene CFTR. To detect more rare alleles the Sanger sequencing is traditionally applied. Lately, highly productive sequencing techniques became available to detect rare mutations. The actual article presents evaluation of efficiency of application of test-system based on technology of target sequencing for detecting mutations unidentified at primary DNA-diagnostic. Besides, in two patients with mucoviscidosis the application of highly productive sequencing techniques permitted to identify previously unknown nonsense mutations Q1038X (c.3112C>T) и W1310X (c.3930G>A).

NGS is a powerful tool for the diagnostics of inherited diseases. A number of studies devoted to the development and validation of targeted NGS panels are published. Here we present not only development and validation of an assay, but report our experience on introduction of a new approach into the real clinical practice. The assay is intended for the diagnostics of frequent newborn inherited diseases: cystic fibrosis, phenylketonuria and galactosemia. The analysis is performed on the Ion PGM™ sequencing platform and allows the detection of single-nucleotide variations as well as copy number variants. We developed the software performing data quality control, providing decision-support variant annotation and generating the medical report that enables clinical application of the assay. Analytical validation of the assay was performed by bi-directional Sanger sequencing of the most part of the targeted region. Clinical validation was performed by multicenter blind testing of clinical and control samples. Sensitivity and specificity of the assay are above 99%. We have developed statements for test ordering, test acquisition form and practical recommendations for the results interpretation. The test has been successfully applied for the confirmatory diagnostics in a clinical laboratory during a year. Thus, the developed assay is a comprehensive ready-to-use CE-IVD solution for clinical diagnostics.

Background Multiplex polymerase chain reaction (PCR) is a common enrichment technique for targeted massive parallel sequencing (MPS) protocols. MPS is widely used in biomedical research and clinical diagnostics as the fast and accurate tool for the detection of short genetic variations. However, identification of larger variations such as structure variants and copy number variations (CNV) is still being a challenge for targeted MPS. Some approaches and tools for structural variants detection were proposed, but they have limitations and often require datasets of certain type, size and expected number of amplicons affected by CNVs. In the paper, we describe novel algorithm for high-resolution germinal CNV detection in the PCR-enriched targeted sequencing data and present accompanying tool. Results We have developed a machine learning algorithm for the detection of large duplications and deletions in the targeted sequencing data generated with PCR-based enrichment step. We have performed verification studies and established the algorithm’s sensitivity and specificity. We have compared developed tool with other available methods applicable for the described data and revealed its higher performance. Conclusion We showed that our method has high specificity and sensitivity for high-resolution copy number detection in targeted sequencing data using large cohort of samples.

A novel combination of two rare pathogenic cystic fibrosis-causing mutations, p.S1159P and p.Y569H, was detected in a patient of 17years age who suffered from pancreatic insufficiency of unknown etiology. Both mutations were previously described in compound heterozygous patients with moderate lung disease, who carried p.F508del on the other allele. However, this newly described combination of alleles is unusually associated with general gastrointestinal manifestations. Both mutations managed to be identified by virtue of using the Ion PGM™ next-generation sequencing (NGS) platform, thereby making our work a “proof of concept” for clinical application of a new NGS technology in conjunction with the developed software for variant annotation.