Single Nucleotide Point Research Articles

Singlet oxygen-based photoelectrochemical detection of single-point mutations in the KRAS oncogene

Single nucleotide point mutations in the KRAS oncogene occur frequently in human cancers, rendering them intriguing targets for diagnosis, early detection and personalized treatment. Current detection methods are based on polymerase chain reaction, sometimes combined with next-generation sequencing, which can be expensive, complex and have limited availability. Here, we propose a novel singlet oxygen (1O2)-based photoelectrochemical detection methodology for single-point mutations, using KRAS mutations as a case study. This detection method combines the use of a sandwich assay, magnetic beads and robust chemical photosensitizers, that need only air and light to produce 1O2, to ensure high specificity and sensitivity. We demonstrate that hybridization of the sandwich hybrid at high temperatures enables discrimination between mutated and wild-type sequences with a detection rate of up to 93.9%. Additionally, the presence of background DNA sequences derived from human cell-line DNA, not containing the mutation of interest, did not result in a signal, highlighting the specificity of the methodology. A limit of detection as low as 112 pM (1.25 ng/mL) was achieved without employing any amplification techniques. The developed 1O2-based photoelectrochemical methodology exhibits unique features, including rapidity, ease of use, and affordability, highlighting its immense potential in the field of nucleic acid-based diagnostics.

PREIMPLANTATION GENETIC TESTING FOR SICKLE CELL DISEASE – A COST EFFECTIVENESS ANALYSIS

Sickle cell disease (SCD) results in an inherited pattern of early morbidity and mortality for those affected, causing significant resource burdens on the healthcare system. Preimplantation genetic testing for monogenic diseases (PGT-M) uses next generation sequencing to identify unaffected embryos for transfer, allowing selection against genetically inherited disease processes. SCD, originating from a known single nucleotide point mutation, offers a precisely targeted opportunity for the use of PGT-M with highly sensitive and specific results.

Generating Single Nucleotide Point Mutations in E. coli with the No-SCAR System.

Genetic manipulation of microbial genomes is highly relevant for studying biological systems and the development of biotechnologies. In E. coli, λ-Red recombineering is one of the most widely used gene-editing methods, enabling site-specific insertions, deletions, and point mutations of any genomic locus. The no-SCAR system combines λ-Red recombineering with CRISPR/Cas9 for programmable selection of recombinant cells. Recombineering results in the transient production of heteroduplex DNA, as only one strand of DNA is initially altered, leaving the mismatched bases susceptible to repair by the host methyl-directed mismatch repair (MMR) system and reduces the efficiency of generating single nucleotide point mutations. Here we describe a method, where expression of cas9 and the MMR-inhibiting mutLE32K variant are independently controlled by anhydrotetracycline- and cumate-inducible promoters from the pCas9CyMutL plasmid. Thus, MMR is selectively inhibited until recombinant cells have undergone replication and the desired mutation is permanently incorporated. By transiently inhibiting MMR, the accumulation of off-target mutations typically associated with MMR-deficient cell types is minimized. Methods for designing the editing template and sgRNA, cloning of the sgRNA, induction of λ-Red and MutLE32K, the transformation of editing oligo, and induction of Cas9 for mutant selection are detailed within.

Druggable sites/pockets of thep53-DNAJA1 protein-protein interaction: In silico modeling and in vitro/in vivo validation.

Mutation of p53 is the most common genetic alteration in human cancer. The vast majority of p53 mutations found in cancer are missense mutations, with some single nucleotide point mutations leading to the accumulation of mutant p53 protein with potential gain of oncogenic function. The mechanism for stabilization and accumulation of missense mutant p53 protein in malignant cells is not fully understood. It is thought that DNAJA1 plays a crucial role as a co-chaperone protein by stabilizing mutant p53 and amplifying oncogenic potential. As such, identifying small molecule inhibitors to disrupt the protein-protein interaction between mutant p53 and DNAJA1 may lead to an effective treatment for preventing carcinogenesis. Studying protein-protein interactions and identifying potential druggable hotspots has historically been limited-protein-protein binding sites require more complex characterization than those of single proteins and the crystal structures of many proteins have not been identified. Due to these issues, identifying salient druggable targets in protein-protein interactions through bench research may take years to complete. However, in silico modeling approaches allow for rapid characterization of protein-protein interfaces and the druggable binding sites they contain. In this chapter, we first review the oncogenic potential of mutant p53 and the crucial role of DNAJA1 in stabilizing missense mutant p53. We then detail our methodology for using in silico modeling and molecular biology to identify druggable protein-protein interaction sites/pockets between mutant p53 and DNAJA1. Finally, we discuss screening for and validating the utility of a small molecule inhibitor identified through our in silico framework. Specifically, we describe GY1-22, a unique compound with activity against mutant p53 that demonstrates therapeutic potential to inhibit cancer cell growth both in vivo and in vitro.

NGS-based mtDNA Profiling Could Reveal Genetic Alterations in Schizophrenia.

Schizophrenia is a complex disease with a putative genetic background. It was hypothesized that impaired mitochondrial function due to genetic alterations in mitochondrial DNA (mtDNA) could contribute to neurological conditions, including mental disorders. The aim of the study was to find out possible pathogenic mutations and/or variants in mtDNA potentially related to schizophrenia development. The study involved 37 patients with paranoid schizophrenia, whose mtDNA profiles were compared to those of 23 healthy controls. Patients and controls were assessed using PANSS (Positive and Negative Syndrome Scale) and General Health Questionnaire (GHQ), respectively. The entire mtDNA was sequenced by the NGS platform (MiSeq®, Illumina). Bioinformatics data were processed by mtDNA Variant Processor and Analyser (Illumina), mtDNA-Server, and SPSS-17. A total of 480 mtDNA variants (single nucleotide replacements, point insertions, and deletions) were found. The polymorphic variant m.1811A>G (MT-RNR2) showed the highest frequency in schizophrenia (24.3%), as compared to the controls (4.3%) (p=0.07). Increased frequency was also found mainly in polymorphisms, belonging to complex 1 genes: MT-ND4 (11251G and 11467G), MT-ND3 (10398G), MT-ND1 (4216С), and MT-ND5 (12611G and 13708А), some of which were associated with mitochondrial dysfunction. Two individual mutations were identified in the patients: a pathogenic one - m.11778 A>G (LHON) and a newly identified, potentially pathogenic - m.4115 Т>C (NADH dehydrogenase 1). Particular mtDNA variants predominantly in complex I, probably serve as a risk genetic background in schizophrenia. The presence of pathogenic mutations in patients with psychotic manifestations expands the clinical scope of mitochondrial diseases and deserves further investigation.

Multi-omics approach to post-CABG renal impairment

Abstract Background Renal impairment is a common complication after CABG (coronary artery bypass graft) surgery associated with an adverse outcome. Purpose To further characterize the molecular framework of the disease through omics analyses. Methods In N=165 CABG patients we performed multi-omics-analyses in preoperatively collected blood and tissue samples as well as 991 creatinine measurements. We used multivariable mixed-model regression analyses to analyse post-operative creatinine increase and to find common genetic polymorphisms, transcripts, metabolites and/or proteins associated with changes in postoperative creatinine increase. Multiple testing was accounted for by setting a 5%-limit on the false discovery rate (FDR) using the Benjamini-Hochberg procedure. Results Post-operative increase of log transformed creatinine was 0.035 (8%); 95% confidence interval (CI) 0.025, 0.045; P<0.001. We identified 55 gene expressions and two proteins associated with post-CABG renal impairment. On the metabolomic and single nucleotide point mutation (SNP) level, no relevant targets were found. The three most important identified gene expressions were MIR3202.1 (beta of log transformed creatinine increase per standard deviation gene expression increase −0.034; 95% CI: −0.048, 0.020; P<0.001), LOC105374386 (−0.032; 95% CI: −0.046, 0.019; P<0.001) and maternal embryonic leucine zipper kinase (MELK) (−0.022; 95% CI: −0.032, 0.013; P<0.001). Expression of all three was associated with a lower risk of post-CABG renal impairment. The same applies to the identified protein CAPRIN2 (−0.042; 95% CI: −0.062, 0.022; P<0.001), while expression of the protein TUBB6 was associated with a higher risk (0.033; 95% CI: 0.017, 0.048; P<0.001). Conclusions In an integrated approach we identified omics-biomarkers for the prediction of renal impairment after CABG surgery. The underlying pathophysiological associations of these genes and proteins are not fully understood. MELK might be an interesting target for further investigations, as it plays a prominent role in cell cycle control, cell proliferation, apoptosis, cell migration and cell renewal. Our results may help to better identify individuals at risk and lay the methodological groundwork for further omics analyses. Funding Acknowledgement Type of funding source: None

Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.

Abstract 2705: Visualization of KRAS point mutations in non-small cell lung cancer tumors with morphological context using the BaseScope in situ hybridization assay

Abstract About 25% of non-small cell lung cancer (NSCLC) patients bear one or more KRAS mutations in their tumors, which is correlated with poor prognosis. The precise identification of somatic mutations in tumors is becoming increasingly important for studying tumor progression and developing targeted therapies. While sequencing technologies allow for mutation-profiling, they do not permit direct visualization and association of genetic alterations with cellular morphology. In addition, DNA mutational status does not predict expression of the mutant allele which may provide information connecting genotype to phenotype. Therefore, a technology for mutation detection at the transcript level directly in the tumor context is desirable. To address this need we developed a specialized RNA in situ hybridization (ISH) method known as BaseScope. The BaseScope assay has a unique signal amplification system that allows for highly sensitive and specific detection of single nucleotide point mutations in tissues. BaseScope probes specific for KRAS G12C, G12A, G12V, G12S and wild type KRAS were designed and expression of each point mutation was assessed in a NSCLC tumor microarray with 48 tumor cores with known KRAS mutation status as determined by DNA sequencing. RNA quality and background signal threshold for each tumor core were determined using PPIB (positive) and dapB (negative) control probes. Using the sequencing data as the gold standard, the BaseScope assay demonstrated 83-100% sensitivity and 97-100% specificity for various KRAS mutations [Table 1]. For KRAS G12C, the assay correctly identified all 6 sequencing-positive cores and identified the rest as negatives. For KRAS G12V, the assay detected 5 of 6 mutated cores with 100% specificity. Interestingly, for KRAS G12S and KRAS G12A mutations, the BaseScope assay demonstrated 100% sensitivity and 97% specificity. Furthermore, it was observed that 100% of the KRAS-mutated tumors showed expression for both wild type and mutant KRAS alleles within these NSCLC tumors. In summary, we demonstrate the development of an RNA ISH assay for point mutations detection with morphological context in FFPE tissues. Unlike current sequencing methods that lack spatial information this assay has the unique ability to identify very small subclones whose frequency within the tumor might fall below the detection limit of sequencing. Performance characteristics of BaseScope KRAS assaysKRAS POINT MUTATIONSNo. of cores with specified mutationsNo. of cores without specified mutationsBaseScope sensitivityBaseScope specificityG12C633100% (6/6)100% (33/33)G12A336100% (3/3)97.2% (35/36)G12V63383% (5/6)100% (33/33)G12S138100% (1/1)97.3% (37/38) Citation Format: Anushka Dikshit, Helen Jarnagin, Emerald Doolittle, Courtney Anderson, Bingqing Zhang, Xiao-Jun Ma. Visualization of KRAS point mutations in non-small cell lung cancer tumors with morphological context using the BaseScope in situ hybridization assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2705.

1216P - Ixovex, a novel oncolytic E1B-mutated adenovirus

BackgroundThere is a great demand for improved oncolytic viruses that selectively replicate inside cancer cells while sparing normal tissues. Here we describe a novel oncolytic adenovirus, Ixovex, that obtains a cancer selective replication phenotype by modulating the level of expression of the different alternatively spliced E1B mRNA isoforms. MethodsIn our work to probe the function of the E1B proteins we generated a virus mutant, the Ixovex virus, in which the SA1 3’ splice acceptor site sequence CAG:GA was mutated to CgG:GA. This single nucleotide point mutation (genomic location 3216) changed aa 399 in the E1B-496R protein from an arginine (AGG) to a glycine (gGG). IxoVex was validated for its viral replication (TCID-50 assay) and cytotoxicity (MTS assay) in a panel of normal and cancer cell lines. Animal studies for anti-tumour efficacy studies were performed in nude mice. ResultsIxovex is a recombinant adenovirus that carries a single point mutation in the E1B-93R 3’ splice acceptor site that results in an overexpression of the E1B-156R splice isoform. Further, the mutation resulted in a loss of E1B-496R (E1B-55K) protein expression and as a consequence a failure of Ixovex to efficiently degrade the p53 tumour suppressor protein. Ixovex significantly inhibited tumour growth and prolonged survival of mice in an immune-deficient lung carcinoma tumour model. In complementation experiments overexpression of E1B-156R was shown to increase the oncolytic index of both Ad5wt and ONYX-015. In contrast to prior art viruses of similar type, Ixovex includes a functional E3B region for better in vivo efficacy. Ixovex virus, throughout this study has been proven to be by far the superior virus in potency compared to ONYX-015. ConclusionsThe decrease in toxicity and the inhibition of replication in normal cells indicate an astounding safety profile of Ixovex. The observation that the ONYX-015 virus replicated better in normal cells compared to Ixovex is intriguing considering that the ONYX-015 virus carries a large deletion that removes the coding sequences and splice signals needed to express the E1B-496R, E1B-93R and E1B-156R proteins. Ixovex significantly inhibit tumour growth in a human lung carcinoma animal model and that ONYX-015 does not. Legal entity responsible for the studyThe authors. FundingIxoGen. DisclosureM. Anwar: Shareholder / Stockholder / Stock options: IxoGen. G. Alusi: Shareholder / Stockholder / Stock options: IxoGen. D. Oberg: Shareholder / Stockholder / Stock options: IxoGen.

Equid alphaherpesvirus 1 from Italian Horses: Evaluation of the Variability of the ORF30, ORF33, ORF34 and ORF68 Genes.

Equid alphaherpesvirus 1 (EHV-1) is an important pathogen of horses. It is spread worldwide and causes significant economic losses. The ORF33 gene has a conserved region that is often used as target in diagnostic PCR protocols. Single nucleotide point (SNP) mutations in ORF30 are usually used to distinguish between neuropathogenic and non-neuropathogenic genotypes. An ORF68 SNP-based scheme has been used for grouping different isolates. Recently, the highest number of variable sites in EHV-1 from the UK has been found in ORF34. In this study, EHV-1 positive samples from Italian horses with a history of abortion were investigated by amplifying and sequencing the ORF30, ORF33, ORF34 and ORF68 genes. Most animals were infected by the neuropathogenic type A2254G. A 118 bp deletion was found at nucleotide positions 701–818 of the ORF68 gene, making impossible to assign the samples to a known group. Sequencing of the ORF34 gene with a newly designed nested PCR showed new SNPs. Analysis of these sequences and of those obtained from genetic databases allowed the identification of at least 12 groups. These data add depth to the knowledge of EHV-1 genotypes circulating in Italy.

The Quality of Genetic Code Models in Terms of Their Robustness Against Point Mutations.

In this paper, we investigate the quality of selected models of theoretical genetic codes in terms of their robustness against point mutations. To deal with this problem, we used a graph representation including all possible single nucleotide point mutations occurring in codons, which are building blocks of every protein-coding sequence. Following graph theory, the quality of a given code model is measured using the set conductance property which has a useful biological interpretation. Taking this approach, we found the most robust genetic code structures for a given number of coding blocks. In addition, we tested several properties of genetic code models generated by the binary dichotomic algorithms (BDA) and compared them with randomly generated genetic code models. The results indicate that BDA-generated models possess better properties in terms of the conductance measure than the majority of randomly generated genetic code models and, even more, that BDA-models can achieve the best possible conductance values. Therefore, BDA-generated models are very robust towards changes in encoded information generated by single nucleotide substitutions.

Who, what, where and how much: tDCS and training effects on working memory

Working memory (WM) is strictly capacity limited and subject to age-related decline. Because WM is a fundamental executive function essential for most cognitive tasks many people are interested in expanding and/or stabilizing WM function. Whereas WM training alone provides mixed results, incorporating transcranial direct current stimulation (tDCS) offers promise across young and older adults. In several within-subjects designs we found that adults benefit from anodal tDCS applied to left or right PFC compared to sham tDCS. However, in single-session per montage studies only subsets of participants benefit. Notably, better educated older adults and higher WM capacity young adults benefit from left or right frontal tDCS, whereas lower WM capacity older adults benefit from lateralized right frontoparietal tDCS. Importantly from an applied perspective, longitudinal designs reveal consistent benefits across participants. Our data indicate that tDCS appears to prolong the return to baseline performance after WM training ends. We see near and far transfer in these multi-session designs suggesting effects have lasting effects on the neural networks underlying WM. There are optimization challenges associated with the large parameter space associated with tDCS. For instance, our data show that tDCS intensity can elicit non-linear benefits to WM indicating that more intensity is not always better. An added layer of complexity is that tDCS intensity interacts with a common single nucleotide point mutation in the catechol-o-methlytransferase (COMT) gene that metabolizes prefrontal dopamine and modulates WM capacity. To complicate matters, these interactions depend on WM task demands: visual or spatial. To optimize longitudinal tDCS for broad usage we must understand by what mechanism individual differences and genetics contribute to the lasting behavioral effects. In summary, once the contributions of individual differences (who), task (what), stimulation site (where) and intensity (how much) are identified, the translational value to a particular individual might be predictable.

Molecular characterisation of equid alphaherpesvirus 1 strains isolated from aborted fetuses in Poland

BackgroundEquid alphaherpesvirus 1 (EHV-1) is one of the main infectious causative agents of abortion in mares and can also be associated with stillbirth, neonatal foal death, rhinopneumonitis in young horses and a neurological disorder called equine herpesvirus myeloencephalopathy (EHM). The neuropathogenicity of the virus was shown to be significantly higher in EHV-1 strains that carry a single nucleotide point (SNP) mutation in the ORF30, which encodes a catalytic subunit of viral DNA polymerase (ORF30 D752). Another gene, ORF68 is frequently used for phylogenetic analysis of EHV-1.Methods27 EHV-1 strains isolated from aborted equine fetuses in Poland, collected between 1993 and 2017, were subjected to PCR targeting the open reading frames (ORFs) 30 and 68 of the EHV-1 genome. PCR products obtained were sequenced and SNPs were analyzed and compared to sequences available in GenBank.ResultsNone of the analyzed sequences belonged to the ORF30 D752neuropathogenic genotype: all EHV-1 belonged to the non-neuropathogenic variant N752. On the basis of ORF68 sequences, the majority of EHV-1 sequences (76.9%) cannot be assigned to any of the known groups; only six sequences (23.1%) clustered within groups II and IV.ConclusionsEHV-1 strains obtained from abortion cases belong to the non-neuropathogenic genotype. Many EHV-1 ORF68 sequences have similar SNPs to those already described in Poland, but a clear geographical distribution was not observed. A single particular ORF68 sequence type was observed in strains isolated from 2001 onwards.

A novel detection procedure for mutations in the 23S rRNA gene of Mycoplasma pneumoniae with peptide nucleic acid-mediated loop-mediated isothermal amplification assay

Rapid and easy detection of a single nucleotide point mutation of bacterial genes, which is directly linked to drug susceptibility, is essential for the proper use of antimicrobial agents. Here, we established a detection method using a peptide nucleic acid mediated loop-mediated amplification (LAMP) assay for macrolide (ML)-susceptible Mycoplasma pneumoniae. This assay specifically detected the absence of missense mutations encoding the central loop of domain V in the gene encoding 23S rRNA, which can reduce the affinity for MLs and subsequently generate ML-resistant strains of M. pneumoniae.Reactions were performed at 62°C for 60min and targeted gene amplifications were detected by real-time turbidity with a turbidimeter and naked-eye inspection of a color change. The assay had an equivalent detection limit of 100.0fg of DNA with the turbidimeter and showed specificity against 54 types of pathogens, whereas amplification was completely blocked, even at 100.0pg of DNA per reaction, in the presence of point mutations at 2063A and 2064A. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures.This method would be a simple and rapid protocol for single nucleotide polymorphism genotyping as point-of-care testing technology without amplification of the sequences carrying the point mutations 2063A and 2064A in ML-resistant M. pneumoniae strains.

Mutation in the Sodium Channel Gene Corresponds With Phenotypic Resistance of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) to Pyrethroids.

The brown dog tick, Rhipicephalus sanguineus sensu lato (Latreille), is a cosmopolitan ectoparasite and vector of pathogens that kill humans and animals. Pyrethroids represent a class of synthetic acaricides that have been used intensely to try to control the brown dog tick and mitigate the risk of tick-borne disease transmission. However, acaricide resistance is an emerging problem in the management of the brown dog tick. Understanding the mechanism of resistance to acaricides, including pyrethroids, is important to adapt brown dog tick control strategies. The main objective of this study was to determine if target-site mutations associated with pyrethroid resistance in other pests could be associated with phenotypic resistance detected in a brown dog tick population from Florida. We amplified segment 6 of the domain III of the voltage-sensitive sodium channel protein, using cDNAs synthesized from pyrethroid-susceptible and pyrethroid-resistant tick strains. A single nucleotide point mutation (SNP) identified in a highly conserved region of domain III S6 in the resistant ticks resulted in an amino acid change from phenylalanine to leucine. This mutation is characteristic of resistance phenotypes in other tick species, and is the first report of this mutation in R. sanguineus. Molecular assays based on this knowledge could be developed to diagnose the risk for pyrethroid resistance, and to inform decisions on integrated brown dog tick management practices.

A Mismatch EndoNuclease Array-Based Methodology (MENA) for Identifying Known SNPs or Novel Point Mutations.

Accurate and rapid identification or confirmation of single nucleotide polymorphisms (SNPs), point mutations and other human genomic variation facilitates understanding the genetic basis of disease. We have developed a new methodology (called MENA (Mismatch EndoNuclease Array)) pairing DNA mismatch endonuclease enzymology with tiling microarray hybridization in order to genotype both known point mutations (such as SNPs) as well as identify previously undiscovered point mutations and small indels. We show that our assay can rapidly genotype known SNPs in a human genomic DNA sample with 99% accuracy, in addition to identifying novel point mutations and small indels with a false discovery rate as low as 10%. Our technology provides a platform for a variety of applications, including: (1) genotyping known SNPs as well as confirming newly discovered SNPs from whole genome sequencing analyses; (2) identifying novel point mutations and indels in any genomic region from any organism for which genome sequence information is available; and (3) screening panels of genes associated with particular diseases and disorders in patient samples to identify causative mutations. As a proof of principle for using MENA to discover novel mutations, we report identification of a novel allele of the beethoven (btv) gene in Drosophila, which encodes a ciliary cytoplasmic dynein motor protein important for auditory mechanosensation.

A valveless rotary microfluidic device for multiplex point mutation identification based on ligation-rolling circle amplification

Genetic variations such as single nucleotide polymorphism (SNP) and point mutations are important biomarkers to monitor disease prognosis and diagnosis. In this study, we developed a novel rotary microfluidic device which can perform multiplex SNP typing on the mutation sites of TP53 genes. The microdevice consists of three glass layers: a channel wafer, a Ti/Pt electrode-patterned resistance temperature detector (RTD) wafer, and a rotary plate in which twelve reaction chambers were fabricated. A series of sample injection, ligation-rolling circle amplification (L-RCA) reaction, and fluorescence detection of the resultant amplicons could be executed by rotating the top rotary plate, identifying five mutation points related with cancer prognosis. The use of the rotary plate eliminates the necessity of microvalves and micropumps to control the microfluidic flow in the channel, simplifying the chip design and chip operation for multiplex SNP detection. The proposed microdevice provides an advanced genetic analysis platform in terms of multiplexity, simplicity, and portability in the fields of biomedical diagnostics.

Identification of two novel HLA-B null alleles using next generation sequencing

Aim Typing of two patients (one of Asian and one of Hispanic descent) using SSOP and SBT resulted in the detection of two new HLA-B null alleles. Detailed analysis of the probe and sequence data showed the new null alleles to be generated by single nucleotide point mutations. To confirm these results, the DNA samples were tested by next generation sequencing (NGS). Methods DNA was extracted from whole blood using the Promega Maxwell 16 Blood DNA Purification kit and Promega Maxwell 16 IVD instrument. Low resolution HLA typing was obtained by performing DNA amplification and probe hybridization using the One Lambda LABType SSO HLA-B Locus kit. High resolution HLA typing was achieved by DNA amplification and subsequent sequencing of the amplified product using the Conexio Genomics SBT Resolver kits. Confirmation testing of the novel HLA-B null alleles was performed by amplifying the DNA samples using the Scisco Genetics NGS HLA kit. Results Results from SSOP, SBT and NGS testing confirmed the presence of two new null alleles. The first null allele from the Hispanic individual is similar to B∗35:01:01:01 with a mutation at position 295 (codon 75) in exon 2. This position appears to be a common polymorphic site for HLA Class I null alleles: A∗02:366N, A∗03:178N, A∗24:60N, B∗08:72N, B∗15:94N, B∗15:294N, B∗44:58N, B∗51:41N, C∗01:69N, C∗03:277N, C∗07:152N, C∗12:84N and C∗17:27N. The nucleotide change (GAG → TAG) results in the creation of a stop codon. The second null allele from the Asian individual is similar to B∗15:01:01:01 with a mutation at position 862 (codon 264) in exon 4. The nucleotide change (GGA → TGA) results in the creation of a stop codon. There are no documented polymorphisms at this position for any other allele. However, A∗02:43N has an insertion at codon 236 that causes a frameshift and premature stop at codon 264. Conclusions Identification of null alleles is important in determining what donors should be evaluated and selected for hematopoietic stem cell transplants. Misidentification could have an impact on graft outcome and lead to complications such as graft-versus-host disease. NGS has shown to be a valuable method for the identification of these kinds of new variants. Furthermore, the variant in exon 4 may suggest that typing outside of the HLA Class I peptide binding domains (exon 2 and 3) should be performed.

Genetic diversity of SCN5A gene and its possible association with the concealed form of Brugada syndrome development in Polish group of patients.

Brugada Syndrome (BS) is an inherited channelopathy associated with a high incidence of sudden cardiac death. The paper presents the discovery of new genetic variants of SCN5A gene which might be associated with the development of a concealed form of Brugada Syndrome. The study involved a group of 59 patients (37 men) with suspected concealed form of Brugada Syndrome. Pharmacological provocation with intravenous ajmaline administration was performed. Six patients with positive test results were subjected to molecular analysis of SCN5A gene with MSSCP method. Additionally, MSSCP genotyping was performed for samples obtained from the family members with Brugada Syndrome, despite the fact that they had negative ajmaline challenge test results. Genetic examinations of the SCN5A gene at 6 positive patients showed 6 known polymorphisms, 8 new single nucleotide point (SNP) variants located at exons, and 12 new single nucleotide point variants located at introns. Among new SNPs localized in SCN5A gene exons three SNPs affected the protein sequence.

Complete genomic landscape of a recurring sporadic parathyroid carcinoma

Parathyroid carcinoma is a rare endocrine malignancy with an estimated incidence of less than 1 per million population. Excessive secretion of parathyroid hormone, extremely high serum calcium level, and the deleterious effects of hypercalcaemia are the clinical manifestations of the disease. Up to 60% of patients develop multiple disease recurrences and although long-term survival is possible with palliative surgery, permanent remission is rarely achieved. Molecular drivers of sporadic parathyroid carcinoma have remained largely unknown. Previous studies, mostly based on familial cases of the disease, suggested potential roles for the tumour suppressor MEN1 and proto-oncogene RET in benign parathyroid tumourigenesis, while the tumour suppressor HRPT2 and proto-oncogene CCND1 may also act as drivers in parathyroid cancer. Here, we report the complete genomic analysis of a sporadic and recurring parathyroid carcinoma. Mutational landscapes of the primary and recurrent tumour specimens were analysed using high-throughput sequencing technologies. Such molecular profiling allowed for identification of somatic mutations never previously identified in this malignancy. These included single nucleotide point mutations in well-characterized cancer genes such as mTOR, MLL2, CDKN2C, and PIK3CA. Comparison of acquired mutations in patient-matched primary and recurrent tumours revealed loss of PIK3CA activating mutation during the evolution of the tumour from the primary to the recurrence. Structural variations leading to gene fusions and regions of copy loss and gain were identified at a single-base resolution. Loss of the short arm of chromosome 1, along with somatic missense and truncating mutations in CDKN2C and THRAP3, respectively, provides new evidence for the potential role of these genes as tumour suppressors in parathyroid cancer. The key somatic mutations identified in this study can serve as novel diagnostic markers as well as therapeutic targets.