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High resolution melting curve analysis targeting the HBB gene mutational hot-spot offers a reliable screening approach for all common as well as most of the rare beta-globin gene mutations in Bangladesh
BMC Genetics volume 19, Article number: 1 (2018)
Bangladesh lies in the global thalassemia belt, which has a defined mutational hot-spot in the beta-globin gene. The high carrier frequencies of beta-thalassemia trait and hemoglobin E-trait in Bangladesh necessitate a reliable DNA-based carrier screening approach that could supplement the use of hematological and electrophoretic indices to overcome the barriers of carrier screening. With this view in mind, the study aimed to establish a high resolution melting (HRM) curve-based rapid and reliable mutation screening method targeting the mutational hot-spot of South Asian and Southeast Asian countries that encompasses exon-1 (c.1 - c.92), intron-1 (c.92 + 1 - c.92 + 130) and a portion of exon-2 (c.93 - c.217) of the HBB gene which harbors more than 95% of mutant alleles responsible for beta-thalassemia in Bangladesh.
Our HRM approach could successfully differentiate ten beta-globin gene mutations, namely c.79G > A, c.92 + 5G > C, c.126_129delCTTT, c.27_28insG, c.46delT, c.47G > A, c.92G > C, c.92 + 130G > C, c.126delC and c.135delC in heterozygous states from the wild type alleles, implying the significance of the approach for carrier screening as the first three of these mutations account for ~85% of total mutant alleles in Bangladesh. Moreover, different combinations of compound heterozygous mutations were found to generate melt curves that were distinct from the wild type alleles and from one another. Based on the findings, sixteen reference samples were run in parallel to 41 unknown specimens to perform direct genotyping of the beta-thalassemia specimens using HRM. The HRM-based genotyping of the unknown specimens showed 100% consistency with the sequencing result.
Targeting the mutational hot-spot, the HRM approach could be successfully applied for screening of beta-thalassemia carriers in Bangladesh as well as in other countries of South Asia and Southeast Asia. The approach could be a useful supplement of hematological and electrophortic indices in order to avoid false positive and false negative results.
The hemoglobinopathies are a group of inherited monogenic disorders of erythrocytes that affect millions of people worldwide [1, 2]. These disorders are caused by mutation(s) of α or β globin gene of hemoglobin that result in a missing or a defective production of the oxygen-transport protein [3]. Beta thalassemia is characterized by an abnormal or absent production of hemoglobin due to mutation(s) in the protein-coding part or the intron of the beta-globin gene or its promoter in chromosome 11 [4,5,6]. There are at least 893 beta-globin gene mutations reported till date (http://globin.bx.psu.edu/cgi-bin/hbvar/query_vars3). Even though large numbers of beta-globin gene mutations have been reported throughout the world, the β-thalassemia patients from each ethnic population carry only a limited number of mutations which comprise of some frequently occurring mutations as well as some rare mutations. For example, five common mutations, namely c.92 + 5G > C, c.92 + 1G > T, c.126_129delCTTT, c.27_28insG and the 619 bp deletion have been reported to account for over 90% of the mutations in beta-thalassemia patients in India [7, 8]. Similar to India, only five mutations, including c.-78A > G, c.52A > T, c.126_129delCTTT, c.216_217insA and c.316-197C > T in HBB gene are responsible for 90% of beta-thalassemia patients in china [9].
The majority of the beta-thalassemia mutations in Bangladeshi population have been reported to originate in the neighboring India, Southeast Asian countries, Sri Lanka, Pakistan etc. [10,11,12,13]. In Bangladesh, the most frequently occurring mutations, namely c.79G > A, c.92 + 5G > C and c.126_129delCTTT; and the less common mutations c.92G > C, c.27_28insG, c.47G > A, c.92G > A, c.46delT, c.92 + 130G > C, and c.51delC have been accounted for more than 95% of beta thalassemia patients [13, 14]. All of these mutations together constitute the mutational hot-spot (c.1 – c.92 of exon 1, c.92 + 1 – c.92 + 130 intron-1 and c.93 – c.217 of exon-2) in the HBB gene of Bangladeshi thalassemia patients. The same hot spot harbors majority of HBB gene mutations in many other countries of South Asia and Southeast Asia. Hence, the hot-spot could be a useful target to screen the beta-globin gene mutations and therefore can serve as a supplement to hematological and electrophoretic indices to overcome certain limitations of these approaches. For instance, the screening of beta-thalassemia carrier which is based solely on hematological or electrophoretic indices may miss a significant number of carriers due to adequacy of mild variant c.79G > A and complex inheritance of other factors such as presence of α-globin gene mutations, mutation in Krüppel-like Factor 1 (KLF1) gene, promoter mutations of gamma-globin gene, iron deficiency anemia, hereditary persistence of fetal Hb etc. [15].
High Resolution Melting (HRM) Analysis is a rapid, high-throughput technique for screening genetic variations in the nucleic acid sequences using the melting properties of double stranded DNA. The HRM analysis enables researchers to rapidly detect and categorize mutations, identify new genetic variants without sequencing. It has been successfully used in detection of mutations in various types of genetic diseases including autosomal recessive, autosomal dominant and X-linked recessive disorders as well as diseases involving somatic mutations [16,17,18,19,20]. Although the HRM analysis has been developed for high throughput screening of mutations in the beta-globin gene in various countries [21, 22], the procedure has not been initiated yet in Bangladesh. The study aimed to devise a rapid mutation screening using HRM curve analysis targeting the hot-spot region. Since the targeted hot-spot harbors most of the common beta-globin gene mutations reported in Bangladesh and other countries of South Asia and Southeast Asia, it could be applied for carrier screening in Bangladesh and some regional countries.
Study participants
A total of 142 transfusion-dependent beta-thalassemia patients in the age range of 1–15 years who came to Dhaka Shisu Hospital (DSH) for follow-up examination for CBC and Hb level followed by blood transfusion were enrolled in the study. The patients had been previously diagnosed with beta-thalassemia by Hb electrophoresis and/or microscopic evaluation of thin blood film. The study also included ninety beta-thalassemia carrier parents and 15 healthy controls who were not thalassemia carriers.
DNA isolation from whole blood
1 mL of blood was collected in EDTA-coated vacutainer. Genomic DNA (gDNA) was extracted from the collected blood using QIAGEN flexigene® DNA kit (Qiagen, Hilden, Germany) following manufacturer's instructions.
Polymerase chain reactions for beta-globin gene amplification
The primers targeting the 428 bp long stretch of HBB gene encompassing the mutational hot-spot region (exon 1, intron 1 and a portion of exon 2) of beta-globin gene in Bangladesh was used for the polymerase chain reactions (PCR) (Table 1) [13, 14]. The PCR amplification was performed in a final reaction volume of 30 μL using 150 ng gDNA as template and the compositions of the PCR components were as follows: 3.0 μL of 10 x PCR buffer (with 1.5 mM MgCl2), 0.9 μL of MgCl2 (25 mM), 6.0 μL of Q-solution (Qiagen), 4.8 μL of dNTP mixture (2.5 mM), 0.6 μL of forward primer (HBB_Ex1F) (10 mM) and 0.6 μL of reverse primer (HBB_Ex2R) (10 mM), 0.3 μL of HotStart Taq DNA polymerase (Qiagen). Finally, the total reaction volume was made 30.0 μL with nuclease-free water. The following thermal cycling profile was used for PCR amplification: initial denaturation at 94 °C for 15 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 40 s and extension at 72 °C for 40 s; and a final extension at 72 °C for 5 min.
Purification of PCR product
Purification of the PCR products was performed using the MinElute® PCR purification kit (Qiagen) following the manufacture's guidelines.
Sequencing of PCR products
Following manufacturer's instruction, the cycle sequencing of the column-purified PCR product was performed using the Big Dye Version 3.1 Cycle Sequencing Kit (Applied Biosystems, Warrington, UK). Thereafter, the cycle sequencing product was purified using the BigDye® XTerminator™ purification kit (Applied Biosystems) and the manufacturer's protocol was followed for the purification steps. Finally, the purified cycle sequencing PCR product was subjected to capillary sequencing in an ABI PRISM 310 Automated Sequencer (Applied Biosystems).
Sequencing data collection and identification of mutations
The sequencing data were collected using ABI PRISM 310 data collection software version 3.1.0 (Applied Biosystems). The collected data were exported in FASTA format which were then analyzed to identify substitution or deletion mutations by using Basic Local Alignment Search Tool (BLAST), which compared the query sequence with the reference (wild type) sequence (NC_000011.10) retrieved from NCBI database.
Real-time PCR-based high resolution melting curve analysis
The real time PCR reactions were performed in a 10 μL reaction volume in a 96-well reaction plate. The PCR mixture contained 5 μL of 2X precision melt supermix (BioRad), 0.2 μL of the forward primer (10 μM), 0.2 μL of the reverse primer (10 μM), 50 ng of genomic DNA in a total of 10 μL reaction volume. The list of primers used for high resolution melt curve analysis is shown in Table 2. The real-time PCR was followed by HRM analysis and the procedures were performed in a CFX96 Touch™ Real-Time PCR machine (BioRad). The real-time PCR with the first set of primers (P1R1) had the following thermal cycling protocol: initial denaturation at 95 °C for 2 min; 40 cycles of denaturation at 94 °C for 10 s, annealing at 60 °C for 15 s and extension at 72 °C for 15 s. Similar to the first set of primers, the thermal cycling protocol for the 2nd set of primers (P2R2) was exactly same except that the annealing temperature for the latter was set to 580c instead of 600c. After the real time PCR was completed, the subsequent melt curve program was identical for both primer sets and it had the following three steps: denaturation at 95 °C for 30 s, renaturation at 60 °C for 30 s, and then melting at 65 °C to 95 °C with an increment of 0.10c per 5 s. After completion of the real time PCR-HRM, the data were analyzed using Precision Melt Analysis™ Software (BioRad). The melt curve shape sensitivity for cluster detection was set to 100%. The difference in Tm threshold for the cluster detection was set to 0.1 and the normalized and the temperature shifted views were used for analysis.
Selection of mutational hot-spot in the HBB gene
Previous studies revealed that most of the beta-globin (HBB) gene mutations (>95%) in Bangladeshi thalassemia patients including three most common mutations, namely c.79G > A, c.92 + 5 G > C and c.126_129delCTTT account for ~85% of the beta-globin gene mutant alleles and these mutations are confined in the exon-1 (c.1 – c.92), the intron-1 (c.92 + 1 – c.92 + 130) and a portion of exon-2 (c.93 – c.217), thus constituting the hot-spot region (Table 3 and Fig. 1). The same hot-spot accounts for over 90% mutations in the HBB gene in India (West Bengal, Uttar Pradesh, Eastern India, and Southern India), Sri Lanka, and Malaysia and over 83%, 82% and 80% of the HBB gene mutations in Thailand, Pakistan, and Mayanmar, respectively [13, 14, 23,24,25,26,27,28]. Therefore, the HBB gene hot-spot region was targeted for mutation screening by the high resolution melting curve (HRM) analysis. With this view in mind, two sets of primers flanking the hot-spot were designed to analyze beta-thalassemia DNA specimens by the HRM analysis. The first set of primers (Forward P1: c.1 – c.21 and Reverse R1: reverse complement of c.92 + 88 – c.92 + 111) could identify the mutations that were flanked in a portion of exon-1 and intron-1, while the second set of primers (Forward P2: c.92 + 88 – c.92 + 111 and Reverse R2: reverse complement of c.207 – c.228) flanked a portion of intron-1 and exon-2 of the hot-spot region. The interfering effect of the SNP c.9 T > C, which is frequently found in Bangladeshi population, was avoided by using a primer (Forward P1: c.1 – c.21) that could overlay the SNP [29]. As the primer overlay the SNP, it did not become a part of the final PCR product, indicating that the SNP c.9 T > C could not influence the HRM results by its interfering effect. Overall, the primer sets targeting the hot-spot region did have a significance to detect majority of the beta-globin gene mutations.
Selection of reference samples with known mutations by sequencing of beta-thalassemia DNA specimens targeting the HBB gene hot-spot
Targeting the hot-spot region of beta-globin gene, a total of 101 beta-thalassemia DNA specimens were subjected to Sanger sequencing. The DNA sequencing data demonstrated that 98% (99/101) of the specimens had either compound heterozygous mutations or homozygous mutations and 2% (2/101) had heterozygous mutations within the hot-spot, indicating the validity of the use of the hot-spot to detect the beta-globin gene mutations in Bangladesh. Accordingly, we could identify a total of twelve different mutations; namely c.27_28insG, c.33C > A, c.46delT, c.47G > A, c.51delC, c.79G > A, c.92G > C, c.92 + 5G > C, c.92 + 130G > C, c.126delC, c.126_129delCTTT and c.135delC. The combination patterns of mutations which had been identified as the genotypes of the recessive disorder are presented in Table 4. Among the above-mentioned 12 mutations, c.33C > A, c.126delC, and c.135delC mutations had not been reported previously for the Bangladeshi population. Based on the mutational information, 40 participants who were parents of beta-thalassemia patients were recruited and their DNA specimens were sequenced to get samples with known heterozygous mutations (Additional file 1: Table S1). Parents of beta-thalassemia participants with rare compound heterozygous mutations c.33C > A and c.51delC were not available in the study. The specimens with known mutations, as manifested by DNA sequencing of beta-thalassemia patients and their parents, were used than for initial evaluation of HRM approach for heterozygous mutations screening, which targeted 10 mutations namely, c.27_28insG, c.46delT, c.47G > A, c.79G > A, c.92G > C, c.92 + 5G > C, c.92 + 130G > C, c.126delC, c.126_129delCTTT and c.135delC of the beta-globin gene.
Furthermore, as a means of cost-saving approach, sixteen out of 101 beta-thalassemia specimens with sequence-based known mutations were selected as references for HRM analysis to see whether the approach could perform direct genotyping of beta-thalassemia patients. The references were sufficient to represent all of the 12 mutations and their common compound heterozygous combination patterns because three reference samples had homozygous c.79G > A, c.92 + 5 G > C, and c.126_129delCTTT mutations and 12 had compound heterozygous mutations, namely c.79G > A plus c.92 + 5G > C, c.47G > A plus c.79G > A, c.79G > A plus c.126_129delCTTT, c.46delT plus c.79G > A, c.27-28insG plus c.79G > A, c.92G > C plus c.92 + 5G > C, c.79G > A plus c.92G > C, c.92 + 5G > C plus c.92 + 130G > C, c.92 + 5G > C plus c.126_129delCTTT, c.79G > A plus c.92 + 130G > C, c.33C > A plus c.51delC, and c.79G > A plus c.126delC and a sample had heterozygous c.135delC deletion.
Screening of heterozygous beta-globin gene mutations by HRM
False negative results due to the presence of mild allele such as c.79G > A is a big concern in the mean corpuscular volume (MCV) ≤80 fL based screening of beta-thalassemia carrier. Furthermore, the electrophoretic parameter HbA2-dependent screening may also give rise to false negative or false positive results based on the coinheritance of the α-globin and Krüppel-like Factor 1 (KLF1) genes mutations, the gamma-globin gene promoter mutations, and iron deficiency anemia. Thus, the establishment of HRM-based screening might be useful for carrier screening to supplement the hematological and electrophoretic indices. Previous studies have demonstrated that c.79G > A, c.92 + 5G > C and c.126_129delCTTT mutations account for 85% of beta-globin related complications. The pre-genotyped specimens were subjected to HRM analysis. The normalized melt curves for c.79G > A, and c.92 + 5G > C mutations are shown in Additional file 2: Figure S1; whereas the normalized melt curves for c.126_129delCTTT mutation are shown in Additional file 3: Figure S2. The temperature shifted difference curves of these three HBB gene mutations are shown in Fig. 2. The difference curves generated by the heterozygous and homozygous alleles could be easily distinguished from the wild type samples based on differences in the melting curve shapes. As shown in Fig. 2a, the difference in relative fluorescence unit (RFU) for homozygous c.79G > A allele was always higher than that for the wild type allele. However, the difference in RFU for heterozygous c.79G > A allele was initially higher, which then dropped below the wild type allele and then finally raised again above the wild type allele. For the homozygous and heterozygous c.92 + 5G > C alleles, the difference in RFU was always lower than that for the wild type allele. However, the heterozygous c.92 + 5G > C allele had a greater reduction in fluorescence intensity compared to the homozygous c.92 + 5G > C allele (Fig. 2a). The allele with deletion mutation c.126_129delCTTT in homozygous state could produce a melt curve that always had a higher fluorescence intensity compared to its wild type counterpart throughout the melting procedure. In contrast, the heterozygous c.126_129delCTTT allele could generate a melt curve with higher fluorescence intensity in the later stages of melting compared to the wild type allele (Fig. 2b).
Moreover, other less frequent heterozygous mutations including c.27_28insG, c.46delT, c.47G > A, c.92G > C and c.92 + 130G > C, and previously unreported mutations in Bangladesh, namely c.126delC and c.135delC were also distinguishable from their respective wild type alleles (Fig. 2c, Fig. 2d, Additional file 4: Figure S3 and Additional file 5: Figure S4). As presented by the normalized and temperature shifted difference curves (Fig. 2c, and Fig. 2d), all of these heterozygous mutational statuses showed a weaker fluorescence intensity during melting compared to their wild type counterparts except c.47G > A, which showed a lower fluorescence intensity during the initial phases of the melting and a higher fluorescence intensity in the latter phases.
After initial testing of pre-genotyped samples, 50 specimens from the parents of beta-thalassemia patients were analyzed without any prior knowledge of their genotypes to validate the assay. All of the specimens had sequence variations from their respective wild type alleles. The sequencing data revealed that 29 out of 50 specimens had c.92 + 5G > C, 18 had c.79G > A, 2 had c.126_129delCTTT and one had c.126delC heterozygous states, respectively (Additional file 6: Figure S5 and Additional file 7: Figure S6). It was noticeable that all c.92 + 5G > C heterozygotes together formed a cluster. Similarly, c.79G > A and c.126_129delCTTT heterozygotes also formed their respective clusters. One c.126delC formed its distinct melt curve which was distinguishable from other heterozygotes. The significance of the approach lies in the fact that it could be applied for mass carrier screening for prevention and control of beta-thalassemia.
Compound heterozygous mutations could be distinguished from wild type alleles by HRM
Our data have already demonstrated that both common as well as rare mutations could be screened using HRM. Next, as a means of cost-saving approach, we wanted to observe whether the compound heterozygous mutations could be separated from their respective wild type alleles for direct genotyping of beta-thalassemia patients. Mutations in the HBB gene other than in the positions c.79 and c.92 + 5 usually occur as co-existing partners either with c.79G > A or c.92 + 5G > C in beta-thalassemia patients. Most of the other HBB gene mutations of beta-thalassemia patients had been detected in close proximity to the common mutations, namely c.79 G > A and c.92 + 5 G > C, suggesting that the compound heterozygous mutations could be screened using a single pair of primers. The compound heterozygous mutations including c.47G > A plus c.79G > A, c.79G > A plus c.92G > C, c.46delT plus c.79G > A, c.27_28insG plus c.79G > A, c.79G > A plus c.92 + 5 G > C, and c.92G > C plus c.92 + 5 G > C generated curves that were distinguishable from the wild type alleles (Fig. 3a). The dsDNA melting patterns generated by combination mutation partners, namely 46delT plus c.79G > A, c.27_28insG plus c.79G > A, c.79G > A plus c.92 + 5 G > C, and c.92G > C plus c.92 + 5 G > C could be unambiguously distinguished from the wild type alleles due to a significant reduction in RFU during melting. On the other hand, the compound heterozygous mutations c.79G > A plus c.92G > C generated a melt curve that had a relatively shorter plateau followed by a longer curved path, which then reached the peak above the wild type RFU path. Moreover, the melting curve pattern generated by c.47G > A plus c.79G > A was more diverse than any other combinations of mutations. It produced the longest plateau in the beginning and then followed the curved path below the wild type allele and finally reached the peak point which lay above the path of wild type allele.
The rare compound heterozygous mutations c.33C > A plus c.51delC could also be distinguished from the wild type allele due to its distinctive melting pattern that had a relatively lower fluorescence intensity in the first half of the melting and had a higher fluorescence intensity in the second half of the melting compared to its wild type counterpart (Fig. 3b). The findings indicate that the compound heterozygous mutations could be screened by HRM.
Direct genotyping of unknown beta-thalassemia samples using HRM
The HRM curve analysis with reference samples of known mutations has already demonstrated that HRM could be a very good approach for distinguishing different types of mutations from the wild type alleles. Combinations of mutations could also be distinguished from one another by HRM. The findings prompted us to investigate whether the HRM approach could be applied for genotyping of beta-thalassemia samples for the confirmation of the disease status. In order to test this hypothesis, we performed HRM analysis on 41 unknown samples without any prior knowledge of their mutational status. The reference samples with known mutations were also run side-by-side to calibrate the data. All of these forty one samples differed in HRM curve patterns from the control wild type alleles. Each one of them had exhibited melting patterns similar to that of the corresponding reference samples with known homozygous, heterozygous or compound heterozygous mutations (Fig. 4, Fig. 5 and Table 5). Among forty one unknown samples, twenty three specimens generated the melt curves similar to one generated by the reference sample having compound heterozygous c.79G > A plus c.92 + 5G > C mutations, eight specimens produced the melt curves that resembled one generated by the reference sample with homozygous c.92 + 5G > C mutation, and two samples generated the melt curves that resembled the melt curve produced by the reference sample having compound heterozygous mutation c.79G > A plus c.126_129delCTTT. Each of the remaining eight unknown samples generated a single melt curve that resembled the melt curve produced by reference samples with the following homozygous mutation c.79G > A and compound heterozygous mutations c.27-28insG plus c.79G > A, c.47G > A plus c.79G > A, c.92 + 5 G > C plusc.92 + 130 G > C, c.92 + 5 G > C plus c.126_129delCTTT, c.79G > A plus c.92G > C, c.46delT plus c.79G > A, and c.92G > C plusc.92 + 5 G > C, respectively. The DNA sequencing data of the unknown samples also validated the HRM result (Table 5).
In summary, the HRM approach based on mutational hot-spot could be useful for screening of carriers as well as disease status of beta-thalassemia in Bangladesh. There might have new HRM patterns other than the references and in such cases the samples would require sequencing for identification of the mutations.
Inherited disorders of Haemoglobin make up the most commonly observed genetic diseases, resulting in significant morbidity and mortality worldwide. It has been reported that between 300,000 to 400,000 children are born with severe hemoglobinopathies each year, and among them 95% are affected with abnormalities of the β-globin gene (HBB) [1]. A study conducted on school children in Bangladesh demonstrated that the beta-thalassemia and hemoglobin E carrier statuses were 4.1% and 6.1% respectively [30], indicating that as many as 8990 thalassemia babies are born each year in Bangladesh with its population of about 160 million and a birth rate of 2.16%, as manifested by Hardy-Weinberg equation. Now, it has been agreed upon at all levels of public and private policy-making to create general awareness and adopt appropriate preventive and control measurements for reduction of beta-thalassemia in Bangladesh. Adoption of mass carrier screening, especially premarital and prenatal screening for carrier detection is required for proper counseling and goal-oriented motivation. In 1970s, the premarital screening programs were initiated in Greece and Cyprus and these proved to be huge successes in decreasing the total number of children being born with β-thalassaemia [31, 32]. Then a number of countries where beta-thalassemia had high prevalence, adopted an approach similar to that of Greece and Cyprus in pursuit of beta-thalassemia prevention [33]. The success of such premarital screening programs depends on the rapidity, reliability and cost-effectiveness of the procedures used to detect the carriers. The conventional strategy for thalassemia screening includes (a) analysis of blood specimens for CBC, where MCV value ≤80 fL indicates thalassemia carriers, (b) analysis of electrophoresis indices for specimens of the suspected carriers, where a specimen with HbA2 > 3.5 is highly considered as a thalassemia carrier, and (c) testing of specimens with HbA2 > 3.5 at the DNA level, where a mutation in any globin gene confirms the carrier status. Each step of the carrier screening has some limitations and we opted to use HRM to overcome those barriers to supplement the conventional methodologies for thalassemia screening [15]. Needless to say, the present approach focused only on beta-thalassemia because it represents the major concerns regarding hemoglobinopathy-related burdens in Bangladesh.
The study has demonstrated that clinically silent or mild mutation c.79G > A could be screened successfully using HRM analysis. This is really important for prevention of beta-thalassemia as the MCV-based screening approach may miss a significant number of E-traits [15]. This is because c.79G > A mutation might not always mean that MCV will be below 80 fL in E-trait. Therefore, if a specimen with MCV ≥ 80 fL is not subjected to further assessment, there is a chance that an E-trait could be missed. The clinically silent c.79G > A mutation is common not only in Bangladesh [13, 14] but also in other South Asian and Southeast Asian countries [34,35,36,37]. It is mentionable here that c.79G > A mutation can exert serious outcomes when it becomes a partner with other severe beta-globin gene mutations, such as c.92 + 5G > C in a compound heterozygous state, which may produce a condition that mimics the β-thalassemia major [34, 38]. Thus the screening which is based exclusively on MCV might not be well suited for preventative measures in Bangladesh and other South Asian and Southeast Asian countries as c.79G > A is a very common mutant HBB gene allele in these regions; indicating that the required screening approach must be a robust, reliable, high throughput and easy-to-perform method like HRM.
In addition to the silent c.79G > A mutation, other common mutations including c.92 + 5G > C and c.126_129delCTTT as well as less frequent heterozygous mutations like c.27_28insG, c.46delT, c.47G > A, c.92G > C, c.92 + 130G > C, c.126delC, and c.135delC were differentiated successfully by our HRM approach. The established HRM method can also supplement the second step of carrier screening approach which is based on hemoglobin A2 quantification. HbA2 quantification is widely used for carrier screening [39]. However, β-globin gene mutations which result in only minimal elevation in HbA2 levels could be missed easily during screening [40]. Moreover, external conditions that influence HbA2 level are known to complicate the interpretation of β-thalassemia screening results. These external conditions include (a) α-thalassemia and iron deficiency anemia which may have influence on lowering HbA2 levels and (b) mutations in the Krüppel-like Factor 1 (KLF1) gene which is known to associate with elevation in HbA2 level [41,42,43]. Additionally, co-inheritance of mutations in β-globin and δ-globin genes (HBD) could lead to HbA2 levels that are normal or even reduced [44]. Consequently, even though the quantification of HbA2 is a useful screening tool, use of HRM could be a supplemental approach that could overcome the limitations of HbA2 quantification only.
Although other DNA-based screening approaches including Denaturing High Performance Liquid Chromatography (DHPLC), Single Strand Conformational Polymorphism (SSCP) and Denaturing Gradient Gel Electrophoresis (DGGE) could provide alternative means of detection of point mutations and small deletions in the beta-globin gene, each of these approaches has some limitations that include technical difficulties, requirement of post amplification downstream processes etc. On the other hand, although the Amplification-Refractory Mutation System (ARMS) PCR has the advantage of being a screening method that can detect several mutations simultaneously, it would require two primers sets for each mutation; one set for the mutant allele and another set for the wild type allele, and in addition, it requires an internal control, thus making the process complicated, time-consuming and costly. Even detection of adjacent mutations would require multiple primers and controls. Moreover, requirement of downstream processes such as gel electrophoresis and the limitation of being able to detect only specified mutations narrow down its scope. On the other hand, gene scanning by High Resolution Melting curve analysis offers advantages of being a closed tube system with no requirement of complicated post amplification processing by chromatography or gel electrophoresis. Moreover, the adjacent mutations could be screened using only one set of primers and even unknown mutations in the amplified region could be distinguished from the wild type alleles. Thus the HRM approach offers numerous advantages over other available methods for screening of mutations.
The present study differs from other HRM-based studies for HBB gene mutation detection in respect of primer designing. The other studies focused on mutation screening by scanning full length HBB gene by HRM using at least one primer pair per 70 bp–200 bp [21, 29], whereas the current study focused only on the designated mutational hot-spot and only two sets of primers were enough to achieve the goal. This study also emphasizes on the application of the approach in the regional countries of South Asia and Southeast Asia using the same mutational hot-spot (Table 3) provided that this may require additional one or two sets of primers to cover other mutations which may have high prevalence in the regional countries. Above all, the approach may draw attention for its higher sensitivity and specificity as well as its rapidity and reliability.
The HRM-PCR profiling of the amplified products not only pointed out the presence of nucleotide variation but also demonstrated that the exact nucleotide identification is possible when the appropriate reference controls are run side-by-side with the samples. Our HRM approach could detect all variant sequences for all 41 blindly-analyzed samples. Thus, direct genotyping of the samples would be unambiguously identified in the presence of appropriate controls including positive references such as genomic DNA from the confirmed cases with the mutant alleles and wild type controls in the reaction systems. However, samples that are different from our reference controls, having new combination with c.79G > A or c.92 + 5G > C or new compound heterozygous combinations are expected to result in different melt curves. Only those samples with new melting curve patterns would require sequencing to identify their genotypes. It can be assumed that the number of such samples would be very few, thus making the genotyping easy, rapid and inexpensive.
In our laboratory settings, it cost $145.00 ($3.50 per sample) for HRM-based screening of forty one samples by targeting the HBB gene hot-spot using two sets of primers that flanked all the major mutations and even some rare mutations. On the other hand, the cost of ARMS PCR for forty one samples was $125.00 ($3.00 per sample) when only one mutation was targeted, indicating that the cost would be much higher for screening of multiple mutations by ARMS PCR than that of the HRM. Finally, detection of mutations by Sanger Sequencing required approximately $565.00 for forty one samples ($13.75 per sample) which was four times higher than that of HRM-based screening. Thus, on the one hand, the HRM-based screening of mutations is a rapid, reliable and high throughput approach, and, on the other hand, it is less expensive than other genotyping approaches.
In conclusion, the established HRM approach could be useful for screening of beta-thalassemia trait and hemoglobin E trait targeting the mutational hot-spot of beta-globin gene in Bangladesh and regional countries. It can be a useful supplement of hematological and electrophoretic indices to avoid false positive or false negative results. The approach should deserve attention for nationwide beta-thalassemia carrier screening for its reliability, accuracy, cost-effectiveness, time-saving, and high-throughput operational platform.
Amplification-Refractory Mutation System
Denaturing Gradient Gel Electrophoresis
Denaturing High Performance Liquid Chromatography
β-globin gene
δ-globin genes
High resolution melting
Krüppel-like Factor
Single Strand Conformational Polymorphism
Modell B, Darlison M. Global epidemiology of haemoglobin disorders and derived service indicators. Bull World Health Organ. 2008;86(6):480–7.
Vichinsky EP. Changing patterns of thalassemia worldwide. Ann N Y Acad Sci. 2005;1054(1):18–24.
Kohne E. Hemoglobinopathies: clinical manifestations, diagnosis, and treatment. Dtsch Arztebl Int. 2011;108(31–32):532.
Cao A, Galanello R. Beta-thalassemia. Genet Med. 2010;12(2):61–76.
Orkin SH, Kazazian HH Jr. The mutation and polymorphism of the human beta-globin gene and its surrounding DNA. Annu Rev Genet. 1984;18(1):131–71.
Galanello R, Origa R. Beta-thalassemia. Orphanet J Rare Dis. 2010;5(1):11.
Thein S, Hesketh C, Wallace R, Weatherall D. The molecular basis of thalassaemia major and thalassaemia intermedia in Asian Indians: application to prenatal diagnosis. Br J Haematol. 1988;70(2):225–31.
Varawalla N, Old J, Sarkar R, Venkatesan R, Weatherall D. The spectrum of β-thalassaemia mutations on the Indian subcontinent: the basis for prenatal diagnosis. Br J Haematol. 1991;78(2):242–7.
Chan V, Chan T, Chebab F, Todd D. Distribution of beta-thalassemia mutations in south China and their association with haplotypes. Am J Hum Genet. 1987;41(4):678.
Kazazian H Jr, Orkin S, Antonarakis S, Sexton J, Boehm CD, Goff S, et al. Molecular characterization of seven beta-thalassemia mutations in Asian Indians. EMBO J. 1984;3(3):593.
Cheng T-C, Orkin SH, Antonarakis SE, Potter MJ, Sexton JP, Markham AF, et al. Beta-thalassemia in Chinese: use of in vivo RNA analysis and oligonucleotide hybridization in systematic characterization of molecular defects. Proc Natl Acad Sci U S A. 1984;81(9):2821–5.
Kimura A, Matsunaga E, Takihara Y, Nakamura T, Takagi Y, Lin S, et al. Structural analysis of a beta-thalassemia gene found in Taiwan. J Biol Chem. 1983;258(5):2748–9.
Chatterjee T, Chakravarty A, Chakravarty S, Chowdhury MA, Sultana R. Mutation Spectrum of β-thalassemia and other Hemoglobinopathies in Chittagong, Southeast Bangladesh. Hemoglobin. 2015;39(6):389–92.
Sultana G, Begum R, Akhter H, Shamim Z, Rahim M, Chubey G. The complete Spectrum of beta (β) thalassemia mutations in Bangladeshi population. Austin Biomarkers Diagn. 2016;3:1–6.
Galanello R. Screening and diagnosis for haemoglobin disorders. 2013.
Krypuy M, Ahmed AA, Etemadmoghadam D, Hyland SJ, Fox SB, Brenton JD, et al. High resolution melting for mutation scanning of TP53 exons 5–8. BMC Cancer. 2007;7(1):168.
Bono C, Nuzzo D, Albeggiani G, Zizzo C, Francofonte D, Iemolo F, et al. Genetic screening of Fabry patients with EcoTILLING and HRM technology. BMC Res Notes. 2011;4(1):323.
Yan J-b, Xu H-p, Xiong C, Ren Z-r, Tian G-l, Zeng F, et al. Rapid and reliable detection of glucose-6-phosphate dehydrogenase (G6PD) gene mutations in Han Chinese using high-resolution melting analysis. J Mol Diagn. 2010;12(3):305–11.
Chen WJ, Dong WJ, Lin XZ, Lin MT, Murong SX, ZY W, et al. Rapid diagnosis of spinal muscular atrophy using high-resolution melting analysis. BMC Med Genet. 2009;10(1):45.
Tricarico R, Crucianelli F, Alvau A, Orlando C, Sestini R, Tonelli F, et al. High resolution melting analysis for a rapid identification of heterozygous and homozygous sequence changes in the MUTYH gene. BMC Cancer. 2011;11(1):305.
Chassanidis C, Boutou E, Voskaridou E, Balassopoulou A. Development of a high-resolution melting approach for scanning Beta globin gene point mutations in the Greek and other Mediterranean populations. PLoS One. 2016;11(6):e0157393.
Saetung R, Ongchai S, Charoenkwan P, Sanguansermsri T. Genotyping of beta thalassemia trait by high-resolution DNA melting analysis. Southeast Asian J Trop Med Public Health. 2013;44(6):1055.
Panigrahi I, Marwaha R. Mutational spectrum of thalassemias in India. Indian J Hum Genet. 2007;13(1):36.
Fisher CA, Premawardhena A, De Silva S, Perera G, Rajapaksa S, Olivieri NA, et al. The molecular basis for the thalassaemias in Sri Lanka. Br J Haematol. 2003;121(4):662–71.
George E, Teh L, Rosli R, Lai M, Tan J. Beta Thalassaemia mutations in Malays: a simplified cost-effective strategy to identify the mutations. Malays J med. Health Sci. 2012;8(1):1–8.
Boonyawat B, Monsereenusorn C, Traivaree C. Molecular analysis of beta-globin gene mutations among Thai beta-thalassemia children: results from a single center study. Appl Clin Genet. 2014;7:253.
Ansari SH, Shamsi TS, Ashraf M, Bohray M, Farzana T, Khan MT, et al. Molecular epidemiology of β-thalassemia in Pakistan: far reaching implications. Int J Mol Epidemiol Genet. 2011;2(4):403.
Harano T, Harano K, Okada S, Shimono KA. Wider molecular spectrum of β-thalassaemia in Myanmar. Br J Haematol. 2002;117(4):988–92.
Lin M, Jiao J-W, Zhan X-H, Zhan X-F, Pan M-C, Wang J-L, et al. High resolution melting analysis: a rapid screening and typing tool for common β-thalassemia mutation in Chinese population. PLoS One. 2014;9(8):e102243.
Khan W, Banu B, Amin S, Selimuzzaman M, Rahman M, Hossain B, et al. Prevalence of beta thalassemia trait and Hb E trait in Bangladeshi school children and health burden of thalassemia in our population. DS (Child) HJ. 2005;21(1):1–7.
Theodoridou S, Alemayehou M, Prappas N, Karakasidou O, Aletra V, Plata E, et al. Carrier screening and prenatal diagnosis of hemoglobinopathies. A study of indigenous and immigrant couples in northern Greece, over the last 5 years. Hemoglobin. 2008;32(5):434–9.
Bozkurt G. Results from the north cyprus thalassemia prevention program. Hemoglobin. 2007;31(2):257–64.
Amato A, Cappabianca MP, Lerone M, Colosimo A, Grisanti P, Ponzini D, et al. Carrier screening for inherited haemoglobin disorders among secondary school students and young adults in Latium, Italy. J Community Genet. 2014;5(3):265–8.
Olivieri NF, Pakbaz Z, Vichinsky E. Hb E/beta-thalassaemia: a common & clinically diverse disorder. Indian J Med Res. 2011;134(4):522.
Mondal SK, Mandal S. Prevalence of thalassemia and hemoglobinopathy in eastern India: a 10-year high-performance liquid chromatography study of 119,336 cases. Asian J. Transfus Sci. 2016;10(1):105.
Moiz B, Hashmi MR, Nasir A, Rashid A, Moatter T, Hemoglobin E. Syndromes in Pakistani population. BMC Blood Disord. 2012;12(1):3.
Weatherall D, Clegg J. Inherited haemoglobin disorders: an increasing global health problem. Bull World Health Organ. 2001;79(8):704–12.
Premawardhena A, Fisher C, Olivieri N, De Silva S, Arambepola M, Perera W, et al. Haemoglobin E β thalassaemia in Sri Lanka. Lancet. 2005;366(9495):1467–70.
Ryan K, Bain BJ, Worthington D, James J, Plews D, Mason A, et al. Significant haemoglobinopathies: guidelines for screening and diagnosis. Br J Haematol. 2010;149(1):35–49.
Ivaldi G, Barberio G, Harteveld CL, Giordano P. HbA2 measurements in β-thalassemia and in other conditions. Thalassemia Reports. 2014;4(2).
Harteveld CL, Higgs DR. α-thalassaemia. Orphanet journal of rare diseases. 2010;5(1):13.
Galanello R, Ruggeri R, Addis M, Paglietti E, Cao A. Hemoglobin A2 in iron deficient 8-thalassemia heterozygotes. Hemoglobin. 1981;5(6):613–8.
Perseu L, Satta S, Moi P, Demartis FR, Manunza L, Sollaino MC, et al. KLF1 gene mutations cause borderline HbA 2. Blood. 2011;118(16):4454–8.
Stephens A, Angastiniotis M, Baysal E, Chan V, Fucharoen S, Giordano P, et al. ICSH recommendations for the measurement of haemoglobin A2. Int J Lab Hematol. 2012;34(1):1–13.
We thank the clinicians and staff form Dhaka Shishu Hospital for their assistance in collecting blood specimens.
Not applicable.
Availability of data and materials
All relevant data are within the paper and supporting information files. Further information is available from the authors on request.
Author information
Authors and Affiliations
KM, FQ, SKS, and MTI conceived of the study and designed the experiment. NS, AB, SKS, MTI and MSI carried out collections of biological samples and data. MTI, SKS, ST, PPB, NB, GSB conducted the experiments and analyzed the data. KM, MA and MTI wrote the manuscript. AKMM, SKQ, TS, BB, SS, MH and GS helped in troubleshooting of the experiments. WAK, MAM, HUS, EKC, AAS, SA, FQ and SSQ provided intellectual input to prepare the project proposal and improve the manuscript. All authors read and approved the manuscript.
Corresponding author
Ethics declarations
Ethics approval and consent to participate
Ethical approval for the study was taken from Bangladesh Medical Research Council (BMRC) of National Ethics Review Committee (NERC), Dhaka, Bangladesh. The approval number for the study was "035 26 02 2017" (Ref: BMRC/NERC/2016–2019/251). Prior to the enrollment, the written informed consents were taken from the parents or guardians of the beta-thalassemia participants and from the parents who were also enrolled in the study.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Additional files
Additional file 1: Table S1.
Mutational status of 40 parents of beta-thalassemia patients recruited for initial evaluation of HRM study. (DOCX 11 kb)
Additional file 2: Figure S1.
Normalized melt curve patterns of homozygous and heterozygous c.79G > A and c.92 + 5G > C mutations. (TIFF 232 kb)
Additional file 3: Figure S2.
Normalized melt curve patterns of homozygous and heterozygous c.126_129delCTTT mutations. (TIFF 173 kb)
Additional file 4: Figure S3.
Normalized melt curve patterns of heterozygous c.27_28insG, c.46delT, c.47G > A and c.92G > C mutations. (TIFF 211 kb)
Additional file 5: Figure S4.
Normalized melt curve patterns of heterozygous c.92 + 130G > C, c.126delC and c.135delC mutations. (TIFF 189 kb)
Additional file 6: Figure S5.
Temperature shifted difference curves of unknown carrier parents subjected to HRM analysis by 1st set of primers. (TIFF 530 kb)
Additional file 7: Figure S6.
Temperature shifted difference curves of unknown carrier parents subjected to HRM analysis by 2nd set of primers. (TIFF 487 kb)
Rights and permissions
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
About this article
Cite this article
Islam, M.T., Sarkar, S.K., Sultana, N. et al. High resolution melting curve analysis targeting the HBB gene mutational hot-spot offers a reliable screening approach for all common as well as most of the rare beta-globin gene mutations in Bangladesh. BMC Genet 19, 1 (2018). https://doi.org/10.1186/s12863-017-0594-3
DOI: https://doi.org/10.1186/s12863-017-0594-3


text:
Tiger-3 15-Nov 06PM
Dear Member,
Online booking of the movie Tiger 3 through satellite will open on 15/11/2023 at 06 PM for the theatre.
Note: Minimum 15 entries are required otherwise shows will be canceled.
There are no reviews yet.
Event Details
Date: November 15, 2023
Start time: 18:00 IST
End time: 21:00 IST
Venue: Palm Greens Club
Email: firstname.lastname@example.com

subset: commoncrawl


text:
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St. Andrew Grove & Party Center
Host your next special family event (wedding, shower, anniversary party, birthday party) in our Upper Hall or Lower Hall (Lower hall available during summer months only). The Picnic Grove (pavilion) is great for graduation parties, family reunions and picnics.
For more information on hall rentals and catering options, please contact our Church Office at 440/843-9149 weekdays from 10:00am to 2:00pm.
Both halls are handicap accessible.
Private parking lot! Wooded setting!
Great location for your next family event!
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text:
Sagging Crawl Space
A sagging crawl space is a sign of major foundation issues that are better addressed sooner rather than later. When your crawl space sags, it's likely due to poorly spaced support columns, columns that are settling, and rotted joists, girders, and posts.
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When you notice any of these signs, it's better to get the root cause fixed as soon as possible before they get worse.
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With the SmartJack® System, we can stabilize and restore sagging floors. Every crawl space jack evenly distributes weight through a precast concrete footing and fill material base to address poor support soils.


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ScanPipe Concepts
A project encapsulates the analysis of software code:
It has a Project workspace, which is a directory that contains the software code files under analysis.
It makes use of one or more code analysis Pipelines scripts to automate the code analysis process.
It tracks Codebase Resources, i.e. its code files and directories
It tracks Discovered Packages, i.e. system and application packages origin and license discovered in the codebase.
In the database, a project is identified by its unique name.
Multiple analysis pipelines can be run on a single project.
Project workspace
A project workspace is the root directory where a project's files are stored.
The following directories exist under the workspace directory:
input/ contains all uploaded files used as the input of a project, such as a codebase archive.
codebase/ contains files and directories - i.e. resources - tracked as CodebaseResource records in the database.
output/ contains any output files created by the pipelines, including reports, scan results, etc.
tmp/ is a scratch pad for temporary files generated during pipelines runs.
A pipeline is a Python script that contains a series of steps, which are executed sequentially to perform a code analysis.
It usually starts with the uploaded input files, which might need to be
extracted first. Then, it generates CodebaseResource
records in the database
Those resources can then be analyzed, scanned, and matched as needed. Analysis results and reports are eventually posted at the end of a pipeline run.
All Built-in Pipelines are located in the scanpipe.pipelines
Each pipeline consists of a Python script and includes one subclass of the
Each step is a method of the Pipeline
The execution order of the steps - or the sequence of steps execution - is
declared through the steps
class attribute.
Refer to Custom Pipelines for details about adding custom pipelines to ScanCode.io.
You can assign one or more pipelines to a project as a sequence.
As mentioned above, pipelines include a group of operations—Pipes—that are combined in a chain-like fashion and executed in orderly manner. Pipes are simply the building blocks of a given pipeline.
For example, the following operations—Steps—are included in the RootFS pipeline, and they are leveraging pipes to accomplish pre-defined tasks:
from scanpipe.pipelines import Pipeline
from scanpipe.pipes import flag
from scanpipe.pipes import rootfs
from scanpipe.pipes import scancode
class RootFS(Pipeline):
def flag_empty_files(self):
Flags empty files.
def scan_for_application_packages(self):
Scans unknown resources for packages information.
All built-in pipes are located in the scanpipe.pipes
Pipes are grouped by type in modules, e.g. codebase
, input
, output
Refer to our Pipes section for information about available pipes and their usage.
Codebase Resources
A project Codebase Resources
are records of its code files and directories.
is a database model and each record is identified by its path
under the project workspace.
The following are some of the CodebaseResource
A status, which is used to track the analysis status for this resource.
A type, such as a file, a directory or a symlink
Various attributes to track detected copyrights, license expressions, copyright holders, and related packages.
Please note that ScanCode-toolkit use the same attributes and attribute names for files.
Discovered Packages
A project Discovered Packages
are records of the system and application packages
discovered in the code under analysis.
is a database model and each record is identified by its Package URL
Package URL
is a fundamental effort to create informative identifiers for
software packages, such as Debian, RPM, npm, Maven, or PyPI packages.
See https://github.com/package-url for more details.
The following are some of the DiscoveredPackage
A type, name, version (all Package URL attributes)
A homepage_url, download_url, and other URLs
Checksums, such as SHA1, MD5
Copyright, license_expression, and declared_license
Please note that ScanCode-toolkit use the same attributes and attribute names for packages.


text:
- Fine Bone China
- Microwave and dishwasher safe
- Includes: Pair of mugs and an attractive keepsake gift box
- Fine Bone China mug is translucent, chip-resistant and elegant
- Each mug holds 360 ml
- Makes a great gift for lovers of coffee, tea, and quality
- Designed in North America by McIntosh
Gustav Klimt (1862-1918)
Merging images of the metaphysical and erotic, Klimt is a leading artist of the Art Nouveau era. In paintings from his Golden Period, Klimt ventures beyond iconographic images to art that exceeds spatial and temporal constraints.
Based in Ottawa, Canada


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If you've got the bedtime blues this lockdown, check out our top tips for an easy bedtime routine here: http://bit.ly/3oKxUYP 💤 #bedtime #bettersleep #wellness
@ukbedstoreIf you're shopping for kids beds for younger children, look no further than mid sleeper beds from the UK Bed Store! Suitable for ages 6 and above and perfect for maximising storage space, whilst also providing children with a fun space for work and play, purchasing one of our mid sleeper beds could be the best decision you make for both you and your child. With more than 40 years experience in the industry, we have built a reputation on offering high quality beds at fantastic prices. What's more, we also deliver our beds to any part of the UK and our team will also be happy to assemble any beds right where you need them. Whatever you need, we'll make sure that finding the perfect beds for your kids is as easy as possible!
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If you've got the bedtime blues this lockdown, check out our top tips for an easy bedtime routine here: http://bit.ly/3oKxUYP 💤 #bedtime #bettersleep #wellness


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From the streets of Ashanti Region of Ghana came Latif Salifu, a talent who sought to become the next big thing on the local and foreign front.
Salifu's formative years started in Buokrom Zongo before journeying to Dunkwa in the Central Region to play for the now defunct Starke FC, then in the national Division Two League.
After an impressive spell with Starke fc, one of the strongest ever production line for Ghana's various national team, Liberty Professionals snapped him up.
The now Division One club offered a perfect platform for his fast rise which eventually landed the smallish winger in the national U-20 team.
Salifu would not be a regular member of the U-20 side but certainly remained in the Ghana side that conquered Africa and the World in 2009.
Ghana swept the board in all the youth competitions continentally and globally by annexing the WAFU Tournament in Nigeria.
The team followed it up with the AFCON glory in Rwanda before capping it with the global glory in Egypt after beating power house, Brazil via penalty shootouts which became Ghana and Africa's first ever trophy.
The now 32-year-old has since struggled to climb the highest echelon of continental or global football and he narrates what has accounted for the difficult rise.
"For some time now I've been home, I've not been playing, not for some time, I'm not going to play football again.
"I have had some injuries and it is not helping my career and that's the reason why I've stopped and I don't want to play again," Latif Salifu told Radio Gold Sports.
"It was my injury from Cote D'Ivoire because I didn't take it serious, I thought it was normal.
"When I came I used to play for Medeama and then back to Berekum Chelsea.
"When I signed for Berekum Chelsea I said no, the way the pain is, I have to go and do another surgery.
"So after the surgery the pains was too much and I decided to play for King Faisal but was for the second round.
"After that I said the pains was too much.
"As at now I have had three surgeries, one on my left leg and the other two on the right leg." Latif Salifu Told Radio Gold Sports.
Latif Salifu previously played for Ghanaian clubs Liberty Professionals, Berekum Chelsea, Medeama and King Faisal with a spell at Ivory Coast club, AS Tanda in between.
This article was written by Adamu Muftawu for Africa-foot.com and relayed by kick442.com as an official media partner.


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Last night was a most beautiful beginning as we come together as a community to seek answers to two burning questions on #antiasianhate: WHY is this happening? WHAT can we all do? No matter what the answers are, we know this much: "Hate cannot drive out hate; only love can do that."
Please read the Report by Lexington Minuteman


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Fair tips legislation scrapped?
With promised fair tips legislation reportedly scrapped, Unite says it must press on with winning in the workplace
Reading time: 5 min
Unite has slammed the government for reportedly scrapping plans to introduce fair tips legislation, first promised by then-business secretary Sajid Javid nearly six years ago.
After Javid's consultation in 2016, hospitality workers then waited more than two years before prime minister Boris Johnson's predecessor Theresa May announced in 2018 that she would introduce legislation to bar restaurants from taking staff tips.
She then failed to take any action in her time in office. In 2019, a pledge to ban restaurants from taking staff tips was included in the Queen's Speech, but again nothing was done.
After years of delays, legislation to ensure hospitality and other workers receive 100 per cent of their tips was back on the table in September last year, when business minister Paul Scully announced that the government would finally bring in the long-promised tips laws.
"Unfortunately, some companies choose to withhold cash from hardworking staff who have been tipped by customers as a reward for good service," Scully said last September.
"Our plans will make this illegal and ensure tips will go to those who worked for it," he added. "This will provide a boost to workers in pubs, cafes and restaurants across the country, while reassuring customers their money is going to those who deserve it."
Speculation mounted that tips legislation would be included in the Queen's Speech next week, but the Financial Times (FT) reported that these plans have been shelved, with one unnamed government minister telling the FT that tips legislation was being "dropped for the foreseeable future".
It was also thought that tips legislation may have been included in a long-promised Employment Bill to strengthen workers' rights, but the Bill has likewise been kicked into the long grass and will reportedly not appear in the Queen's Speech.
The Employment Bill was first promised by prime minister Boris Johnson in the Tory party manifesto during the last general election in 2019 as a way to improve workers' rights post-Brexit.
The news that the Employment Bill, including promised tips legislation, has vanished into thin air was met by fury from unions.
Commenting, TUC general secretary France O'Grady said, "If the Government fails to bring forward an Employment Bill at next week's Queen's Speech it will betray some of the lowest-paid and most vulnerable workers in Britain.
"Fair tips are just the thin end of the wedge," she added. "Without new legislation workers will be denied a host of other vital rights and protections.
"These include fair notice for shifts and payment for cancelled shifts, flexible working rights, and protection from pregnancy discrimination."
Unite general secretary Sharon Graham said the latest news about the uncertain fate of tips legislation and the wider Employment Bill shows that Unite must press on with its strategy of winning in the workplace.
"Every year this government promises action to ensure fair tipping – and then does precisely nothing to deliver on that promise," she said. "That is why workers are turning to Unite because we are winning for them, just as we did at Pizza Express.
"A hospitality worker can lose thousands of pounds a year from their earnings when the employer refuses to hand over their tips. In a sector notorious for long hours and low wages, tipping misappropriation is another abuse. If the government won't fix it, Unite will."
Last month, Unite members working for Pizza Express declared victory for fair tips after an internal Pizza Express committee scrapped a tips allocation plan that saw low-paid waiting staff lose up to £2000 a year.
The plan, suddenly introduced in May by Pizza Express bosses last year, saw the percentage of card tips that wait staff receive slashed from 70 per cent to 50 per cent, with the other 50 per cent going to back-of-house kitchen staff.
Unite members campaigned heavily against the latest tips injustice from Pizza Express, and thanks to their efforts, in March, an internal Pizza Express committee, established because of pressure from Unite, scrapped the policy.
Now, the committee, which is made up of staff representatives across Pizza Express, have agreed to return to the previous system, where wait staff receive 70 per cent of card tips and kitchen staff receive 30 per cent.
By Hajera Blagg


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Natalie Tan's Book of Luck and Fortune (Large Print / Library Binding)
Unavailable for purchase.
Lush and visual, chock-full of delicious recipes, Roselle Lim's magical debut novel is about food, heritage, and finding family in the most unexpected places.
At the news of her mother's death, Natalie Tan returns home. The two women hadn't spoken since Natalie left in anger seven years ago, when her mother refused to support her chosen career as a chef. Natalie is shocked to discover the vibrant neighborhood of San Francisco's Chinatown that she remembers from her childhood is fading, with businesses failing and families moving out. She's even more surprised to learn she has inherited her grandmother's restaurant.
About the Author
Roselle Lim is a Filipino-Chinese writer living on the north shore of Lake Erie. She loves to write about food and magic. When she isn't writing, she is sewing, sketching, or pursuing the next craft project.


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Redshift Okta Integration: Testing User Permissions
Okta is the preferred Identity as a Service (IDaaS) solution for thousands of organizations worldwide. Here at QloudX, we too have a few large customers who have chosen Okta as their unified identity platform for every application they use throughout the company. All employees get an Okta account which they use to sign into any company apps they need.
And so, when we started using Redshift for analytics, users started asking, "Why can't I use my Okta credentials to sign into Redshift?" Until then, every user was manually created within Redshift itself. As you can imagine, this approach has many issues:
- Redshift users are completely disjointed from the company-wide "official" user directory.
- Keeping the hundreds of user accounts in-sync across the Redshift clusters in dev, stage & prod is tedious & error-prone.
Soon, we started exploring whether it's even possible to use Okta to log in to Redshift. Fortunately, we found an excellent post on the AWS Big Data Blog that walks you through exactly how to achieve this:
Federate Amazon Redshift access with Okta as an identity provider
This post was instrumental in our success. Without it, we would have spent days experimenting without much progress.
Managing User Permissions
We have hundreds of users, groups & schemas in our Redshift. Ensuring that someone logging into Redshift via Okta did not inadvertently get more permissions than they should have, was very important for us.
To test this, we started by developing a couple of not-so-trivial queries that would show us exactly what permissions a user & group has.
User Access to Schemas
This first query neatly lists out users, schemas & what permissions the users have on the schemas:
SELECT u.usename AS user, s.schemaname AS schema,
has_schema_privilege(u.usename, s.schemaname, 'usage') AS usage_permission,
has_schema_privilege(u.usename, s.schemaname, 'create') AS create_permission
FROM pg_user u CROSS JOIN (SELECT DISTINCT schemaname FROM pg_tables) s
WHERE u.usename IN ('email@example.com', 'firstname.lastname@example.com', 'firstname.lastname@example.com')
AND s.schemaname IN ('testing_okta');
The query is essentially using Redshift's HAS_SCHEMA_PRIVILEGE function to get the user's privileges. Notice how the usernames are email addresses; that's how the users are created in Redshift when they log in via Okta for the first time.
This query produces an output like this:
user | schema | usage_permission | create_permission |
firstname.lastname@example.com | testing_okta | true | true |
email@example.com | testing_okta | true | true |
email@example.com | testing_okta | false | false |
The "usage" permission on a schema allows users to access objects in the schema & the "create" permission allows users to create objects in the schema.
Redshift Group Permissions
This next query lists out the permissions that members of a Redshift group have. Since Redshift groups are used as the primary means of restricting permissions (by creating Okta groups by the same name), this was critical information for us to ensure the least privilege after Okta integration.
SELECT schema, table_name, g.groname AS group,
CASE WHEN charindex('r', split_part(split_part(array_to_string(relacl,
'|'), g.groname, 2), '/', 1)) > 0 THEN 'SELECT ' ELSE '' END ||
CASE WHEN charindex('a', split_part(split_part(array_to_string(relacl,
'|'), g.groname, 2), '/', 1)) > 0 THEN 'INSERT ' ELSE '' END ||
CASE WHEN charindex('w', split_part(split_part(array_to_string(relacl,
'|'), g.groname, 2), '/', 1)) > 0 THEN 'UPDATE ' ELSE '' END ||
CASE WHEN charindex('d', split_part(split_part(array_to_string(relacl,
'|'), g.groname, 2), '/', 1)) > 0 THEN 'DELETE ' ELSE '' END
AS permisssions FROM (
SELECT n.nspname AS schema, c.relname AS table_name, c.relacl
FROM pg_user u1 CROSS JOIN pg_class c
LEFT JOIN pg_namespace n ON c.relnamespace = n.oid
LEFT JOIN pg_user u2 ON c.relowner = u2.usesysid
WHERE c.relowner = u1.usesysid AND n.nspname = 'testing_okta'
) JOIN pg_group g ON array_to_string(relacl, '|')
LIKE '%' || g.groname || '%'
WHERE relacl IS NOT NULL AND g.groname = 'testing_okta';
This query essentially decodes the pg_class.relacl
value using this, & presents the results in a human-friendly manner, like so:
schema | table_name | group | permisssions |
testing_okta | my_table | testing_okta | SELECT INSERT UPDATE DELETE |
Having verified all permissions, both at the user & group level, we were at last able to roll out this Redshift Okta integration to production!
If you too are looking to integrate Okta with Redshift, I hope this article, the AWS blog post, & the queries provided here make your journey as smooth as possible. 😊
About the Author ✍🏻
Harish KM is a Principal DevOps Engineer at QloudX & a top-ranked AWS Ambassador since 2020. 👨🏻💻
With over a decade of industry experience as everything from a full-stack engineer to a cloud architect, Harish has built many world-class solutions for clients around the world! 👷🏻♂
With over 20 certifications in cloud (AWS, Azure, GCP), containers (Kubernetes, Docker) & DevOps (Terraform, Ansible, Jenkins), Harish is an expert in a multitude of technologies. 📚
These days, his focus is on the fascinating world of DevOps & how it can transform the way we do things! 🚀


text:
"The clown Pierrot sings of Columbine,
A creature fair as highest angels be.
To think that such a heart could e'er be mine;
The clown beset by cupid's bow is me.
My lady is above all things revered,
And cut from thread which dreams are woven of,
But still she looked upon Pierrot, mere,
And promised him her sacramental love.
I wish upon you all delight as this –
Her dear Pierrot has been blessed to reap.
But now, enough rejoicing in my bliss;
I still my tongue a while, the lady speaks."
"Bellow forth your love, Pierrot,
Tell of my unblemished graces;
Flourishing in apt affection –
To your words that reach all places.
Truly, gods have smiled upon you,
Furnishing your heart with passion;
Plain as you might be in essence,
Unassuming and oft outdone.
Venerate me as you promised,
Lest I stray to a suitor new.
Love me, and be lost, Pierrot;
As you sing for me, I love you."
"Happily, Columbine, poetry pleases you –
Even to fall to the arms of a commoner;
But beauty, vanity, prominent majesty,
All of it calls to you, all of it beckons you.
Pierrot dissipates, living in monochrome,
Wasting his sentience crafting his arias.
Wouldn't you rather be living in eminence,
Beautiful, luminous, glistening beside me?
Follow me, Columbine, elegant, ever-free;
Blossoming, glorious, shine as a firefly,
Enliven this colourless jester no longer.
Fall to me: Harlequin, worshipping all of me."
"Colourless, perhaps, but loyal,
Pale, but never straying from me;
Songs of love, he has to offer,
Still you speak of absent beauty?
Yet you see my heart's ambition –
And you promise luminescence
Where this simple fool has sonnets,
Ballads, choruses: naught but words.
Hold me not too dear, Pierrot,
For all my love was vanity.
I exchange your songs for sequins,
Into the arms of Harlequin."
"How cruelly does this story culminate
That I end without a blithe word spoken;
Sweet Columbine absconds to live her fate,
And the poor Pierrot, he is broken.
Now my laughter, smeared in sombre lipstick
Is twisted to a still, despondent frown;
Lost amid the comic and the tragic,
And who has pity for a grieving clown?
Upon his knees, the poor Pierrot weeps –
For Columbine, whose love he bears within.
Forsake him here to tend his sorrows, deep,
As inky tears malign his silver skin."


text:
Working with Task Runner
Task Runner is a task agent application that polls AWS Data Pipeline for scheduled tasks and executes them on Amazon EC2 instances, Amazon EMR clusters, or other computational resources, reporting status as it does so. Depending on your application, you may choose to:
Allow AWS Data Pipeline to install and manage one or more Task Runner applications for you. When a pipeline is activated, the default
object referenced by an activity runsOn field is automatically created. AWS Data Pipeline takes care of installing Task Runner on an EC2 instance or on the master node of an EMR cluster. In this pattern, AWS Data Pipeline can do most of the instance or cluster management for you. -
Run all or parts of a pipeline on resources that you manage. The potential resources include a long-running Amazon EC2 instance, an Amazon EMR cluster, or a physical server. You can install a task runner (which can be either Task Runner or a custom task agent of your own devise) almost anywhere, provided that it can communicate with the AWS Data Pipeline web service. In this pattern, you assume almost complete control over which resources are used and how they are managed, and you must manually install and configure Task Runner. To do so, use the procedures in this section, as described in Executing Work on Existing Resources Using Task Runner.


text:
The late payment challenge
Time to end the scourge of late payments, says Phil Hall, head of public affairs & public policy at the AAT (Association of Accounting Technicians).
Time to end the scourge of late payments, says Phil Hall, head of public affairs & public policy at the AAT (Association of Accounting Technicians).
With almost a quarter of insolvencies (23%) being caused by late payment issues, it would be difficult for even the most sceptical to deny that late payment is a significant issue for the British economy.
Of course, whilst collapsing businesses are the ultimate price paid for late payment, the impact is much, much wider. Those companies that do manage to absorb late payment are still likely to face myriad problems as a result.
For example, the loss of income can stop small businesses from investing and growing, damage productivity and in many cases can have mental health implications on both business owners and sometimes other members of staff.
There is broad agreement on the problems and consequences of late payments for individuals, employers and the economy. But agreement on an effective solution remains elusive.
The voluntary Prompt Payment Code was introduced in 2012 and hailed as a potential solution by many. This voluntary code requires large companies to pay their suppliers within a maximum of 60 days – hardly a challenge for most businesses in the 21st Century.
Despite the less than challenging nature of the Code, only 2,000 companies have signed it. Of these, some are in breach of the code because they have payment terms beyond 60 days. There is very little enforcement of the code and no financial penalties are imposed for a failure to comply.
Belatedly recognising the shortcomings of the Code, since April 2017, the government has required companies to report on their payment practices on a half-yearly basis. Although this may provide some useful data for policy makers it has no discernible value in terms of changing behaviour.
There is a legal obligation to report the data but there is no legal action if that data provides great cause for concern i.e. theoretically if only 1% of all invoices are paid within agreed terms (irrespective of length) then no action whatsoever will be taken against that company. This point was far from uniquely demonstrated by Grainer & Worrall Engineering who paid suppliers within agreed payment terms in just 4% of cases for the six months from June to December 2017.
A year ago, the much-heralded post of "Small Business Commissioner" was created. This was supposed to be based on the Australian Small Business Commissioner model i.e. a Commissioner with teeth to impose fines. Instead our Commissioner has no powers to impose fines and is widely regarded as toothless.
It's not surprising that the SME community appears to have little confidence in his ability to make a positive difference. 84% of SMEs stated they did not believe the Commissioner would have any impact on their business according to polling by Close Brothers Invoice Financing in 2018.
In summary, the past decade or so has been characterised by a series of initiatives, legislative tweaks, voluntarism and reliance on big employers to do the right thing, all of which have failed to address the problem of late payments.
AAT has a strong interest in improving matters because 60% of its 140,000 members either work for or run their own SME and its 4,250 licensed accountants provide tax and accountancy services to more than 400,000 British SMEs. It's a big issue for AAT members and an even bigger issue for members clients.
The time has come for Government to legislate to end the scourge of late payments once and for all.
AAT has made three key recommendations to the Department for Business, Energy & Industrial Strategy (BEIS) that should resolve the problem. These are:
The campaign to deliver these changes has already gained substantial support from the SME community as well as the fashion, construction and recruitment industries.
Political support has been similarly impressive. The Green Party, Liberal Democrats and Scottish National Party back AAT's campaign together with almost three quarters (73%) of Labour and Conservative MPs according to a recent YouGov poll.
The Chartered Institute of Credit Management (CICM) who operate the existing Prompt Payment Code, and the Small Business Commissioner also appear supportive.
Diverse and comprehensive backing for AAT's recommendations for payment reform makes it increasingly difficult for the Government to continue to drag its heels and back the status quo.


text:
New 2024 Toyota Camry LE Sedan
8-Speed Automatic
Vehicle Summary
Stock # 24HT1078
Condition new
Exterior MIDNIGHT BLACK METALLIC
Interior Black
Engine 2.5L I4 DOHC 16V
Fuel Type Gasoline
Drivetrain FWD
Transmission 8-Speed Automatic
Location Diehl of Hermitage
Vehicle Description
Black w/Fabric Seat Trim, Blind Spot Monitor w/Rear Cross Traffic Alert, Heated Exterior Mirrors w/Turn Signal Indicators, 4-Wheel Disc Brakes, 6 Speakers, ABS brakes, Air Conditioning, Alloy wheels, AM/FM radio: SiriusXM, Apple CarPlay/Android Auto, Auto High-beam Headlights, Automatic temperature control, Brake assist, Bumpers: body-color, Delay-off headlights, Driver door bin, Driver vanity mirror, Dual front impact airbags, Dual front side impact airbags, Electronic Stability Control, Emergency communication system: Safety Connect (1-year trial), Exterior Parking Camera Rear, Fabric Seat Trim, Four wheel independent suspension, Front anti-roll bar, Front Bucket Seats, Front Center Armrest, Front dual zone A/C, Front reading lights, Fully automatic headlights, Illuminated entry, Knee airbag, Low tire pressure warning, Occupant sensing airbag, Outside temperature display, Overhead airbag, Overhead console, Panic alarm, Passenger door bin, Passenger vanity mirror, Power door mirrors, Power driver seat, Power steering, Power windows, Radio data system, Radio: Audio, Rear anti-roll bar, Rear seat center armrest, Rear side impact airbag, Rear window defroster, Remote keyless entry, Security system, Speed control, Speed-sensing steering, Split folding rear seat, Steering wheel mounted audio controls, Tachometer, Telescoping steering wheel, Tilt steering wheel, Traction control, Trip computer, Variably intermittent wipers, and Wheels: 17" x 7.5J Alloy. Black w/Fabric Seat Trim, Blind Spot Monitor w/Rear Cross Traffic Alert, Heated Exterior Mirrors w/Turn Signal Indicators.Midnight Black Metallic 2024 Toyota Camry LE 4D Sedan FWD 8-Speed Automatic 2.5L I4 DOHC 16V28/39 City/Highway MPG
Crash Safety Ratings
You May Also Qualify For
Uber Driver Bonus Cash
Effective Dates: 01-03-2024 - 01-06-2025
Customer must: 1. Purchase or Lease a New, unused or unlicensed Camry, Avalon, Prius Family, Sequoia, Sienna (as specified above) from ground stock inventory through a participating Toyota Dealer. 2. Sign and Complete the Uber Driver Incentive Certificate. New, unused and unlicensed Toyota vehicle purchased from dealers ground stock inventory.
$750 -
Olympic/Paralympic Partner Incentive Offer
Effective Dates: 01-03-2024 - 01-06-2025
Customer must be associated with *US Olympic Athlete, *US Paralympic Athlete, *NGB (National Governing Body) Employee, *USOPP (United States Olympic and Paralympic Properties) employee or USOPC (United States Olympic & Paralympic Committee). Customer must be issued a TMNA authorized Toyota Sponsored Olympic* Consumer Cash Request Form.Non-transferable (not transferable within household). Must be 18 years of age and have a valid driver's license.
$500 -
Vehicle Special Purchase Program
Effective Dates: 04-02-2024 - 04-30-2024
The Buyer may not combine any other TMS, GST, SET or Dealer Incentives with the Toyota Vehicle Special Purchase Program such as, but not limited to, special financing, subvented lease rates or other cash incentives not listed on Attachment A. An exception is the Buyer may combine the Military, Recent College Graduate, or T-DRIVE incentives (when available) with the Toyota Vehicle Special Purchase Program Attachment A incentives
Vehicle Features
- Wheels: 17" x 7.5J Alloy
- Tires: P215/55R17 AS
- Wheels w/Silver Accents
- Steel Spare Wheel
- Compact Spare Tire Mounted Inside Under Cargo
- Clearcoat Paint
- Body-Colored Front Bumper w/Black Rub Strip/Fascia Accent and Metal-Look Bumper Insert
- Body-Colored Rear Bumper
- Chrome Side Windows Trim and Black Front Windshield Trim
- Body-Colored Door Handles
- Body-Colored Power Side Mirrors w/Manual Folding
- Fixed Rear Window w/Defroster
- Light Tinted Glass
- Variable Intermittent Wipers
- Galvanized Steel/Aluminum Panels
- Black Grille
- Trunk Rear Cargo Access
- Auto On/Off Projector Beam Led Low Beam Daytime Running Auto High-Beam Headlamps w/Delay-Off
- LED Brakelights
- Headlights-Automatic Highbeams
- Front Bucket Seats -inc: 8-way power-adjustable driver seat w/power lumbar support and 6-way adjustable front passenger seat
- Driver Seat
- 6-Way Passenger Seat -inc: Manual Recline, Height Adjustment and Fore/Aft Movement
- 60-40 Folding Bench Front Facing Fold Forward Seatback Rear Seat
- Manual Tilt/Telescoping Steering Column
- Front Cupholder
- Rear Cupholder
- Remote Releases -Inc: Power Cargo Access and Power Fuel
- Cruise Control w/Steering Wheel Controls
- Distance Pacing
- Dual Zone Front Automatic Air Conditioning
- HVAC -inc: Underseat Ducts
- Illuminated Glove Box
- Driver Foot Rest
- Interior Trim -inc: Piano Black Instrument Panel Insert and Chrome/Metal-Look Interior Accents
- Full Cloth Headliner
- Urethane Gear Shifter Material
- Day-Night Rearview Mirror
- Driver And Passenger Visor Vanity Mirrors w/Driver And Passenger Auxiliary Mirror
- Full Floor Console w/Covered Storage, Mini Overhead Console w/Storage and 1 12V DC Power Outlet
- Front Map Lights
- Fade-To-Off Interior Lighting
- Full Carpet Floor Covering
- Carpet Floor Trim and Carpet Trunk Lid/Rear Cargo Door Trim
- Cargo Area Concealed Storage
- Radio w/Seek-Scan, Clock, Speed Compensated Volume Control, Steering Wheel Controls and Radio Data System
- Window Grid And Roof Mount Antenna
- 2 LCD Monitors In The Front
- Engine: 2.5L I-4 DOHC 16-Valve -inc: VVT-i intake and exhaust
- Transmission: 8-Speed Direct Shift ECT-i Automatic -inc: sequential shift mode
- Engine Auto Stop-Start Feature
- Front-Wheel Drive
- 2.80 Axle Ratio
- Battery w/Run Down Protection
- Gas-Pressurized Shock Absorbers
- Front And Rear Anti-Roll Bars
- Electric Power-Assist Speed-Sensing Steering
- 15.8 Gal. Fuel Tank
- Single Stainless Steel Exhaust
- Strut Front Suspension w/Coil Springs
- Multi-Link Rear Suspension w/Coil Springs
- 4-Wheel Disc Brakes w/4-Wheel ABS, Front Vented Discs, Brake Assist and Hill Hold Control
- Lane Tracing Assist (LTA)
- Side Impact Beams
- Collision Mitigation-Front
- Tire Pressure Monitor System (TPMS) Low Tire Pressure Warning
- Dual Stage Driver And Passenger Front Airbags
- Curtain 1st And 2nd Row Airbags
- Airbag Occupancy Sensor
- Rear Child Safety Locks
- Outboard Front Lap And Shoulder Safety Belts -inc: Rear Center 3 Point, Height Adjusters and Pretensioners
- Back-Up Camera
Welcome To
Diehl Automotive
Massillon · Robinson · Butler · Grove City · Moon · Sharon · Beaver Falls
Diehl Automotive Group Puts Customers First With Family Approach to Business.
Purchasing a brand-new automobile is one of the largest investments most people will make in their life, which often times creates some apprehension and nervousness. However, the car-buying process should also be an exciting and enjoyable experience. The friendly, experienced team of automotive professionals at Diehl Automotive goes out of their way to eliminate any uneasiness you might have and provide expert guidance that leads to you comfortably choosing the perfect vehicle for your schedule and budget. Our family is very close and we know how important it is to take care of each other. We approach our business the same way. We're here to take care of our customers.
Crash Safety Ratings
Factory Warranty
3 Years / 36,000 Miles
5 Years / 60,000 Miles
5 Years / Unlimited Miles
Roadside Assistance
2 Years / Unlimited Miles
2 Years / 25,000 Miles
Awards & Accolades
Consumer Guide Automotive
Best Buys
New vehicle pricing includes all offers and incentives. Tax, Title and Tags not included in vehicle prices shown and must be paid by the purchaser. Some prices may include manufacturer-to-dealer offers that are not compatible with manufacturer special finance programs and/or lease offers. While great effort is made to ensure the accuracy of the information on this site, errors do occur so please verify information with a customer service rep. This is easily done by calling us at (724) 608-3340 or by visiting us at the dealership.
**With approved credit. Terms may vary. Monthly payments are only estimates derived from the vehicle price with a 72 month term, 5.99% interest and 20% down-payment.
*Number of views in last 30 days
Based on 2024 EPA mileage ratings. Use for comparison purposes only. Your actual mileage will vary depending on how you drive and maintain your vehicle.
All advertised vehicles are subject to actual dealer availability. Certain vehicles listed may not be available, or may have different prices. *All calculated payments are subject to credit approval and include tax, tag, title, and applicable fees. Advertised price excludes tax, tag, registration, title, processing fee and dealer installed accessories. Vehicle option and pricing are subject to change. Prices include all dealer incentives. Pricing and availability varies by dealership. Please check with your dealer for more information. Prices do not include dealer charges, such as advertising, that can vary by manufacturer or region, or costs for selling, preparing, displaying or financing the vehicle. Images displayed may not be representative of the actual trim level of a vehicle. Colors shown are the most accurate representations available. However, due to the limitations of web and monitor color display, we cannot guarantee that the colors depicted will exactly match the color of the car. Information provided is believed accurate but all specifications, pricing, and availability must be confirmed in writing (directly) with the dealer to be binding. Neither the Dealer nor Dealer Venom is responsible for any inaccuracies contained herein and by using this application you the customer acknowledge the foregoing and accept such terms.


text:
Managing Unread Reply Notifications
Notifications help keep you informed when you have unread text messages in your Text-Em-All account. You may set up text, email, and browser notifications to keep you up to date. This article will walk you through how to set up and manage your notifications.
Set up new unread text reply notifications
- Navigate to your Settings > Notifications page
- Click Add Unread Message Notification and select the text number you'd like to enable for notifications. You can also just click on the text number you wish to edit if it already appears in the list.
- Click the checkbox to the left of the notification type that you'd like to enable: email, text, browser, or all three.
- By default, we set new notifications to send every 15 minutes, from 9:00 AM to 9:00 PM Sunday through Saturday. To change these parameters click the blue change link.
5. Click Save to confirm your selections.
Unread text reply notifications settings can also be accessed on the Inbox and Text Settings page by clicking the three-dot menu on any text number and selecting Manage Notifications on the drop-down menu that appears.
Note: By default, daily email notifications will be sent at 9:00 AM every day. However, you can manage these alerts directly within your account to change the time interval, times of day, and days of the week that you'd like to receive notifications.
Set a notification schedule
By default email notifications are sent at 9:00AM every day and text messages are set to trigger every 15 minutes. However, you can update these alert schedules in the same place you turn on/off notifications.
- Navigate to your Settings > Notifications page
- Click on the text number whose notifications you would like to manage.
- Click change next to the item you wish to edit.
- You can edit the frequency and days/times of the week. If you want to always get notifications, click the toggle button to toggle off the time/date settings and always get notifications.
- When finished click Save.
Pause Notifications
You may pause text and email notifications at any time without losing your existing settings. To pause notifications:
- Navigate to your Settings > Notifications page
- Click on the text number whose notifications you would like to pause.
- Click the Pause button under the notifications you wish to pause.
- Click Save.
Set Notification Destinations
Through the Settings > Notifications page, you can also manage the destinations where alerts are sent.
- Email notifications are sent to the email address on file for the account. You can change the email address that receives notifications through the Settings > Profile page.
- Text notifications must be sent to a cell phone number. You can edit this text number at any time.
- Notifications to your cell phone will come from the same number each time. Replies to this number are not monitored.


text:
Top 5 Offbeat Places to visit In Maldives
When someone speaks about the Maldives, the first thing that comes to everyone's mind is its stunning islands and beachy areas. In truth, there are plenty of such locations you can visit on a Maldives tour and admire the tropical environment here. Overall, there are several interesting locales in the Maldives, from the fishing hamlets and sea shacks in the South to the beaches at the Northern stretch.
Nevertheless, Maldives is home to many more exciting places with unique elements and histories. While you should add sites like Victory Monument and Tsunami Monument in your itinerary, give the following offbeat destinations a try as well.
Obsah / Contents
1) Sea of Stars, Vaadhoo Island
While searching for Maldives attractions, you would find Vaadhoo Island in Thrillophilia review and similar platforms. While there are many fun places to see at this spot, the Sea of Stars, in particular, is a fun spot to go to.
The waters at this beach are home to multiple bioluminescent sea plankton. Here, the quantity of these underwater species is so high that they appear as bright-blue stars in the waters.
For the best view of this glowy phenomenon, you should visit during May to November. Reaching the place is easy and affordable, too; just take a speedboat from Malé to the island.
2) Maafushi, Kaafu Atoll
Maafushi in the Maldives is not a top location when it comes to experiencing the 5-star opulence and fun of hotels and resorts. However, the place is home to many island-getaway spots and local guesthouses.
Therefore, if you are drawn to a more relaxed yet interesting experience of Maldives while getting in touch with the local lifestyle, visit Maafushi.
The area was ravaged back in 2004 due to the Indian Ocean Tsunami. Currently, the officials are rebuilding it and you can visit the sailor huts and fishing jetties in the area.
There are beautiful quiet beaches at this spot with a serene ambience and natural beauties like the palms and clear waters. So, you would have a comfortable time at this spot.
3) Hukuru Miskiiy, Malé
One of the best places you must go to when in the Maldives is the oldest Mosque at Hukuru Miskiiy. This historic spot is located smack-dab in the middle of modern architecture and locations. Yet, when you enter the space, you would step back in time to the old-school lifestyle and history of Maldives.
The mosque has been well-preserved since its origin in 1656. To note, the establishment has its face towards the setting sun instead of the general northeast direction towards Mecca.
The walls made of coral stone are visually appealing and still display the scriptures and patterns. Plus, you would also get to see elaborate wood carvings within the walls of Hukuru Miskiiy.
4) National Museum, Malé
While going to a museum is not necessarily a unique choice while visiting any city, it is not a popular choice when in the Maldives. So, instead of enjoying your time at the beaches and resort area, you should add The National Museum in Malé to your Maldives tour package.
This is present inside the palace at the Sultan Park and the interiors are well-preserved over the years. Here, you would get to see many of the costumes and ornaments of the Kings and Queens of the old put-on display.
Additionally, several photographs, manuscripts, sculptures, and musical instruments are also present in the museum.
5) Ithaa Undersea Restaurant, Conrad Maldives Rangali Island, Alif Dhaal Atoll
One of the notable unique attractions in the Maldives is the underwater eating area in Hilton Maldives Resort and Spa. This spot allows for only 14 visitors at a time, so getting the chance to attend is exclusive. You have to make a reservation first.
When you get the chance to enter the premises, you would notice a 270-degree panoramic view of the underwater marine life here.
You have to climb down one spiral-shaped staircase at a jetty's end; the pavilion here is thatched. To enter, you would have to climb through a curved tunnel-like roof from above into the restaurant.
Enjoy different cuisines like fusion Maldivian delicacies with contemporary Asian and Western touches at this spot.
Overall, there are many exciting areas to visit and admire in the Maldives, starting from the common to unique locations. The latter options make for interesting memories, and you can travel to them with your companions or alone. You can interact with the locals at these spots and gain a full-scale experience of the Maldivian culture.


text:
DETROIT (AP) — U.S. safety regulators are investigating why windshield wipers can fail on almost 194,000 Freightliner big trucks.
The National Highway Traffic Safety Administration says in documents posted Friday that Freightliner maker Daimler Trucks has received four complaints, 12 reports from the field and more than 1,800 warranty claims due to the problem.
In one case a driver reported the wipers stopped working during a rain storm, causing him to lose vision and lose control of his truck.
The investigation covers Freightliner Cascadia trucks from the 2015 and 2016 model years.
The agency says it will conduct an engineering analysis on the wiper system performance. It also will study comparable vehicles.
A message was left Friday seeking comment from a Daimler Trucks spokesman.


text:
I am currently living in Boston and somehow surviving the many blizzards we have recently faced! After receiving my Masters in Education through UMass Boston and completing the Boston Teacher Residency teaching accreditation program last summer, I am now happily teaching in a 3rd grade inclusion classroom at the Eliot K-8 Innovation Public School in the North End.
Emily Galindo