Genotyping Procedure Research Articles

Genotyping single point mutations in rd1 and rd8 mice using melting curve analysis of qPCR fragments

PCR is tolerant to single nucleotide mismatches. Therefore, genotyping of point mutations by PCR requires special conditions for the amplification of allele-specific PCR fragments. MS-PCR (mutagenically separated PCR) is an improved version of ARMS (amplification refractory mutation system) in which additional nucleotide mismatches near the mutation site are used to separate the wt fragments from the mutant fragments in a single-tube PCR. In the originally described procedure, the resulting fragments are resolved on agarose gels according to differences in size introduced by different lengths of the allele-specific primers. In order to evaluate the PCR fragments by melting curve analysis, we enlarged the difference in the melting temperatures of the fragments of the two alleles by increasing the GC content of the longer allele-specific primer resulting in a higher melting temperature of the corresponding fragment. Using the murine retinal degeneration mutations rd1 and rd8 as an example, we show that such primers result in an easy to handle genotyping procedure: qPCR followed by melting curve analysis. In summary, MS-PCR is a simple and easy-to-use method for detecting single nucleotide variants.

Partial protein binding of uracil and thymine affects accurate dihydropyrimidine dehydrogenase (DPD) phenotyping

Fluorouracil is among the most used antimetabolite drugs for the chemotherapeutic treatment of various types of gastrointestinal malignancies. Dihydropyrimidine dehydrogenase (DPYD) genotyping prior to fluorouracil treatment is considered standard practice in most European countries. Yet, current pre-therapeutic DPYD genotyping procedures do not identify all dihydropyrimidine dehydrogenase (DPD)-deficient patients. Alternatively, DPD activity can be estimated by determining the DPD phenotype by quantification of plasma concentrations of the endogenous uracil and thymine concentrations and their respective metabolites dihydrouracil (DHU) and dihydrothymine (DHT). Liquid chromatography - mass spectrometry (LC-MS) detection is currently considered as the most adequate method for quantification of low-molecular weight molecules, although the sample preparation method is highly critical for analytical outcome. It was hypothesized that during protein precipitation, the recovery of the molecule of interest highly depends on the choice of precipitation agent and the extent of protein binding in plasma. In this work, the effect of protein precipitation using acetonitrile (ACN) compared to strong acid perchloric acid (PCA) on the recovery of uracil, thymine, DHU and DHT is demonstrated. Upon the analysis of plasma samples, PCA precipitation showed higher concentrations of uracil and thymine as compared to ACN precipitation. Using ultrafiltration, it was shown that uracil and thymine are significantly (60–65 %) bound to proteins compared to DHU and DHT. This shows that before harmonized cut-off levels of DPD phenotyping can be applied in clinical practice, the analytical methodology requires extensive further optimization.

Molecular epidemiology of hepatitis B, hepatitis C, and HIV-1 co-infections in Ethiopia: Implications for disease burden and intervention strategies

BackgroundHepatitis B virus (HBV) exhibits high prevalence rates within Ethiopia. The genetic diversity of HBV, marked by mixed genotype infections, may hold significant implications for the trajectory of disease and responses to treatment. Ethiopia grapples with a substantial public health challenge posed by co-infections involving HBV, hepatitis C virus (HCV), and human immunodeficiency virus 1 (HIV-1), particularly among vulnerable populations. MethodsA comprehensive investigation into HBV, HCV, and HIV-1 co-infection was conducted. A total of 7,789 blood samples were meticulously analyzed, among which 815 exhibited HBV positivity. Among the HBV-positive samples, 630 were subjected to genotyping procedures, resulting in the identification of a prevalent trend of mixed infections characterized by HBV genotypes A/E/F (67.30%). Serological assessments were performed on 492 specimens to ascertain the presence of HCV and HIV-1 co-infections, revealing respective co-infection rates of 13.02% for HBV/HIV, 3.31% for HBV/HCV, and 2.07% for triple infection. ResultsThe investigation revealed the intricate prevalence of co-infections in Ethiopia, notably underlining the continued transmission of viruses. The prominent occurrence of mixed HBV genotypes A/E/F suggests dynamic viral interactions and ongoing transmission pathways. These findings accentuate the necessity for targeted interventions and enhanced patient care, as co-infections carry significant clinical complexities. ConclusionsThis study furnishes crucial insights into the molecular epidemiology of HBV, HCV, and HIV-1 co-infections in Ethiopia. The acquired knowledge can contribute to the advancement of strategies for clinical management and the formulation of public health interventions aimed at ameliorating the burden of viral infections within the nation.

Naturally sterile Mus spretus hybrids are suitable for the generation of pseudopregnant embryo transfer recipients

For the preparation of embryo transfer recipients, surgically vasectomized mice are commonly used, generated by procedures associated with pain and discomfort. Sterile transgenic strains provide a nonsurgical replacement, but their maintenance requires breeding and genotyping procedures. We have previously reported the use of naturally sterile STUSB6F1 hybrids for the production of embryo transfer recipients and found the behavior of these recipients to be indistinguishable from those generated by vasectomized males. The method provides two substantial 3R impacts: refinement (when compared with surgical vasectomy) and reduction in breeding procedures (compared with sterile transgenic lines). Despite initial promise, the 3Rs impact of this innovation was limited by difficulties in breeding the parental STUS/Fore strain, which precluded the wider distribution of the sterile hybrid. The value of a 3R initiative is only as good as the uptake in the community. Here we, thus, select a different naturally sterile hybrid, generated from strains that are widely available: the B6SPRTF1 hybrid between C57BL/6J and Mus spretus. We first confirmed its sterility by sperm counting and testes weight and then trialed the recovery of cryopreserved embryos and germplasm within three UK facilities. Distribution of sperm for the generation of these hybrids by in vitro fertilization was found to be the most robust distribution method and avoided the need to maintain a live M. spretus colony. We then tested the suitability of B6SPRTF1 sterile hybrids for the generation of embryo transfer recipients at these same three UK facilities and found the hybrids to be suitable when compared with surgical vasectomized mice and a sterile transgenic strain. In conclusion, the potential 3Rs impact of this method was confirmed by the ease of distribution and the utility of sterile B6SPRTF1 hybrids at independent production facilities.

Genetic Diversity of Legionella pneumophila Isolates from Artificial Water Sources in Brazil

Legionella pneumophila (Lp) is a Gram-negative bacterium found in natural and artificial aquatic environments and inhalation of contaminated aerosols can cause severe pneumonia known as Legionnaires’ Disease (LD). In Brazil there is hardly any information about this pathogen, so we studied the genetic variation of forty Legionella spp. isolates obtained from hotels, malls, laboratories, retail centers, and companies after culturing in BCYE medium. These isolates were collected from various sources in nine Brazilian states. Molecular identification of the samples was carried out using Sequence-Based Typing (SBT), which consists of sequencing and analysis of seven genes (flaA, pilE, asd, mip, mompS, proA, and neuA) to define a Sequence Type (ST). Eleven STs were identified among 34/40 isolates, of which eight have been previously described (ST1, ST80, ST152, ST242, ST664, ST1185, ST1464, ST1642) and three were new STs (ST2960, ST2962, and ST2963), the former identified in five different cooling towers in the city of São Paulo. The ST1 that is widely distributed in many countries was also the most prevalent in this study. In addition, other STs that we observed have also been associated with legionellosis in other countries, reinforcing the potential of these isolates to cause LD in Brazil. Unfortunately, no human isolates could be characterized until presently, but our observations strongly suggest the need of surveillance implementation system and control measures of Legionella spp. in Brazil, including the use of more sensitive genotyping procedures besides ST.

Comparing Genotyping Accuracy Using Buccal Swabs versus Tail Biopsies by PCR in B6;C3-Tg(Prnp-SNCA*A53T)83Vle and B6;C3-Tg(Prnp-SNCA*A53T)83Vle Sncatm1Mjff Mice.

Genotyping is a common and necessary procedure performed on genetically modified animals to distinguish carriers from noncarriers of the variants of interest. Established methods involve collection of tissues such as tips of tails or notches of ears. Noninvasive methods have been described but not widely adopted for reasons including inertia to change, needs to adjust PCR protocols, and the lack of validation; noninvasive genotyping methods are a refinement on animal welfare, but questions remain regarding how they compare with invasive methods in terms of genotyping accuracy rate and reproducibility. To gain answers to these questions, we compared the detection accuracy of the transgene and determination of zygosity in B6;C3-Tg(Prnp-SNCA*A53T)83Vle and B6;C3-Tg(Prnp-SNCA*A53T)83Vle Snca tm1Mjff neonatal mice between tail biopsies and buccal swabs. Moreover, we weighed and observed mice following genotyping to see if any clinical differences can be discerned. Weight data did not support statistically significant differences in mice undergoing different genotyping procedures and control. No statistically significant difference was found between using buccal swabs or tail biopsies for genotyping with PCR or quantitative PCR. None of the pups swabbed was rejected by the dam. Our findings indicate that buccal swabbing is a more humane and feasible alternative to tail biopsies for high-throughput genotyping.

A Single-Step Genome-Wide Association Study for Semen Traits of Egyptian Buffalo Bulls.

The present study aimed to contribute to the limited research on buffalo (Bubalus bubalis) semen traits by incorporating genomic data. A total of 8465 ejaculates were collected. The genotyping procedure was conducted using the Axiom® Buffalo Genotyping 90 K array designed by the Affymetrix Expert Design Program. After conducting a quality assessment, we utilized 67,282 SNPs genotyped in 192 animals. We identified several genomic loci explaining high genetic variance by employing single-step genomic evaluation. The aforementioned regions were located on buffalo chromosomes no. 3, 4, 6, 7, 14, 16, 20, 22, and the X-chromosome. The X-chromosome exhibited substantial influence, accounting for 4.18, 4.59, 5.16, 5.19, and 4.31% of the genomic variance for ejaculate volume, mass motility, livability, abnormality, and concentration, respectively. In the examined genomic regions, we identified five novel candidate genes linked to male fertility and spermatogenesis, four in the X-chromosome and one in chromosome no. 16. Additional extensive research with larger sample sizes and datasets is imperative to validate these findings and evaluate their applicability for genomic selection.

Two-step method for rapid isolation of genomic DNA and validation of R81T insecticide resistance mutation in Myzus persicae

Isolation and amplification of nucleic acid (DNA) is considered a vital and potent instrument in molecular biological research. However, its functioning outside of a laboratory setting is difficult because of complex procedures that demand expert personnel and expensive equipment in addition to the fulfillment of several additional requirements. DNA isolation from minute insects is sometimes difficult, making diagnostic and genotyping procedures problematic. Thus, the current work offers a high-throughput, cost-effective, straightforward, and faster approach for isolating DNA from the aphid Myzus persicae. Intriguingly, two-step DNA extraction process yielded a high yield of extremely pure genomic DNA and required only 10 s to complete. PCR investigation aiming at amplifying the non-synonymous R81T region on the loop D site of the nAChR gene of M. persicae was subsequently utilized to successfully validate the recovered DNA. Moreover, the proposed method was compared in terms of yield and purity with conventionally used DNA isolation methods including, phenol:chloroform, salt out, and commercially available kits. In conclusion, this newly developed method would enable researchers to quickly process many biological samples used to analyze genetic diversity, mutant screening, and large spectrum diagnosis both in laboratory and field conditions.

Expert consensus on the standardization and clinical application of genotyping for human leukocyte antigens

The genes encoding human leukocyte antigens (HLA) are located in the region of 6p21.31 of the human chromosome, accounting for 1/3 000 of the human genome. The area is seen as a structurally complex, with the characteristics of multi-gene inheritance, high polymorphism, haplotype inheritance, linkage disequilibrium, racial diversity, and regional distribution. In recent years, with the continuous innovation of HLA genotyping technology, new issues and challenges have emerged in terms of the standardization of the technology platform, the implementation of the performance validation and verification plan for clinical testing projects, quality control of the entire testing process, analysis and interpretation of results, consultation and selection of donors, and clinical application. This consensus, in combination with the current status of HLA genotyping technology in clinical practice in China, systematically sorts out the key contents in the standardization and clinical application of the HLA genotyping technology platform. The first part proposes the basic requirements for the standardization of the HLA typing technology platform from the perspectives of personnel, instrumentation, reagents and consumables, experimental methods, environmental settings, and introduces the methods and performance indicators for confirming the performance of HLA genotyping with next-generation sequencing (NGS) technology. The second part elaborates on the key points of quality control during the entire process of HLA genotyping procedure, quality control of result reporting and releasing, and management of result interpretation and bioinformatics systems. The last part describes the clinical consultation and application promotion of HLA genotyping. After widely soliciting revisions and discussions from domestic experts in the field of HLA and transplantation, the consensus aims to promote the development and clinical application of the HLA gene in related medical field.

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

Rapid genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) using MinION nanopore sequencing.

The global distribution and constant evolution are challenges for the control of porcine reproductive and respiratory syndrome virus (PRRSV), one of the most important viruses affecting swine worldwide. Effective control of PRRSV benefits from genotyping, which currently relies on Sanger sequencing. Here we developed and optimized procedures for real-time genotyping and whole genome sequencing of PRRSV directly from clinical samples based on targeted amplicon- and long amplicon tiling sequencing using the MinION Oxford Nanopore platform. Procedures were developed and tested on 154 clinical samples (including lung, serum, oral fluid and processing fluid) with RT-PCR Ct values ranging from 15 to 35. The targeted amplicon sequencing (TAS) approach was developed to obtain sequences of the complete ORF5 (main target gene for PRRSV genotyping) and partial ORF4 and ORF6 sequences of both PRRSV-1 and PRRSV-2 species. After only 5 min of sequencing, PRRSV consensus sequences with identities to reference sequences above 99% were obtained, enabling rapid identification and genotyping of clinical PRRSV samples into lineages 1, 5 and 8. The long amplicon tiling sequencing (LATS) approach targets type 2 PRRSV, the most prevalent viral species in the U.S. and China. Complete PRRSV genomes were obtained within the first hour of sequencing for samples with Ct values below 24.9. Ninety-two whole genome sequences were obtained using the LATS procedure. Fifty out of 60 sera (83.3%) and 18 out of 20 lung samples (90%) had at least 80% of genome covered at a minimum of 20X sequence depth per position. The procedures developed and optimized in this study here are valuable tools with potential for field application during PRRSV elimination programs.

Construction of an individual identification panel for horses using insertion and deletion markers.

Individual identification and paternity testing are important for avoiding inbreeding in the management of small populations of wild and domestic animals. In horse racing industries, they are extremely important for identifying and registering individuals and doping control to ensure fair competition. In this study, we constructed an individual identification panel for horses by using insertion and deletion (INDEL) markers. The panel included 39 INDEL markers selected from a whole-genome INDEL database. Genotyping of 89 Thoroughbreds showed polymorphisms with minor allele frequencies (MAFs) of 0.180-0.489 in all markers. The total probability of exclusion for paternity testing, power of discrimination, and probability of identity were 0.9994271269, >0.9999999999, and 0.9999999987, respectively. The panel was applied to 13 trios (sires, dams, and foals), and no contradictions were observed in genetic inheritance among the trios. When this panel was applied to the trios (52 trios) containing false fathers, an average of 7.3 markers excluded parentage relationships. In addition, genomic DNA extracted from the urine of six horses was partially genotyped for 39 markers, and 6-28 markers were successfully genotyped. The newly constructed panel has two advantages: a low marker mutation rate compared with short tandem repeats and a genotyping procedure that is as simple as short tandem repeat typing compared with single nucleotide variant typing. This panel can be applied for individual identification, paternity determination, and urine-sample identification in Thoroughbred horses.

Fetal Hemoglobin Altering Effects of KLF1, BCL11A rs11886868 and XmnI-HBG2 on Transfusion Dependent Beta Thalassemia Patients: Preeliminary Study

Aim: High fetal hemoglobin value is one of the quantitative trait in beta thalassemia and may effect transfusion dependency status of beta thalassemic cases. There are population-based differences about known genetic modifiers of different fetal hemoglobin values. We aimed to find if high fetal hemoglobin value are caused by XmnI-HBG2 polymorphism, rs11886868 of BCL11A or KLF1 whole gene mutations. Material and Methods: Genotyping procedure of thirty well re-defined and characterized transfusion dependent beta thalassemia patients was conducted via either sanger sequencing or and PCR-RFLP. Statistic analysis of groups and multiple logistic regression analysis of related genotypes were performed. Results: We found strong correlations between transfusion dependency and fetal hemoglobin levels (P<0.05). IVS.I.110 (G>A) homozygous mutation was found to be predominant in HBB gene. Lower fetal hemoglobin levels were seen in IVS.I.110 (G>A) homozygous group (P<0.05). Total count of variations among the three modifier genes BCL11A polymorphism was leading first. We did not observe any statistically significant relationship in patients with beta thalassemia major patients who have high fetal hemoglobin values between three modifiers group (P>0.05). Conclusion: This is the first research report from Turkey in terms of 3 different modifiers were analysed and evaluated. Since some cases have more than one variations in these three modifiers, involving higher sample size may overcome this challenge. Other genomic alterations rather than XmnI-HBG2, variations of BCL11A rs11886868 and mutation profile of KLF1 gene, which could decrease or abolish the effect of gamma globin repressors, may have more direct role with high fetal hemoglobin levels in patients with transfusion dependent beta thalassemia in Turkey.

Correlation Between Patients with Wounds and Isolated Pseudomonas aeruginosa and Staphylococcus aureus at Barau Dikko Teaching Hospital, Kaduna, Nigeria

This study was carried out to determine the correlation between patients with wounds and isolated Pseudomonas aeruginosa and Staphylococcus aureus in clinics at Barau Dikko Teaching Hospital, Kaduna, Nigeria. The Socio-demographic, medical and drug histories and characteristics of the wounds from each consented patient were taken using a questionnaire along with sixty samples of the patient’s wound swab samples. Isolation of Pseudomonas aeruginosa and Staphylococcus aureus from the wound swab samples was carried out using standard phenotypic and genotypic procedures. Out of the 60 samples collected, 30 (50.0%) each were from general out-patient and inpatients. The higher percentage 12 (20.0%) and 39 (65.0%) were patients in the age group between 61 and above and male patients respectively. Regarding the patient’s occupations, the higher percentage 20 (33.3%) of the patients were businessmen and women. The patient’s wound location indicated that a higher percentage of 38 (63.3%) wounds were located on the leg. Also, only 13 (21.7%) patients had diabetes and 44 (73.3%) of the wound patients were receiving antimicrobials; the commonest being metronidazole 11 (18.33%), followed by mupirocin/Supirocin 9 (15.6%). A total of eleven isolates each of Pseudomonas aeruginosa and Staphylococcus aureus were isolated from the sixty wound swab samples of the in-and out-patients. Association between antimicrobial agents use and the organisms showed significant difference (P < 0.05), while the association between sex, diabetes status, duration of the wound and the isolation of Pseudomonas aeruginosa and Staphylococcus aureus from the wound of patients showed no statistically significant difference (P > 0.05).

Genetic Characterization of Pathogenic Escherichia Coli Isolated from Smallholding Dairy Buffaloes, their Products, and Some Environmental Sources

This study was conducted to evaluate the potential existence of pathogenicEscherichia coli in smallholding dairy buffaloes, their products as well as in some environmental sources. In total, 150 samples including an equal sample size (n = 25) of raw milk, Kareish cheese, water, feed, udder and rectal swabs were examined. Phenotypic and genotypic procedures were used to characterize the bacterial population. Twenty-eight isolates were confirmed as E. coli from the examined samples (18.66%). The recovered isolates were categorized as Enterohemorrhagic E. coli (n=11),Enteropathogenic E. coli(n=9), Enterotoxigenic E. coli(n= 5)and Enteroinvasive E. coli (n=3). All isolates were resistant to erythromycin followed by oxacillin (89.28%), and nalidixic acid (67.85%), while 96% of the recovered strains displayed high sensitivity to imipenem. A great proportion of the recovered E. coli (67.9%) exhibited multidrug resistance. The majority of isolates (92.9%) harbored stx1either alone or in association with stx2 which present in 60.7% (17/28) while eaeA genes were detected in 50% among the recovered isolates in association with stx1 or combined with stx2 (n =11). Twelve isolates (42.86%) expressed all the examined beta lactamase encoding genes; while only 25% of the isolates harbored all tested beta lactamase genes in common with stx1, stx2 and eaeA genes.The results herein indicated that the pathogenicE. coli could be present in the examined samples which pose a relevant threat and negative impact on animal as well as consumer health. The study emphasizes the necessity of developing long-term strategies to assure dairy food safety.

Collection of genetic data at scale for a nationally representative population: the UK Millennium Cohort Study.

A DNA bank has been created from the Millennium Cohort Study (MCS) saliva samples. A total of 23,336 samples are available, from 9,259 cohort members (4,630 males and 4,629 females), 8,898 mothers and 5,179 fathers. There are 4,533 mother, child, father 'triads'. This paper describes the collection of the saliva samples from cohort members and their biological parents in the MCS. It analyses response rates and predictors of response, and details the DNA extraction, genotyping and imputation procedures performed on the data.

Low frequency of HPV positivity in breast tumors among patients from south-central Poland

BackgroundSome studies suggest that Human Papilloma Virus (HPV) infection is important factor in carcinogenesis of breast tumors. This study’ objective was to analyze HPV prevalence in breast cancers of patients from south-central Poland.Materials and methodsThe study was performed based on archival paraffin embebbed and formalin fixed blocks in the group of 383 patients with breast cancer. HPV prevalence and its genotype were assessed, respectively by: nested PCR (with two groups of primers: PGMY09/PGMY11 and GP5+/GP6+), quantitative PCR (qPCR). Tumors were classified as HPV positive in case of at least one positive result in nested PCR and positive results in genotyping procedure. For all HPV positive tissues P16 immunostaining was applied in order to confirm active viral infection.ResultsIn the group of 383 breast cancers, HPV positivity was found in 17 samples (4.4%) in nested PCR. All these samples were subjected to HPV genotyping. This analysis revealed presence of HPV type 16 into two tumors (0.5%). In these two cancers, P16 overexpression was reported.ConclusionIn breast tumors of patients from south-central Poland in Poland, HPV positivity is demonstrated in very low percentage of cases.

BDNF Val66Met polymorphism is associated with negative symptoms in early-onset schizophrenia spectrum and other psychotic disorders

Background and ObjectivesTo investigate the clinical characteristics of adolescents with early-onset full psychotic disorders either with Brain-derived neurotrophic factor (BDNF) Val66Met (rs6265) or DRD2/ANKK1 Taq1A (rs1800497) polymorphisms. Method101 cases with early-onset schizophrenia (EOS) or other psychotic spectrum disorders (SSD) and 150 healthy controls were included in the current study. Using the Positive and Negative Symptom Scale (PANSS), patient subgroups were compared for their psychotic symptoms, age-onset, duration of untreated illness, and family history of psychiatric disorders. The real-time polymerase chain reaction (RT-PCR) was implemented for genotyping procedures. ResultsStudy groups and patient subgroups were similar regarding their sociodemographic characteristics (16.4 ± 2.6 years, 62.2% male for all participants). Results did not reveal any difference between patients and healthy controls for DRD2/ANKK1 Taq1A and BDNF Val66Met genotypes. Patients with A1A2 (Gly/Lys) allele reported higher rates of substance use compared to A2A2 (Glu/Glu) counterparts. Other clinical variables were found similar. EOS/SSD group with Val66Met heterozygote allele revealed lower levels of negative symptoms than Val/Val homozygotes. Conversely, the age onset of full psychotic disorder, total positive symptom score, and total general psychopathology score of PANSS were found comparable between Val/Met and Val/Val groups. In the logistic regression model, Glu/Lys genotypes remained significant for the presence of substance use. ConclusionThe interaction between substance use and the DRD2 gene should be investigated for the development of early-onset psychotic disorders. BDNF Val66Met polymorphism also could be a disease-modifying factor for the presence of negative symptoms.

Single Nucleotide Polymorphism rs17173608 in the Chemerin Encoding Gene: Is It a Predictor of Insulin Resistance and Severity of Coronary Artery Disease in Non-Obese Type 2 Diabetes?

(1) Background: Chemerin, or the RARRES2 (Retinoic Acid Receptor Responder 2) gene, is found to be associated with an increased incidence of insulin resistance, endothelial dysfunction, type 2 diabetes (T2D), and coronary artery disease (CAD). This study investigates associations of RARRES2 rs17173608 with insulin resistance and the severity of CAD in non-obese T2D patients in relation to the clinical and genetic factors. (2) Methods: A total of 300 patients with T2D and CAD were recruited in this study. The associations of insulin resistance and the severity of CAD with RARRES2 rs17173608 and clinical factors were assessed. The genotyping procedures were performed using the TaqMan method. The significant associations (p ≤ 0.05) from preliminary tests were employed to carry out the secondary analysis. (3) Results: RARRES2 rs17173608 (TT, TG, and GG polymorphisms in the preliminary analysis; TG and GG polymorphisms in a secondary analysis) was associated with insulin resistance and the severity of CAD in both the preliminary and secondary analysis (all p-values were < 0.05). Additionally, in the secondary analysis, FPG and ACEI were also associated with insulin resistance and the severity of CAD (all p-values were < 0.05). (4) Conclusion: From the preliminary findings, rs17173608 is a significant predictor of insulin resistance and the severity of CAD.

High-throughput insertional mutagenesis reveals novel targets for enhancing lipid accumulation in Nannochloropsis oceanica

The microalga Nannochloropsis oceanica is considered a promising platform for the sustainable production of high-value lipids and biofuel feedstocks. However, current lipid yields of N. oceanica are too low for economic feasibility. Gaining fundamental insights into the lipid metabolism of N. oceanica could open up various possibilities for the optimization of this species through genetic engineering. Therefore, the aim of this study was to discover novel genes associated with an elevated neutral lipid content. We constructed an insertional mutagenesis library of N. oceanica, selected high lipid mutants by five rounds of fluorescence-activated cell sorting, and identified disrupted genes using a novel implementation of a rapid genotyping procedure. One particularly promising mutant (HLM23) was disrupted in a putative APETALA2-like transcription factor gene. HLM23 showed a 40%-increased neutral lipid content, increased photosynthetic performance, and no growth impairment. Furthermore, transcriptome analysis revealed an upregulation of genes related to plastidial fatty acid biosynthesis, glycolysis and the Calvin-Benson-Bassham cycle in HLM23. Insights gained in this work can be used in future genetic engineering strategies for increased lipid productivity of Nannochloropsis.