Search

Patients who carry Rhesus (RH) blood group variants may develop Rh alloantibodies requiring matched red blood cell transfusions. Serologic reagents for Rh variants often fail to specifically identify variant Rh antigens and are in limited supply. Therefore, red blood cell genotyping assays are essential for managing transfusions in patients with clinically relevant Rh variants. Well-characterized DNA reference reagents are needed to ensure quality and accuracy of the molecular tests. Eight lyophilized DNA reference reagents, representing 21 polymorphisms in RHD and RHCE, were produced from an existing repository of immortalized B-lymphoblastoid cell lines at the Center for Biologics Evaluation and Research/US Food and Drug Administration. The material was validated through an international collaborative study involving 17 laboratories that evaluated each DNA candidate using molecular assays to characterize RHD and RHCE alleles, including commercial platforms and laboratory-developed testing, such as Sanger sequencing, next-generation sequencing, and third-generation sequencing. The genotyping results showed 99.4% agreement with the expected results for the target RH polymorphisms and 87.9% for RH allele agreement. Most of the discordant RH alleles results were explained by a limited polymorphism coverage in some genotyping methods. Results of stability and accelerated degradation studies support the suitability of these reagents for use as reference standards. The collaborative study results demonstrate the qualification of these eight DNA reagents for use as reference standards for RH blood group genotyping assay development and analytical validation., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. All rights reserved.)

Objectives: Genotyping of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in monitoring viral evolution and transmission during the pandemic. The quality of the sequence data obtained from these genotyping efforts depends on several factors, including the quantity/integrity of the input material, the technology, and laboratory-specific implementation. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in genomic epidemiology studies. We aimed to establish clear and broadly applicable recommendations for reliable virus genotyping., Methods: We established and used a sequencing data analysis workflow that reliably identifies and removes technical artefacts; such artefacts can result in miscalls when using alternative pipelines to process clinical samples and synthetic viral genomes with an amplicon-based genotyping approach. We evaluated the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination., Results: We found that at least 1000 viral genomes are necessary to confidently detect variants in the SARS-CoV-2 genome at frequencies of ≥10%. The broad applicability of our recommendations was validated in over 200 clinical samples from six independent laboratories. The genotypes we determined for clinical isolates with sufficient quality cluster by sampling location and period. Our analysis also supports the rise in frequencies of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was facilitated by travel during the summer of 2020., Conclusions: We present much-needed recommendations for the reliable determination of SARS-CoV-2 genome sequences and demonstrate their broad applicability in a large cohort of clinical samples., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The glycoprotein encoded by the ACKR1 gene expresses the Duffy blood group antigens and is a receptor for malaria parasites. We recently described 18 long-range ACKR1 alleles in an autochthonous population of a malaria endemic region. Extending this work, we sequenced the gene in a 53-sample repository established by the US Food and Drug Administration (FDA) as reference reagents for blood group genotyping. The FDA samples have been characterized for 19 genes; however, long-range haplotype information for these genes, including ACKR1, was lacking. We used a hybrid approach, novel for this type of gene, to characterize ACKR1 by combining two next-generation sequencing technologies, the short-read massively parallel sequencing and the long-read nanopore sequencing. The expedient integration of data from both next-generation sequencing systems were necessary and sufficient to allow determination of all 25 long-range ACKR1 alleles found in the 53 samples accurately. All 25 alleles identified in our current FDA cohort were novel and, unexpectedly, none had been observed among the 18 alleles in our previous study. The alleles will be useful for validation, calibration, and proficiency testing of red cell genotyping. The lack of any overlap between the ACKR1 alleles in the two studies documents differences in mutation rate and recombination frequency among populations. The exact haplotype and their interethnic or interpopulation dissimilarities can influence disease susceptibility and therapy., (Published by Elsevier Inc.)

α1-antitrypsin deficiency (AATD) remains the only readily identified genetic cause of chronic obstructive pulmonary disease (COPD). Furthermore, there is growing evidence that even a moderate deficiency increases the risk of lung disease among smokers. Despite these facts, the uptake of testing for AATD in at-risk populations remains low for many reasons, and a lack of clarity among clinicians regarding the most appropriate diagnostic techniques presents a major deterrent. This Personal View addresses the benefits of diagnosis, the technical basis of the available diagnostic methods, and possible clinical confounders for each test. We include a series of unusual cases encountered at our National Centre of Expertise to provide context. The topics covered should equip clinicians with the core knowledge required to confidently assess patients for AATD., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Pharmacogenetic testing increasingly is available from clinical and research laboratories. However, only a limited number of quality control and other reference materials currently are available for the complex rearrangements and rare variants that occur in the CYP2D6 gene. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Cell Repositories (Camden, NJ), has characterized 179 DNA samples derived from Coriell cell lines. Testing included the recharacterization of 137 genomic DNAs that were genotyped in previous Genetic Testing Reference Material Coordination Program studies and 42 additional samples that had not been characterized previously. DNA samples were distributed to volunteer testing laboratories for genotyping using a variety of commercially available and laboratory-developed tests. These publicly available samples will support the quality-assurance and quality-control programs of clinical laboratories performing CYP2D6 testing., (Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Extended blood group genotyping is an invaluable tool used for prevention of alloimmunization. Genotyping is particularly suitable when antigens are weak, specific antisera are unavailable, or accurate phenotyping is problematic because of a disease state or recent transfusions. In addition, genotyping facilitates establishment of mass-scale patient-matched donor databases. However, standardization of genotyping technologies has been hindered by the lack of reference panels. A well-characterized renewable reference panel for standardization of blood group genotyping was developed. The panel consists of genomic DNA lyophilized and stored in glass vials. Genomic DNA was extracted in bulk from immortalized lymphoblastoid cell lines, generated by Epstein-Barr virus transformation of peripheral blood lymphocytes harvested from volunteer blood donors. The panel was validated by an international collaborative study involving 28 laboratories that tested each DNA panel member for 41 polymorphisms associated with 17 blood group systems. Overall, analysis of genotyping results showed >98% agreement with the expected outcomes, demonstrating suitability of the material for use as reference. Highest levels of discordance were observed for the genes CR1, CD55, BSG, and RHD. Although limited, observed inconsistencies and procedural limitations reinforce the importance of reference reagents to standardize and harmonize results. Results of stability and accelerated degradation studies support the suitability of this panel for use as reference reagent for blood group genotyping assay development and standardization., (Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: The WHO European Region (EUR) has adopted the goal of eliminating measles and rubella but individual countries perform differently in achieving this goal. Measles virus spread across the EUR by mobile groups has recently led to large outbreaks in the insufficiently vaccinated resident population. As an instrument for monitoring the elimination process and verifying the interruption of endemic virus transmission, molecular surveillance has to provide valid and representative data. Irrespective of the country's specific situation, it is required to ensure the functionality of the laboratory surveillance that is supported by the WHO Global Measles and Rubella Laboratory Network., Aims: To investigate whether the molecular surveillance in the EUR is adequate for the challenges in the elimination phase, we addressed the quality assurance of molecular data, the continuity and intensity of molecular monitoring, and the analysis of transmission chains., Sources: Published articles, the molecular External Quality Assessment Programme of the WHO, the Centralized Information System for Infectious Diseases of the WHO EUR and the WHO Measles and Rubella Nucleotide Surveillance databases served as information sources., Content: Molecular proficiency testing conducted by the WHO in 2016 has shown that the expertise for measles and rubella virus genotyping exists in all parts of the EUR. The analysis of surveillance data reported nationally to the WHO in 2013-2016 has revealed some countries with outbreaks but not sufficiently representative molecular data. Long-lasting supranational MV transmission chains were identified., Implications: A more systematic molecular monitoring and recording of the transmission pattern for the whole EUR could help to create a meaningful picture of the elimination process., (Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. All rights reserved.)

The rpoB gene codes for the RNA polymerase β subunit, which is the target of rifampicin, an essential drug in the treatment of tuberculosis and other mycobacterial infections. This gene is present in all bacteria, but its length and nucleotide sequence vary between bacterial species, including mycobacteria. Mutations in the rpoB gene alter the structure of this protein and cause drug resistance. To describe the resistance-associated mutations, the scientific and medical communities have been using, since 1993, a numbering system based on the Escherichia coli sequence annotation. Using E. coli reference for describing mutations in mycobacteria leads to misunderstandings, particularly with the increasing use of whole genome sequencing, which brought an alternative numbering system based on the Mycobacterium tuberculosis rpoB sequence. We propose using a consensus numbering system for the reporting of resistance mutations based on the reference genomes from the species interrogated (such as strain H37Rv for M. tuberculosis). This manuscript provides the necessary figures and tables allowing researchers, microbiologists and clinicians to easily convert other annotation systems into one common language., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Personalized medicine has gained increasing importance in clinical oncology, and several clinically important biomarkers are implemented in routine practice. In an effort to guarantee high quality of molecular testing in France, three subsequent external quality assessment rounds were organized at the initiative of the National Cancer Institute between 2012 and 2014. The schemes included clinically relevant biomarkers for metastatic colorectal (KRAS, NRAS, BRAF, PIK3CA, microsatellite instability) and non-small cell lung cancer (EGFR, KRAS, BRAF, PIK3CA, ERBB2), and they represent the first multigene/multicancer studies throughout Europe. In total, 56 laboratories coordinated by 28 regional molecular centers participated in the schemes. Laboratories received formalin-fixed, paraffin-embedded samples and were asked to use routine methods for molecular testing to predict patient response to targeted therapies. They were encouraged to return results within 14 calendar days after sample receipt. Both genotyping and reporting were evaluated separately. During the three external quality assessment rounds, mean genotype scores were all above the preset standard of 90% for all biomarkers. Participants were mainly challenged in case of rare insertions or deletions. Assessment of the written reports showed substantial progress between the external quality assessment schemes on multiple criteria. Several essential elements such as the clinical interpretation of test results and the reason for testing still require improvement by continued external quality assessment education., (Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

The use of ancestry informative markers (AIMs) in forensic analysis is of considerable utility since ancestry inference can progress an investigation when no identification has been made of DNA from the crime-scene. Short-amplicon markers, including insertion deletion polymorphisms, are particularly useful in forensic analysis due to their mutational stability, capacity to amplify degraded samples and straightforward amplification technique. In this study we report the completion of H952 HGDP-CEPH panel genotyping with a set of 46 AIM-Indels. The study adds Central South Asian and Middle Eastern population data, allowing a comparison of patterns of variation in Eurasia for these markers, in order to enhance their use in forensic analyses, particularly when combined with sets of ancestry informative SNPs. Ancestry analysis using principal component analysis and Bayesian methods indicates that a proportion of classification error occurs with European-Middle East population comparisons, but the 46 AIM-Indels have the capability to differentiate six major population groups when European-Central South Asian comparisons are made. These findings have relevance for forensic ancestry analyses in countries where South Asians form much of the demographic profile, including the UK, USA and South Africa. A novel third allele detected in MID-548 was characterized - despite a low frequency in the HGDP-CEPH panel samples, it appears confined to Central South Asian populations, increasing the ability to differentiate this population group. The H952 data set was implemented in a new open access SPSmart frequency browser - forInDel: Forensic Indel browser., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

In this study, a new STR 25-plex typing system, including 23 autosomal STRs (D1S1656, D2S1338, D2S441, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D22S1045, CSF1PO, FGA, Penta D, Penta E, TH01, TPOX, vWA) and a Y-STR locus of DYS391 and amelogenin, was developed. The included 24 STRs belonged to the main international DNA databases (CODIS, ISSL, ESS-extended, UCL, GCL and NCIDD) except D6S1043 (specially chosen for Chinese population). Developmental validation indicated that the STR 25-plex typing system was reproducible, accurate, sensitive and robust. The sensitivity testing of the system was such that a full profile was obtainable even with 125pg of human DNA. Specificity testing was demonstrated by the lack of cross-reactivity with a variety of commonly encountered animal species and microbial pool. For the stability testing, full profiles can been obtained with humic acid concentration≤60ng/μL and hematin<500μM. Also, this multiplex system is suitable for mixture study. All of the minor alleles were called for ratios of 1:1, 1:3 and 3:1 of the mixture with the system. In addition, the whole PCR amplification can finish within 1h, making the system suitable for fast-detection. For the forensic evaluation of the multiplex system, 23 autosomal STRs included followed the Hardy-Weinberg equilibrium. A total of 268 alleles were detected for the 23 autosomal STR loci among 200 individuals. Since 23 autosomal STRs were independent from each other, CMECduo was 0.99999916563607 and CMECtri was 0.99999999986525. All the forensic efficiency parameters demonstrated that this multiplex system is highly polymorphic and informative in the Han population of China., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: The costs associated with developing high density microarray technologies are prohibitive for genotyping animals when there is low economic value associated with a single animal (e.g. prawns). DNA pooling is an attempt to address this issue by combining multiple DNA samples prior to genotyping. Instead of genotyping the DNA samples of the individuals, a mixture of DNA samples (i.e. the pool) from the individuals is genotyped only once. This greatly reduces the cost of genotyping. Pooled samples are subject to greater genotyping inaccuracies than individual samples. Wrong genotyping will lead to wrong biological conclusions. It is thus required to calibrate the resulting genotypes (allele frequencies)., Methods: We present a regression based approach to translate raw array output to allele frequency. During training, few pools and the individuals that constitute the pools are genotyped. Given the genotypes of individuals that constitute the pool, we compute the true allele frequency. We then train a regression algorithm to produce a mapping between the raw array outputs to the true allele frequency. We test the algorithm using pool samples withheld from the training set. During prediction, we use this map to genotype pools with no prior knowledge of the individuals constituting the pools., Results and Discussion: After data quality control we have available a dataset comprised of 912 pools. We estimate allele frequency using three approaches: the raw data, a commonly used piecewise linear transformation, and the proposed local-global learner fusion method. The resulting RMS errors for the three approaches are 0.135, 0.120, and 0.080 respectively., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

Purpose: To optimise a polymerase chain reaction (PCR) based DNA sequencing technique for genotyping polyoma virus in clinical specimens obtained from renal transplant patients., Materials and Methods: A hundred and thirty (106 peripheral blood and 24 urine) clinical specimens collected from renal transplant patients were included in the study for detecting the presence of  DNA of BK virus (BKV), JC virus (JCV) by PCR targeting the viral protein 1 (VP1) gene. PCR based DNA sequencing was performed to determine the genotypes of polyoma virus and subjected to bioinformatics analysis to determine the amino acid sequences and screen for mutations in the VP1 gene., Results: Polyoma virus was detected in 23 (17.69%) specimens of which 19 (82.60%) were positive for BK virus, 3 (13.04%) for JC virus and 1 for both BK and JC virus. PCR based DNA sequencing detected BK virus genotype I in 12 (50%), genotype IV in 8 (33.3%) and JC virus in 4 (16.6%) clinical specimens. BKV genotype I was the predominant genotype (64.2% in peripheral blood and 33.33% in urine) prevalent in south India. Six novel mutations were found--at position 29, 30 to 47 of BKV genotype I; at position 11 and 15 of BKV genotype IV and at position 2 and 30 of JCV., Conclusion: BKV genotype I is the prominent genotype in India and novel mutations detected in the VP1 gene of BKV and JCV are being reported for the first time in literature.

Background: Gilbert's syndrome is one of the most common metabolic syndromes in the human population characterised by mild unconjugated hyperbilirubinemia resulting from reduced activity of the bilirubin conjugating enzyme UDP-glucuronosyltransferase (UGT1A1). Although Gilbert's syndrome is usually quite benign UGT1A1(TA)n genotyping is important in exclusion of more serious causes of hyperbilirubinemia and since it has significant implications for personalised medicine. The aim of our study was to develop plasmid based reference materials which could be used for UGT1A1(TA)n genotyping., Methods: Plasmids were generated using recombinant DNA technology and their number of repeats as well as the entire sequence verified by Sanger sequencing. Their suitability as reference materials was tested using sizing by capillary electrophoresis and denaturing high performance liquid chromatography., Results: Plasmids containing all four different alleles (TA)5, (TA)6, (TA)7 and (TA)8 that are present in the human population as well as a plasmid with (TA)4 repeats were successfully generated., Conclusions: Prepared plasmid reference materials allow the creation of all possible UGT1A1(TA)n polymorphism genotypes and can serve as an efficient substitute for the human genomic DNA reference material in routine genotyping and in the development of new genotyping tests., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Accurate human papillomavirus (HPV) typing is essential for evaluating and monitoring HPV vaccines in cervical cancer screening and in epidemiological surveys. In our country, different HPV DNA detection and genotyping methodologies have been established for diagnosing and monitoring HPV-related disease in clinical practice and for research. However, there is a lack of reference materials to standardize the methods for HPV detection and genotyping. In this study, we constructed candidate reference materials comprising 15 targets (13 types of high-risk HPV, two types of low-risk HPV). We evaluated whether the candidate reference materials could be used as the reference for HPV detection and genotyping using quantitative real-time polymerase chain reaction. Standard curves for the wide linear range (10(1)-10(6)copies/μL) produced high correlation regression coefficient R(2) of 0.99. The reaction efficiencies were 96.3% to 101.2% for the standard curves, indicating highly efficient reactions. Specific genotypes were detected in single or multiple mixed samples. Our results suggest that these reference materials may provide useful standards for standardizing quality assurance for different HPV-typing assays and for proficiency testing in diagnostic laboratories., (Copyright © 2014. Published by Elsevier B.V.)

Background: PCR-based technology is indispensable for genetic diagnosis. On the other hand, allele dropout is one significant cause of genotyping errors. Most allele dropout mechanisms are related to annealing failure caused by single nucleotide variant (SNV) situated inside the primer sequences. Here, we demonstrate a novel allele dropout mechanism caused by a non-primer-binding-site SNV., Methods: We demonstrate that the apparent homozygosity of NM&#95;000137.1(FAH):c.1035&#95;1037del was caused by allele dropout., Results: The non-primer-binding-site SNV causes a strong secondary hairpin structure formation of the PCR products and leads to amplification failure. SNV check of the primer sequences per se during primer design is not adequate to avoid allele dropout., Conclusions: The next-generation primer design software should analyze the secondary structure of primers and template sequence taking SNV in both sequences into account in order to avoid genotyping errors., (Copyright © 2013 Elsevier B.V. All rights reserved.)