Allele-specific Polymerase Chain Reaction Assay Research Articles

Fine mapping and candidate gene analysis of CRA8.1.6, which confers clubroot resistance in turnip (Brassica rapa ssp. rapa).

Clubroot disease poses a significant threat to Brassica crops, necessitating ongoing updates on resistance gene sources. In F2 segregants of the clubroot-resistant inbred line BrT18-6-4-3 and susceptible DH line Y510, the genetic analysis identified a single dominant gene responsible for clubroot resistance. Through bulk segregant sequencing analysis and kompetitive allele-specific polymerase chain reaction assays, CRA8.1.6 was mapped within 110 kb (12,255-12,365 Mb) between markers L-CR11 and L-CR12 on chromosome A08. We identified B raA08g015220.3.5C as the candidate gene of CRA8.1.6. Upon comparison with the sequence of disease-resistant material BrT18-6-4-3, we found 249 single-nucleotide polymorphisms, seven insertions, six deletions, and a long terminal repeat (LTR) retrotransposon (5,310 bp) at 909 bp of the first intron. However, the LTR retrotransposon was absent in the coding sequence of the susceptible DH line Y510. Given the presence of a non-functional LTR insertion in other materials, it showed that the LTR insertion might not be associated with susceptibility. Sequence alignment analysis revealed that the fourth exon of the susceptible line harbored two deletions and an insertion, resulting in a frameshift mutation at 8,551 bp, leading to translation termination at the leucine-rich repeat domain's C-terminal in susceptible material. Sequence alignment of the CDS revealed a 99.4% similarity to Crr1a, which indicate that CRA8.1.6 is likely an allele of the Crr1a gene. Two functional markers, CRA08-InDel and CRA08-KASP1, have been developed for marker-assisted selection in CR turnip cultivars. Our findings could facilitate the development of clubroot-resistance turnip cultivars through marker-assisted selection.

PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay: A highly sensitive method for detection of MYD88 L265P mutation.

Agarose gel-based conventional and real-time allele-specific polymerase chain reaction (AS-PCR) assays are currently used for sensitive detection and quantification of MYD88 L265P mutation. Visual inspection of an agarose gel can often be ambiguous. We propose a new allele-specific quantification PCR (AS-qPCR) assay, PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay, that uses Intercalating Nucleic Acid (INA®) technology for increased affinity and specificity. This study compares PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay with conventional AS-PCR. We included a total of 102 peripheral and bone marrow blood samples from 94 patients with a lymphoproliferative disorder. Droplet digital PCR (ddPCR) was used as a third method in case of discrepancy. A positive percent agreement of 100% (95% CI 0.92-1.0) and a negative percent agreement of 98% (95% CI 0.90-1.0) were found between the conventional AS-PCR and the AS-qPCR methods. Including the ddPCR results to validate the discrepant cases, the sensitivity and specificity of PlentiPlex™ MYD88 Waldenström lymphoma qPCR Assay were 1.0 (95% CI 0.97-1.0) and 1.0 (95% CI 0.96-1.0), respectively. Our data demonstrate that PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay is a fast, highly sensitive, and specific method for the detection of MYD88 L265P compared with conventional AS-PCR.

Developing DPYD Genotyping Method for Personalized Fluoropyrimidines Therapy.

Fluoropyrimidine drugs are widely used in chemotherapy to treat solid tumors. However, severe toxicity has been reported in 10% to 40% of patients. The DPYD gene encodes the rate-limiting enzyme dihydropyrimidine dehydrogenase responsible for fluoropyrimidine catabolism. The DPYD variants resulting in decreased or no enzyme activity are associated with increased risk of fluoropyrimidine toxicity. This study aims to develop a pharmacogenetic test for screening DPYD variants to guide fluoropyrimidine therapy. A multiplex allele-specific polymerase chain reaction (AS-PCR) assay, followed by capillary electrophoresis, was developed to detect 5 common DPYD variants (c.557A > G, c.1129-5923C > G, c.1679T > G, c.1905 + 1G > A, and c.2846A > T). Deidentified population samples were used for screening positive controls and optimizing assay conditions. Proficiency testing samples with known genotypes were analyzed for test validation. All variants detected were confirmed by Sanger sequencing. From the deidentified population samples, 5 samples were heterozygous for c.557A > G, 2 samples were heterozygous for c.1129-5923C > G (HapB3), and 1 sample was heterozygous for c.2846A > T. The 20 proficiency samples matched with their assigned genotypes, including 13 wild-type samples, 3 samples heterozygous for c.1679T > G, 2 samples heterozygous for c.1905 + 1G > A, and 2 samples heterozygous for c.2846A > T. One of the 3 patient samples was heterozygous for c.1129-5923C > G (HapB3). All the variants detected by the multiplex AS-PCR assay were concordant with Sanger sequencing results. A robust multiplex AS-PCR assay was developed to rapidly detect 5 variants in the DPYD gene. It can be used for screening DPYD variants to identify patients with increased risk of toxicity when prescribed fluoropyrimidine therapy.

Correlation of Rheumatoid Arthritis with Interleukin-38 and its Polymorphism (rs6743376)

Among autoimmune disorders, rheumatoid arthritis is one of the most prevalent. The IL-1 family and the IL36 subfamily both include interleukin-38. IL-38 is anti-inflammatory cytokine. IL-38 is essential for inflammation control and host defense. In the Pakistani population, this research intends to evaluate the relationship flanked by IL-38 levels and its polymorphism (rs 6743376) C>A and rheumatoid arthritis. Eighty samples in total were included and divided into sick and control groups. IL-38 levels were measured through ELISA. For the SNP of IL-38, an allele-specific polymerase chain reaction assay was applied. All subjects' serum levels of CRP, RF, and ACCP were assessed. 70% of patients had negative CRP tests, while 30% had positive CRP tests. All of the patients' RF and ACCP tests came back positive. As the p-value was smaller than (0.001), a significant difference was observed in the mean ESR test values between patients and controls. IL-38 concentration was higher in the RA having a mean value of 26.8621. In comparison to homozygous allele (CC) and heterozygous allele (AC), patients with homozygous allele (AA) are more common. Contrarily, the control group revealed that the homozygous allele (CC) was more prevalent than the homozygous allele (AA) and heterozygous allele (AC). When compared to homozygous allele (CC) and heterozygous allele (AC), IL-38 rs6743376 (A/A) is related with a higher risk of developing RA illness. In contrast to heterozygous (A/A) allele, homozygous (C/C) allele and heterozygous (A/C) allele are protective in RA. According to this study, IL-38 homozygous alleles (CC) and heterozygous alleles (AC) are associated with a lower probability of developing RA than heterozygous alleles (AA).

Polymorphisms and expression levels of TNP2, SYCP3, and AZFa genes in patients with azoospermia.

Azoospermia (the total absence of sperm in the ejaculate) affects approximately 10% of infertile males. Despite diagnostic advances, azoospermia remains the most challenging issue associated with infertility treatment. Our study evaluated transition nuclear protein 2 (TNP2) and synaptonemal complex protein 3 (SYCP3) polymorphisms, azoospermia factor a (AZFa) microdeletion, and gene expression levels in 100 patients with azoospermia. We investigated a TNP2 single-nucleotide polymorphism through polymerase chain reaction (PCR) restriction fragment length polymorphism analysis using a particular endonuclease. An allele-specific PCR assay for SYCP3 was performed utilizing two forward primers and a common reverse primer in two PCR reactions. Based on the European Academy of Andrology guidelines, AZFa microdeletions were evaluated by multiplex PCR. TNP2, SYCP3, and the AZFa region main gene (DEAD-box helicase 3 and Y-linked [DDX3Y]) expression levels were assessed via quantitative PCR, and receiver operating characteristic curve analysis was used to determine the diagnostic capability of these genes. The TNP2 genotyping and allelic frequency in infertile males did not differ significantly from fertile volunteers. In participants with azoospermia, the allelic frequency of the SYCP3 mutant allele (C allele) was significantly altered. Deletion of sY84 and sY86 was discovered in patients with azoospermia and oligozoospermia. Moreover, SYCP3 and DDX3Y showed decreased expression levels in the azoospermia group, and they exhibited potential as biomarkers for diagnosing azoospermia (area under the curve, 0.722 and 0.720, respectively). These results suggest that reduced SYCP3 and DDX3Y mRNA expression profiles in testicular tissue are associated with a higher likelihood of retrieving spermatozoa in individuals with azoospermia. The homozygous genotype TT of the SYCP3 polymorphism was significantly associated with azoospermia.

Standardization of a multiplex assay to identify weak D types in a mixed-race Brazilian population.

RH allele variability is caused by several types of variants, resulting in altered RhD and RhCE phenotypes. Most of the weak D phenotypes in European-derived populations are weak D types 1, 2, or 3, which are not involved in alloimmunization episodes. However, the Brazilian population is racially diverse, and the accuracy of molecular and serologic tests developed in recent years has allowed for the identification of other RH variants, that are common in the Brazilian population, such as weak D type 38 or weak partial 11, the latter involved in alloimmunization cases. Furthermore, patients with these two weak D variants must be transfused with D- red blood cell units, as do patients with weak D type 4 or DAR, which are also common D variants in Brazil. Weak D type 38 and weak partial 11 can be serologically misclassified as weak D types 1, 2, or 3 in patients, based on European experience, or as D- in donors. Additionally, pregnant women may unnecessarily be identified as requiring Rh immune globulin. RhCE phenotypes are reliable indicators of RhD variants. For individuals with the Dce phenotype, the preferred approach is to specifically search for RHD*DAR. However, when encountering DCe or DcE phenotypes, we currently lack a developed method that assists us in rapidly identifying and determining the appropriate course of action for the patient or pregnant woman. Two multiplex assays were proposed: one for the identification of RHD*weak partial 11, RHD*weak D type 38, and RHD*weak D type 3 and another for RHD*weak D type 2 and RHD*weak D type 5. The multiplex assays were considered valid if the obtained results were equivalent to those obtained from sequencing. Expected results were obtained for all tested samples. The proposed multiplex allele-specific polymerase chain reaction assays can be used in the molecular investigation of women of childbearing age, patients, and blood donors presenting a weak D phenotype with DCe or DcE haplotypes in a mixed-race population, such as Brazil.

Detection of homozygous wildtype V1016V using allele-specific polymerase chain reaction in Aedes albopictus

Abstract. Setiawan AR, Fadila SZ, Sucipto TH, Fauziyah S, Madaniyah S, Dewi EC, Naw SW, Tukiran, Cahyaningrum SE. 2023. Detection of homozygous wildtype V1016V using allele-specific polymerase chain reaction in Aedes albopictus. Biodiversitas 24: 62-67. Aedes sp. is a carrier of several viruses that can infect humans and cause diseases such as zika, yellow fever, chikungunya, and dengue fever. Symptoms of dengue infection vary, consisting of classic dengue fever (DD), dengue hemorrhagic fever (DHF), and dengue shock syndrome. Insecticide spray can be used to manage Aedes mosquitoes chemically. Insecticide substances target nervous system proteins. Voltage-gated sodium channels (VGSC) are rendered inactive by pyrethroid binding. Knockdown is a signal indicating an insect has been knocked down in response to a specific insecticide. However, using insecticides for a long time can cause mosquitoes to become resistant. The pesticide resistance of mosquitoes is known as knockdown resistance (kdr). This study aims to detect kdr mutations (V1016G) in two male Aedes albopictusmosquitos named A1 and A2 collected from settlements in Kranggan, Sawahan, Surabaya, East Java, Indonesia, using allele-specific polymerase chain reaction (AS-PCR) assay. RNA was extracted from the two mosquito samples using an RNA extraction kit. After that, the extracted RNA was tested for kdr mutations using the AS-PCR method. After assaying, both samples are homozygous wildtype (V1016V) because the results showed bands appearing from samples A1 and A2 at 60 bp. On the other hand, this study has the potential to serve as preliminary monitoring for the program controlling vectors.

Validation of KASP-SNP markers in cassava germplasm for marker-assisted selection of increased carotenoid content and dry matter content.

Provitamin A biofortification and increased dry matter content are important breeding targets in cassava improvement programs worldwide. Biofortified varieties contribute to the alleviation of provitamin A deficiency, a leading cause of preventable blindness common among pre-school children and pregnant women in developing countries particularly Africa. Dry matter content is a major component of dry yield and thus underlies overall variety performance and acceptability by growers, processors, and consumers. Single nucleotide polymorphism (SNP) markers linked to these traits have recently been discovered through several genome-wide association studies but have not been deployed for routine marker-assisted selection (MAS). This is due to the lack of useful information on markers’ performances in diverse genetic backgrounds. To overcome this bottleneck, technical and biological validation of the loci associated with increased carotenoid content and dry matter content were carried out using populations independent of the marker discovery population. In the present study, seven previously identified markers for these traits were converted to a robust set of uniplex allele-specific polymerase chain reaction (PCR) assays and validated in two independent pre-breeding and breeding populations. These assays were efficient in discriminating marker genotypic classes and had an average call rate greater than 98%. A high correlation was observed between the predicted and observed carotenoid content as inferred by root yellowness intensity in the breeding (r = 0.92) and pre-breeding (r = 0.95) populations. On the other hand, dry matter content-markers had moderately low predictive accuracy in both populations (r< 0.40) due to the more quantitative nature of the trait. This work confirmed the markers’ effectiveness in multiple backgrounds, therefore, further strengthening their value in cassava biofortification to ensure nutritional security as well as dry matter content productivity. Our study provides a framework to guide future marker validation, thus leading to the more routine use of markers in MAS in cassava improvement programs.

P414 Diagnostic allele-specific PCR for the identification of Candida auris clades and common resistance mutations

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Candida auris (C. auris) is an opportunistic pathogenic yeast that emerged worldwide during the past decade. This fungal pathogen poses a significant public health threat due to common multidrug resistance (MDR), alarming hospital outbreaks, and frequent misidentification. Genomic analyses have identified five distinct clades that are linked to five geographic areas of origin and characterized by differences in several phenotypic traits such as virulence and drug resistance. Typing of C. auris strains and the identification of clades can be a powerful tool in molecular epidemiology and might be of clinical importance by estimating outbreak and MDR potential. As C. auris has caused global outbreaks, including in low-income countries, typing C. auris strains quickly and inexpensively is highly valuable. We report five allele-specific multiplex polymerase chain reaction (AS-multiplex PCR) assays for the identification of C. auris and each of the five described clades of C. auris based on conserved mutations in the internal transcribed spacer (ITS) rDNA region and a clade-specific gene cluster. Additionally, we developed AS-PCR assays for the identification of SNPs in FKS1 and ERG11 that are commonly linked to echinocandin and azole resistance respectively. This PCR method provides a fast, cheap, sequencing-free diagnostic tool for the identification of C. auris, C. auris clades, and common resistance mutations.

ASSOCIATION BETWEEN SPORT RELATED POLYGENIC PROFILE AND MAGNITUDE OF PHYSICAL PERFORMANCES CHANGES FOLLOWING RESISTANCE TRAINING AMONG NOVICE FIELD HOCKEY ATHLETES

Background: Studies have shown that inter-individual variation in response to resistance training is attributed to genetic variation. Aim: To correlate total genotype scores (TGS) with the magnitude of physical performances (skeletal muscle mass, muscular strength, muscular power, performance decrement, and VO2max) change, following 8 weeks of resistance training. Methods: We included forty-five (N=45) participants (age = 16.53 ± .5 years old, body mass = 61.03 ± 6.67 kg, height = 1.67±.05 m) and randomly assigned into; high intensity resistance training (n=15), moderate intensity resistance training (n=15) and a control group (n=15). They were prescribed with the same upper and lower body exercise routines. Pre- and post-test physical performances were measured. Genotyping was conducted using in-house designed allele-specific polymerase chain reaction assays. TGS for nine SNPs: Angiotensin Converting Enzyme, ACE rs1799752; Alpha-acitin-3, ACTN3 rs1815739; Adrenergic Receptor B3, ADRB3 rs4994; Angiotensinogen, AGT rs699; Bradykinin Receptor B2, BDKRB2 rs1799722; Peroxisome Proliferator-Activated Receptor Alpha, PPARA rs4253778; Peroxisome Proliferator-Activated Receptor Gamma Co-Activator 1-Alpha, PPARGC1A rs8192678; Thyrotropin Releasing Hormone Receptor, TRHR rs7832552 and Vascular Endothelial Growth Factor, VEGF rs1870377,were calculated and assigned with strength-power and endurance quality. Pearson correlation analysis was employed to investigate the correlation between TGS, and magnitude of physical performances change following intervention. Result: There is a significant correlation between strength-power TGS with lower body muscular strength (r= .65, p&lt;.01) and power (r= -.69, p&lt;.01) following moderate intensity resistance training. Conclusion: In this study, it is demonstrated that participants with higher strength-power TGS, gained greater lower body strength improvement and lesser muscular power decrement even with moderate intensity as compared to high intensity resistance training. Therefore, personalising training based on athlete’s genetic profile may optimise performance.

Linkage analysis and residual heterozygotes derived near isogenic lines reveals a novel protein quantitative trait loci from a Glycine soja accession.

Modern soybean [Glycine max (L.) Merr] cultivars have low overall genetic variation due to repeated bottleneck events that arose during domestication and from selection strategies typical of many soybean breeding programs. In both public and private soybean breeding programs, the introgression of wild soybean (Glycine soja Siebold and Zucc.) alleles is a viable option to increase genetic diversity and identify new sources for traits of value. The objectives of our study were to examine the genetic architecture responsible for seed protein and oil using a recombinant inbred line (RIL) population derived from hybridizing a G. max line (‘Osage’) with a G. soja accession (PI 593983). Linkage mapping identified a total of seven significant quantitative trait loci on chromosomes 14 and 20 for seed protein and on chromosome 8 for seed oil with LOD scores ranging from 5.3 to 31.7 for seed protein content and from 9.8 to 25.9 for seed oil content. We analyzed 3,015 single F4:9 soybean plants to develop two residual heterozygotes derived near isogenic lines (RHD-NIL) populations by targeting nine SNP markers from genotype-by-sequencing, which corresponded to two novel quantitative trait loci (QTL) derived from G. soja: one for a novel seed oil QTL on chromosome 8 and another for a novel protein QTL on chromosome 14. Single marker analysis and linkage analysis using 50 RHD-NILs validated the chromosome 14 protein QTL, and whole genome sequencing of RHD-NILs allowed us to reduce the QTL interval from ∼16.5 to ∼4.6 Mbp. We identified two genomic regions based on recombination events which had significant increases of 0.65 and 0.72% in seed protein content without a significant decrease in seed oil content. A new Kompetitive allele-specific polymerase chain reaction (KASP) assay, which will be useful for introgression of this trait into modern elite G. max cultivars, was developed in one region. Within the significantly associated genomic regions, a total of eight genes are considered as candidate genes, based on the presence of gene annotations associated with the protein or amino acid metabolism/movement. Our results provide better insights into utilizing wild soybean as a source of genetic diversity for soybean cultivar improvement utilizing native traits.

Association of TNF-α rs1800629 with Adult Acute B-Cell Lymphoblastic Leukemia.

TNF–α influences lymphomagenesis by upregulating proinflammatory and antiapoptotic pathways. In this study, we evaluated the frequency of TNF–α rs1800629 (–308 G>A) polymorphism in newly diagnosed adult patients with acute lymphoblastic leukemia (ALL) and its correlation with age at diagnosis, gender and subtype of ALL. In this case control study, a total of 330 individuals were recruited, including 165 newly diagnosed adult patients with ALL, from the Radiation and Isotope Center in Khartoum (RICK) and 165 healthy normal controls. TNF–α rs1800629 polymorphism was tested through allele-specific polymerase chain reaction (PCR) assay. The frequency of the rs1800629 GA genotype was high (70.9% vs. 60%, OR = 1.84) in the patient group as compared to healthy controls, whereas GG and AA genotypes did not exhibit any statistically significant difference between controls and patients. Based on subtype, GG and GA rs1800629 genotypes showed increased risk of B-ALL (OR 0.46 and 2.12, respectively), whereas rs1800629 GG, GA and AA genotypes did not show any disease association with T-ALL (p > 0.05). Age at diagnosis and gender did not exhibit any association of rs1800629 with ALL in the patient group. In conclusion, rs1800629 is associated with high risk of adult B-ALL, with an insignificant effect of age at diagnosis and gender.

Discovery of the New Leaf Rust Resistance Gene Lr82 in Wheat: Molecular Mapping and Marker Development.

Breeding for leaf rust resistance has been successful worldwide and is underpinned by the discovery and characterisation of genetically diverse sources of resistance. An English scientist, Arthur Watkins, collected pre-Green Revolution wheat genotypes from 33 locations worldwide in the early part of the 20th Century and this collection is now referred to as the ‘Watkins Collection’. A common wheat genotype, Aus27352 from Yugoslavia, showed resistance to currently predominating Australian pathotypes of the wheat leaf rust pathogen. We crossed Aus27352 with a leaf rust susceptible wheat selection Avocet S and a recombinant inbred line (RIL) F6 population of 200 lines was generated. Initial screening at F3 generation showed monogenic segregation for seedling response to leaf rust in Aus27352. These results were confirmed by screening the Aus27352/Avocet S RIL population. The underlying locus was temporarily named LrAW2. Bulked segregant analysis using the 90K Infinium SNP array located LrAW2 in the long arm of chromosome 2B. Tests with molecular markers linked to two leaf rust resistance genes, Lr50 and Lr58, previously located in chromosome 2B, indicated the uniqueness of LrAW2 and it was formally designated Lr82. Kompetitive allele-specific polymerase chain reaction assays were developed for Lr82-linked SNPs. KASP_22131 mapped 0.8 cM proximal to Lr82 and KASP_11333 was placed 1.2 cM distal to this locus. KASP_22131 showed 91% polymorphism among a set of 89 Australian wheat cultivars. We recommend the use of KASP_22131 for marker assisted pyramiding of Lr82 in breeding programs following polymorphism check on parents.

EP009: Identification of a novel exonic deletion in the GALNS gene causing Morquio syndrome

Mucopolysaccharidosis (MPS) IVA or Morquio syndrome is a rare lysosomal storage disorder caused by N-acetylgalactosamine-6-sulfatase deficiency. The condition is characterized by intracellular accumulation of the glycosaminoglycans keratan sulfate and chondroitin-6-sulfate, which classically leads to progressive skeletal and joint abnormalities, short stature, cardiorespiratory compromise, impaired vision, hearing loss, and hepatomegaly. The diagnosis is made by the identification of reduced N-acetylgalactosamine-6-sulfatase activity as well as detection of compound heterozygous or homozygous pathogenic variants in GALNS. We present a case of two sisters with the severe classic phenotype of Morquio syndrome who were born to healthy non-consanguineous parents of East Indian background. Patient 1 first presented at age 1.5 years with trouble walking and abnormal gait with a subsequent skeletal survey suggestive of dysostosis multiplex. She was diagnosed at age 2 years with Morquio syndrome based on reduced leukocyte N-acetylgalactosamine-6-sulfatase activity of 2.8 nmol/17hr/mg protein (reference range 45-443 nmol/17hr/mg protein). Medical history for patient 1 includes normal cognition, truncal shortening, scoliosis, distal joint laxity, cervical arthropathy, gibbus deformity, corneal clouding, and obstructive sleep apnea. Patient 2, the younger sister, was diagnosed with MPS IV at age 3 following a history of frequent falls and identification of genu valgum and early dysostosis. Given the family history, she was tested for Morquio syndrome and N-acetylgalactosamine-6-sulfatase leukocyte activity was <0.062nmol/17hr/mg protein (reference range 45-433 nmol/17hr/mg protein). Patient 2 has a history of cervical C1-C2 instability and occipitocervical stenosis, severe bilateral genu valgum, hip dysplasia, leg length discrepancy, pectus carinatum and corneal clouding. Both children, now at age 11 and 10 years respectively, need to use a wheelchair for any distance outside the home, despite orthopedic procedures. Prior genetic testing for MPS IVA, which utilized next generation sequencing (NGS), did not identify sequence variants associated with MPS IVA, however it was noted that a large region of the GALNS gene corresponding to exon 9 was not covered in the sequencing data. After establishing care with our center, repeat enzyme testing identified absent N-acetylgalactosamine-6 sulfatase activity for patient 1 and significantly reduced activity of 0.18 nmol/17hr/mg protein for patient 2 (reference range 49-255 nmol/17hr/mg protein; affected range < 7 nmol/17hr/mg protein). Subsequent polymerase chain reaction (PCR) amplification of the coding exons for GALNS produced an appropriate product for all exons with the exception of exon 9 and we hypothesized that a deletion encompassing exon 9 was present in our patients. Therefore, an allele specific PCR assay was designed to confirm the exon 9 deletion and determine the precise deletion breakpoints (c.899-397_1003-18632del) for our patients. The reference mRNA sequence was NM_000512.4 with codon 1 corresponding to the start ATG. To our knowledge, the pathogenic deletion identified in our patients (c.899-397_1003-18632del) has not been reported in the literature. Various deletions encompassing at least one exon have been reported in GALNS, however none encompassing only exon 9. NGS is increasingly utilized as a first-tier test for lysosomal storage diseases via targeted gene panels or exome/genome sequencing. The inability of this testing to detect large deletions or rearrangements, especially in the heterozygous state, could lead to a delayed or even missed diagnosis. For example, if the deletion described in this case was in cis with missense change in GALNS, NGS would label the patient as a heterozygote rather than affected. Our case highlights the benefit of biochemical analysis for lysosomal storage disorders in cases with equivocal or incomplete molecular analysis. Molecular analysis is useful to confirm a biochemical diagnosis and assist with genetic counseling and future prenatal testing and carrier testing. Recognizing limitations of molecular testing is important to ensure accurate diagnosis and treatment for individuals with Morquio syndrome in a timely manner.

Multiple insecticide resistance mechanisms in urban population of Anopheles coluzzii (Diptera: culicidae) from Lagos, South-West Nigeria

Malaria is a major public health challenge in Africa with Nigeria accounting for the highest burden of the disease in the world. Vector control has proved to be a highly effective component of malaria control, however, the development and spread of insecticide resistance in major vectors of malaria have been a major challenge. This study assessed resistance mechanisms in Anopheles coluzzii populations from Kosofe, Lagos mainland and Ojo Local Government Areas in Lagos, Nigeria where An. gambiae s.l is resistant to DDT and Permethrin. WHO susceptibility bioassay test was used in determining resistance status of An. coluzzii to discriminating doses of DDT and Permethrin while synergist assay was used to assess the involvement of monooxygenases in resistance development. Sub-species of An. gambiae s.l (An. gambiae and An. coluzzii) were identified using polymerase chain reaction (PCR) and Restriction Fragment Length Polymorphism (PCR-RFLP) while Allele-Specific Polymerase Chain Reaction (AS-PCR) assay was used to detect knockdown mutation (kdr-West; L1014F). Biochemical assays were used in determining the activities of metabolic enzymes. High DDT resistance was recorded in An. coluzzii populations from the three sites. Mortality rate of mosquitoes exposed confirmed Permethrin resistance in Kosofe (50%) and Lagos mainland (48%) but resistance was suspected in Ojo (96%). All specimens tested were confirmed as An. coluzzii with low kdr frequency; 11.6%, 16.4% and 6.7% in Kosofe, Lagos mainland and Ojo respectively. Pre-exposure to synergist (PBO) before exposure to Permethrin led to increased mortality in all populations. Esterase activity was insignificantly overexpressed in Kosofe (p = 0.849) and Lagos mainland (p = 0.229) populations. In contrast, GST activity was significantly lower in populations from Lagos mainland (63.650 ± 9.861; p = 0.007) and Ojo (91.765 ± 4.959; p = 0.042) than Kisumu susceptible strains (120.250 ± 13.972). Monooxygenase activity was higher in Lagos mainland (2.371 ± 0.261) and Ojo (1.361 ± 0.067) populations, albeit significantly in Lagos mainland (p = 0.007) only. Presence of target-site mutation in all populations, increased mortality with pre-exposure to PBO and elevated monooxygenase in Lagos mainland population were confirmed. Multiple resistance mechanisms in some urban populations of An. coluzzii from Lagos, Nigeria calls for appropriate resistance management strategies.

Determination of MYD88L265P mutation fraction in IgM monoclonal gammopathies

AbstractWe describe a novel method for the detection of MYD88L265P mutation using a competitive allele-specific polymerase chain reaction (Cast-PCR) assay. This assay has a sensitivity of 1 × 10−3, is applicable in reactions containing very low amounts of DNA (as low as 20 pg), and allowed the detection of MYD88L265P somatic mutation in both tumor-derived DNA (tDNA) and cell-free DNA (cfDNA). In addition, using the Cast-PCR assay, we were able to determine the mutation allele fraction (MAF) in each tested sample. We then analyzed baseline tDNA and cfDNA samples from 163 patients (53 with immunoglobulin M monoclonal gammopathy of undetermined significance and 110 with Waldenström's macroglobulinemia [WM], of whom 54 were asymptomatic and 56 were symptomatic) and also in sequential samples of 37 patients. MAF in both cfDNA and tDNA was higher among patients with symptomatic compared with asymptomatic WM and in those with asymptomatic WM compared with those with immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance. In addition, the evaluation of sequential samples showed that MAF decreased after treatment, whereas it increased in patients who relapsed or progressed to symptomatic WM. Thus, Cast-PCR is a highly sensitive, cost-effective diagnostic tool for MYD88L265P detection, applicable in both tDNA and cfDNA samples, that also provides a quantitative evaluation of the tumor load in patients with IgM monoclonal gammopathies.

Detection of Knockdown-Resistance Mutations (V1016G and F1534C) in Dengue Vector from Urban Park, Surabaya, Indonesia

An urban park is potentially a source of vector-borne disease transmission due to it being a natural and artificial mosquito breeding habitats combined with people's continuous presence. Thus, this study aims to screen the occurrence of knockdown-resistance (kdr) mutant alleles (V1016G and F1534C) in mosquito populations collected from urban parks in Surabaya, Indonesia. Cross sectional study was conducted in July 2019. A total of 28 ovitraps were installed in seven urban parks, having four ovitraps installed in each park. In total, 1,662 eggs were collected, and only 187 emerged into adult mosquitoes, consisting of 97 Aedes (Stegomyia) aegypti and 90 Aedes (Stegomyia) albopictus. All-female adult mosquitoes (n=55) were tested using allele-specific polymerase chain reaction assay (AS-PCR) to detect voltage gated sodium channel (VGSC) gene mutations. This study found no mutations in Valine to Glysine mutation in point 1016 (V1016G) and Phenylalanine to Cysteine in point 1534 (F1534C) alleles in both two species. All of mosquito samples have wild type genotype of kdr alleles (V1016V and F1534F). Data were analysed using R Studio 1.4 Version by Genetics package. Results showed that the frequency of resistant alleles (G1016 and C1534) was zero, and the frequency of susceptible allele was 1 (V1016 and F1534). Insecticide bioassay could not be established due to the limited number of adult mosquitoes, so insecticide resistance status could not be determined. However, this study can be used as preliminary monitoring for the vector control program.

Diagnostic Allele-Specific PCR for the Identification of Candida auris Clades.

Candida auris is an opportunistic pathogenic yeast that emerged worldwide during the past decade. This fungal pathogen poses a significant public health threat due to common multidrug resistance (MDR), alarming hospital outbreaks, and frequent misidentification. Genomic analyses have identified five distinct clades that are linked to five geographic areas of origin and characterized by differences in several phenotypic traits such as virulence and drug resistance. Typing of C. auris strains and the identification of clades can be a powerful tool in molecular epidemiology and might be of clinical importance by estimating outbreak and MDR potential. As C. auris has caused global outbreaks, including in low-income countries, typing C. auris strains quickly and inexpensively is highly valuable. We report five allele-specific polymerase chain reaction (AS-PCR) assays for the identification of C. auris and each of the five described clades of C. auris based on conserved mutations in the internal transcribed spacer (ITS) rDNA region and a clade-specific gene cluster. This PCR method provides a fast, cheap, sequencing-free diagnostic tool for the identification of C. auris, C. auris clades, and potentially, the discovery of new clades.

Smoking and P53 polymorphism association with chromosomal aberration in lung cancer

Tobacco exposure is the primary leading cause for cancer of the oral cavity, the airways and lung. The carcinogens in cigarette smoke have been linked as an environmental risk factor for causing genetic mutations in tumor suppressor, including TP53 (p53) which has been linked as a causative for occurrence and progression in lung cancer. The aim of the current case-control study is to assess the presence and frequency of chromosomal aberrations between smokers and non-smokers in case and control group. Further, analysis was also extended to include the p53 codon 72 Arg/Pro mutation analysis between the groups of study cohort. In methods, the case-control epidemiological study included a total of 100 cases and controls who were further categorized as smokers and non-smokers for analysis. Chromosomal aberration assay was done on peripheral blood lymphocytes and studied using Geimsa stain, while analysis of the p53 codon 72 Arg/Pro mutation was done by allele-specific polymerase chain reaction assay. The chromosomal aberration assessed include structural abnormalities like breaks, fragment, dysenteric and gaps, as well as numerical aneuploidies. Results showing the number of chromosomal aberrations was higher among smoker cases as compared to controls, and numerical aneuploidy was also detected only among this cohort. The mean of chromosomal aberrations observed between cases and controls and smokers as well as non-smokers was significant at p < 0.0001. In case of the p53 genotyping result, the frequency of the Arg/Arg allele was higher among controls as compared to cases and the difference significant at p < 0.005. Among female participants, the difference between Arg/Arg and Pro/Arg was also found to be significant between cases and controls. Combined analysis of the p53 codon 72 alleles and mean of chromosomal aberrations between cases and controls was found to be significant at p < 0.005. Our study found an association between risk of lung cancer as well as occurrence of chromosomal aberrations. In case of the p53 polymorphism, the significance was detected only among females. In Conclusion, the studied p53 variant and chromosomal aberration along with smoking has shown significant association with increased risk of lung cancer.

Conversion and Validation of Uniplex SNP Markers for Selection of Resistance to Cassava Mosaic Disease in Cassava Breeding Programs

Cassava mosaic disease (CMD) is a major viral disease adversely affecting cassava production in Africa and Asia. Genomic regions conferring resistance to the disease have been mapped in African cassava germplasm through biparental quantitative trait loci (QTL) mapping and genome-wide association studies. To facilitate the utilization of these markers in breeding pipelines to support selections, proof-of-concept technical and biological validation research was carried out using independent pre-breeding and breeding populations. Kompetitive Allele-Specific Polymerase Chain Reaction (KASP) assays were designed from three single nucleotide polymorphism (SNP) markers linked to a major resistance locus on chromosome 12 (S12_7926132, S12_7926163) and a minor locus on chromosome 14 (S14_4626854). The designed assays were robust and easy to score with &gt;99% genotype call rate. The overall predictive accuracy (proportion of true positives and true negatives) of the markers (S12_7926132 and S14_4626854) was 0.80 and 0.78 in the pre-breeding and breeding population, respectively. On average, genotypes that carried at least one copy of the resistant allele at the major CMD2 locus had a significantly higher yield advantage. Nevertheless, variation was observed in prediction accuracies for the major locus (S12_7926132) among sub-families from the two populations, suggesting the need for context-specific utilization, for example, by screening for co-segregation of favorable SNP alleles with resistance in the parents being used for crosses. Availability of these validated SNP markers on the uniplex KASP genotyping platform represents an important step in translational genetics toward marker-assisted selection to accelerate introgression of favorable resistant alleles in breeding populations.