High-resolution Melting Technology Research Articles

Gene polymorphism in IL17A and gene-gene interaction in the IL23R/IL17A axis are associated with susceptibility to coronary artery disease

AimsStudies have confirmed that the IL-23R/IL-17A axis plays an important role in the development of autoimmune and inflammatory diseases. However, its role in coronary artery disease (CAD) remains unclear. Here, we conducted a large sample case-control study to investigate the association between the IL23R/IL17A axis and CAD in the Chinese Han population. MethodsTwo SNPs, rs2275913: G>A (IL17A) and rs6682925: T>C (IL23R), were genotyped in 3042 CAD cases and 3216 controls using the high-resolution melt technology (HRM). Logistic regression analyses were used to adjust the traditional risk factors for CAD and perform the gene interaction analyses. Multiple linear regression analyses were used to study the relationships between the selected SNPs and the levels of serum lipids. In addition, meta-analysis also was performed for the association between rs6682925 and rs2275913 with CAD in different popolations. ResultsOur case-control and meta-analysis for single SNPs demonstrated that the frequencies of the alleles and the distribution of the genotypes had no significant differences in CAD cases compared with controls. In the stratified analysis, we observed that the frequency of the IL17A rs2275913-A allele was more epidemic in early-onset CAD than in the controls (Padj = 0.005, OR = 1.209, 95% CI: 1.059–1.382), and the minor allele C of rs6682925 was associated with a decreased level of serum total cholesterol under a recessive model (Padj = 0.011). We demonstrated a significant interaction between rs6682925 and rs2275913 and CAD in the Chinese Han population. Four genotypes (CTGG, CCAA, CCAG, CCGG) were significantly associated with CAD (Padj = 2.94 × 10−4, OR = 0.619, 95% CI: 0.478–0.803; Padj = 0.01, OR = 1.808, 95% CI: 1.152–1.869; Padj = 6 × 10−6, OR = 2.179, 95% CI: 1.558–3.049; Padj = 0.001, OR = 1.883, 95% CI: 1.282–2.762, respectively). ConclusionOur study found no single SNP of rs2275913 in IL17A and rs6682925 in IL23R was associated with CAD in the Chinese population, but the interaction of them were significantly associated with CAD susceptibility, highlighting the key role of the IL-23R/IL-17A axis in the development of CAD. In addition, we also found rs2275913 was associated with early-onset CAD and rs6682925 was associated with total cholesterol levels, which will contribute to the clinical stratified management of this common disease.

Rapid Detection of Antimicrobial Resistance in Mycoplasma genitalium by High-Resolution Melting Analysis with Unlabeled Probes.

ABSTRACTWith looming resistance to fluoroquinolones in Mycoplasma genitalium, public health control strategies require effective antimicrobial resistance (AMR) diagnostic methods for clinical and phenotypic AMR surveillance. We developed a novel AMR detection method, MGparC-AsyHRM, based on the combination of asymmetric high-resolution melting (HRM) technology and unlabeled probes, which simultaneously performs M. genitalium identification and genotypes eight mutations in the parC gene that are responsible for most cases of fluoroquinolone resistance. These enhancements expand the traditional HRM from the conventional detection of single-position mutations to a method capable of detecting short fragments with closely located AMR positions with a high diversity of mutations. Based on the results of clinical sample testing, this method produces an accordance of 98.7% with the Sanger sequencing method. Furthermore, the specificity for detecting S83I, S83N, S83R, and D87Y variants, the most frequently detected mutations in fluoroquinolone resistance, was 100%. This method maintained a stable and accurate performance for genomic copies at rates of ≥20 copies per reaction, demonstrating high sensitivity. Additionally, no specific cross-reactions were observed when testing eight common sexually transmitted infection (STI)-related agents. Notably, this work highlights the significant potential of our method in the field of AMR testing, with the results suggesting that our method can be applied in a range of scenarios and to additional pathogens. In summary, our method enables high throughput, provides excellent specificity and sensitivity, and is cost-effective, suggesting that this method can be used to rapidly monitor the molecular AMR status and complement current AMR surveillance.IMPORTANCE Mycoplasma genitalium was recently added to the antimicrobial-resistant (AMR) threats “watch list” of the U.S. Centers for Disease Control and Prevention because this pathogen has become extremely difficult to treat as a result of increased resistance. M. genitalium is also difficult to culture, and therefore, molecule detection is the only method available for AMR testing. In this work, we developed a novel AMR detection method, MGparC-AsyHRM, based on the combination of asymmetrical HRM technology and unlabeled probes, and it simultaneously performs M. genitalium identification and genotypes eight mutations in the parC gene that are responsible for most cases of fluoroquinolone resistance. The MGparC-AsyHRM method is a high-throughput, low-cost, simple, and culture-free procedure that can enhance public health and management of M. genitalium infections and AMR control, providing a strong complement to phenotypic AMR surveillance to address the spread of fluoroquinolone resistance.

Detection and quantification of cashew in commercial tea products using High Resolution Melting (HRM) analysis.

Tea, a popular aromatic infusion and food supplement, prepared from Camellia sinensis (L.) Kuntze leaves, is often subjected to adulteration with various undeclared inorganic and plant-derived materials. Cashew (Anacardium occidentale L.) nut husk is one of the most common plant tea adulterants. To date, there are limited DNA-based technologies for tea authentication and quantitative detection of adulterants. Herein, we used a universal plant DNA barcoding marker coupled with High Resolution Melting (Bar-HRM) analysis to authenticate tea products from cashew ground nut. Additionally, cashew-specific markers coupled with HRM technology were used to detect and quantify adulteration of tea with cashew DNA. This methodology can reliably detect admixtures as low as 1% v/v cashew in commercial tea products. Overall, our results demonstrate that the HRM technology is a strong molecular approach in tea authentication, capable of detecting very low adulterations in DNA admixtures. PRACTICAL APPLICATION: In this study, we established the use of high-resolution DNA-based technologies for the detection of cashew adulteration in tea, even in very low quantities. The technology could be applied to a greater range of plant-based tea adulterants. This work is expected to facilitate the traceability and authenticity of tea products and form the basis for the development of strategies against fraudulent practices.

A method for the unbiased quantification of reassortment in segmented viruses

Reassortment of segmented viruses can be an important source of genetic diversity underlying viral evolution and emergence. Methods for the quantification of reassortment have been described but are often cumbersome and best suited for the analysis of reassortment between highly divergent parental strains. While it is useful to understand the potential of divergent parents to reassort, outcomes of such heterologous reassortment are driven by differential selection acting on the progeny and are typically strain specific. To quantify reassortment robustly, a system free of differential selection is needed. We have generated such a system for influenza A virus and for mammalian orthoreovirus by constructing well-matched parental viruses carrying small genetic tags. The method utilizes high-resolution melt technology for the identification of reassortant viruses. Ease of sample preparation and data analysis enables streamlined genotyping of a large number of virus clones. The method described here thereby allows quantification of the efficiency of reassortment and can be applied to diverse segmented viruses.

High-Resolution Melting (HRM) Analysis Reveals Genotypic Differentiation of Venturia inaequalis Populations in Greece

During the recent years, High Resolution Melting (HRM) analysis has been developed as a rapid and accurate tool in plant disease diagnostics, species identification and SNP genotyping. This approach has been applied to analyze the genetic diversity in several plant species with molecular markers, including single sequence repeats (SSR). However, no studies have been carried out to investigate the variation of SSR in plant pathogenic fungi by using the HRM technology. In this report, the genetic structure of Venturia inaequalis populations in Greece was investigated for the first time by using six microsatellite markers. The developed HRM protocol was able to generate genotype-specific melting curves, consistent with the haploid nature of the fungus. Compared to the more complexed genome of diploid plants, several limitations were avoided. The shape of the melting curves revealed differences between the genotypes in each SSR marker and showed that all the genotypes could be easily distinguished. The genetic analysis of apple scab populations revealed high genetic variation within the populations (96%), while only 4% of the total gene diversity was attributed to among-population variation. The isolates were grouped into three populations according to the principal coordinate analysis (PCoA).

Application of High Resolution Melting Curve Analysis in Detection of SLC4A1 Gene Mutation in Patients with Hereditary Spherocytosis

To investigate the feasibility and clinical significance of high resolution melting(HRM) curve analysis to detect SLC4A1 gene D38A and K56E mutations in the patients with hereditary spherocytosis(HS). Peripheral blood was collected from 23 cases of HS for routine tests and their genomic DNA was extracted by routine technique. Specific primers of mutation sites D38A and K56E of SLC4A1 gene were designed. The HRM method was used to analyze all the samples, and then the results of HRM were verified with DNA sequencing technology. Among 23 specimens of HS patients, 6 cases of heterozygous mutant gene were detected by HRM technology, including 3 cases of D38A mutation and 3 cases of K56E mutation, which were confirmed by DNA sequencing. The HRM technology can correctly detect 2 common mutation sites including D38A and K56E in SLC4A1 gene in an efficient, fast, and reliable way, which not only can be used for clinical diagnosis, but also expected to be a new method for clinical researchers to define gene mutation spectrum in HS patients.

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

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

High Resolution Melting as a rapid, reliable, accurate and cost-effective emerging tool for genotyping pathogenic bacteria and enhancing molecular epidemiological surveillance: a comprehensive review of the literature.

Rapid, reliable and accurate molecular typing methods are essential for outbreaks detection and infectious diseases control, for monitoring the evolution and dynamics of microbial populations, and for effective epidemiological surveillance. The introduction of a novel method based on the analysis of melting temperature of amplified products, known as High Resolution Melting (HRM) since 2002, has found applications in epidemiological studies, either for identification of bacterial species or molecular typing, as well as an extensive and increasing use in many research fields. HRM method is based on the use of saturating third generation dyes, advanced real-time PCR platforms, and bioinformatics tools. To describe, by a comphrehensive review of the literature, the use, application and usefulness of HRM for the genotyping of bacterial pathogens in the context of epidemiological surveillance and public health. A literature search was carried out during July-August 2016, by consulting the biomedical databases PubMed/Medline, Scopus, EMBASE, and ISI Web of Science without limits. The search strategy was performed according to the following keywords: high resolution melting analysis and bacteria and genotyping or molecular typing. All the articles evaluating the application of HRM for bacterial pathogen genotyping were selected and reviewed, taking into account the objective of each study, the rationale explaining the use of this technology, and the main results obtained in comparison with gold standards and/or alternative methods, when available. HRM method was extensively used for molecular typing of both Gram-positive and Gram-negative bacterial pathogens, representing a versatile genetic tool: a) to evaluate genetic diversity and subtype at species/subspecies level, based also on allele discrimination/identification and mutation screening; b) to recognize phylogenetic groupings (lineage, sublineage, subgroups); c) to identify antimicrobial resistance; d) to detect and screen for mutations related to drug-resistance; e) to discriminate gene isoforms. HRM method showed, in almost all instances, excellent typeability and discriminatory power, with high concordance of typing results obtained with gold standards or comparable methods. Conversely, for the evaluation of genetic determinants associated to antibiotic-resistance or for screening of associated mutations in key gene fragments, the sensitivity and specificity was not optimal, because the targeted amplicons did not encompass all the crucial mutations. Despite the recent introduction of sequencing-based methods, the HRM method deserves consideration in research fields of infectious diseases, being characterized by low cost, rapidity, flexibility and versatility. However, there are some limitations to HRM assays development, which should be carefully considered. The most common application of HRM for bacterial typing is related to Single Nucleotide Polymorphism (SNP)-based genotyping with the analysis of gene fragments within the multilocus sequence typing (MLST) loci, following an approach termed mini-MLST or Minim typing. Although the resolving power is not totally correspondent to MLST, the Simpson's Index of Diversity provided by HRM method typically >0.95. Furthermore, the cost of this approach is less than MLST, enabling low cost surveillance and rapid response for outbreak control. Hence, the potential of HRM technology can strongly facilitate routine research and diagnostics in the epidemiological studies, as well as advance and streamline the genetic characterization of bacterial pathogens.

Gene silencing of Nox4 by CpG island methylation during hepatocarcinogenesis in rats.

ABSTRACTThe association between the downregulation of genes and DNA methylation in their CpG islands has been extensively studied as a mechanism that favors carcinogenesis. The objective of this study was to analyze the methylation of a set of genes selected based on their microarray expression profiles during the process of hepatocarcinogenesis. Rats were euthanized at: 24 h, 7, 11, 16 and 30 days and 5, 9, 12 and 18 months post-treatment. We evaluated the methylation status in the CpG islands of four deregulated genes (Casp3, Cldn1, Pex11a and Nox4) using methylation-sensitive high-resolution melting technology for the samples obtained from different stages of hepatocarcinogenesis. We did not observe methylation in Casp3, Cldn1 or Pex11a. However, Nox4 exhibited altered methylation patterns, reaching a maximum of 10%, even during the early stages of hepatocarcinogenesis. We observed downregulation of mRNA and protein of Nox4 (97.5% and 40%, respectively) after the first carcinogenic stimulus relative to the untreated samples. Our results suggest that Nox4 downregulation is associated with DNA methylation of the CpG island in its promoter. We propose that methylation is a mechanism that can silence the expression of Nox4, which could contribute to the acquisition of neoplastic characteristics during hepatocarcinogenesis in rats.

Detection of Mutations in SF3B1 in Chronic Lymphocytic Leukaemia Patients By Reverse Transcription (Rt) Polymerase Chain Reaction and High Resolution Melt Curve Analysis, an Alternative Approach to Next Generation Sequencing for Routine Molecular Diagnostic Laboratories?

Genetic makers such as TP53, ATM mutations and IgH somatic hypermutation status have been used to predict clinical behaviour and response to therapy in Chronic Lymphocytic Leukaemia (CLL) for the last decade. Recently identified genetic markers including splicing factor 3 B1 (SF3B1), NOTCH and BirC3 are also significant but their role cannot be fully evaluated until a robust assay is available for a routine molecular diagnostic laboratoryThe cell spliceosome is involved in the precise excision of introns from pre-mRNA and is composed of five subunits of which SF3B1 is a core component. Patient samples with SF3B1 mutations display enhanced intron retention in a small subset of transcripts involved in cancer related regulation such as cell cycle control, apoptosis and angiogenesis. Studies of CLL patient samples has shown that those with mutations in SF3B1 have been associated with shorter treatment free and overall survival.The screening methodology for SF3B1 mutations through deep sequencing, though very sensitive, is not available in routine diagnostic laboratories. Approximately 90% of CLL-associated SF3B1 mutations occur is 336bp region of the cDNA exome amenable to PCR amplification. We designed primers flanking this region and following reverse transcription (RT) PCR amplification, detected the products on standard agarose gels. Following bidirectional Sanger sequencing, we compared the sequences obtained with reference SF3B1 cDNA/amino acid code. We assessed the impact of the mutations by the polyphen database and compared this to the immunophenotype and cytogenetic data as well as response to treatment through minimal residual disease (MRD) tracking. We subsequently designed an assay for detection of sequence variants in the SF3B1 region of interest by High Resolution Melt (HRM) curve analysis. This has enabled us to identify genetic variants of the SF3B1 region prior to sequencing. The application of this HRM pre-sequencing screening assay has resulted in a significant reduction in the number of samples requiring sequencing.The patient cohort consisted of 52 patients enrolled on a Phase II, multi centre study of fludarabine, cyclophosphamide and rituximab (FCR) and included patients < 65 yrs, WHO status of 0, 1 or 2 without a deletion 17p. All samples were collected and tested prior to treatment, at the end of treatment (after 4 or 6 cycles when MRD negative), 6 monthly post treatment for 5 years for minimal residual disease and then annually by 6 colour immunophenotyping. Patients (51/52, no RNA available for 1 patient) were screened prior to treatment for the presence of SF3B1 mutations. Five of 51 (9.8 %) patients had SF3B1 mutations detectable by this methodology shown in the table below.Table 1Pt.Nucleotide changeAmino acid changePolyphenIGHV M=mutatedU=unmutatedCytogeneticsImmunophenotypingMRD1c.2146A>Gp.K700EDamagingU V4-6*01NormalCD5 -veNot tracked due to neg CD52c.2179G>Cp.A711PDamagingU V1-69*01NormalNeg @ 3 mts Pos @ 16 mts3c.2267G>Ap.G740EDamagingM V3-74*01del 13qCD5 weak +ve CD38 +veNeg @ 3 mts Pos @ 12 mts4c.2146A>Gp.K700EDamagingU V1-69*01del 13qNever MRD neg5c.2032C>Tp.H662YDamagingU V3-11*01del 13qCD38 +veWithdrawnPatients with mutations in SF3B1 all had favourable cytogenetics (normal or del 13q) and the majority (4/5) had unmutated IGHV genes. We detected two patients with a mutation at c.2146A>G, p.K700E and one at c.2267G>A, pG740E which have been described previously and two novel mutations at c.2179G>C, p.A711P and c.2032C>T, p.H662Y. All mutations detected were damaging as per the polyphen database. Two patients have weak or no expression of CD5. One patient has never achieved MRD negativity, and two patients who became MRD negative at 3 months reverted to MRD positivity 9 and 13 months later respectively. One patient's MRD levels could not be tracked due to negative CD5 expression and was followed by a less sensitive IgH clonality assay (sensitivity approx. 1%) and one patient was withdrawn from the study.In summary, we have developed a simple PCR based technique to detect SF3B1 mutations in CLL patient samples with high disease burden which can be applied in routine molecular diagnostic laboratories without access to next generation sequencing. The use of HRM technology resulted in a > 90% reduction in the number of samples requiring sequencing. DisclosuresNo relevant conflicts of interest to declare.

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria.

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs. One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures. SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status. While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele. In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs. This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

Allelic variants in durum wheat (Triticum turgidum L. var. durum) DREB genes conferring tolerance to abiotic stresses.

To identify potential and useful markers able to discriminate promising lines of durum wheat (Triticum turgidum L. var durum) tolerant to salt and drought stresses, nucleotide sequences of Dehydration-Responsive-Element Binding Factor (DREB) genes were used to design primers probed with High Resolution Melting technology for the identification of allelic variants. DREB1, DREB2, DREB3, DREB4 and DREB5 conserved regions corresponding to EREBP/AP2 domain and containing the conserved core sequence (5'-TACCGACAT-3'), the protein site directly involved in DNA recognition, were analyzed. The validated primers were probed on four lines of durum wheat differentially tolerant to salt and drought stresses treated with solutions containing different salt concentrations. Some SNPs mutations were identified in the highly tolerant durum cultivar Jennah Khetifa treated with the maximum salt concentration (1.5 M). The SNPs mutations identified were non-synonymous (nsSNPs) causing changes in peptide sequences. These concerned amino acid residues directly involved in the maintenance of protein geometry, the recognition of the specific cis-element, and the contacts between the protein and DNA. A validation of the found SNPs was carried out by analyzing the regressions between DREBs SNPs allelic variants and some morpho-physiological characters in a RIL population, deriving from a cross between the two durum wheat genotypes utilized for SNPs detection, grown under contrasting environments. Several phenotypical characters have been assessed in the progeny across all the localities evaluating the different performances under different stress levels and related with SNPs occurrence. Significant relations between SNPs variants and morpho-physiological characteristics were found in the progeny growth in very severe drought environments, suggesting a role of the identified SNPs in conferring a superior capability to adverse stress conditions and, at the same time, the key role of these genes in empowering salt tolerance.

Sequence-based detection of mutations in cadherin 1 to determine the prevalence of germline mutations in patients with invasive lobular carcinoma of the breast.

BackgroundLoss of cadherin 1 (CDH1) expression, which is normally involved in cell adhesion and maintenance of tissue architecture, is a hallmark of invasive lobular carcinoma (ILCA). Because hereditary cancers may require different risk reduction, counseling and treatment options than sporadic cancer, it is critical to determine the prevalence of germline CDH1 mutations in patients with ILCA.MethodsAll patients with ILCA (n = 100) previously enrolled in the Clinical Breast Care Project were identified. Genomic DNA was isolated from peripheral blood samples and DNA variants were detected for each exon of CDH1 using high-resolution melting technology followed by direct sequencing.ResultsWithin the 100 samples screened, four nonsynonymous variants were detected: A592T in one Hispanic patient, A617T in two patients, both African American, P825L in a Causasian patient whose grandmother had stomach cancer, and G879S in a Caucasian patient. Further evaluation of A617T in an additional 165 African American patients found that 11 patients, none with ILCA, carried this variant including one patient who was homozygous for the variant.ConclusionsCDH1 mutations are infrequent in patients with ILCA, and the variants that were detected have been classified as non-pathogenic. These data suggest that ILCA does not have a significant hereditary component and do not support CDH1 gene mutation testing in patients with ILCA.

The TRHR Gene Is Associated with Hypothalamo-Pituitary Sensitivity to Levothyroxine

Background: Thyroidectomized patients need variable doses of levothyroxine (LT₄) to obtain target thyroid-stimulating hormone (TSH) levels. Individual feedback set-points have been hypothesized and the influence of several genes in the regulation of the pituitary-thyroid axis has been demonstrated. Objectives: We hypothesized that genetic variants of the TRHR gene could be associated with a different hypothalamo-pituitary sensitivity to thyroid hormone feedback. Methods: We retrospectively analyzed 84 thyroidectomized patients with no residual thyroid function and undetectable thyroglobulin levels. Patients were evaluated under LT₄ resulting in TSH levels detectable but <0.5 μIU/ml. The two SNPs rs3134105 and rs3110040 were identified as informative markers of the TRHR gene. Genotyping was performed using high-resolution melting technology. Genotype distribution was compared between the patients and 99 euthyroid controls. Results: The selected SNPs were in linkage disequilibrium and only rs3134105 was further considered. A significant difference between the three possible genotypes for rs3134105 was found for TSH (p = 0.04) and free thyroxine (fT₄)/TSH ratio (p = 0.02). Moreover, despite similar serum concentrations of free triiodothyronine (fT₃) and fT₄, carriers of at least one A allele of rs3134105 had significantly lower serum TSH levels (p = 0.01) as well as higher fT₃/TSH (p = 0.01) and fT₄/TSH ratios (p < 0.01). Conclusions: We demonstrated an association between serum TSH levels and discrete alleles of the TRHR gene in totally thyroidectomized patients under LT₄ therapy. Therefore, the TRHR gene seems to be a determinant of hypothalamo-pituitary sensitivity to LT₄.

Investigation of JAK1 and STAT3 polymorphisms and their gene-gene interactions in nonspecific digestive disorder of rabbits.

Acting in a cellular signaling pathway, Janus kinase 1 (JAK1) and signal transducer and activator of transcription 3 (STAT3) play critical roles in several kinds of physiological and pathological responses, including inflammatory response. In this study, we detected three coding single nucleotide polymorphisms (cSNPs) of JAK1 and four cSNPs of STAT3 in New Zealand White rabbits. We analyzed their association with genetic resistance to nonspecific digestive disorder (NSDD) based on a case-control study (253 cases and 227 controls). The rabbits were genotyped for c.1421 C>T and c.3036 G>A in JAK1 using restriction fragment length polymorphisms and for c.831 T>C and c.399 G>A of STAT3 using high-resolution melting technology. The frequencies of the alleles and genotypes differed significantly between the case and control groups (P<0.05). The case-control association analysis revealed that in JAK1, allele C increased the risk of NSDD (OR: 1.60, 95% CI 1.166-2.185, P=0.003), whereas allele A played a potentially protective role against NSDD (OR: 0.74, 95% CI 0.572-0.952, P=0.019). We used five inheritance models to evaluate the importance of the associated genotypes. Under the dominant inheritance model, the association analysis suggested that the CT/TT genotype increased the risk of NSDD (OR: 1.65, 95% CI 1.13-2.40, P=0.009). A haplotype analysis showed that allele H2 (the two cSNPs: CG; OR: 1.354, 95% CI 1.050-1.747, P=0.019) markedly increased the risk of the disorder. The association analysis revealed that in STAT3, allele G played a potentially protective role against NSDD (OR: 0.74; 95% CI 0.569-0.951; P=0.019), whereas allele C increased the risk of NSDD (OR: 1.37; 95% CI 1.059-1.760; P=0.016). Recessive inheritance was shown to be the best fitting model for c.399 and c.831. Under the recessive inheritance model, the association analysis suggested that the G/G genotype increased the risk of NSDD (OR: 1.73; 95% CI 1.07-2.80; P=0.025). A haplotype analysis showed that H1' (the two cSNPs: AC; OR: 1.365; CI 1.059-1.760; P=0.016) significantly increased the risk of the disorder. The CATMOD program (SAS 9.2) and the multifactor dimensionality reduction method were used to analyze the genetic interactions between JAK1 and STAT3. Data on the genetic interactions revealed that the JAK1 and STAT3 risk alleles described above contribute to NSDD susceptibility in combination with each other, and that model (c1421, c3036, c831) was the best model (OR: 2.7262; 95% CI: 4.7408-5.1986; P<0.0001). To our knowledge, this is the first report of the genetic polymorphisms of the JAK1 and STAT3 genes and their associations with the incidence of NSDD in rabbits.

Promoter methylation status of MGMT, hMSH2, and hMLH1 and its relationship to corresponding protein expression and TP53 mutations in human esophageal squamous cell carcinoma

To determine the relevance of O-6-methylguanine-DNA methyltransferase (MGMT), human mutS homolog 2 (hMSH2), and human mutL homolog 1 (hMLH1) in TP53 mutations in esophageal squamous cell carcinoma, we employed methylation-sensitive high-resolution melting technology and methylation-specific polymerase chain reaction (PCR) to analyze promoter hypermethylation of MGMT, hMSH2, and hMLH1, respectively, in 51 paired tumors and their adjacent normal tissues. The protein expression of the three proteins was also evaluated by Western blot analysis, and the PCR products of TP53, from exon 5 to exon 8, were directly sequenced to measure the mutation spectrum. Esophageal tumor tissues embraced statistically higher MGMT and hMSH2 promoter methylation level than normal tissue. The promoter methylation status of MGMT and hMSH2 corresponds positively with the protein expression level of MGMT and hMSH2. However, such relevance was not found for hMLH1. Furthermore, TP53 mutation status was well associated with MGMT and hMSH2 promoter methylation status, indicating that silencing of the two genes could lead to TP53 mutation in ESCC.

Impact of Mutation Type and Amplicon Characteristics on Genetic Diversity Measures Generated Using a High-Resolution Melting Diversity Assay

We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutation type, position, and concentration on HRM scores. The impact of amplicon length and G/C content on HRM scores was also evaluated. Different mutation types affected HRM scores to varying degrees (1-bp deletion < 1-bp change < 3-bp insertion < 9-bp insertion). The impact of mutations on HRM scores was influenced by amplicon length and the position of the mutation within the amplicon. Mutations were detected at concentrations of 5% to 95%, with the greatest impact at 50%. The G/C content altered melting temperature values of amplicons but had no impact on HRM scores. These data are relevant to the design of assays that measure genetic diversity using HRM technology.

High resolution melting analysis for the identification of novel mutations in DKC1 and TERT genes in patients with dyskeratosis congenita

Dyskeratosis congenita (DC) is a rare inherited bone-marrow failure syndrome with high clinical heterogeneity. Cells derived from DC patients present short telomeres at early ages, as a result of mutations in genes encoding components of the telomerase complex (DKC1, TERC, TERT, NHP2 and NOP10), or the shelterin complex (TINF2). However, mutations have been identified only in around 50% of the cases, indicating that other genes could be involved in the development of this disease. Indeed, mutations in TCBA1 or chromosome segment C16orf57 have been described recently. We have used HRM technology to perform genetic analysis in the above mentioned genes, in Spanish patients showing both, some clinical features of DC and short telomeres. The mutations have been identified by PCR amplification of DC genes followed by high resolution melting (HRM) and direct DNA sequencing analysis. We have identified seven new families with DC, three with X-linked DC and four with autosomal dominant DC, in which we have found two novel mutations in DKC1 (p.His68Arg and p.Lys390del) and four novel mutations in TERT gene (p.Pro530Leu, p.Arg698Trp, p.Arg971His and p.Arg698Gln). The results show that the use of HRM analysis enables a rapid and inexpensive identification of mutations in dyskeratosis congenita associated genes.

Genetic screening of Fabry patients with EcoTILLING and HRM technology

BackgroundAnderson-Fabry disease (FD) is caused by a deficit of the α-galactosidase A enzyme which leads to the accumulation of complex sphingolipids, especially globotriaosylceramide (Gb3), in all the cells of the body, causing the onset of a multi-systemic disease with poor prognosis in adulthood. In this article, we describe two alternative methods for screening the GLA gene which codes for the α-galactosidase A enzyme in subjects with probable FD in order to test analysis strategies which include or rely on initial pre-screening.FindingsWe analyzed 740 samples using EcoTILLING, comparing two mismatch-specificendonucleases, CEL I and ENDO-1, while conducting a parallel screening of the same samples using HRM (High Resolution Melting). Afterwards, all samples were subjected to direct sequencing. Overall, we identified 12 different genetic variations: -10C>T, -12G>A, -30G>A, IVS2-76_80del5, D165H, C172Y, IVS4+16A>G, IVS4 +68 A>G, c.718_719delAA, D313Y, IVS6-22C>T, G395A. This was consistent with the high genetic heterogeneity found in FD patients and carriers. All of the mutations were detected by HRM, whereas 17% of the mutations were not found by EcoTILLING. The results obtained by EcoTILLING comparing the CEL I and ENDO-1 endonucleases were perfectly overlapping.ConclusionOn the basis of its simplicity, flexibility, repeatability, and sensitivity, we believe thatHRM analysis of the GLA gene is a reliable presequencing screening tool. This method can be applied to any genomic feature to identify known and unknown genetic alterations, and it is ideal for conducting screening and population studies.