Buccal Swab Samples Research Articles

Single nucleotide polymorphism discrimination and genotyping based on cascade strand displacement reaction mediated label-free Cas12a system

Single nucleotide polymorphism (SNP) discrimination plays an important role in precision medicine by providing insights into an individual’s genetic information. The low abundance of the mutant target and the minimal impact caused by SNP on the whole nucleic acid sequence remain a challenge during discrimination. Herein, a cascade strand displacement reaction mediated label-free Cas12a sensing platform is established for SNP analysis. Split G-quadruplex (G4) motif is recruited as label-free signal output for Cas12a sensing system, and hence overcomes relying of fluorescence labeled substrates like conventional method. The established platform exhibits excellent single base difference discrimination ability attributing to the cascade strand displacement process, during which, single-base mismatch will affect the strand-exchange rate significantly. The limit of detection reaches 1 copy / test after integration with isothermal preamplification. The proposed method can discriminate as low as 0.1 % single base variation and perform robustly in biological matrixes. Human buccal swab samples are successfully genotyped with high accuracy.

From Mother to Child: Epigenetic Signatures of Hyperglycemia and Obesity during Pregnancy.

In utero exposure to maternal hyperglycemia and obesity can trigger detrimental effects in the newborn through epigenetic programming. We aimed to assess the DNA methylation levels in the promoters of MC4R and LPL genes from maternal blood, placenta, and buccal swab samples collected in children born to mothers with and without obesity and Gestational Diabetes Mellitus (GDM). A total of 101 Caucasian mother-infant pairs were included in this study. Sociodemographic characteristics, clinical parameters, physical activity, and adherence to the Mediterranean diet were evaluated in the third trimester of pregnancy. Clinical parameters of the newborns were recorded at birth. A negative relationship between MC4R DNA methylation on the fetal side of the GDM placenta and birth weight (r = -0.630, p = 0.011) of newborns was found. MC4R DNA methylation level was lower in newborns of GDM women (CpG1: 2.8% ± 3.0%, CpG2: 3.8% ± 3.3%) as compared to those of mothers without GDM (CpG1: 6.9% ± 6.2%, CpG2: 6.8% ± 5.6%; p < 0.001 and p = 0.0033, respectively), and it was negatively correlated with weight (r = -0.229; p = 0.035), head circumference (r = -0.236; p = 0.030), and length (r = -0.240; p = 0.027) at birth. LPL DNA methylation was higher on the fetal side of the placenta in obese patients as compared to normal-weight patients (66.0% ± 14.4% vs. 55.7% ± 15.2%, p = 0.037), and it was associated with maternal total cholesterol (r = 0.770, p = 0.015) and LDL-c (r = 0.783, p = 0.012). These results support the role of maternal MC4R and LPL methylation in fetal programming and in the future metabolic health of children.

Epigenetic Age Estimation by Detecting DNA Methylation Status in Buccal Swabs.

The analysis of DNA methylation (DNAm) levels at specific CpG sites represents one of the most promising molecular techniques for estimating an individual's age. To date, a considerable number of studies have reported the development of age prediction models on the basis of DNAm in body fluids, with only a few utilizing buccal swabs. The objective of this study was to identify age-dependent methylation CpG sites in three different genes (HOXC4, TRIM59, and ELOVL2) in buccal swab samples from the Chinese Han population. A total of 461 buccal swabs, with an age range of 0.4-80.8 years, were divided into a training set (n=325) and a validation set (n=136). Samples were analyzed by pyrosequencing in order to identify age-related genes with correlation coefficient. A random forest regression model was ultimately proposed, including eight CpGs in three genes, with a mean absolute error (MAE) of 2.119 years. The model performs independent validation set with an MAE of 4.391 years. Our findings illustrate that buccal swabs present a suitable alternative to biological traces for age prediction based on DNAm pattern using pyrosequencing and random forest regression, offering the additional advantage of being collected noninvasively.

Cancer-Related Cognitive Impairment Associated with APOE rs7412 and BDNF rs6265 in Breast Cancer Survivors

ObjectivesCancer-related cognitive impairment (CRCI) is a highly prevalent and debilitating symptom reported by breast cancer survivors (BCS). The etiology of CRCI remains unclear, leading to poor symptom management. Building from prior studies, BCS with the C/C genotype of apolipoprotein E (APOE) rs7412 and the T/T genotype of brain-derived neurotrophic factor (BDNF) rs6265 were hypothesized to experience more severe CRCI. Therefore, we investigated the relationships between the severity of CRCI and polymorphisms of APOE and BDNF among BCS. MethodsThis was a subanalysis of data from a larger descriptive, correlational, and cross-sectional study. Subjective and objective CRCI were measured using the Patient-Reported Outcomes Measurement Information System and CANTAB Cambridge Cognitive assessment, respectively. Buccal swab samples were collected to evaluate the single nucleotide polymorphisms. Multivariable generalized linear regression models were used to analyze data. ResultsAPOE rs7412 and BDNF rs6265 were significantly associated with lower self-reported cognitive abilities in a total of 353 BCS. Age was positively associated with self-reported cognitive scores, indicating that younger BCS perceived lower cognitive abilities. Individuals carrying genotype of C/T for APOE with the C/C or C/T for BDNF showed positive associations with cognitive abilities. ConclusionsYounger BCS with the C/C genotype for APOE rs7412 and the T/T genotype for BDNF rs6265 may be at risk for CRCI. Knowledge regarding predictive markers for CRCI symptoms is essential for precision symptom management. Further investigation with a longitudinal and translational design is necessary to explore the etiologies for CRCI. Implications for Nursing PracticeIntegrating genetic phenotyping into routine clinical practice will provide nurses with unique opportunities to understand individual susceptibilities, and how symptoms may trigger other symptoms. Further, findings from these innovative investigations will provide symptom interventionists and implementation scientists with critical data to optimize individualized strategies for symptom prevention, detection, and management.

Buccal Swab Samples from Japanese Brown Cattle Fed with Limonite Reveal Altered Rumen Microbiome.

The areas of the Mount Aso grasslands in Kumamoto, Japan, are the primary location for the breeding of the Kumamoto strain of Japanese Brown cattle (JBRK). Although Aso limonite, deposited by volcanic ash and magma, has been commonly fed to pregnant JBRK in this area, the mechanisms of its salutary effects on pregnant JBRK have not yet been elucidated. Approximately 100 days before the expected day of calf delivery, seven JBRK (four supplemented with limonite and three controls without limonite) were assigned to this study, from which a buccal swab was collected at the highest rumination every 30 days for 90 days. DNA extracted from these swabs was then analyzed using a 16S rRNA gene amplicon sequence analysis. Statistically significant differences between the two groups were discovered through beta-diversity analysis, though results from alpha-diversity analysis were inconclusive. The microbiota identified were classified into six clusters, and three of the main clusters were core-rumen bacteria, primarily cellulose digestion in cluster 1, oral bacteria in cluster 2, and non-core-rumen bacteria in cluster 3. In the limonite group, core-rumen bacteria decreased while non-core-rumen bacteria increased, suggesting that limonite feeding alters rumen microbiota, particularly activation of non-core-rumen microbiota.

Multidrug resistance 1 (MDR1) gene mutation in dogs

The multidrug resistance (MDR1) gene mutation in the domestic dog (Canis lupus familiaris) is an inherited condition most frequently observed in herding breeds. Dogs with the mutated gene are at risk of neurological toxicosis and life-threatening reactions if certain drugs are administered. Determining the MDR1 genotype of a dog via blood or buccal swab sampling will assist owners and veterinary professionals in understanding their risk of multidrug sensitivity. Some of the drugs known to be dangerous when administered to a dog with the mutation are used in everyday practice. To ensure patient safety, the student and registered veterinary nurse should have an understanding of this condition, particularly when dealing with the dog breeds at higher risk. This article aims to provide student and registered veterinary nurses with a comprehensive and logical analysis of the MDR1 gene mutation in dogs.

Association of CHAT Gene Polymorphism rs3793790 and rs2177370 with Donepezil Response and the Risk of Alzheimer's Disease Continuum.

Genetic variation plays an important role in drug response, there are few relevant studies on patients with Alzheimer's disease continuum (ADC). This study focused on the associations between two single nucleotide polymorphisms (SNPs) (rs3793790 and rs2177370) located in the CHAT gene and donepezil response in ADC patients, and further evaluated the associations between the two SNPs and ADC. According to 2018 National Institute on Aging and Alzheimer's Association (NIA-AA) standard, amyloid β-protein positive (Aβ+) and negative (Aβ-) patients were recruited according to the Aβ-PET/CT standard. rs3793790 and rs2177370 were genotyped in buccal swab samples by using the MassARRAY system. We used the Mini Mental State Examination (MMSE) in Chinese version, caregiver evaluation, and prescribing behavior to assess therapeutic response during the 9-month period. Using logistic regression models, we analyzed the relationship between the two SNPs and donepezil response in 58 Aβ+ patients treated with donepezil alone at the initial diagnosis of ADC. We also explored a probable link between the two SNPs and ADC in 147 Aβ+ and 73 Aβ- patients using a logistic regression analysis. The chance of donepezil response was higher in patients with the G allele of rs3793790 and/or the A allele of rs2177370 than in those without (odds ratio (OR) 6.83, 95% confidence interval (CI): 1.64-28.49). Additionally, the rs3793790 variant was not associated with ADC, whereas the A allele in rs2177370 increased 1.51-fold the ADC risk (OR 2.51, 95% CI: 1.28-4.95). The genetic variants of rs3793790 and rs2177370 were associated with the donepezil response, and rs2177370 may have a moderate relationship with the risk of ADC.

The Effects of Smoking on Telomere Length, Induction of Oncogenic Stress, and Chronic Inflammatory Responses Leading to Aging.

Telomeres, potential biomarkers of aging, are known to shorten with continued cigarette smoke exposure. In order to further investigate this process and its impact on cellular stress and inflammation, we used an in vitro model with cigarette smoke extract (CSE) and observed the downregulation of telomere stabilizing TRF2 and POT1 genes after CSE treatment. hTERT is a subunit of telomerase and a well-known oncogenic marker, which is overexpressed in over 85% of cancers and may contribute to lung cancer development in smokers. We also observed an increase in hTERT and ISG15 expression levels after CSE treatment, as well as increased protein levels revealed by immunohistochemical staining in smokers' lung tissue samples compared to non-smokers. The effects of ISG15 overexpression were further studied by quantifying IFN-γ, an inflammatory protein induced by ISG15, which showed greater upregulation in smokers compared to non-smokers. Similar changes in gene expression patterns for TRF2, POT1, hTERT, and ISG15 were observed in blood and buccal swab samples from smokers compared to non-smokers. The results from this study provide insight into the mechanisms behind smoking causing telomere shortening and how this may contribute to the induction of inflammation and/or tumorigenesis, which may lead to comorbidities in smokers.

Comparing skin swabs, buccal swabs, and toe clips for amphibian genetic sampling, a case study with a small anuran (Acris blanchardi).

Multiple methods for collecting genetic samples from amphibians exist, each with their own implications for study design, animal welfare, and costs. Toe clipping is one common method, but there is ongoing debate regarding its potential detriment. Less invasive methods should be implemented, if efficacious, as amphibians are a particularly vulnerable vertebrate group. Skin and buccal swabbing are less invasive methods for genetic sampling, but the potential for contamination and a lower yield of DNA may exist. To compare these methods, we gathered skin swabs, buccal swabs, and toe clips from the same individuals of a relatively small anuran species, Blanchard's Cricket Frog (Acris blanchardi). We then compared DNA yield, DNA purity, amplification success rate, and genotypic data quality among sample types. We found toe clips and buccal swabs generated similar DNA yield and purity, with skin swabs yielding significantly less DNA of significantly lower purity than the other sample types. Amplification success rate was significantly higher using toe clips compared to the other sample types, though buccal swab samples amplified more readily than skin swabs. Genotypic data from toe clips and buccal swabs did not differ significantly in quality, but skin swab data quality was significantly lowest among sample types. Thus, skin swabbing could produce erroneous data in some situations, but buccal swabbing is likely an effective substitute to toe clipping, even for small species. Our results can help future researchers select which genetic sampling method might best suit their research needs.

Oral microbiome dysbiosis among cigarette smokers and smokeless tobacco users compared to non-users

Tobacco use significantly influences the oral microbiome. However, less is known about how different tobacco products specifically impact the oral microbiome over time. To address this knowledge gap, we characterized the oral microbiome of cigarette users, smokeless tobacco users, and non-users over 4 months (four time points). Buccal swab and saliva samples (n = 611) were collected from 85 participants. DNA was extracted from all samples and sequencing was carried out on an Illumina MiSeq, targeting the V3–V4 region of the 16S rRNA gene. Cigarette and smokeless tobacco users had more diverse oral bacterial communities, including a higher relative abundance of Firmicutes and a lower relative abundance of Proteobacteria, when compared to non-users. Non-users had a higher relative abundance of Actinomyces, Granulicatella, Haemophilus, Neisseria, Oribacterium, Prevotella, Pseudomonas, Rothia, and Veillonella in buccal swab samples, compared to tobacco users. While the most abundant bacterial genera were relatively constant over time, some species demonstrated significant shifts in relative abundance between the first and last time points. In addition, some opportunistic pathogens were detected among tobacco users including Neisseria subflava, Bulleidia moorei and Porphyromonas endodontalis. Overall, our results provide a more holistic understanding of the structure of oral bacterial communities in tobacco users compared to non-users.

Dual toeholds regulated CRISPR-Cas12a sensing platform for ApoE single nucleotide polymorphisms genotyping

Single nucleotide polymorphisms (SNPs) are closely associated with many biological processes, including genetic disease, tumorigenesis, and drug metabolism. Accurate and efficient SNP determination has been proved pivotal in pharmacogenomics and diagnostics. Herein, a universal and high-fidelity genotyping platform is established based on the dual toeholds regulated Cas12a sensing methodology. Different from the conventional single stranded or double stranded activation mode, the dual toeholds regulated mode overcomes protospacer adjacent motif (PAM) limitation via cascade toehold mediated strand displacement reaction, which is highly universal and ultra-specific. To enhance the sensitivity for biological samples analysis, a modified isothermal recombinant polymerase amplification (RPA) strategy is developed via utilizing deoxythymidine substituted primer and uracil-DNA glycosylase (UDG) treatment, designated as RPA-UDG. The dsDNA products containing single stranded toehold domain generated in the RPA-UDG allow further incorporation with dual toeholds regulated Cas12a platform for high-fidelity human sample genotyping. We discriminate all the single-nucleotide polymorphisms of ApoE gene at rs429358 and rs7412 loci with human buccal swab samples with 100% accuracy. Furthermore, we engineer visual readout of genotyping results by exploiting commercial lateral flow strips, which opens new possibilities for field deployable implementation.

A developmental validation of the Quick TargSeq 1.0 integrated system for automated DNA genotyping in forensic science for reference samples.

Analysis of short tandem repeats (STRs) is a global standard method for human identification. Insertion/Deletion polymorphisms (DIPs) can be used for biogeographical ancestry inference. Current DNA typing involves a trained forensic worker operating several specialized instruments in a controlled laboratory environment, which takes 6-8h. We developed the Quick TargSeq 1.0 integrated system (hereinafter abbreviated to Quick TargSeq) for automated generation of STR and DIP profiles from buccal swab samples and blood stains. The system fully integrates the processes of DNA extraction, polymerase chain reaction (PCR) amplification, and electrophoresis separation using microfluidic biochip technology. Internal validation studies were performed using RTyper 21 or DIP 38 chip cartridges with single-source reference samples according to the Scientific Working Group for DNA Analysis Methods guidelines. These results indicated that the Quick TargSeq system can process reference samples and generate STR or DIP profiles in approximately 2h, and the profiles were concordant with those determined using traditional STR or DIP analysis methods. Thus, reproducible and concordant DNA profiles were obtained from reference samples. Throughout the study, no lane-to-lane or run-to-run contamination was observed. The Quick TargSeq system produced full profiles from buccal swabs with at least eight swipes, dried blood spot cards with two 2-mm disks, or 10ng of purified DNA. Potential PCR inhibitors (i.e., coffee, smoking tobacco, and chewing tobacco) did not appear to affect the amplification reactions of the instrument. The overall success rate and concordance rate of 153 samples were 94.12% and 93.44%, respectively, which is comparable to other commercially available rapid DNA instruments. A blind test initiated by a DNA expert group showed that the system can correctly produce DNA profiles with 97.29% genotype concordance with standard bench-processing methods, and the profiles can be uploaded into the national DNA database. These results demonstrated that the Quick TargSeq system can rapidly generate reliable DNA profiles in an automated manner and has the potential for use in the field and forensic laboratories.

Advancing tuberculosis diagnosis and management in cynomolgus macaques using Xpert MTB/RIF ultra assay

The detection and management of Mycobacterium tuberculosis complex (MTBC) infection, the causative agent of tuberculosis (TB), in macaques, including cynomolgus macaques (Macaca fascicularis), are of significant concern in research and regions where macaques coexist with humans or other animals. This study explored the utility of the Xpert MTB/RIF Ultra assay, a widely adopted molecular diagnostic tool to diagnose tuberculosis (TB) in humans, to detect DNA from the Mycobacterium tuberculosis complex in clinical samples obtained from cynomolgus macaques. This investigation involved a comprehensive comparative analysis, integrating established conventional diagnostic methodologies, assessing oropharyngeal-tracheal wash (PW) and buccal swab (BS) specimen types, and follow-up assessments at 3-month, 6-month, and 12-month intervals. Our results demonstrated that the Xpert MTB/RIF Ultra assay was able to detect MTBC in 12 of 316 clinical samples obtained from cynomolgus macaques, presenting a potential advantage over bacterial culture and chest radiographs. The Xpert MTB/RIF Ultra assay exhibited exceptional sensitivity (100%) at the animal level, successfully detecting all macaques positive for M. tuberculosis as confirmed by traditional culture methods. The use of PW samples revealed that 5 positive samples from 99 (5.1%) were recommended for testing, compared to 0 samples from 99 buccal swab (BS) samples (0.0%). In particular, the definitive diagnosis of TB was confirmed in three deceased macaques by MTB culture, which detected the presence of the bacterium in tissue autopsy. Our findings demonstrate that the implementation of the Xpert MTB/RIF Ultra assay, along with prompt isolation measures, effectively reduced active TB cases among cynomolgus macaques over a 12-month period. These findings highlight the advance of the Xpert MTB/RIF Ultra assay in TB diagnosis and its crucial role in preventing potential outbreaks in cynomolgus macaques. With its rapidity, high sensitivity, and specificity, the Xpert MTB/RIF Ultra assay can be highly suitable for use in reference laboratories to confirm TB disease and effectively interrupt TB transmission.

Estimation of Human Chronological Age from Buccal Swab Samples through a DNA Methylation Analysis Approach of a Five-Locus Multiple Regression Model.

Recent advancements in forensic genetics have facilitated the extraction of additional characteristics from unidentified samples. This study delves into the predictive potential of a five-gene (ELOVL2, FHL2, KLF14, C1orf132, and TRIM59) methylation rate analysis for human age estimation using buccal swabs collected from 60 Italian volunteers. The methylation levels of specific CpG sites in the five genes were analyzed through bisulfite conversion, single-base extension, and capillary electrophoresis. A multivariate linear regression model was crafted on the training set, then the test set was employed to validate the predictive model. The multivariate predictive model revealed a mean absolute deviation of 3.49 years in the test set of our sample. While limitations include a modest sample size, the study provides valuable insights into the potential of buccal swab-based age prediction, aiding in criminal investigations where accurate age determination is crucial. Our results also highlight that it is necessary to investigate the effectiveness of predictive models specific to biological tissues and individual populations, since models already proven effective for other populations or different tissues did not show the same effectiveness in our study.

Mitochondrial point heteroplasmy: insights from deep-sequencing of human replicate samples

BackgroundHuman mitochondrial heteroplasmy is an extensively investigated phenomenon in the context of medical diagnostics, forensic identification and molecular evolution. However, technical limitations of high-throughput sequencing hinder reliable determination of point heteroplasmies (PHPs) with minor allele frequencies (MAFs) within the noise threshold.ResultsTo investigate the PHP landscape at an MAF threshold down to 0.1%, we sequenced whole mitochondrial genomes at approximately 7.700x coverage, in multiple technical and biological replicates of longitudinal blood and buccal swab samples from 11 human donors (159 libraries in total). The results obtained by two independent sequencing platforms and bioinformatics pipelines indicate distinctive PHP patterns below and above the 1% MAF cut-off. We found a high inter-individual prevalence of low-level PHPs (MAF < 1%) at polymorphic positions of the mitochondrial DNA control region (CR), their tissue preference, and a tissue-specific minor allele linkage. We also established the position-dependent potential of minor allele expansion in PHPs, and short-term PHP instability in a mitotically active tissue. We demonstrate that the increase in sensitivity of PHP detection to minor allele frequencies below 1% within a robust experimental and analytical pipeline, provides new information with potential applicative value.ConclusionsOur findings reliably show different mutational loads between tissues at sub-1% allele frequencies, which may serve as an informative medical biomarker of time-dependent, tissue-specific mutational burden, or help discriminate forensically relevant tissues in a single person, close maternal relatives or unrelated individuals of similar phylogenetic background.

The Importance of Confirming False Homozygosity in Pretransplant HLA Typing Results of Patients with Hematologic Malignancies.

Loss of heterozygosity (LOH) on chromosome 6p, where the HLA genes are located, can result in incorrect homozygosity findings during HLA genotyping in patients with hematologic malignancies. The degree of HLA compatibility between donor and recipient is crucial in hematopoietic stem cell transplantation. Therefore, we present a case of false homozygosity in HLA genotyping due to LOH on chromosome 6p in a patient diagnosed with acute myeloid leukemia (AML). HLA molecular typing was conducted on both peripheral blood and buccal swab samples. The analysis included sequence-based typing (SBT) and next-generation sequencing-based typing. Additionally, chromosomal microarray analysis (CMA) was performed. A 68-year-old male presented with anemia and thrombocytopenia. Subsequent bone marrow examination confirmed AML. High-resolution HLA genotyping of Peripheral blood during blast crisis revealed homozygosity at the -A, -B, and -C loci. Conventional karyotyping showed a normal karyotype, 46,XY[20]. Retesting of HLA genotyping one week later confirmed the homozygous results. Subsequently, HLA typing was repeated using buccal swab specimens, confirming heterozygosity at all 4 HLA loci. CMA on peripheral blood samples during blast crisis revealed a large terminal region of copy-neutral LOH spanning approximately 43.5 Mb in the chromosome region 6p25.3p21.1. LOH at the HLA gene locus can significantly impact donor selection, potentially leading to the selection of mistakenly identified homozygous donors. Clinicians and laboratory personnel should be aware of these issues to prevent erroneous HLA typing results in patients with hematologic malignancies. It is advisable to confirm the HLA typing of recipients with hematologic malignancies whenever homozygosity is detected at any locus. This can be achieved through careful interpretation of low peaks in SBT, and by using buccal swab samples or peripheral blood collected after achieving remission.

CRISPR/Cas13a-based single-nucleotide polymorphism detection for reliable determination of ABO blood group genotypes.

The ABO blood group plays an important role in blood transfusion, linkage analysis, individual identification, etc. Serologic methods of blood typing are gold standards for the time being, which require stable typing antisera and fresh blood samples and are labor intensive. At present, reliable determination of ABO blood group genotypes based on single-nucleotide polymorphisms (SNPs) among A, B, and O alleles remains necessary. Thus, in this work, CRISPR/Cas13a-mediated genotyping for the ABO blood group by detecting SNPs between different alleles was proposed. The ABO*O.01.01(c.261delG) allele (G for the A/B allele and del for the O allele) and ABO*B.01(c.796C > A) allele (C for the A/O allele and A for the B allele) were selected to determine the six genotypes (AA, AO, BB, BO, OO, and AB) of the ABO blood group. Multiplex PCR was adapted to simultaneously amplify the two loci. CRISPR/Cas13a was then used to specifically differentiate ABO*O.01.01(c.261delG) and ABO*B.01(c.796C > A) of A, B, and O alleles. Highly accurate determination of different genotypes was achieved with a limit of detection of 50 pg per reaction within 60 min. The reliability of this method was further validated based on its applicability in detecting buccal swab samples with six genotypes. The results were compared with those of serological and sequencing methods, with 100% accuracy. Thus, the CRISPR/Cas13a-mediated assay shows great application potential in the reliable identification of ABO blood group genotypes in a wide range of samples, eliminating the need to collect fresh blood samples in the traditional method.

Split G-quadruplex based PfAgo sensing platform for nucleotide mutation discrimination and human genotyping.

A PfAgo-G4 sensing platform exploiting G4 as a signal reporter was proposed, validated, and optimized. By introducing two mismatches at the Link strand, a universal nucleotide design rule was established for accurate single nucleotide polymorphism discrimination with PfAgo-G4. The FUT2 gene was then successfully and accurately genotyped using human buccal swab samples.

A GENOME-WIDE ASSOCIATION STUDY IN A ROMANIAN COLORECTAL CANCER COHORT IDENTIFIES GENETIC MARKERS ASSOCIATED WITH SUSCEPTIBILITY TO SARS-COV-2 INFECTIONS

Colorectal cancer ranks second in the incidence of cancer deaths worldwide. Our objectives are to identify genetic markers associated with colorectal cancer susceptibility using a genome-wide association study and to evaluate the identified genetic markers in the context of associated SARS-CoV-2 infections in the Romanian population. The study population consisted of 576 unrelated histopathologically confirmed colorectal cancer (CRC) cases and 1,069 controls consisting of patients admitted for medical conditions, excluding cancer. The DNA was extracted from buccal swab samples and analyzed by deCODE Genetics (Reykjavik, Iceland). We used the NHGRI catalog database to identify 85 unique variants from a systematic literature review for variants associated with CRC. Two of the tested variants in the Romanian GWAS reached genomewide significance (p-value lower than 5 × 10-8), but one of them did not match the other criteria. The top three markers were located on chr3, and the following two were located on chr2 and chr12, respectively. The Romanian GWAS results for CRC were compared to established markers linked to the measurement of COVID-19 symptoms. This allowed us to identify loci that may be useful in the future for assessing prognosis, treatment, and outcomes. We confirmed that established colorectal cancer SNPs are linked to colorectal cancer risk in the Romanian population while also examining the potential genetic connections between colorectal cancer predisposition and SARS-CoV-2 infection and symptoms.

Scalable noninvasive amplicon-based precision sequencing (SNAPseq) for genetic diagnosis and screening of β-thalassemia and sickle cell disease using a next-generation sequencing platform.

β-hemoglobinopathies such as β-thalassemia (BT) and Sickle cell disease (SCD) are inherited monogenic blood disorders with significant global burden. Hence, early and affordable diagnosis can alleviate morbidity and reduce mortality given the lack of effective cure. Currently, Sanger sequencing is considered to be the gold standard genetic test for BT and SCD, but it has a very low throughput requiring multiple amplicons and more sequencing reactions to cover the entire HBB gene. To address this, we have demonstrated an extraction-free single amplicon-based approach for screening the entire β-globin gene with clinical samples using Scalable noninvasive amplicon-based precision sequencing (SNAPseq) assay catalyzing with next-generation sequencing (NGS). We optimized the assay using noninvasive buccal swab samples and simple finger prick blood for direct amplification with crude lysates. SNAPseq demonstrates high sensitivity and specificity, having a 100% agreement with Sanger sequencing. Furthermore, to facilitate seamless reporting, we have created a much simpler automated pipeline with comprehensive resources for pathogenic mutations in BT and SCD through data integration after systematic classification of variants according to ACMG and AMP guidelines. To the best of our knowledge, this is the first report of the NGS-based high throughput SNAPseq approach for the detection of both BT and SCD in a single assay with high sensitivity in an automated pipeline.