scholarly journals Prevalence of UL97 gene mutations and polymorphisms in cytomegalovirus infection in the colon associated with or without ulcerative colitis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Satoshi Tamura ◽  
Satoshi Osawa ◽  
Natsuki Ishida ◽  
Takahiro Miyazu ◽  
Shinya Tani ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Gene Mutations ◽  
Pcr Amplification ◽  
Kinase Domain ◽  
Resistance Mutations ◽  
Cmv Infection ◽  
Nested Polymerase Chain Reaction ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ul97 Gene

AbstractCytomegalovirus (CMV) reactivation in the colon is common in patients with severe ulcerative colitis (UC). Ganciclovir (GCV) resistance conferring CMV UL97 gene mutations have been reported in recent years. However, the prevalence of UL97 gene mutations in GCV-naive CMV infection in the colon remains unknown. We investigated the prevalence of CMV UL97 gene mutations in patients with colonic CMV infection associated with or without UC. Twenty-two GCV-naive patients with colonic CMV infection, 15 with UC and 7 with other diseases, were enrolled. Frozen biopsy samples or formalin-fixed paraffin-embedded samples were used for nested polymerase chain reaction (PCR) amplification of the UL97 gene. Sanger DNA sequencing was performed. In comparison with AD169 reference strain, natural polymorphisms were frequently detected in codons N68D (100%), I244V (100%), and D605E (86.4%). Seven polymorphisms were detected infrequently (< 10%) outside the kinase domain. However, no known GCV resistance mutations were found. There seemed to be no difference between the ratio of polymorphisms in patients with and without UC. In conclusions, we did not detect UL97 gene mutations associated with GCV resistance in GCV-naive patients with or without UC. Consistent with previous reports, D605E polymorphism may be used as a genetic marker for CMV in East Asian countries.

scholarly journals Prevalence of UL97 gene mutations in cytomegalovirus reactivation in the colon associated with or without ulcerative colitis

2021 ◽  
Author(s):  
Satoshi Tamura ◽  
Satoshi Osawa ◽  
Natsuki Ishida ◽  
Takahiro Miyazu ◽  
Shinya Tani ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Gene Mutations ◽  
Pcr Amplification ◽  
Resistance Mutations ◽  
Cmv Infection ◽  
Nested Polymerase Chain Reaction ◽  
Cytomegalovirus Reactivation ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ul97 Gene

Abstract Cytomegalovirus (CMV) reactivation in the colon is common in patients with severe ulcerative colitis (UC). Ganciclovir (GCV) resistance conferring CMV UL97 gene mutations have been reported in recent years. However, the prevalence of UL97 gene mutations in GCV-naive CMV infection in the colon remains unknown. We investigated the prevalence of CMV UL97 gene mutations in patients with colonic CMV infection associated with or without UC. Twenty-two GCV-naive patients with colonic CMV infection, 15 with UC and 7 with other diseases, were enrolled. Frozen biopsy samples or formalin-fixed paraffin-embedded samples were used for nested polymerase chain reaction (PCR) amplification of the UL97 gene. Sanger DNA sequencing was performed. UL97 mutations were frequently detected in codons T75A (95.5%), Q126L (86.4%), and D605E (86.4%), and less frequently (< 10%) in L228P, D263G, A53S, R137C, A140V, G188S, A674T, and T675A. However, no known GCV resistance mutations were found. There seemed to be no difference between the ratio of mutations in patients with and without UC. In conclusions, we did not detect UL97 gene mutations associated with GCV resistance in GCV-naive patients with or without UC. T75A, Q126L, and D605E mutations may be used as genetic markers for CMV in East Asian countries.

scholarly journals Prevalence of UL97 gene mutations in cytomegalovirus reactivation in the colon associated with or without ulcerative colitis

2020 ◽  
Author(s):  
Satoshi Tamura ◽  
Satoshi Osawa ◽  
Natsuki Ishida ◽  
Takahiro Miyazu ◽  
Satoshi Suzuki ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Gene Mutations ◽  
Pcr Amplification ◽  
Japanese Patients ◽  
Resistance Mutations ◽  
Cmv Infection ◽  
Nested Polymerase Chain Reaction ◽  
Cytomegalovirus Reactivation ◽  
Significant Difference ◽  
Ul97 Gene

Abstract Background: Cytomegalovirus (CMV) reactivation in the colon is common in patients with severe ulcerative colitis (UC) and in immunocompromised patients, and may be associated with poor prognosis. Ganciclovir (GCV) resistance conferring CMV UL97 gene mutations have been reported in recent years. However, the prevalence of UL97 gene mutations in GCV-naive CMV colitis remains to date unknown. The present study aimed to investigate the prevalence of CMV UL97 gene mutations in Japanese patients with colonic CMV infection associated with or without UC.Methods: Twenty-two GCV-naive patients with colonic CMV infection, 15 with UC and 7 with other diseases, were enrolled. Frozen biopsy samples or formalin-fixed paraffin-embedded samples were used for nested polymerase chain reaction (PCR) amplification of the UL97 gene. Sanger DNA sequencing of the PCR products was performed.Results: UL97 mutations were frequently detected in codons T75A (95.5%), Q126L (86.4%), and D605E (86.4%), and less frequently (<10%) in L228P, D263G, A53S, R137C, A140V, G188S, A674T, and T675A. However, no known GCV resistance mutations were found. There was no significant difference between the ratio of mutations in patients with and without UC.Conclusions: We did not detect UL97 gene mutations associated with GCV resistance in GCV-naive patients with or without UC. In contrast, T75A, Q126L, and D605E mutations may be used as genetic markers for CMV in East Asian countries.

KIT Gene Mutations and Patterns of Protein Expression in Mucosal and Acral Melanoma

2012 ◽  
Vol 16 (2) ◽  
pp. 135-142 ◽  
Author(s):  
Suzan Abu-Abed ◽  
Nancy Pennell ◽  
Teresa Petrella ◽  
Frances Wright ◽  
Arun Seth ◽  
...  
Keyword(s):  
Protein Expression ◽  
Kinase Inhibitors ◽  
Case Series ◽  
Gene Mutations ◽  
Mucosal Melanoma ◽  
Acral Melanoma ◽  
Formalin Fixed Paraffin ◽  
Kit Gene ◽  
Formalin Fixed Paraffin Embedded ◽  
Optimized Conditions

Background: Recently characterized KIT (CD117) gene mutations have revealed new pathways involved in melanoma pathogenesis. In particular, certain subtypes harbor mutations similar to those observed in gastrointestinal stromal tumors, which are sensitive to treatment with tyrosine kinase inhibitors. Objective: The purpose of this study was to characterize KIT gene mutations and patterns of protein expression in mucosal and acral melanoma. Methods: Formalin-fixed, paraffin-embedded tissues were retrieved from our archives. Histologic assessment included routine hematoxylin-eosin stains and immunohistochemical staining for KIT. Genomic DNA was used for polymerase chain reaction-based amplification of exons 11 and 13. Results: We identified 59 acral and mucosal melanoma cases, of which 78% showed variable levels of KIT expression. Sequencing of exons 11 and 13 was completed on all cases, and 4 (6.8%) mutant cases were isolated. Conclusion: We successfully optimized conditions for the detection of KIT mutations and showed that 8.6% of mucosal and 4.2% of acral melanoma cases at our institution harbor KIT mutations; all mutant cases showed strong, diffuse KIT protein expression. Our case series represents the first Canadian study to characterize KIT gene mutations and patterns of protein expression in acral and mucosal melanoma.

scholarly journals Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Tim A. D. Smith ◽  
Omneya A. AbdelKarem ◽  
Joely J. Irlam-Jones ◽  
Brian Lane ◽  
Helen Valentine ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Candidate Genes ◽  
Pcr Amplification ◽  
The Cancer Genome Atlas ◽  
Analysis Tool ◽  
Endogenous Control ◽  
Formalin Fixed Paraffin ◽  
Stability Ranking ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Abstract Quantitative real time polymerase chain reaction (qPCR) data are normalised using endogenous control genes. We aimed to: (1) demonstrate a pathway to identify endogenous control genes for qPCR analysis of formalin-fixed paraffin-embedded (FFPE) tissue using bladder cancer as an exemplar; and (2) examine the influence of probe length and sample age on PCR amplification and co-expression of candidate genes on apparent expression stability. RNA was extracted from prospective and retrospective samples and subject to qPCR using TaqMan human endogenous control arrays or single tube assays. Gene stability ranking was assessed using coefficient of variation (CoV), GeNorm and NormFinder. Co-expressed genes were identified from The Cancer Genome Atlas (TCGA) using the on-line gene regression analysis tool GRACE. Cycle threshold (Ct) values were lower for prospective (19.49 ± 2.53) vs retrospective (23.8 ± 3.32) tissues (p < 0.001) and shorter vs longer probes. Co-expressed genes ranked as the most stable genes in the TCGA cohort by GeNorm when analysed together but ranked lower when analysed individually omitting co-expressed genes indicating bias. Stability values were < 1.5 for the 20 candidate genes in the prospective cohort. As they consistently ranked in the top ten by CoV, GeNorm and Normfinder, UBC, RPLP0, HMBS, GUSB, and TBP are the most suitable endogenous control genes for bladder cancer qPCR.

scholarly journals Improved RT-PCR Amplification for Molecular Analyses with Long-term Preserved Formalin-fixed, Paraffin-embedded Tissue Specimens

2006 ◽  
Vol 54 (7) ◽  
pp. 773-780 ◽  
Author(s):  
Kiyohiro Hamatani ◽  
Hidetaka Eguchi ◽  
Keiko Takahashi ◽  
Kazuaki Koyama ◽  
Mayumi Mukai ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Rt Pcr ◽  
Molecular Analyses ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Tissue Specimens ◽  
Formalin Fixed

scholarly journals Targeted Mutational Analysis of Cortisol-Producing Adenomas

2021 ◽  
Author(s):  
Juilee Rege ◽  
Jessie Hoxie ◽  
Chia-Jen Liu ◽  
Morgan N Cash ◽  
James M Luther ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Mutational Analysis ◽  
Cushing Syndrome ◽  
Gene Mutations ◽  
Amplicon Sequencing ◽  
Formalin Fixed Paraffin ◽  
Mutation Profile ◽  
Somatic Gene ◽  
Formalin Fixed Paraffin Embedded ◽  
Aldosterone Producing Adenoma

Abstract Background Somatic gene mutations have been identified in only about half of cortisol-producing adenomas (CPA). Affected genes include PRKACA, GNAS, PRKAR1A, and CTNNB1. Objective To expand our understanding of the prevalence of somatic mutations in CPA from patients with overt Cushing syndrome (OCS) and “subclinical” mild autonomous cortisol excess (MACE), with an immunohistochemistry (IHC)‒guided targeted amplicon sequencing approach using formalin-fixed paraffin-embedded (FFPE) tissue. Method We analyzed FFPE adrenal tissue from 77 patients (n=12 men, 65 women) with either OCS (n=32) or MACE (n=45). Using IHC for 17α-hydroxylase/17,20-lyase (CYP17A1) and 3β-hydroxysteroid dehydrogenase (HSD3B2), we identified 78 CPA (32 OCS-CPA and 46 MACE-CPA). Genomic DNA was isolated from the FFPE CPA and subjected to targeted amplicon sequencing for identification of somatic mutations. Results Somatic mutations were identified in 71.8% (56/78) of the CPA. While PRKACA was the most frequently mutated gene in OCS-CPA (14/32, 43.8%), somatic genetic aberrations in CTNNB1 occurred in 56.5% (26/46) of the MACE-CPA. Most GNAS mutations were observed in MACE-CPA (5/7,71.4%). No mutations were observed in PRKAR1A. In addition to the known mutations, we identified one previously unreported mutation in PRKACA. Two patients with MACE harbored two adjacent tumors within the same adrenal gland: one patient had two CPA, and the other patient had a CPA and an aldosterone-producing adenoma (identified by IHC for aldosterone synthase). Conclusion Comprehensive FFPE IHC-guided gene-targeted sequencing approach identified somatic mutations in 71.8% of the CPA. OCS-CPA demonstrated a distinct mutation profile compared to MACE-CPA.

scholarly journals BCL-1 Gene Rearrangements in Iranian Non-Hodgkin Lymphoma Patients

2015 ◽  
Vol 8 (8) ◽  
pp. 108
Author(s):  
Manoush Tohidirad ◽  
Mehrdad Asghari Estiar ◽  
Azim Rezamand ◽  
Saeid Ghorbian ◽  
Sasan Andalib ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
Pcr Amplification ◽  
B Cell Lymphoma ◽  
Gene Rearrangements ◽  
Non Hodgkin Lymphoma ◽  
Formalin Fixed Paraffin ◽  
Mantle Cell ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

<p>In the present study, our aim was to assess the incidence of BCL-1 gene rearrangements in formalin-fixed paraffin embedded (FFPE) tissue in patients with non-Hodgkin lymphomas (NHL). The BIOMED-2 protocol was applied to assess the BCL-1 gene rearrangements in NHL patients. PCR amplification was carried out on FFPE in 100 patients with B-cell lymphoma including 89 cases with diffused large B-cell lymphoma (DLBCL) (15 cases under 18 years old) and 11 cases with mantle cell lymphoma (MCL). Out of the 100 patients, 19 cases (19%) were identified to have concurrent translocation involving BCL-1. The significant association was seen between BCL-1 gene rearrangements and the lymphomas in patients older than 55 years (P&lt;0.05). Out of 100 cases, 80 cases were positive and 20 cases were negative regarding CD20. No significant association was found between DLBCL lymphoma in patients under 18 years old and BCL-1 gene rearrangements (P&gt;0.05). In addition, the positive and negative expressions of LCA/CD45 marker were 76% (76/100) and 26% (26/100), respectively. Our findings revealed that BCL-1 gene rearrangement assays using BIOMED-2 protocol can be considered as a valuable approach in detection of the lymphomas.</p>

scholarly journals KIT Somatic Mutations and Immunohistochemical Expression in Canine Oral Melanoma

Animals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2370
Author(s):  
Ginevra Brocca ◽  
Beatrice Poncina ◽  
Alessandro Sammarco ◽  
Laura Cavicchioli ◽  
Massimo Castagnaro
Keyword(s):  
Pcr Amplification ◽  
Mean Value ◽  
Single Nucleotide ◽  
Oral Melanoma ◽  
Pathway Activation ◽  
Formalin Fixed Paraffin ◽  
Frequent Event ◽  
Formalin Fixed Paraffin Embedded ◽  
Exon 11 ◽  
Formalin Fixed

Canine oral melanoma (COM) is an aggressive neoplasm with a low response to therapies, sharing similarities with human mucosal melanomas. In the latter, significant alterations of the proto-oncogene KIT have been shown, while in COMs only its exon 11 has been adequately investigated. In this study, 14 formalin-fixed, paraffin-embedded COMs were selected considering the following inclusion criteria: unequivocal diagnosis, presence of healthy tissue, and a known amplification status of the gene KIT (seven samples affected and seven non-affected by amplification). The DNA was extracted and KIT target exons 13, 17, and 18 were amplified by PCR and sequenced. Immunohistochemistry (IHC) for KIT and Ki67 was performed, and a quantitative index was calculated for each protein. PCR amplification and sequencing was successful in 97.62% of cases, and no single nucleotide polymorphism (SNP) was detected in any of the exons examined, similarly to exon 11 in other studies. The immunolabeling of KIT was positive in 84.6% of the samples with a mean value of 3.1 cells in positive cases, yet there was no correlation with aberration status. Our findings confirm the hypothesis that SNPs are not a frequent event in KIT activation in COMs, with the pathway activation relying mainly on amplification.

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