Screening JAK2 Exon 12 in JAK2 V617F Negative Patients with Myeloproliferative Disorders Reveals a New Splice Site Mutation

Abstract Background. The identification of the somatic mutation V617F in exon 14 of the Janus kinase 2 gene (JAK2) has simplified the diagnosis of many patients affected with typical chronic myeloproliferative diseases (MPDs). In patients without the V617F the molecular basis of MPD are still unclear but, recently, mutations in exon 12 of the JAK2 gene have been identified in a minority of patients, associated with a selective increase in erythropoiesis resulting in polycythemia vera (PV) or idiopathic erythrocytosis (IE) (Scott et al, NEJM 2007). Aim. To determine the JAK2 exon 12 mutational status in a group of JAK2 V617F negative patients with PV or IE. Methods. Genomic DNA was extracted from peripheral blood leukocytes from 85 MPD patients (PV or IE) included in the present study. All the samples were tested for JAK2 V617F mutation by allele specific polymerase chain reaction (ASO-PCR) and those negative for V617F mutant allele were subjected to real time quantitative PCR (RQ-PCR) using hybridization probes. Subsequently, all JAK2 V617F negative samples by both ASO-PCR and RQ-PCR where subjected to direct sequencing to exclude JAK2 exon 12 mutations. Results. JAK2 V617F was positive in 78 (91.7%) of the 85 patients, however, in two of these patients the V617F mutation was only detected upon subjecting genomic DNA to RQ-PCR which revealed low levels of the mutant allele, 2.65 % and 3.98%. Analysis of the JAK2 exon 12 in the seven JAK2 V617F negative patients detected three previously described mutations: a duplication (V536-1546) and two deletions (H539-K540del+542K and R541-E543 delinsK)and a previously unreported splice site mutation detected in intron 12 (IVS12nt6 T-C). To our knowledge this was the first description of intronic mutations in the JAK2 gene. No mutations were detected in the remaining 3 (3.6%) patients. Conclusions. In this cohort study JAK2 mutations were observed in 82 (96,5%), of the 85 patients, of these 78 (95.12%) had the V617F allele. In two cases V617F was detected at low levels (2.65% and 3.98%) only by RQ-PCR, highlighting the need for sensitive techniques to detect somatic mutations. One of the patients, a young male with erythrocytosis and low serum Epo levels, revealed a previously unreported splicing mutation (IVS12 nt6: T-C). This study illustrates the heterogeneity at the DNA level in PV patients which may assist in better understanding the genotype and phenotype relationship in MPD patients and assist in further delineating the role of JAK2 in these disorders.

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A Novel Splice Site Mutation in the SERPING1 Gene Leads to Haploinsufficiency by Complete Degradation of the Mutant Allele mRNA in a Case of Familial Hereditary Angioedema

Journal of Clinical Immunology ◽

10.1007/s10875-014-0042-3 ◽

2014 ◽

Vol 34 (5) ◽

pp. 521-523 ◽

Cited By ~ 8

Author(s):

Roger Colobran ◽

Ricardo Pujol-Borrell ◽

Manuel Hernández-González ◽

Mar Guilarte

Keyword(s):

Splice Site ◽

Hereditary Angioedema ◽

Mutant Allele ◽

Splice Site Mutation ◽

Site Mutation ◽

Complete Degradation ◽

Serping1 Gene

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Relationship between HUWE1 Mutation and Functionality in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-118294 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4512-4512

Author(s):

Lisa J Crawford ◽

David Campbell ◽

Ken I Mills ◽

Alexandra E Irvine

Keyword(s):

Multiple Myeloma ◽

Cell Line ◽

Cell Lines ◽

Splice Site ◽

Mutant Cell ◽

Splice Site Mutation ◽

Missense Mutations ◽

Site Mutation ◽

Mutational Status ◽

Expression And Activity

Abstract Background Proteasome inhibitors have provided significant therapeutic advance in the treatment of Multiple Myeloma (MM), however resistance and dose limiting side effects remain a clinical challenge. Recent research has focused on developing strategies to target other enzymes within the ubiquitin proteasome system, with the aim of overcoming resistance and toxicity. We have previously reported deregulated expression of the E3 ligase HUWE1 in MM and demonstrated that knockdown or inhibition of HUWE1 leads to a decrease in MYC activity (Blood 2016, 128:240; 2017, 130:3077). HUWE1 is a large HECT domain E3 ligase that is involved in the regulation of key proteins such as p53, MYC and MCL-1. Mutations in HUWE1 have recently been identified in MM patients, significantly associated with the t(11;14) subgroup (blood-2018-03-840132). A recurrently mutated splice site mutation in the DUF913 (domain of unknown function 913) of HUWE1 was identified in 18% of patients, the remainder are predominantly missense mutations distributed across the coding sequence (CDS). Other studies have demonstrated that point mutations in the dimerization interface of HUWE1, which acts to regulate its activity, result in hyper-activation of HUWE1 (eLife 2017;6:e21036; Sci Rep 2017;7:15050). While 7% of the mutations reported in MM patients are found in this region, the effect of the majority of mutations on the functional significance of HUWE1 has yet to be determined. The aim of this study was to analyse HUWE1 expression and activity in HUWE1 mutant MM cell lines. Methods HUWE1 mutational status was analysed in a publically available dataset of MM cell lines (www.keatslab.org). Inhibitors of HUWE1 (BI8622, BI8626; described in EMBO Mol Med 2014, 6:1525-1541) were purchased from Syngene. The effect of the inhibitors on HUWE1 mutant MM cell lines was assessed using CellTitre-glo. HUWE1 auto-ubiquitination activity was analysed using UbiQapture-Q and Western blotting. Results HUWE1 mutations were identified in 6 out of 69 MM cell lines. HUWE1 mutational status was confirmed in 5 cell lines (U266, XG-1, XG-2, KMS-27, H1112) by Sanger sequencing. In agreement with MM patient data, HUWE1 mutations were predominantly found in cell lines expressing the t(11;14) translocation (4/6 cell lines) and are distributed in a similar manner. H1112 cells harbor the recurrent splice site mutation observed in patients, whereas the other cell lines contain missense mutations across the CDS. HUWE1 protein expression in mutant cell lines was compared with expression in 5 MM cell lines expressing wild type (WT) HUWE1 (JJN3, MM.1S, ANBL-6, KMS-18, OPM-2). No significant difference in expression was observed in the majority of HUWE1 mutant cell lines, however, HUWE1 expression was significantly lower in the H1112 cell line (p=0.002) compared to HUWE1 WT cell lines. Accordingly, HUWE1 auto-ubiquitination activity was reduced only in H1112 cells. XG-2 and U266 displayed similar sensitivity to HUWE1 inhibitors as HUWE1 WT cell lines (IC50 12-18 µM vs 9-20 µM), while XG-1, H1112 and KMS-27 were less sensitive (IC50 20-33 µM). The effect of HUWE1 on substrate proteins (e.g. MYC) varies depending on tumor type. In HUWE1 WT MM cell lines, inhibition of HUWE1 leads to significantly decreased expression of MYC and MCL-1 in (p<0.01), through increased proteasomal degradation. A similar decrease in MYC and MCL-1 expression is observed in XG-2, and in MCL-1 in U266 cells (which lack expression of c-MYC). Conversely, no significant effect on MYC or MCL-1 expression was seen in XG-1 cells, while barely detectable levels of MYC and MCL-1 were observed in H1112 and KMS-27 cells, suggesting altered or absent HUWE1 activity in these cell lines. Conclusion HUWE1 has recently been identified as a mutational driver in t(11;14) MM, however, little is known about the functional consequence of HUWE1 mutations. Here we show that the H1112 cell line, representative of the most commonly occurring HUWE1 mutation in MM, leads to reduced expression and activity of HUWE1 and is associated with low expression of HUWE1 substrates MYC and MCL-1. Conversely, expression of HUWE1 and activity against selected substrates remains unchanged in XG-2 and U266 cells. Moreover, recent studies demonstrate that certain HUWE1 mutations lead to enhanced catalytic activity. In summary, the pathogenicity of HUWE1 mutations in MM is likely to depend on the type and location of the mutation. Disclosures No relevant conflicts of interest to declare.

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Allelic Expression Imbalance of JAK2 V617F Mutant Contributes to Phenotypic Variation in BCR-ABL Negative Chronic Myeloproliferative Disorders.

Blood ◽

10.1182/blood.v110.11.4651.4651 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4651-4651

Author(s):

Myung-Geun Shin ◽

Hye-Ran Kim ◽

Hyeoung-Joon Kim ◽

Mi-Ji Kim ◽

Hee-Nam Kim ◽

...

Keyword(s):

Gene Expression ◽

Genomic Dna ◽

Mutant Allele ◽

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Allelic Expression ◽

Jak2 V617f Mutation ◽

Allelic Expression Imbalance ◽

Mutation Status ◽

V617f Mutation

Abstract Background: An acquired somatic mutation in the JAK2 gene (V617F) could present the primary causative lesion in BCR-ABL-negative chronic myeloproliferative disorders (CMPD). However, some of the clinical and genetic data implied that the pathophysiological role of JAK2 V617F mutation in the CMPD would be more complex. Quantitative assessment of JAK2 V617F mutation has shown substantial heterogeneity in the genomic DNA. Recently, it has been reported that allelic variation in gene expression is common in the human genome. To test the hypothesis that JAK2 V617F mutant allele could be increased in cDNA, we examined JAK2 V617F mutation status in the genomic DNA and cDNA from a total of 78 patients with BCR-ABL-negative CMPD. Patients and Methods: Enrolled patients with BCR-ABL-negative CMPD in this study comprised 42 cases of essential thrombocythemia (ET), 26 polycythemia vera (PV), 7 idiopathic myelofibrosis (MF) and 3 unclassifiable (UC) CMPD. A 364-bp PCR product containing JAK2 V617F mutation was bidirectionally sequenced from total BM cells. A quantitative real time PCR-based allelic discrimination assay and pyrosequencing (Pyrosequencer PSQ96) were developed for quantitative analysis of JAK2 V617F mutation status. Homozygous JAK2 V617F mutation was defined if the mutant peak was more than 50% of total peak area. Results: The proportion of mutant alleles ranged from 36% to 100% in real-time PCR and pyrosequencing analysis. Patients with MF had higher percentages of JAK2 mutant alleles than patients with ET (MF > PV > ET). The prevalence of homozygous V617F mutations was significantly higher in PV patients (73%) than in patients with ET (17%). Allelic expression imbalance of heterozygous JAK2 mutation was common in patients with PV and ET. Interestingly, allelic expression imbalance in patients with MF was not remarkably impaired, but preferential expression of JAK2 mutant allele showed threefold increase from the cDNA compared with the genomic DNA from patients with PV and ET. Conclusion: Allelic imbalance in the gene expression of JAK2 V617F mutant could provide the underlying mechanisms to elucidate phenotypic variation in BCR-ABL negative CMPD.

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Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method

Diagnostics ◽

10.3390/diagnostics10030153 ◽

2020 ◽

Vol 10 (3) ◽

pp. 153 ◽

Cited By ~ 1

Author(s):

Gustavo Barcelos Barra ◽

Ticiane Henriques Santa Rita ◽

Ana Luisa Santa Cruz Almeida ◽

Rafael Henriques Jácomo ◽

Lídia Freire Abdalla Nery

Keyword(s):

Whole Blood ◽

Mutant Allele ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Absolute Quantification ◽

Janus Kinase ◽

Molecular Diagnostic ◽

Janus Kinase 2 ◽

Cq Method ◽

V617f Mutation

Detection of the Janus Kinase-2 (JAK2) V617F mutation is a diagnostic criterion for myeloproliferative neoplasms, and high levels of mutant alleles are associated with worse outcomes. This mutation is usually tested on blood DNA by allele-specific qPCR (AS-qPCR) and measured using absolute quantification. However, some automated DNA extractions co-extracts of PCR inhibitors from blood and qPCR absolute quantification need increased efforts in order to maintain standard curves. JAK2 V617F can also be detected in serum using droplet digital PCR (ddPCR), a specimen with less inhibitors and favorable to automated extractions, but ddPCR instruments are not wide available as qPCR thermocyclers. Here, we evaluate whether JAK2 V617F could be accurately quantified by AS-qPCR using the 2-∆∆Cq method on blood DNA and validate the assay using gold-standard molecular diagnostic protocols. Next, we apply the validated method to assess if the mutation could be reliably detected/quantified in serum. JAK2 V617F could be quantified by AS-qPCR using the 2-∆∆Cq method—the assay was highly accurate (bias of 1.91%) compared to a commercial kit, highly precise (total CV% of 0.40%, 1.92%, 11.12% for samples with 93%, 54%, and 2.5% of mutant allele), highly sensitive (limit of detection of 0.15%), and demonstrated a linear detection response from 1.1% to 99.9%. Serum presented a higher mutant allele burden compared to the paired whole blood (mean of 4%), which allows for an increased JAK2 mutant detection rate and favors increased JAK2 V617F high-throughput analysis.

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A Novel Splice-Site Variation in COL5A1 Causes Keratoconus in an Indian Family

Journal of Ophthalmology ◽

10.1155/2019/2851380 ◽

2019 ◽

Vol 2019 ◽

pp. 1-5 ◽

Cited By ~ 1

Author(s):

Qinghong Lin ◽

Lin Zheng ◽

Zhengwei Shen ◽

Liming Jie

Keyword(s):

Splice Site ◽

Genomic Dna ◽

Family Members ◽

Splice Site Mutation ◽

Site Mutation ◽

Acceptor Splice Site ◽

Blood Leukocytes ◽

Indian Family ◽

South Indian ◽

Gene Functional Analysis

Objective. This study aims to clarify the association between keratoconus (KC) and potential pathogenic genetic variants in a three-generation South Indian family. Methods. In the present study, a three-generation KC family, which comprised 10 affected patients and nine unaffected individuals, was recruited. The family history and necessary ophthalmological exams, such as visual acuity and slit-lamp, were performed for all participants. Genomic DNA was extracted from peripheral blood leukocytes, and whole exome sequencing (WES) was performed using the genomic DNA of the proband (III:4) and two other family members (III:2, III:3). The acceptor-splice-site mutation was validated and verified using polymerase chain reaction (PCR) and Sanger sequencing. Gene functions and pathways associated with the identified mutations were subjected to in silico analysis. Results. A novel COL5A1 acceptor-splice-site mutation IVS50-4C > G was found in the 10 affected individuals in the three-generation KC family, but this was not found in any of the unaffected family members or unrelated healthy individuals. Gene functional analysis using the SpliceMan and ExonScan software predicted that the splice-site mutation was potentially associated with KC pathogenesis. This mutation might affect the assembly of the collagen triple helix. Conclusion. The present study confirmed the association between the COL5A1 gene and KC and identified a novel COL5A1 acceptor-splice-site mutation (IVS50-4C > G) in intron 50, which may affect the splicing of the adjacent exon 50.

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