Genotyping Of Mutations Research Articles

Rules for resolving Mendelian inconsistencies in nuclear pedigrees typed for two-allele markers.

Gene-mapping studies, regularly, rely on examination for Mendelian transmission of marker alleles in a pedigree as a way of screening for genotyping errors and mutations. For analysis of family data sets, it is, usually, necessary to resolve or remove the genotyping errors prior to consideration. At the Center of Inherited Disease Research (CIDR), to deal with their large-scale data flow, they formalized their data cleaning approach in a set of rules based on PedCheck output. We scrutinize via carefully designed simulations that how well CIDR’s data cleaning rules work in practice. We found that genotype errors in siblings are detected more often than in parents for less polymorphic SNPs and vice versa for more polymorphic SNPs. Through computer simulations, we conclude that some of the CIDR’s rules work poorly in some circumstances, and we suggest a set of modified data cleaning rules that may work better than CIDR’s rules.

PI3K pathway mutations are associated with longer time to local progression after radioembolization of colorectal liver metastases.

PurposeTo establish the relationship between common mutations in the MAPK and PI3K signaling pathways and local progression after radioembolization.Materials and MethodsRetrospective review of a HIPAA-compliant institutional review-board approved database identified 40 patients with chemo-refractory colorectal liver metastases treated with radioembolization who underwent tumor genotyping for hotspot mutations in 6 key genes in the MAPK/PI3K pathways (KRAS, NRAS, BRAF, MEK1, PIK3CA, and AKT1). Mutation status as well as clinical, tumor, and treatment variables were recorded. These factors were evaluated in relation to time to local progression (TTLP), which was calculated from time of radioembolization to first radiographic evidence of local progression. Predictors of outcome were identified using a proportional hazards model for both univariate and multivariate analysis with death as a competing risk.ResultsSixteen patients (40%) had no mutations in either pathway, eighteen patients (45%) had mutations in the MAPK pathway, ten patients (25%) had mutations in the PI3K pathway and four patients (10%) had mutations in both pathways. The cumulative incidence of progression at 6 and 12 months was 33% and 55% for the PI3K mutated group compared with 76% and 92% in the PI3K wild type group. Mutation in the PI3K pathway was a significant predictor of longer TTLP in both univariate (p=0.031, sHR 0.31, 95% CI: 0.11-0.90) and multivariate (p=0.015, sHR=0.27, 95% CI: 0.096-0.77) analysis. MAPK pathway alterations were not associated with TTLP.ConclusionsPI3K pathway mutation predicts longer time to local progression after radioembolization of colorectal liver metastases.

Application of high resolution melting analysis in the molecular diagnosis of inherited diseases

The basic principle of high resolution melting (HRM) is that DNA-saturated fluorescent dye is added into the PCR reaction system; the PCR amplicon is denatured by heating in a certain temperature range; specialized instrumentsdetect degeneration double-stranded DNA fluorescence signal and draw the melting curve and then wild-type and heterozygous mutants are differentiatedaccording to different melting curve shape. HRM technology is widely used in gene mutation scanning because of its advantages of simplicity, accuracy, fastness, low cost, short cycle time and high throughput.In addition, compared with other mutation screening methods, HRM analysis is a real closed-tube method, which indicates that high-resolution melting analysis is directly performed after PCR reaction and HRM technology is more convenient.In conclusion , HRM technology is a new technology for mutation screening and genotyping.(Chin J Lab Med, 2017, 40: 146-148) Key words: Genetic diseases, inborn; Polymerase chain reaction; Mutation; Genotyping techniques; High resolution melting

Levels of insecticide resistance to deltamethrin, malathion, and temephos, and associated mechanisms in Aedes aegypti mosquitoes from the Guadeloupe and Saint Martin islands (French West Indies)

BackgroundIn the Guadeloupe and Saint Martin islands, Aedes aegypti mosquitoes are the only recognized vectors of dengue, chikungunya, and Zika viruses. For around 40 years, malathion was used as a mosquito adulticide and temephos as a larvicide. Since the European Union banned the use of these two insecticide molecules in the first decade of the 21st century, deltamethrin and Bacillus thuringiensis var. israelensis are the remaining adulticide and larvicide, respectively, used in Guadeloupe. In order to improve the management of vector control activities in Guadeloupe and Saint Martin, we investigated Ae. aegypti resistance to and mechanisms associated with deltamethrin, malathion, and temephos.MethodsAe. aegypti mosquitoes were collected from six different localities of Guadeloupe and Saint Martin. Larvae were used for malathion and temephos bioassays, and adult mosquitoes for deltamethrin bioassays, following World Health Organization recommendations. Knockdown resistance (Kdr) genotyping for V1016I and F1534C mutations, and expression levels of eight enzymes involved in detoxification mechanisms were examined in comparison with the susceptible reference Bora Bora strain.ResultsResistance ratios (RR50) calculated for Ae. aegypti larvae showed high resistance levels to temephos (from 8.9 to 33.1-fold) and low resistance levels to malathion (from 1.7 to 4.4-fold). Adult females displayed moderate resistance levels to deltamethrin regarding the time necessary to affect 50% of individuals, varying from 8.0 to 28.1-fold. Molecular investigations on adult mosquitoes showed high resistant allele frequencies for V1016I and F1534C (from 85 to 96% and from 90 to 98%, respectively), as well as an overexpression of the glutathione S-transferase gene, GSTe2, the carboxylesterase CCEae3a, and the cytochrome genes 014614, CYP6BB2, CYP6M11, and CYP9J23.ConclusionsAe. aegypti populations from Guadeloupe and Saint Martin exhibit multiple resistance to organophosphates (temephos and malathion), and pyrethroids (deltamethrin). The mechanisms associated with these resistance patterns show strong frequencies of F1534C and V1016I Kdr mutations, and an over-expression of CCEae3a, GSTe2, and four cytochrome P450 genes (014614, CYP9J23, CYP6M11, CYP6BB2). These results will form the baseline for a deeper understanding of the insecticide resistance levels and associated mechanisms of Ae. aegypti populations and will be used to improve vector control strategies in Guadeloupe and Saint Martin.

Association of janus kinase 2 with primary Budd–Chiari syndrome in Egyptian patients

Objective The aim of this study was to evaluate the association of janus kinase 2 (JAK2) V617F mutation with primary Budd–Chiari syndrome (BCS) in Egyptian patients. Background BCS is a rare disorder caused by thrombosis of the hepatic veins or the terminal portion of the inferior vena cava. JAK2 V617F mutation is an objective tool for diagnosing BCS patients. Patients and methods This study was conducted on 50 patients: 35 patients with primary BCS, and 15 age-matched and sex-matched healthy individuals who were taken as the control group. Patient and control groups were subjected to full history taking, general clinical examination, and laboratory investigations including measurement of complete blood count; liver function tests – serum aspartate aminotransferase and serum alanine aminotransferase (ALT), total and direct bilirubin, serum albumin, prothrombin time, partial thromboplastin time, and international normalized ratio; and renal function tests – serum blood urea and serum creatinine. Genotyping of JAK2 mutation was performed by PCR-RFLP method to diagnose the underlying etiology of BCS. Results There was a statistically significant difference between patients and controls regarding hemoglobin, platelets, ALT, aspartate aminotransferase, direct and total bilirubin, albumin, prothrombin time, partial thromboplastin time, international normalized ratio, urea, and creatinine. There was no statistically significant difference between studied patients and control groups regarding JAK2 mutations. Mutant form of JAK2 had significantly increased platelets count and ALT serum levels than wild form, whereas there was no significant difference regarding other laboratory parameters. Conclusion JAK2 V617F mutation was not significantly different between patients and controls.

P3.02b-018 Detection of Epidermal Growth Factor Receptor Mutations in Circulating Cell-Free DNA versus Tumor Biopsy: Topic: EGFR Biomarkers

Epidermal growth factor receptor (EGFR) mutations are predictive marker of EGFR-tyrosine kinase inhibitor (TKI) therapy. We compared the sensitivity of EGFR mutation detection techniques between matched tumor tissue and peripheral blood sample in patients with lung adenocarcinoma. We collected the paired samples from plasma and paraffin-embedded tumor tissue in 202 patients before EGFR-TKIs. DNA extraction was performed using the QIAamp MinElute virus spin kit and EGFR mutation analysis was done by two detection methods. One is the PNAClampTM which is the PNA-based PCR clamping that selectively amplifies only the mutated target DNA sequence as minor portion in mixture with the major wild type DNA sequences. The other is the PANAMutyperTM EGFR kit, which use PNA clamping-assisted fluorescence melting curve analysis to perform mutation detection and genotyping. The degree of agreement was evaluated by Cohen's kappa value. The EGFR mutation rates by PANAMutyperTMR and PNAClampTM were 51.0% vs. 47.0% in tissue, and 22.2% vs. 11.4% in plasma sample, respectively. Overall concordance rates and the degree of agreement between tissue and plasma samples was better in PANAMutyperTMR (69.3%, k=0.393, p<0.001) than PNAClampTM (62.3%, k=0.211, p<0.001). Sensitivity of plasma EGFR mutations was higher (41.7% vs. 22.1%, p<0.001) and false negative rate was lower in PANAMutyperTMR test (58.3% vs. 77.9%, p<0.001). Response to EGFR-TKI was correlated with plasma EGFR mutation by PANAMutyperTMR (p=0.025) unlike PNAClampTM (p=0.829). The sensitivity and concordance rate of PANAMutyperTMR test were better than standard PNAClampTM test. So this technique can be useful to detect EGFR mutation in circulating cell-free DNA sample.

Clinical Profile and BRAF Status of 30 Japanese Patients with Adult Langerhans Cell Histiocytosis

Background: Langerhans cell histiocytosis (LCH) is a rare inflammatory myeloid neoplasm characterized by aberrant expansion of CD1a+CD207+ Langerhans-like cells with a variable infiltration of immune cells. Although the precise pathophysiology is to be elucidated, a frequent occurrence of oncogenic BRAF-V600E mutation in high-risk patients (Badalian-Very, et al. Blood. 2010) has made it one of the candidates of major driver mutations in LCH as well as in hairy cell leukemia. Its onset in adulthood is rather infrequent as compared with that in childhood, and the clinical profile and treatment outcome of adult LCH is poorly documented. Here, we report the clinical features and BRAF status of Japanese patients with adult LCH in a single institution.Methods: Between 2005 and 2016, 30 adult LCH patients with histopathological diagnosis and/or characteristic radiological findings were referred to our hospital for evaluation and treatment. In almost all cases, we re-evaluated the involved organs and determined the clinical subtypes. For detection of BRAF-V600E, cell-free DNA (cfDNA) was prepared from plasma of patients and was subjected to genotyping of BRAF mutations by allele-specific Q-PCR, which was specifically designed for detection of BRAF-V600E with a 3'-phosphate-modified oligonucleotide blocker as previously described (Kobayashi M and Tojo A. Blood. 2014). Mutant BRAF load was estimated from the standard curve in each assay and was expressed as the percentage of mutant alleles to total number of alleles. Considering the safety and efficacy at outpatient clinic without hospitalization, we adopted 9 cycles of JLSG Special C regimen as a first-line therapy for the patients who require treatment. The protocol is consisted of 6mg vinblastine (VBL) on Day1, 60mg oral prednisolone (PSL) on Day1-5, and 20mg/m2 methotrexate (MTX) on Day15 with 1.5mg/kg daily 6-mercaptopurine (6MP) (Morimoto A, et al. Int J Hematol. 2013).Results: Patients' median age at diagnosis was 43 (range 24-66) and 50% was female. Fourteen patients (47%) had a single organ disease (single-system; Ss) and others had multi-organ disease (multi-system; Ms). Bone (50%) and skin (29%) were main lesions in Ss patients, who were younger (median 39) than Ms patients (median 44). Nine patients (30%) developed diabetes insipidus caused by pituitary lesions. Pulmonary involvement was found in 6 patients (20%), and all except one were heavy smokers at the onset or relapse of LCH in these patients. Among Ms patients, there were 8 high-risk patients with lung, liver, spleen or bone marrow involvement. We performed ASQ-PCR analysis of BRAF mutation in 20 patients (12 Ms patients and 8 Ss patients), and only 4 Ms patients were positive for V600E mutant. Twenty-three patients received Special C regimen and 4 refractory patients were followed by 2-chlorodeoxyadenosine (2-CdA) as a salvage therapy. After Special C regimen with or without a salvage therapy, five patients had relapsed. Three of them received once more Special C regimen and two among them showed improvement. During their clinical courses, only two Ms patients, who were positive for BRAF-V600E and received 2-CdA, died of LCH progression and a cerebrovascular event, respectively.Conclusion: Since adult LCH is quite rare and most likely ignored by physicians, the diagnosis and the clinical intervention is generally delayed. Nonetheless, most of patients benefited from the low-intensity chemotherapy and their prognosis is rather favorable. Recent findings of MAPK and PI3K pathway mutation in LCH will facilitate the understanding of its pathogenesis and development of its novel molecular therapy. DisclosuresNo relevant conflicts of interest to declare.

Evidence of a multiple insecticide resistance in the malaria vector Anopheles funestus in South West Nigeria.

BackgroundKnowing the extent and spread of insecticide resistance in malaria vectors is vital to successfully manage insecticide resistance in Africa. This information in the main malaria vector, Anopheles funestus sensu stricto, is completely lacking in the most populous country in Africa, Nigeria. This study reports the insecticide susceptibility status and the molecular basis of resistance of An. funestus as well as its involvement in malaria transmission in Akaka-Remo, a farm settlement village in southwest Nigeria.Results Plasmodium infection analysis using TaqMan protocol coupled with a nested PCR revealed an infection rate of 8% in An. funestus s.s. from Akaka-Remo. WHO susceptibility tests showed this species has developed multiple resistance to insecticides in the study area. Anopheles funestus s.s. population in Akaka-Remo is highly resistant to organochlorines: dieldrin (8%) and DDT (10%). Resistance was also observed against pyrethroids: permethrin (68%) and deltamethrin (87%), and the carbamate bendiocarb (84%). Mortality rate with DDT slightly increased (from 10 to 30%, n = 45) after PBO pre-exposure indicating that cytochrome P450s play little role in DDT resistance while high mortalities were recorded after PBO pre-exposure with permethrin (from 68 to 100%, n = 70) and dieldrin (from 8 to 100%, n = 48) suggesting the implication of P450s in the observed permethrin and dieldrin resistance. High frequencies of resistant allele, 119F in F0 (77%) and F1 (80% in resistant and 72% in susceptible) populations with an odd ratio of 1.56 (P = 0.1859) show that L119F-GSTe2 mutation is almost fixed in the population. Genotyping of the A296S-RDL mutation in both F0 and F1 samples shows an association with dieldrin resistance with an odd ratio of 81 (P < 0.0001) (allelic frequency (R) = 76% for F0; for F1, 90 and 10% were observed in resistant and susceptible populations, respectively) as this mutation is not yet fixed in the population.ConclusionThe study reports multiple insecticide resistance in An. funestus from Akaka Remo. It is, therefore, necessary to pay more attention to this major malaria vector for effective malaria control in Nigeria.

Association of HFE gene mutations with nonalcoholic fatty liver disease in the Iranian population.

To determine whether the HFE gene variants H63D and C282Y are associated with NAFLD in persons with type 2 diabetes, we conducted a case-control study including 145 case of NAFLD patients with a history of type 2 diabetes and 145 matching control. The genomic DNA was extracted from the peripheral venous blood and the genotyping of HFE gene mutations was analyzed using the PCR-RFLP technique. Statistical analysis was performed using SPSS 12.0 software by χ2 test, t test and ANOVA (P&lt;0.05). Data showed no increased frequency of HFE mutations in persons with type 2 diabetes and no association between H63D mutation and NAFLD in the study population. Also, we analyzed index of physiological variables including FBS, lipid profile (TC, TG, LDL-C, and HDL-C), BMI, HbA1c, and micro albuminuria and Cr levels). Data showed there are no relationship between these indexes and HFE gene mutations and either NAFLD as a complication of diabetes. But our results showed a relationship between C282Y mutation and NAFLD in persons with type 2 diabetes. C282Y mutation might be a genetic marker of NAFLD in Iranian population.

Predominant mutations in a hotspot of SERPINC1 associated with venous thromboembolism in the Chinese population: a case-control study

BackgroundAntithrombin plays a key anticoagulant role. The prevalence of antithrombin deficiency is thought to be very low in patients with thromboembolism. However, increasing evidence suggests that antithrombin deficiency may be underestimated. We aimed to uncover predominant mutations in SERPINC1 in the Chinese population. MethodsIn this case-control study, we selected individuals with venous thromboembolism from the biobank of Hubei Clinical Research Center of Thrombosis and Haemostasis in China and controls from the same area and the same time period. The controls had no history of thrombotic diseases. We included 1304 individuals with venous thromboembolism and 1334 controls from China. We also enrolled a cohort of white individuals from Spain, including 165 patients with antithrombin deficiency, 86 patients with venous thromboembolism, and 100 controls. Re-sequencing of SERPINC1 was performed in 190 Chinese patients with venous thromboembolism. Genotyping of predominant mutations was performed in the whole study population with a PCR-RFLP method using BstUI. Plasma antithrombin antigen concentrations, activity, and thrombin generation potential were measured. We obtained odds ratios (ORs) using logistic regression controlling for other risk factors for venous thromboembolism. This study was approved by the Ethics Committee of the Union Hospital affiliated with Huazhong University of Science and Technology. Written informed consent for research was obtained from each individual included in the study. FindingsWe found no hotspot-mutations in any of the individuals of the white Spanish cohort. A hotspot-mutation involving residues 294 and 295 of antithrombin was revealed in the Chinese cohort. We identified the c.883G>A (p.Val295Met) mutation in 11 patients but in one control. We found the c.881G>T (p.Arg294Leu) mutation in six patients and the c.880C>T (p.Arg294Cys) mutation in two patients, whereas we identified the c.881G>A (p.Arg294His) mutation in one healthy control. Overall, these mutations significantly increased the risk of venous thromboembolism (OR=11·4, 95% CI 2·6–49·4; p=0·001). Carriers of these mutations had normal antithrombin antigen (79·1–116·0 U/dL) and functional activity (75·3–136·3 U/dL), but had a significantly increased endogenous thrombin potential (2·0–2·7 fold). InterpretationOur results support the existence in the Chinese population of a hotspot in SERPINC1 that significantly increases the risk of venous thromboembolism without affecting functional assays of antithrombin. FundingNational Natural Science Foundation of China (81370622 and 81400099).

Synthesis of Clickable Coating Polymers by Postpolymerization Modification: Applications in Microarray Technology.

In this paper, we report on the postpolymerization modification (PPM) of a polymer to introduce new functionalities that enable click chemistry reactions for microarray applications. The parent polymer, named copoly(DMA-NAS-MAPS), is composed of N,N-dimethylacrylamide (DMA), a monomer that self-adsorbs onto different materials through weak interactions such as hydrogen bonding or van der Waals forces, 3-(trimethoxysilyl)propyl methacrylate (MAPS) that strengthens the stability of the coating through the formation of covalent bonds with siloxane groups on the surface to be coated, and N-acryloyloxysuccinimide (NAS), an active ester group, highly reactive toward nucleophiles, which enables bioprobe immobilization. This copolymer has been widely exploited to coat surfaces for microarray applications but exhibits some limitations because of the potential hydrolysis of the active ester (NHS ester). The degradation of the NHS ester hampers the use of this coating in some situations, for example, when probe immobilization cannot be accomplished through a microspotting situation, but in large volumes, for example, in microchannel derivatization or micro-/nanoparticle functionalization. To overcome the limitations of NHS esters, we have developed a family of polymers that originate from the common copolymer precursor, by reacting the active ester contained in the polymer chain with a bifunctional amine. In particular, the functional groups introduced in the polymer using PPM enable click chemistry reactions such as azide/alkyne or thiol/maleimide "click" reactions, with suitably modified biomolecules. The advantages of such reactions are quantitative yields, orthogonality of functional groups, and insensitivity of the reaction to pH. The new click functionalities, inserted with quantitative yields, improve the stability of the coating, enabling the attachment of biomolecules directly from a solution and avoiding the spotting of reduced volumes (pL) of probes. Finally, we have demonstrated the applicability of the click surfaces in a highly effective solid-phase PCR for the genotyping of the G12D KRAS mutation.

Abstract 3157: Serial droplet digital PCR (ddPCR) of plasma cell-free DNA (cfDNA) as pharmacodynamic (PD) biomarker in Phase 1 clinical trials for patients (pts) with KRAS mutant non-small cell lung cancer (NSCLC)

Abstract Introduction: Phase 1 clinical trials of novel therapeutics have historically focused on toxicity, but increasingly are doubling as efficacy studies in biomarker-enriched populations. Given the small sample sizes (∼3-6 patients per dose), response on imaging may be a coarse marker of therapeutic effect. Here we piloted serial ddPCR of plasma cfDNA as a PD marker in a phase I combination study of a MEK inhibitor and a CDK 4/6 inhibitor in patients with RAS mutated cancers. Methods / Results: Twenty-five pts with RAS-mutated cancer (incl. 17 patients with KRAS-mutant NSCLC) have been enrolled to date in a phase I dose escalation trial of the MEK inhibitor PD-0325901 with the CDK4/6 inhibitor palbociclib (NCT02022982). Plasma for cfDNA genotyping was collected at baseline prior to therapy and at the beginning of cycle 2. Plasma genotyping for KRAS G12X mutations was performed using a validated and highly quantitative droplet digital PCR assay. Pts were enrolled in 5 dose level cohorts ranging from 75 mg palbociclib daily (3 weeks on, 1 week off) with 2 mg PD-0325901 BID (3 weeks on 1week off) to 125 mg palbociclib daily with 8 mg PD-0325901 BID (Table). KRAS mutations were detected in 14/24 pts at baseline (59%, median 1402 copies/mL plasma, range: 11-93000), consistent with the previously reported sensitivity of 64%. A second blood draw at cycle 2 was obtained for all 14 pts. A positive plasma response, defined as decrease of KRAS G12X mutants from first to second dose, was observed in 6 pts (range -6% - -100%) with the most plasma responders (n = 4 pts) at the maximum administered dose. At lower administered doses, there was a median increase in plasma KRAS mutant levels. Conclusions: Increasing dose levels resulted in more consistent decreases in KRAS mutation in cfDNA, consistent with a dose-dependent pharmacodynamic effect.These results highlight the potential value of serial plasma ddPCR as a PD marker in early phase clinical trials. Dose LevelPalbociclib (QD, 3 wks on 1 wk off) mgPD-0325901 (BID, 3 wks on 1 wk off) mgN Enrolled (N analyzed)Median Plasma change KRAS mut(%)17523 (1)+2427543 (3)+60 (range +31 - +150)310048 (4)+150 (range -6 - +341)412544 (2)+9 (range -45 - +61)512587 (4)-27 (range -7 - -43) Citation Format: Cloud P. Paweletz, Geoffrey R. Oxnard, Nora Feeney, John F. Hilton, Leena Gandhi, Khanh T. Do, Adrienne Anderson, Andrew Wolanski, Alexander Tejeda, Jessie M. English, Paul T. Kirschmeier, Pasi A. Jänne, Geoffrey I. Shapiro. Serial droplet digital PCR (ddPCR) of plasma cell-free DNA (cfDNA) as pharmacodynamic (PD) biomarker in Phase 1 clinical trials for patients (pts) with KRAS mutant non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3157.

Abstract 1376: Ultra-sensitive picodroplet digital PCR assay for multiplex genotyping of mutations in epidermal growth factor receptor (EGFR) in non-small cell lung cancer patients

Abstract BACKGROUND: The epidermal growth factor receptor (EGFR) mutations have been used as a reliable predictor of response to EGFR tyrosine kinase inhibitor (TKI) treatment. Two common EGFR mutations (L858R, and exon19 deletion) and EGFR T790M mutation are clinically important for decision-making in clinical practice. Picodroplet digital PCR (ddPCR) has recently emerged as an accurate and ultra-sensitive method for tumor genotyping and have a great advantage over conventional mutation detection methods. We have previously reported far more frequent incidence of pretreatment EGFR T790M mutation than previous reports utilizing ddPCR method. Here, we developed a multiplex ddPCR assay to detect three clinically relevant EGFR mutations in one reaction. METHODS: Positive and negative control plasmids for the EGFR mutation assay were prepared by cloning DNA fragments containing wild-type or mutant EGFR. Genomic DNAs from lung cancer cell lines H1975, PC-9/ZD, A549 and wild-type human genomic DNA were digested with CviQ1, and then were used to quantitatively assess each EGFR mutant sequence in the multiplex assay panels. We then evaluated the utility of multiplex ddPCR to detect the three clinically relevant mutations in EGFR from FFPE samples of patients with non-small cell lung cancer. 45 FFPE samples were also genotyped for EGFR mutations by conventional qPCR-based methods for validation. The concordance between duplex and multiplex ddPCR assay was also evaluated. RESULTS: Serial dilutions experiments using genomic DNA harboring EGFR mutations revealed a linear performance with an analytical sensitivity of approximately 0.01% for each mutation. All of the 33 EGFR-activating mutations detected in FFPE samples by conventional qPCR-based method were also detected by multiplex ddPCR assay. Owing to its ultra-high sensitivity, additional T790M mutation at ultra-low allele frequency (&amp;lt;0.1%) was also detected in the same reaction. The regression analysis between duplex and multiplex assay demonstrated a correlation coefficient (R2) of 0.9986 for L858R, 0.9844 for exon19 deletion, and 0.9959 for T790M, respectively. CONCLUSIONS: Using ddPCR technology, we established multiplex and ultra-sensitive genotyping platform for three clinically relevant EGFR mutations. Results of a proof-of-principle study using clinical samples indicated that the clinical utility of multiplex ddPCR assay to screen for multiple EGFR-activating mutations concurrently with low-frequency T790M mutation. Citation Format: Yasuhiro Koh, Tomoya Kawaguchi, Masaru Watanabe, Shun-ichi Isa, Masahiko Ando, Akihiro Tamiya, Akihito Kubo, Hideo Saka, Sadanori Takeo, Hirofumi Adachi, Tsutomu Tagawa, Seiichi Kakegawa, Motohiro Yamashita, Kazuhiko Kataoka, Yukito Ichinose, Yukiyasu Takeuchi, Kazuhiro Sakamoto, Akihide Matsumura. Ultra-sensitive picodroplet digital PCR assay for multiplex genotyping of mutations in epidermal growth factor receptor (EGFR) in non-small cell lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1376.

Genotyping of Intron Inversions and Point Mutations in Exon 14 of the FVIII Gene in Iranian Azeri Turkish Families with Hemophilia A.

Hemophilia A (HA) is an inherited X-linked bleeding disorder caused by a variety of mutations that are distributed throughout the large FVIII gene (F8). The most common mutations in studied populations with severe HA are introns 22 and 1 inversions, gross exon deletions and point mutations in exon 14. The aim of this study was to define the frequency of these common mutations in Iranian population of Azeri Turkish in North West of Iran. Fifty patients with severe HA and forty-three female potential carriers were genotyped by inverse shifting polymerase chain reaction (IS-PCR), long-range PCR, multiplex PCR, and sequencing methods for the detection of Intron 22 and 1 inversions, gross exon deletions, and exon 14 point mutations, respectively. F8 intron 22 inversion was detected in 22 (44%) out of 50 patients. Moreover, we detected one intron 1 inversion (2%), and one point mutation in exon 14 (2%). In this population, 52% of the patients with hemophilia A did not show to carry a mutation in the analyzed regions by three mentioned methods. F8 intron 22 inversion was the major causative mutation in nearly 50% of severe HA cases in an Azerbaijani Turkish population, which is similar to the incidence of other populations. IS-PCR is a robust, rapid, efficient, and cost-effective method for the genetic analysis of patients with severe HA and for HA carrier detection, especially in developing countries.

Multimodal MRI features predict isocitrate dehydrogenase genotype in high-grade gliomas.

High-grade gliomas with mutations in the isocitrate dehydrogenase (IDH) gene family confer longer overall survival relative to their IDH-wild-type counterparts. Accurate determination of the IDH genotype preoperatively may have both prognostic and diagnostic value. The current study used a machine-learning algorithm to generate a model predictive of IDH genotype in high-grade gliomas based on clinical variables and multimodal features extracted from conventional MRI. Preoperative MRIs were obtained for 120 patients with primary grades III (n = 35) and IV (n = 85) glioma in this retrospective study. IDH genotype was confirmed for grade III (32/35, 91%) and IV (22/85, 26%) tumors by immunohistochemistry, spectrometry-based mutation genotyping (OncoMap), or multiplex exome sequencing (OncoPanel). IDH1 and IDH2 mutations were mutually exclusive, and all mutated tumors were collapsed into one IDH-mutated cohort. Cases were randomly assigned to either the training (n = 90) or validation cohort (n = 30). A total of 2970 imaging features were extracted from pre- and postcontrast T1-weighted, T2-weighted, and apparent diffusion coefficient map. Using a random forest algorithm, nonredundant features were integrated with clinical data to generate a model predictive of IDH genotype. Our model achieved accuracies of 86% (area under the curve [AUC] = 0.8830) in the training cohort and 89% (AUC = 0.9231) in the validation cohort. Features with the highest predictive value included patient age as well as parametric intensity, texture, and shape features. Using a machine-learning algorithm, we achieved accurate prediction of IDH genotype in high-grade gliomas with preoperative clinical and MRI features.

Serum Amyloid A Type 1 Gene Polymorphism in Egyptian Children with Familial Mediterranean Fever

Background: Since spontaneous inflammation is an important contributor to familial Mediterranean fever (FMF), genetic variants mediating inflammation are of interest. We investigated gene variants in the acute-phase serum amyloid A type 1 (SAA1), a sensitive marker of inflammatory activity, and their association with susceptibility and severity of FMF. Methods: The genotypes of 2 single-nucleotide polymorphisms within exon 3 of SAA1 (2995C/T and 3010C/T) were determined in 105 Egyptian children with FMF and in 125 controls by polymerase chain reaction-restriction fragment length polymorphism. Genotyping of the causative MEFV mutations was performed by reverse hybridization. Results: The M694I mutation was the most frequent allele (42.8%), followed by V726A (18.6%), M680I (17.1%), E148Q (11.9%) and M694V (9.0%). The frequency of the SAA1 α, β and γ alleles was not significantly different between FMF patients and controls. The genotype frequency of SAA1 α/α was higher in patients than in healthy subjects (21.0 vs. 14.4%) although it did not reach statistical significance. The clinical manifestations including age at disease onset, number of FMF attacks, colchicine dose and severity score were not related to genotypes of SAA1. However, M694V mutation and female gender were significantly associated with severity. Conclusion: The genetic polymorphism of SAA1 is not associated with susceptibility and severity of FMF in Egyptian children.

Single cell genotyping of exome sequencing-identified mutations to characterize the clonal composition and evolution of inv(16) AML in a CBL mutated clonal hematopoiesis

We recently described the development of an inv(16) acute myeloid leukemia (AML) in a CBL mutated clonal hematopoiesis. Here, we further characterized the clonal composition and evolution of the AML based on the genetic information from the bulk specimen and analyses of individual bone marrow cells for mutations in CAND1, PTPRT, and DOCK6. To control for allele dropout, heterozygous polymorphisms located close to the respective mutation loci were assessed in parallel. The clonal composition concluded from exome sequencing suggested a proliferation advantage associated with the acquisition of mutations in CAND1, PTPRT, and DOCK6. Out of 102 single cell sequencing reactions on these mutations and the respective polymorphisms, analyses yielded conclusive results for at least 2 mutation sites in 12 cells. The single cell genotyping not only confirmed the co-occurrence of the PTPRT, CAND1 and DOCK6 mutations in the same AML clone but also revealed a clonal hierarchy, as the PTPRT mutation was likely acquired after the CAND1 and DOCK6 mutations. This insight had not been possible based solely on the exome sequencing data and suggests that the mutation in PTPRT, which encodes a STAT3-inhibiting protein tyrosine phosphatase, contributed to the AML development at a later stage by enhancing proliferation.

Molecular prevalence of multiple genetic disorders in Border collies in Japan and recommendations for genetic counselling

Reproductive management is necessary to prevent deleterious genetic disorders in purebred dogs, but comprehensive studies aimed at prevention of multiple underlying genetic disorders in a single breed have not been performed. The aims of this study were to examine mutant allele frequencies associated with multiple genetic disorders, using Border collies as a representative breed, and to make recommendations for prevention of the disorders. Genotyping of known mutations associated with seven recessive genetic disorders was performed using PCR assays. More than half (56%) of the Border collies had no mutant alleles associated with any of the seven disorders, suggesting that these disorders can be removed from the population over several generations. Since frequencies of each mutant allele differed among disorders, reproductive management should be performed after the establishment of prevention schemes that are appropriate for each disorder, the type and specificity of genetic test available, and the effective population size in each breeding colony.

Setup of a Protocol of Molecular Diagnosis of β-Thalassemia Mutations in Tunisia using Denaturing High-Performance Liquid Chromatography (DHPLC).

β-Thalassemia is one of the most prevalent worldwide autosomal recessive disorders. It presents a great molecular heterogeneity resulting from more than 200 causative mutations in the β-globin gene. In Tunisia, β-thalassemia represents the most prevalent monogenic hemoglobin disorder with 2.21% of carriers. Efficient and reliable mutation-screening methods are essential in order to establish appropriate prevention programs for at risk couples. The aim of the present study is to develop an efficient method based on the denaturing high-performance liquid chromatography (DHPLC) in which the whole β-globin gene (HBB) is screened for mutations covering about 90% of the spectrum. We have performed the validation of a DHPLC assay for direct genotyping of 11 known β-thalassemia mutations in the Tunisian population. DHPLC assay was established based on the analysis of 62 archival β-thalassemia samples previously genotyped then validated with full concordance on 50 tests with blind randomized samples previously genotyped with DNA sequencing and with 96% of consistency on 40 samples as a prospective study. Compared to other genotyping techniques, the DHPLC method can meet the requirements of direct genotyping of known β-thalassemia mutations in Tunisia and to be applied as a powerful tool for the genetic screening of prenatal and postnatal individuals.

Enhancement of PCR Detection Limit by Single-Tube Restriction Endonuclease-PCR (RE-PCR).

Polymerase chain reaction (PCR) is widely used in biological research and diagnostics because of its high sensitivity and specificity. However, the sensitivity of PCR is strongly influenced by topological characteristics of the template. Supercoiled templates are known to inhibit PCR, whereas linearized forms of the same supercoiled templates facilitate PCR. This study was conducted to compare the PCR efficiency of circular supercoiled DNA templates to their restriction endonuclease (RE)-mediated linearized forms. Additionally, we also evaluated the possibility of RE digestion of the circular supercoiled templates within the complete PCR buffer. Following a systematic approach, we demonstrated that circular supercoiled templates could be efficiently linearized by RE in the complete PCR buffer itself. This allowed linearization of circular supercoiled templates and their subsequent amplification in the PCR buffer in a single-tube format. Using this extremely simple RE-PCR approach, we documented up to tenfold increases in detection efficiency of PCR with two different circular supercoiled templates of clinical origin, including an international calibration standard. This inexpensive and easy approach to increasing PCR sensitivity can be easily adapted to any standard PCR protocol aimed at amplifying circular supercoiled genomes. Apart from its application in the development of sensitive clinical diagnostic PCR assays for a large number of organisms, this method could also prove to be very useful in simplifying the existing protocols for other applications where pre-PCR restriction digestion is required, such as mutation detection, genotyping, and selective template amplification.