High-resolution Melting Screening Research Articles

Combined immunodeficiency caused by pathogenic variants in the ZAP70 C-terminal SH2 domain.

ZAP-70, a protein tyrosine kinase recruited to the T cell receptor (TCR), initiates a TCR signaling cascade upon antigen stimulation. Mutations in the ZAP70 gene cause a combined immunodeficiency characterized by low or absent CD8+ T cells and nonfunctional CD4+ T cells. Most deleterious missense ZAP70 mutations in patients are located in the kinase domain but the impact of mutations in the SH2 domains, regulating ZAP-70 recruitment to the TCR, are not well understood. Genetic analyses were performed on four patients with CD8 lymphopenia and a high resolution melting screening for ZAP70 mutations was developed. The impact of SH2 domain mutations was evaluated by biochemical and functional analyses as well as by protein modeling. Genetic characterization of an infant who presented with pneumocystis pneumonia, mycobacterial infection, and an absence of CD8 T cells revealed a novel homozygous mutation in the C-terminal SH2 domain (SH2-C) of the ZAP70 gene (c.C343T, p.R170C). A distantly related second patient was found to be compound heterozygous for the R170C variant and a 13bp deletion in the ZAP70 kinase domain. While the R170C mutant was highly expressed, there was an absence of TCR-induced proliferation, associated with significantly attenuated TCR-induced ZAP-70 phosphorylation and a lack of binding of ZAP-70 to TCR-ζ. Moreover, a homozygous ZAP-70 R192W variant was identified in 2 siblings with combined immunodeficiency and CD8 lymphopenia, confirming the pathogenicity of this mutation. Structural modeling of this region revealed the critical nature of the arginines at positions 170 and 192, in concert with R190, forming a binding pocket for the phosphorylated TCR-ζ chain. Deleterious mutations in the SH2-C domain result in attenuated ZAP-70 function and clinical manifestations of immunodeficiency.

HRM screening of the UBC9 gene encoding the SUMO-E2-conjugating enzyme- case-control study in breast cancer.

UBC9 (E2) small ubiquitin-like modifier conjugating enzyme plays a key role in the post-translational modification of proteins named sumoylation. Defects in small ubiquitin-like modifier modification may contribute to breast carcinogenesis. In the present work, we examined UBC9genetic variation. UBC9genetic variation was analyzed by using the high resolution melting (HRM) method. HRM study was conducted on 173-182healthy women and 188-190women with breast cancer. During HRM screening, we analysed three known single-nucleotide polymorphisms in introns: rs4984806, rs909916and rs909917, and one known single nucleotide polymorphism rs8063in exon 7, in a non-coding region. The genotype frequencies for all polymorphisms were in accordance with Hardy- Weinberg equilibrium among the control subjects and breast cancer patients. The linkage disequilibrium analysis displayed that there was one polymorphism block, which consisted of three single nucleotide polymorphisms: rs909916, rs909917and rs4984806. We identified two common haplotypes CCG and TTC, but we did not find significant differences in the distribution of these haplotypes between cases and controls. Our study showed no differences in the occurrence of indicated polymorphisms in the UBC9gene in a group of healthy women compared to women with breast cancer. These results suggest that the polymorphisms of the UBC9gene- rs4984806, rs909916, rs909917and rs8063can be not associated with breast cancer risk.

Familial Left Ventricular Non-Compaction Is Associated With a Rare p.V407I Variant in Bone Morphogenetic Protein 10.

Left ventricular non-compaction (LVNC) is a heritable cardiomyopathy characterized by hypertrabeculation, inter-trabecular recesses and thin compact myocardium, but the genetic basis and mechanisms remain unclear. This study identified novel LVNC-associated mutations inNOTCH-dependent genes and investigated their mutational effects.Methods and Results:High-resolution melting screening was performed in 230 individuals with LVNC, followed by whole exome and Sanger sequencing of available family members. Dimerization of bone morphogenetic protein 10 (BMP10) and its binding to BMP receptors (BMPRs) were evaluated. Cellular differentiation, proliferation and tolerance to mechanical stretch were assessed in H9C2 cardiomyoblasts, expressing wild-type (WT) or mutant BMP10 delivered by adenoviral vectors. Rare variants, p.W143*-NRG1and p.V407I-BMP10, were identified in 2 unrelated probands and their affected family members. Although dimerization of mutant V407I-BMP10 was preserved like WT-BMP10, V407I-BMP10 pulled BMPR1a and BMPR2 receptors more weakly compared with WT-BMP10. On comparative gene expression and siRNA analysis, expressed BMPR1a and BMPR2 receptors were responsive to BMP10 treatment in H9C2 cardiomyoblasts. Expression of V407I-BMP10 resulted in a significantly lower rate of proliferation in H9C2 cells compared with WT-BMP10. Cyclic stretch resulted in destruction and death of V407I-BMP10 cells. The W143*-NRG1and V470I-BMP10variants are associated with LVNC. Impaired BMPR-binding ability, perturbed proliferation and differentiation processes and intolerance to stretch in V407I-BMP10 mutant cardiomyoblasts may underlie myocardial non-compaction.

H3F3 mutation status of giant cell tumors of the bone, chondroblastomas and their mimics: a combined high resolution melting and pyrosequencing approach

Behjati et al recently described recurrent mutations of H3F3 genes in giant cell tumors of the bone and chondroblastomas. Both these entities belong to the spectrum of giant cell-rich bone lesions, often presenting a diagnostic challenge for the pathologist. Our aim was to investigate the value of searching for H3F3 mutations in the diagnosis of giant cell tumors of the bone and giant cell-rich chondroblastomas. Two hundred eighty-one bone lesion samples, including 170 giant cell tumors of the bone, 26 chondroblastomas and 85 other giant cell-rich and/or epiphyseal tumors, were analyzed. Mutation status was determined using first high resolution melting screening and then mutation profiling pyrosequencing. Mutational status was compared with clinical data and, for giant cell tumors of the bone, with p63 immunostaining status. As histone methylation changes have been reported in association with H3F3 mutations, the methylation status of lysine 37 was investigated. H3F3A and H3F3B were found in 85% of giant cell tumors of the bone and 88% of chondroblastomas. In addition to the major G35W mutation, we found two rare H3F3A mutations: one G35R and one G35V. Among the other tumors studied, we only found H3F3A gene mutations in two cases of ‘dedifferentiated chondrosarcoma mimicking giant cell tumor of the bone’. A H3F3B mutation was also observed in one case of dedifferentiated chondroblastoma. P63 expression in giant cell tumors of the bone seems to be associated with H3F3 gene mutations (P=0.004). H3F3 mutations did not correlate with clinical data, outcome or methylation changes in Lysin 37. In conclusion, H3F3 mutations are sensitive and specific markers of giant cell tumors of the bone and chondroblastomas. High resolution melting and pyrosequencing procedures are high-performance tools in this context. Determination of H3F3 mutation will allow reclassification of some entities belonging to the spectrum of giant cell-rich lesions.

Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing.

BackgroundFunctions of most genes predicted in the soybean genome have not been clarified. A mutant library with a high mutation density would be helpful for functional studies and for identification of novel alleles useful for breeding. Development of cost-effective and high-throughput protocols using next generation sequencing (NGS) technologies is expected to simplify the retrieval of mutants with mutations in genes of interest.ResultsTo increase the mutation density, seeds of the Japanese elite soybean cultivar Enrei were treated with the chemical mutagen ethyl methanesulfonate (EMS); M2 seeds produced by M1 plants were treated with EMS once again. The resultant library, which consisted of DNA and seeds from 1536 plants, revealed large morphological and physiological variations. Based on whole-genome re-sequencing analysis of 12 mutant lines, the average number of base changes was 12,796 per line. On average, 691 and 35 per line were missense and nonsense mutations, respectively. Two screening strategies for high resolution melting (HRM) analysis and indexed amplicon sequencing were designed to retrieve the mutants; the mutations were confirmed by Sanger sequencing as the final step. In comparison with HRM screening of several genes, indexed amplicon sequencing allows one to scan a longer sequence range and skip screening steps and to know the sequence information of mutation because it uses systematic DNA pooling and the index of NGS reads, which simplifies the discovery of mutants with amino acid substitutions.ConclusionsA soybean mutant library with a high mutation density was developed. A high mutation density (1 mutation/74 kb) was achieved by repeating the EMS treatment. The mutation density of our library is sufficiently high to obtain a plant in which a gene is nonsense mutated. Thus, our mutant library and the indexed amplicon sequencing will be useful for functional studies of soybean genes and have a potential to yield useful mutant alleles for soybean breeding.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2079-y) contains supplementary material, which is available to authorized users.

Development of a High-Resolution Melting Curve Analysis Screening Test for SRSF2 Splicing Factor Gene Mutations in Myelodysplastic Syndromes

Somatic mutations of the spliceosome machinery have been recently identified by whole genome analysis in hematologic diseases, such as myelodysplastic syndrome, chronic lymphocytic leukemia, myeloproliferative neoplasms, acute myeloid leukemia, and advanced forms of mastocytosis, and also in nonhematologic conditions. SRSF2 is a member of the serine/arginine-rich family pre-mRNA splicing factors that plays a role in mRNA export from the nucleus and translation. We describe a high-resolution melting (HRM) curve analysis to screen for SRSF2 hotspot mutations in a fast, sensitive, and reliable way. Fifty bone marrow samples from patients with myelodysplastic syndrome were analyzed by the HRM assay and by direct sequencing. HRM screening identified four melting patterns corresponding to a negative (wild-type) group and three different mutated groups. Each mutated group was identified according to the positive control used: P95H, P95L, and P95R, respectively. An HRM mutated pattern was identified in seven patients. Positive and negative results from HRM were compared with direct sequencing results with a sensitivity and specificity of 100% (95% CI, 0.56-1, and 95% CI, 0.89-1, respectively). Analytical sensitivity analysis revealed a detection threshold of up to 1:9 (mutated/wild type) dilution. This rapid screening method may provide useful information for clinical decision making and be helpful to optimize laboratory resources and reduce turnaround time.

The origin of the giant liver fluke, Fascioloides magna (Trematoda: Fasciolidae) from Croatia determined by high-resolution melting screening of mitochondrial cox1 haplotypes

The high-resolution melting (HRM) method, recently optimized as a reliable technique for population study of the European Fascioloides magna populations, was applied to determine an origin of F. magna individuals from Croatia. The structure and frequency of mitochondrial cytochrome c oxidase subunit I (439 bp; cox1) haplotypes of 200 Croatian flukes coming from 19 red deer (Cervus elaphus elaphus) livers were screened and compared with recently determined reference samples of F. magna from all European foci-Italy, Czech Republic, and Danube floodplain forests. While the reference haplotypes Ha1 and Ha2 were specific for flukes from the first European focus of fascioloidosis, the Natural Park La Mandria in Italy, the remaining three haplotypes (Ha3, Ha4, and Ha5) represented parasites from the second focus, Czech Republic. Besides, Ha3 and Ha4 were found also in the third, latest, and still expanding European focus, the Danube floodplain forests. The HRM screening of cox1 haplotypes of Croatian F. magna individuals resulted in classification of samples into the two mitochondrial haplogroups characterized by well-distinguished melting curves. They corresponded to Ha3 and Ha4 reference haplotypes that confirmed the Danube origin of F. magna from Croatia. The results support the theory that the Danube floodplain forests population of F. magna represents uniform genetic pool of the parasite. The spread of F. magna alongside the Danube River down to Croatia was possible due to suitable ecological conditions for definitive and intermediate hosts present in this unique biotope.

Validation of a quantitative PCR-high-resolution melting protocol for simultaneous screening ofCOL1A1andCOL1A2point mutations and large rearrangements: Application for diagnosis of osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a connective tissue disorder mostly characterized by autosomal dominant inheritance. Over 1,100 causal mutations have been identified scattered along all exons of genes encoding type I collagen precursors, COL1A1 and COL1A2. Because of the absence of mutational hotspots, Sanger sequencing is considered the gold standard for molecular analysis even if the workload is very laborious and expensive. To overcome this issue, different prescreening methods have been proposed, including DHPLC and biochemical studies on cultured dermal fibroblasts; however, both approaches present different drawbacks. Moreover, in case of patients who screen negative for point mutations, an additional screening step for complex rearrangements is required; the added causative variants expected from this approach are about 1-2%. The aim of this study was to optimize and validate a new protocol that combines quantitative PCR (qPCR) and high-resolution melting (HRM) curve analysis to reduce time and costs for molecular diagnosis. Results of qPCR-HRM screening on 57 OI patients, validated by DHPLC-direct sequencing and multiplex ligation-dependent probe amplification (MLPA), indicate that all alterations identified with the mentioned methodologies are successfully detected by qPCR-HRM. Moreover, HRM was able to discriminate complex genotypes and homozygous variants. Finally, qPCR-HRM outperformed direct sequencing and DHPLC-MLPA in terms of rapidity and costs.

Diagnostic Testing for IDH1 and IDH2 Variants in Acute Myeloid Leukemia : An Algorithmic Approach Using High-Resolution Melting Curve Analysis

Isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations and polymorphism are reported in 5% to 15% of acute myeloid leukemia (AML) cases, with G105 and R132 of IDH1 and R140 and R172 of IDH2 known to be clinically significant. Current Sanger sequencing assays to detect IDH mutations are labor intensive and not cost effective for clinical testing of low-frequency mutations. Therefore, we developed clinical assays using high-resolution melting (HRM) analysis to screen for all four variants listed above, followed by Sanger sequencing confirmation. The sensitivities of the assays were 7.3% and 7.9% for the detection of IDH2 and IDH1 variants, respectively, against the background of wild-type transcripts. Comparison of HRM to Sanger sequencing on 146 AML bone marrow samples for validation showed near-perfect concordance for all positive and negative results for IDH1 (98%) and IDH2 (94%). Postvalidation clinical implementation of upfront HRM screening (N = 106), using a more conservative algorithm to avoid false-negative results, reduced the number of Sanger sequencing tests by 73% (IDH1) and 78% (IDH2). Of the variant calls made by HRM in postvalidation clinical samples, Sanger confirmed the presence of a variant in 62% (IDH1) and 44% (IDH2) of the samples. In conclusion, our HRM assays are rapid, convenient, and versatile assays for screening and confirmation of alterations in IDH1 and IDH2.

Abstract 3183: Dysregulated expression and function of Plexin-B1 in ovarian cancers

Abstract Background: Ovarian cancer has a tendency to spread within the peritoneal cavity early in the disease course. Understanding the metastatic pathways of ovarian cancer is thus of high importance for devising ovarian cancer treatment. Plexin-B1 is a cognate receptor of Semaphorin 4D (Sema4D). This study aims at investigating the role of Plexin-B1 in ovarian cancer. Materials and Methods: Expression of Plexin-B1 and its ligand in ovarian epithelial cell lines was examined by western blot and qPCR. Their expression in benign, borderline, and malignant tumors was revealed by immunohistochemistrty of paraffin embedded tissues. High-resolution melting (HRM) curve analysis was used to screen for mutation in Rac binding region of PLXNB1. Recombinant Sema4D was purified and used to treat OVCA420 cells which express high level of Plexin-B1. siRNA was used to knock-down Plexin-B1 in OVCA420. Cell migration and invasion ability was assayed by transwell migration and invasion assay. Results: Plexin-B1 was most highly overexpressed in serous ovarian cancer cell lines OVCA420, OVCA429, and SW626, whereas Sema4D was ovexpressed in OVCAR3, TOV21G, and PAI. In contrast, Plexin-B1 and Sema4D were not expressed in immortalized normal ovarian epithelial cell lines. Immunohistochemical studies also revealed high Plexin B1 expression in ovarian borderline tumors and invasive cancers while no Plexin B1 expression was detectable in benign tumor. On the other hand, downregulation of Plexin-B1 was observed in the metastatic foci compared with their corresponding primary tumors. There is also a tendency of higher Plexin-B1 expression in serous histotype than in other histotypes. A preliminary HRM screening of Plexin-B1 cDNA from 28 ovarian cancers detected no mutation in the Rac binding region. Knock-down of Plexin-B1 by siRNA caused increased migration and invasion of ovarian cancer cells. In contrast to Sema4D, treatment of ovarian cancer cells OVCA420 by follicle-stimulating hormone which enhances ovarian cancer growth, led to Plexin-B1 downregulation. Conclusion: Our study suggested that Plexin-B1 may contribute to early ovarian carcinogenesis. Its dyregulation in ovarian cancers is paradoxically associated with altered cancer cell invasion and migration. The role of Plexin B1 in ovarian cancer progression is complex. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3183.

Rapid detection of SMARCB1 sequence variation using high resolution melting

BackgroundRhabdoid tumors are rare cancers of early childhood arising in the kidney, central nervous system and other organs. The majority are caused by somatic inactivating mutations or deletions affecting the tumor suppressor locus SMARCB1 [OMIM 601607]. Germ-line SMARCB1 inactivation has been reported in association with rhabdoid tumor, epitheloid sarcoma and familial schwannomatosis, underscoring the importance of accurate mutation screening to ascertain recurrence and transmission risks. We describe a rapid and sensitive diagnostic screening method, using high resolution melting (HRM), for detecting sequence variations in SMARCB1.MethodsAmplicons, encompassing the nine coding exons of SMARCB1, flanking splice site sequences and the 5' and 3' UTR, were screened by both HRM and direct DNA sequencing to establish the reliability of HRM as a primary mutation screening tool. Reaction conditions were optimized with commercially available HRM mixes.ResultsThe false negative rate for detecting sequence variants by HRM in our sample series was zero. Nine amplicons out of a total of 140 (6.4%) showed variant melt profiles that were subsequently shown to be false positive. Overall nine distinct pathogenic SMARCB1 mutations were identified in a total of 19 possible rhabdoid tumors. Two tumors had two distinct mutations and two harbored SMARCB1 deletion. Other mutations were nonsense or frame-shifts. The detection sensitivity of the HRM screening method was influenced by both sequence context and specific nucleotide change and varied from 1: 4 to 1:1000 (variant to wild-type DNA). A novel method involving digital HRM, followed by re-sequencing, was used to confirm mutations in tumor specimens containing associated normal tissue.ConclusionsThis is the first report describing SMARCB1 mutation screening using HRM. HRM is a rapid, sensitive and inexpensive screening technology that is likely to be widely adopted in diagnostic laboratories to facilitate whole gene mutation screening.

White matter lesions in spastic paraplegia with mutations in SPG5/ CYP7B1

Hereditary spastic paraplegias (HSPs) are relatively frequent disorders presenting great genetic heterogeneity. The recent identification of mutations in SPG5/CYP7B1 in six autosomal recessive kindred linked to the SPG5 locus on chromosome 8q prompted us to test the relative frequency of SPG5/CYP7B1 variants in 12 families and in sporadic HSP patients by high-resolution melting screening combined with direct sequencing. We present two patients who harbored three mutations (including two novel variants) in SPG5/CYP7B1 and white matter involvement evidenced at brain MRI. In HSP patients in whom no other genes were mutated, screening of SPG5/CYP7B1 seems to have a low diagnostic yield in autosomal recessive (8%) and sporadic (<1%) cases, even in those with complicated clinical features.