Sanger Sequencing Results Research Articles

Molecular testing for H. pylori clarithromycin and quinolone resistance: a prospective Chinese study.

In China, there is a high prevalence of antibiotic-resistant Helicobacter pylori infections in the population. The aim of the study was to assess a new ARMS-PCR test for detection of H. pylori clarithromycin resistance (CR) and quinolone resistance (QR) mutations and evaluate the spectrum of antibiotic resistance in patients from three Chinese provinces. Sanger sequencing and multiplex ARMS-PCR were used to detect H. pylori CR and QR bacteria in gastric biopsy samples. Among the 1,182 patients enrolled with gastritis, 643 (54.4%) were positive for H. pylori. Of these, 371 (57.7%) had antibiotic-resistant strains, comprising 236 (63.6%) with a single drug antibiotic-resistant strain and 135 (36.4%) with multiple drug-resistant strains. Following Sanger sequencing analysis of 23S rRNA and gyrA gene for mutations (antibiotic resistance markers), rates of CR, QR, and multidrug resistance (CR and QR) were 19.9, 12.0, and 25.8%, respectively. The 23S rRNA CR mutation A2143G (286, 96.9%) and the gyrA QR mutations C261A (85, 31.5%) and G271A (71, 26.3%) were common. Benchmarking against Sanger sequencing results, multiplex ARMS-PCR test had a high diagnostic sensitivity and specificity for detection of CR (96 and 93%), QR (95 and 92%) and multidrug resistance (95 and 95%). Based on our findings, the high incidence of single and multiple antibiotic resistance requires the routine checking of antibiotic resistance in all patients with suspected H. pylori infections. Multiplex ARMS-PCR is a simple and rapid test that can be now used for more efficient treatment of H. pylori infections and reduces the misuse of antibiotics.

Discrimination of a single nucleotide polymorphism in the haptoglobin promoter region, rs5472, using a competitive fluorophore-labeled probe hybridization assay following loop-mediated isothermal amplification.

Personalized peptide vaccination, which involves activation of the host immune system against cancer cells using personalized peptide vaccines (PPVs), can improve overall survival in multiple cancer types. However, the clinical efficacies of PPVs vary for unknown reasons. Recently, a single nucleotide polymorphism (NG_012651.1:g.4461_5460[4960A>G]) in the haptoglobin promoter region, rs5472, was significantly associated with clinical response of PPV. Therefore, rs5472 is expected to be a predictive biomarker for PPV therapy. Here, we described a single nucleotide discrimination method for rs5472 analysis by combining the loop-mediated isothermal amplification and quenching probe methods. In evaluation of saliva samples, this method showed high concordance with the results of Sanger sequencing (100%, n=36). Importantly, this method did not require calculation of melting temperature for single nucleotide discrimination and could therefore be carried out on a simple instrument. Accordingly, this method may be more robust and applicable to near-patient testing.

Evaluation of clinical findings and neurofibromatosis type 1 bright objects on brain magnetic resonance images of 60 Turkish patients with NF1 gene variants.

Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene. This retrospective study aims to evaluate the clinical manifestations and brain magnetic resonance images (MRI) analysis in 60 genetically confirmed NF1 patients. The results of next-generation sequencing (NGS), Sanger sequencing, and MLPA of NF1 gene were evaluated. A total of 54 different variants were identified. Fourteen out of them were novel variants (25.9%). Patients who complied with NIH criteria had most frequently frameshift variants (11/32 patients), and those with only CALMs had missense variants (9/28 patients). Neurofibromatosis type 1 bright objects (NBOs) on T2-weighted MRI were detected in 42 patients (42/56; 75%). These brain lesions were detected mostly in basal ganglia and in cerebellar vermis. NBOs were detected more in the patients who complied with NIH criteria (80.6%) compared to those who were only CALMs (68%). While frameshift variants (33.3%) were the most common type variants in the patients who had NBOs, the most common variants were splicing (35.7%) and missense (35.7%) variants in the patients whose MRIs were normal. Frameshift variants (11/28 patients; 39.3%) were the most common in the patients with more than one brain locus involvement. Therefore, we consider that frameshift variants may be associated with increased incidence of NBOs and involvement of more than one brain locus. In addition, NBOs may occur less frequently in the patients with splicing variants. To our knowledge, this is the first study evaluated the relationship between NF1 gene variants and NBOs. Future studies may help us understand the etiology of NBOs.

Rapid and Simple Detection of Isoniazid-Resistant Mycobacterium tuberculosis Utilizing a DNA Chromatography-Based Technique.

Despite the availability of anti-tuberculosis drugs, the treatment of tuberculosis has been complicated by drug-resistant tuberculosis. The early detection of drug resistance makes early treatment possible. However, the available tools are mainly for rifampicin resistance detection, and the existing isoniazid resistance detection method is expensive, highly technical, and complicated, making it unsustainable for use in developing nations. This study aimed to develop a simple, rapid, and low-cost diagnostic kit for isoniazid-resistant tuberculosis using the single-stranded tag hybridization method to target an isoniazid resistance-conferring mutation. Specificity and sensitivity were assessed using DNA extracted from 49 isoniazid-resistant and 41 isoniazid-susceptible Mycobacterium tuberculosis clinical isolates cultured in mycobacterial growth indicator tubes. Positive signals were observed on mutant and wild-type lines with 100% sensitivity and specificity compared with Sanger sequencing results. In contrast, no positive signal was observed for non-tuberculosis mycobacteria. The detection limit of this method was 103 CFU or less. The STH-PAS system for isoniazid-resistant M. tuberculosis detection developed in this study offers a better alternative to conventional phenotypic isoniazid resistance determination, which will be of both clinical and epidemiological significance in resource-limited nations.

A custom amplicon sequencing approach to detect resistance associated mutations and sequence types in Mycoplasma genitalium

BackgroundMycoplasma genitalium resistance to antibiotic treatments is increasing, with very limited treatment alternatives on the horizon. Surveillance via sequencing of multiple M. genitalium loci would allow: monitoring of known antibiotic resistance mutations, associations between resistance/treatment failure and specific mutations, and strain typing for epidemiological purposes. In this study we assessed the performance of a custom amplicon sequencing approach, which negates the cost of library preparation for next generation sequencing. MethodsFifty-two M. genitalium positive samples (cervical, vaginal, anal and rectal swabs, and urine) were used. Three regions associated with M. genitalium antibiotic resistance (23S rRNA, parC and gyrA genes) were targeted, in conjunction with a locus used for differentiation of sequence types in the mgpB gene, and findings compared to Sanger sequencing. ResultsAmplicon sequencing provided adequate sequence read coverage (>30×) for the majority of samples for 23S rRNA gene (96%) and mgpB (97%), parC (78%) and gyrA (75%). Single nucleotide polymorphisms (SNPs) were characterised in samples for 23S rRNA gene (94%), parC (56%) and gyrA (4%). Unlike Sanger sequencing, mixed mutations could be identified by the amplicon sequencing method, and ratios of mutation types determined. All results, with one exception, were concordant to Sanger sequence results. Sequence diversity in the mgpB region was represented by 15 sequence types, 4 being observed in multiple samples. ConclusionsWe have demonstrated the utility of this custom amplicon sequencing approach for generating highly informative datasets with the capacity to identify and determine ratios of mixed sequences. The use of this customisable amplicon sequencing method enables cost effective, scalable amplicon sequencing of multiple target regions of interest in M. genitalium.

Molecular analysis of 76 Chinese hemophilia B pedigrees and the identification of 10 novel mutations.

BackgroundHemophilia B (HB) is an X‐linked recessive inherited bleeding disorder caused by mutations in the F9 gene that lead to plasma factor IX deficiency. To identify the causative mutations in HB, a molecular analysis of HB pedigrees in China was performed.MethodsUsing next‐generation sequencing (NGS) and an in‐house bioinformatics pipeline, 76 unrelated HB pedigrees were analyzed. The mutations identified were validated by comparison with the results of Sanger sequencing or Multiplex Ligation‐dependent Probe Amplification assays. The pathogenicity of the causative mutations was classified following the American College of Medical Genetics and Genomics guidelines.ResultsThe mutation detection rate was 94.74% (72/76) using NGS. Of the 76 HB pedigrees analyzed, 59 causative variants were found in 72 pedigrees, with 38 (64.41%) missense mutations, 9 (15.25%) nonsense mutations, 2 (3.39%) splicing mutations, 5 (8.47%) small deletions, 4 (6.78%) large deletions, and 1 intronic mutation (1.69%). Of the 59 different F9 mutations, 10 were novel: c.190T>G, c.199G>T, c.290G>C, c.322T>A, c.350_351insACAATAATTCCTA, c.391+5delG, c.416G>T, c.618_627delAGCTGAAACC, c.863delA, and c.1024_1027delACGA. Of these 10 novel mutations, a mosaic mutation, c.199G>T(p.Glu67Ter), was identified in a sporadic HB pedigree. Using in‐silico analysis, these novel variants were predicted to be disease‐causing. However, no potentially causative mutations were found in the F9 coding sequences of the four remaining HB pedigrees. In addition, two HB pedigrees carrying additional F8/F9 mutations were discovered.ConclusionThe identification of these mutations enriches the spectrum of F9 mutations and provides further insights into the pathogenesis of HB in the Chinese population.

Deciphering Aspergillus fumigatus cyp51A-mediated triazole resistance by pyrosequencing of respiratory specimens.

BackgroundInfections caused by triazole drug-resistant Aspergillus fumigatus are an increasing problem. The sensitivity of standard culture is poor, abrogating susceptibility testing. Early detection of resistance can improve patient outcomes, yet tools for this purpose are limited.ObjectivesTo develop and validate a pyrosequencing technique to detect resistance-conferring cyp51A polymorphisms from clinical respiratory specimens and A. fumigatus isolates.MethodsMethod validation was performed by Sanger sequencing and pyrosequencing of 50 A. fumigatus isolates with a spectrum of triazole susceptibility patterns. Then, 326 Aspergillus quantitative PCR (qPCR)-positive respiratory samples collected over a 27 month period (January 2017–March 2019) from 160 patients at the UK National Aspergillosis Centre were assessed by cyp51A pyrosequencing. The Sanger sequencing and pyrosequencing results were compared with those from high-volume culture and standard susceptibility testing.ResultsThe cyp51A genotypes of the 50 isolates analysed by pyrosequencing and Sanger sequencing matched. Of the 326 Aspergillus qPCR-positive respiratory specimens, 71.2% were reported with no A. fumigatus growth. Of these, 56.9% (132/232) demonstrated a WT cyp51A genotype and 31.5% (73/232) a resistant genotype by pyrosequencing. Pyrosequencing identified the environmental TR34/L98H mutation in 18.7% (61/326) of the samples in contrast to 6.4% (21/326) pan-azole resistance detected by culture. Importantly, pyrosequencing detected resistance earlier than culture in 23.3% of specimens.ConclusionsThe pyrosequencing assay described could detect a wide range of cyp51A polymorphisms associated with triazole resistance, including those not identified by commercial assays. This method allowed prompt recognition of resistance and the selection of appropriate antifungal treatment when culture was negative.

Abstract 2343: The incidence of BRAF mutation in non-small cell lung cancers is low in Korean patients

Abstract Introduction: BRAF mutation is present in about 8% of all cancer. Oncogenic activity of BRAF V600E mutation was established in lung cancers. Dabrafenib and trametinib combination therapy is preferred first line therapy for advanced or metastatic lung cancer with BRAF V600E mutation. However, there is only little basic knowledge of BRAF mutational status among Korean population. In this study, BRAF mutation, particularly BRAF V600E mutational status was explored among Korean non-small cell lung cancer patients. The clinical and pathologic characteristics of BRAF V600E mutation in non-small cell lung cancers were also investigated. Materials and Methods: The final cohort was 378 cases of primary non-small cell lung cancers. Fromalin-fixed paraffin embedded (FFPE) tissue blocks were used. PNA-mediated clamping PCR for BRAF V600, BRAF V600E real-time PCR and immunohistochemistry for mutation specific monoclonal antibody of Ventana VE1 were performed on all 378 cases of non-small cell lung cancers. For positive cases, direct sequencing was performed for validation. Results: BRAF V600 mutation was detected in five cases using PNA clamping method among 378 non-small cell carcinoma cases. By using BRAF real-time PCR, a BRAF V600E mutation was detected in 3 patients among total 378 cohort. There were 2 discordant cases between PNA clamping and real-time PCR. On immunohistochemistry, 3 patients showed positive staining for tumor cytoplasm and all positive cases also had positive results in real-time PCR. Direct Sanger sequencing was done in 5 cases that had any positive results for 3 methods. The results of Sanger sequencing was the same with those of real-time PCR and immunohistochemistry. Direct sequencing using PAN PCR product was performed on 2 negative cases for validation. On sequencing using PNA clamping PCR product, all cases showed mutation for BRAF gene other than V600E genotype. Finally, there were 3 cases (0.8%) of BRAF V600E mutation and 2 cases of BRAF mutation (0.5%) other than V600E genotype. Among BRAF mutated patients, there were 2 never smokers and 3 ever smokers. All patients harboring BRAF mutation had no concomitant EGFR or ALK aberrations. Conclusion: BRAF mutation incidence is relatively lower than that of Western data set and similar to that of Japanese report. Immunohistochemistry for Ventana VE1 antibody can be a useful screening method to detect BRAF V600E mutation. Citation Format: Ahrong Kim, Hyo Yeong Ahn, Chang Hun Lee, Young Dae Kim, Min Ki Lee, Yeon Joo Jeong, So Jeong Lee. The incidence of BRAF mutation in non-small cell lung cancers is low in Korean patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2343.

Whole‑exome sequencing identifies a novel mutation of SLC20A2 (c.C1849T) as a possible cause of hereditary multiple exostoses in a Chinese family.

Although the main causative genes for hereditary multiple exostoses (HME) are exostosin (EXT)-1 and EXT-2, there are numerous patients with HME without EXT-1 and EXT-2 mutations. The present study aimed to identify novel candidate genes for the development of HME in patients without EXT-1 and EXT-2 mutations. Whole-exome sequencing was performed in a Chinese family with HME and without EXT-1 and EXT-2 mutations, followed by a combined bioinformatics pipeline including annotation and filtering processes to identify candidate variants. Candidate variants were then validated using Sanger sequencing. A total of 1,830 original variants were revealed to be heterozygous mutations in three patients with HME which were not present in healthy controls. Two mutations [c.C1849T in solute carrier family 20 member 2 (SLC20A2) and c.G506A in leucine zipper and EF-hand containing transmembrane protein 1 (LETM1)] were identified as possible causative variants for HME through a bioinformatics filtering procedure and harmful prediction. Sanger sequencing results confirmed these two mutations in all patients with HME. A mutation in SLC20A2 (c.C1849T) led to a change in an amino acid (p.R617C), which may be involved in the development of HME by inducing metabolic disorders of phosphate and abnormal proliferation and differentiation in chondrocytes. In conclusion, the present study revealed two mutations [SLC20A2 (c.C1849T) and LETM1 (c.G506A) in a Chinese family with HME. The mutation in SLC20A2 (c.C1849T)] was more likely to be involved in the development of HME.

Detection of GNAQ/ 11 Mutations in Uveal Melanoma Patients' FFPE DNA with Droplet Digital PCR

To quantitatively detect GNAQ/11 mutations in uveal melanoma (UM) by droplet digital PCR (ddPCR). Formaldehyde-fixed paraffin-embedded (FFPE) tumor samples were taken from 78 UM patients with enucleation in West China Hospital between 2009 and 2015. None of the patients received radiotherapy or chemotherapy before enucleation. A retrospective study was conducted to detect GNAQ/11 mutation in UM by ddPCR. To compare the consistency of the results of the two detection methods, DNA sequencing was performed on the target gene by Sanger sequencing. 78 patients with UM were studied retrospectively. GNAQ/11 mutations in uveal melanoma was detected by ddPCR. The consistency of the results of the two detection methods was analyzed. GNAQ/11 mutations frequency was 91.9%. The consistency test between Sanger sequencing and ddPCR of GNAQ/11 mutations in 74 patients with UM was conducted. Kappa coefficient=0.436, P=0.001. The error rate of Sanger sequencing results was significantly higher in the heterogeneous group than in the homogeneous group (12/37 vs. 3/16, P=0.53), but the difference was not statistically significant. The results of ddPCR and Sanger sequencing showed good consistency, and the mutation ratio of GNAQ/11 in UM was significantly different. GNAQ/11 mutation frequency in UM patients detected by ddPCR was close to the reported frequency. It is more recommended to use ddPCR with high sensitivity to detect gene mutations in samples of tumor tissue DNA derived from FFPE. Sanger sequencing is prone to false negative results.

Screening for deafness-associated mitochondrial 12S rRNA mutations by using a multiplex allele-specific PCR method.

Mitochondrial 12S rRNA A1555G and C1494T mutations are the major contributors to hearing loss. As patients with these mutations are sensitive to aminoglycosides, mutational screening for 12S rRNA is therefore recommended before the use of aminoglycosides. Most recently, we developed a novel multiplex allele-specific PCR (MAS-PCR) that can be used for detecting A1555G and C1494T mutations. In the present study, we employed this MAS-PCR to screen the 12S rRNA mutations in 500 deaf patients and 300 controls from 5 community hospitals. After PCR and electrophoresis, two patients with A1555G and one patient with C1494T were identified, this was consistent with Sanger sequence results. We further traced the origin of three Chinese pedigrees. Clinical evaluation revealed variable phenotypes of hearing loss including severity, age at onset and audiometric configuration in these patients. Sequence analysis of the mitochondrial genomes from matrilineal relatives suggested the presence of three evolutionarily conserved mutations: tRNACys T5802C, tRNALys A8343G and tRNAThr G15930A, which may result the failure in tRNAs metabolism and lead to mitochondrial dysfunction that was responsible for deafness. However, the lack of any functional variants in GJB2, GJB3, GJB6 and TRMU suggested that nuclear genes may not play active roles in deafness expression. Hence, aminoglycosides and mitochondrial genetic background may contribute to the clinical expression of A1555G/C1494T-induced deafness. Our data indicated that the MAS-PCR was a fast, convenience method for screening the 12S rRNA mutations, which was useful for early detection and prevention of mitochondrial deafness.

Gene panel for Mendelian strokes

BackgroundMendelian stroke causes nearly 7% of ischaemic strokes and is also an important aetiology of cryptogenic stroke. Identifying the genetic abnormalities in Mendelian strokes is important as it would facilitate...

A novel missense mutation of CRYBB1 causes congenital cataract in a Chinese family.

To identify the pathogenic gene and mutation site of a Chinese family with congenital cataract. Eight family members and 100 controls were employed, and targeted exome sequencing was used to identify the genetically pathogenic factor of the proband. Targeted next-generation sequencing identified a novel missense mutation c.209A>C (p.Q70P) of CRYBB1 gene in the family. Sanger sequencing results showed that this heterozygous mutation was a causative mutation, which was not found in unaffected family members and healthy controls. Bioinformatics predicts that the effect of this mutation on protein function is probably harmful. We demonstrate that c.209A>C of CRYBB1 gene is a pathogenic mutation in the family of congenital nuclear cataract in this study. This is the first report that this mutation leads to congenital nuclear cataract, which broadens the mutation spectrum of CRYBB1 gene in congenital nuclear cataract.

VE1 Immunohistochemistry Improves the Limit of Genotyping for Detecting BRAFV600E Mutation in Papillary Thyroid Cancer.

Detection of BRAFV600E is useful for making diagnosis and risk stratification of papillary thyroid carcinoma (PTC). Molecular testing, however, is not always available for routine clinical use. To assess the clinical utility and reliability of VE1 immunohistochemistry (IHC) for detecting BRAFV600E mutation in PTC, VE1 IHC was performed on the tissue microarrays of 514 patients with PTC and was compared with Sanger sequencing results. Of 514 PTC cases, 433 (84.2%) were positive for VE1 expression. Among 6 discordant cases between VE1 IHC and Sanger sequencing, 3 initial VE1-false negative cases turned out to be true false negative on repeat testing, and 3 VE1-false positive cases showed BRAFV600E mutation using digital PCR analysis. PTCs with low variant allele fraction were positive for VE1 IHC but were not detected using sequencing. VE1 IHC showed 99.3% sensitivity, 100% specificity, 100% positive predictive value, and 96.4% negative predictive value. The BRAFV600E mutation was significantly associated with older age, multifocality, extrathyroidal extension, lymph node metastasis, and advanced tumor stage. In conclusion, VE1 IHC is a reliable method for detecting BRAFV600E mutation in PTC specimens.

Identification and clinical implications of a novel pathogenic variant in the GJB2 gene causes autosomal recessive non-syndromic hearing loss in a consanguineous Iranian family.

Mutations in the GJB2 gene, which encodes the connexin26 protein and is involved in inner ear homeostasis, are the most common cause of autosomal recessive non-syndromic hearing loss (ARNSHL) in many populations. This study was aimed to determine the molecular etiology in a consanguineous Iranian family affected by profound ARNSHL. A comprehensive family history was obtained, and clinical evaluations and pedigree analysis were performed in the family with 4 affected members. After extraction of genomic DNA, the entire coding region of GJB2 was directly sequenced in all family members. In silico analyses were also performed using available software tools. Sanger sequencing results showed a novel rare homozygous variant (c.109_110insG) in the GJB2 gene. This frameshift variant in exon 2 of the GJB2 gene fulfills the criteria of being categorized as pathogenic according to the American College of Medical Genetics and Genomics (ACMG) guideline. Confirmation was done with the co-segregation study and checking the frequency of the novel variant in 100 ethnically matched normal control subjects. The present study suggests that investigation of GJB2 mutations may still be useful to determine the etiology of HL in Iran.

Application of PCR reverse dot blot in non-syndromic deafness gene detection

Objective:To detect 20 common deafness gene mutations in non- syndromic deafness patients in China using PCR- RDB, and analyze and summarize the mutation data to explore the clinical value of this method. Method:The PCR- RDB and Sanger sequencing were used to detect 20 common mutations of four deafness genes（GJB2, GJB3, SLC26A4 and mtDNA） in 500 patients with non- syndromic hearing loss . The Sanger sequencing was used to compare the sensitivity, specificity, positive predictive value, negative predictive value, and total coincidence rate of the deafness mutation detected by PCR- RDB. Result:A total of 500 samples were detected. 147 wild- type samples, 81 homozygous mutant samples, 240 heterozygous mutant samples, 32 composite heterozygous mutant samples were detected using the PCR- RDB within the range of 20 gene mutations, which were identical to the Sanger sequencing results. GJB2 c.235delC and SLC26A4 c.919- 2 A>G are the most common hotspot mutations in this study, followed by mtDNA m. 1555 A>G. Compared with the Sanger sequencing method, the sensitivity, specificity, positive predictive value, negative predictive value, and total coincidence rate of the real- time fluorescence PCR melting curve method were 100%, and the Kappa value was one. Conclusion:PCR reverse dot-blot hybridization is a simple, rapid, sensitive and specific method for detecting 20 mutations of 4 common deafness genes in Chinese population, it is expected to be used in clinical detection of deafness genes in the future.

Establishment of SHOX CNE9/10 knock-out osteosarcoma U2OS cell and its effects on cell growth and apoptosis

BackgroundOsteosarcoma is a common malignant tumor of musculoskeletal stromal cells. Osteosarcoma clinical behavior depends mostly on the histologic grade, the site of primary tumor, the response to chemotherapy, and the presence of pulmonary metastases. The aim of this study was to knockout SHOX CNE9/10 in U2OS osteosarcoma cells and to analyze the effects on cell growth and apoptosis.Material/MethodsU2OS cells with CNE9 knockout and U2OS cells with CNE10 knockout were established via the CRISPR/Cas9 system. Sanger sequencing was used to detect the success of the knockdown experiment. Western blotting and quantitative polymerase chain reaction were used to detect the expression levels of short stature homeobox-containing gene (SHOX) protein and messenger RNA (mRNA) after knockdown of CNE9 and CNE10. The cell viability and apoptotic rate were detected by the Cell Counting Kit-8 method and by flow cytometry.ResultsThe Sanger sequencing results showed that the knockdown experiment was successful. The levels of SHOX mRNA and protein were significantly reduced after knocking down CNE9 and CNE10. Knockdown of CNE9 and CNE10 significantly increased the growth and inhibited the apoptosis of U2OS osteosarcoma cells. CNE9/CNE10 knockdown U2OS cells were successfully constructed.ConclusionsKnockdown of CNE9 and CNE10 promoted U2OS cell growth and inhibited apoptosis by decreasing SHOX expression. This CNE9/CNE10 knockout U2OS cell model could provide a bridge for the research on SHOX and CNEs in osteosarcoma.

A report of Hb Fontainebleau [α21 (B2) Ala > Pro] as a result of founder effect phenomenon

ObjectiveTo date, more than 400 alpha chain variants or point mutations in the α-globin genes that lead to single amino acid substitutions have been described. Here, we state a rare alpha-globin gene variant Hb Fontainebleau [a21 (B2) Ala>Pro] identified in heterozygote state in two families from the north of Iran during premarital thalassemia screening program. MaterialTwo first cousin couples with reduced hematological indices compatible with α-thalassemia were referred to Lab for premarital screening of thalassemia. At first CBC and capillary electrophoresis tests were applied. For molecular investigation genomic DNA was isolated from peripheral blood samples. Then, multiplex-Gap PCR and Sanger sequencing techniques were applied to discover common α- globin deletions and probable point mutations, respectively. ResultsIn the capillary electrophoresis results, one unknown peak was observed to the left of Hb A. Sanger sequencing results revealed that all cases are heterozygote for Hb Fontainebleau (HBA2: c.64G4C, p.Ala21Pro). Family history analysis has shown that all presented cases have belonged to the families which are originally from India. ConclusionThe presence of Hb Fontainebleau in Mazandaran province and in subjects with Indian origin may indicate the founder effect phenomenon. DNA analysis of subjects with reduced hematological indices is recommended for finding common and rare mutations to predict childbirth with thalassemia in premarital screening programs.

Rapid and sensitive screening and identification of CRISPR/Cas9 edited rice plants using quantitative real-time PCR coupled with high resolution melting analysis

Gene-editing techniques, such as TALEN and CRISPR/Cas9, have been widely used for target DNA editing in many research fields. However, how to rapidly screen and identify the expected gene-edited products with high efficiency and low cost is still a difficult task. Here, we report the development and optimization of one such method that combines quantitative PCR with high-resolution melting analysis (qPCR-HRM) to screen and identify CRISPR/Cas9-edited rice plants. The results showed that gene-edited rice plants with small target DNA in/dels or even single base pair insertion/deletions could be successfully identified. The sensitivity of the qPCR-HRM method is as low as 1%, which is satisfying to be used for quantitative evaluation of gene-editing efficiency. Sanger sequencing results confirmed that the qPCR-HRM method also has high accuracy. It is concluded that the developed qPCR-HRM method is reproducible, accurate, and efficient for quick screening and identification of gene-edited rice plants.

Исследование частоты встречаемости двойной ВИЧ-инфекции в России

Objective. To develop and validate the method of dual HIV infection detection based on the results of Sanger sequencing and to evaluate of dual HIV infection frequency in Russia. Materials and methods. We carried out the sequencing of HIV-1 pol gene fragment in model samples of dual HIV infection, the calculation of degeneracy bases index (DB-index), and the index of synonymity (SM-index) and determination of their threshold values for dual HIV infection detection. We analyzed nucleotide sequences of the pol gene fragment in HIV-1 samples (n = 1561) taken from Russian patients. Results. The threshold values of 34 for the DB-index and 0.05 for the SM-index allow detecting effectively the dual HIV infection using reverse transcriptase (RT) and protease-reverse transcriptase (PR-RT) fragments of the pol gene correspondingly. At total 21 (1.35%) dual HIV-infected samples (18 of which were collected in 2014 and later) were revealed in the collection studied; 12 of them were obtained from patients residing in the Central Federal District. An inverse correlation between the likelihood of dual HIV infection detecting and the duration of the patient's infection was found. In total, 11 out of 21 samples with dual HIV infection had mutations associated with drug resistance; 7 of them (33.3%) had substitutions that were not associated with natural polymorphism of HIV-1. Conclusion. The problem of dual HIV infection in Russia is actual and requires further study. Key words: HIV, dual HIV infection, drug resistance, nucleotide sequence, DB-index, SM-index, PR-RT region