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PubMed2005

Common human SCN5A polymorphisms have altered electrophysiology when expressed in Q1077 splice variants.

BACKGROUND: Eight common (>0.5%) polymorphisms of SCN5A have been described in the US population. Every human also continuously generates two wild-type (WT) splice variants, one with a glutamine residue at position 1077 (Q1077) and one lacking this glutamine (Q1077del). One polymorphism (H558R) has been studied in both splice variants, five polymorphisms (R34C, R481W, S524Y, P1090L,V1951L) have not been previously studied, and two polymorphisms (S1103Y and R1193Q) have been studied in only one of the two splice variants. OBJECTIVES: The purpose of this study was to examine the electrophysiologic molecular phenotype of the eight common polymorphisms in the two human splice variants of SCN5A. METHODS: Currents from 16 channels (all polymorphisms in both splice variants) were determined by voltage clamp and compared with WT after expression in HEK-293 cells. RESULTS: Six of eight polymorphisms showed a distinct phenotype that depended upon the background splice variant used for expression. Only R34C and V1951L showed no functional differences. S524Y showed a dramatic reduction in current density in the Q1077 background similar to that previously described for H558R. Four other polymorphisms (R481W, P1090L, S1103Y, R1193Q) showed shifts in activation, inactivation, or recovery that depended upon the splice variants. Shifts of a similar magnitude have been reported for arrhythmia syndrome mutations and are thought to be pathogenic. CONCLUSION: The majority of common human SCN5A polymorphisms have a distinct molecular phenotype that depends upon the splice variant background. These findings have implications for the interpretation of previous studies of arrhythmia mutations. The significance of these findings for clinical arrhythmia remains to be elucidated.

Alternative Splicing↗

PubMed2026

Clinical characteristics and prognostic impact of multiple pathogenic variants across the genetic spectrum of arrhythmogenic and dilated cardiomyopathies.

BACKGROUND: Arrhythmogenic and dilated cardiomyopathies (ACM and DCM, respectively) are genetically heterogeneous disorders of the right and/or left ventricles associated with an increased risk of major arrhythmic events (MAE) and end-stage heart failure (ESHF). In arrhythmogenic right ventricular cardiomyopathy (ARVC), the presence of >1 pathogenic or likely pathogenic (P/LP) variant is associated with worse outcomes. Whether this phenomenon occurs for non-desmosomal arrhythmogenic left ventricular cardiomyopathy (ALVC)/DCM genes is unknown. OBJECTIVE: This study aimed to evaluate the impact of single vs multiple P/LP variants on arrhythmic and heart failure outcomes across the ACM/DCM genetic spectrum. METHODS: We retrospectively analyzed 1054 genotype-positive patients with ≥1 P/LP variant in a definitive or strong evidence ARVC- or ALVC/DCM-causative gene. Primary endpoints were MAE (sustained ventricular tachycardia, ventricular fibrillation, aborted cardiac arrest, appropriate implantable cardioverter-defibrillator therapy, and sudden cardiac death) and ESHF (transplant or heart failure death). RESULTS: Of the 1054 patients, 27 (3%) harbored >1 P/LP variants (21 with ≥1 ALVC/DCM gene; 6 with >1 ARVC gene). MAE occurred in 20% of single-variant patients compared with 48% and 50% of those with >1 P/LP variants in ≥1 ALVC/DCM- and >1 ARVC-susceptibility gene(s), respectively. ESHF occurred in 9%, 29%, and 17% of patients, respectively. On adjusted analysis, >1 P/LP variants in ≥1 ALVC/DCM-susceptibility (MAE hazard ratio [HR], 2.46 [1.28-4.72]; P = .01 and ESHF HR, 3.15 [1.35-7.37], P = .01) and >1 ARVC-susceptibility gene(s) (MAE HR, 2.67 [1.28-8.72], P = .03) were independent predictors of the primary endpoints. CONCLUSION: Multiple P/LP variants confer an increased risk of arrhythmic events and heart failure across the ACM/DCM spectrum.

Arrhythmogenic cardiomyopathy↗

PubMed2026

Functional screening of ZIP8 naturally occurring variants identifies pathogenic mutations and trafficking defects.

The rapid expansion of human genomic data has revealed a large number of naturally occurring variants, creating a major challenge for functional annotation. The human metal transporter SLC39A8 (ZIP8) is a clinically important divalent metal transporter, yet most of its documented variants remain uncharacterized. Here, we developed a workflow to functionally evaluate ZIP8 variants by integrating laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) with scaled-up cell-based transport assays. Using this method, we systematically analyzed 33 naturally occurring missense variants located in the extracellular domain (ECD) of ZIP8. The assay enables direct quantification of intracellular metal accumulation with substantially improved throughput (∼150 samples per hour). Functional screening identified 14 potential pathogenic variants with significantly reduced transport activity. Comparison with computational predictions revealed a moderate correlation between activity and AlphaMissense pathogenicity scores (R2 = 0.423), while an error rate of ∼20% for AlphaMissense underscores the need for experimental validation. Flow cytometry analysis showed that most loss-of-function variants exhibit impaired trafficking of the protein to the cell surface possibly due to mutation-caused protein misfolding or instability. Structural mapping of activity-compromised variants, together with functional assessment of the ZIP8-ECD, highlights the importance of this domain in ZIP8 expression and intracellular protein trafficking. Together, this work establishes a scalable approach for functional screening of metal transporter variants and provides new insights into the structure-function relationships of ZIP8.

Journal Article↗

PubMed2006

Chromatographic and electrophoretic characterization of protein variants.

Almost all proteins are expressed in several variants, also known as isoforms. Individual protein variants differ by modifications of the individual amino acid side chains, or the N- or C-terminus. Typical modifications are glycosylation, phosphorylation, acetylation, methylation, deamidation or oxidation. It is of utmost interest to either get a quantitative picture of the variants of a particular protein or to separate the variants in order to be able to identify their molecular structure. Protein variants are present in native as well as in recombinant proteins. In the case of protein production it is interesting, how variants are generated during fermentation, purification processes, storage, and how present individual variants influence the biological activity. This review provides a comparison of chromatographic and electrophoretic separation methods to analyze and to prepare protein variants.

Chromatography↗

PubMed2005

Identification of human herpesvirus 6 variants A and B by primer-specific real-time PCR may help to revisit their respective role in pathology.

BACKGROUND: Human herpesvirus 6 (HHV-6) isolates are classified into two variants, termed HHV-6A and HHV-6B, on the basis of distinct genetic, antigenic and biological characteristics, but the specific pathogenicity of each variant remains poorly understood. OBJECTIVES: To design a rapid, sensitive and specific real-time variant-specific PCR (VS-PCR) method to differentiate both variants in biological specimens. STUDY DESIGN: The VS-PCR was adapted from a real-time PCR assay, based on TaqMan technology, previously developed for the genome quantitation of both HHV-6 variants [Gautheret-Dejean A, Manichanh C, Thien-Ah-Koon F, Fillet AM, Mangeney N, Vidaud M, et al. Development of a real-time polymerase chain reaction assay for the diagnosis of human herpesvirus-6 infection and application to bone marrow transplant patients. J Virol Meth 2002;100:27-35], a consensual reverse primer (Taq2) being changed into two variant-specific primers named H6A and H6B. This method was applied to a large set of biological specimens obtained in different pathological contexts. RESULTS: The sensitivity threshold was about 10 copies/well for HHV-6A-specific PCR (PCR-A) and 1 copy/well for HHV-6B-specific PCR (PCR-B). Both assays showed a linear dynamic range from 10 to 100,000 copies of HHV-6 DNA. Regarding the specificity and the capacity of discrimination of each assay, one variant could be detected and identified in the presence of more than 1000 times higher concentrations of the other variant in virus mixtures. The comparison of the results obtained with this VS-PCR with those previously obtained with a classic PCR method allowed us to validate our new technique on a wide panel of biological samples, including numerous patients with severe HHV-6-related symptoms. The high prevalence of HHV-6B was confirmed in healthy individuals and immunocompromised patients. HHV-6A was identified in distinct samples from several patients exhibiting neurological disorders. CONCLUSIONS: We developed a new VS-PCR assay, able to differentiate HHV-6A and HHV-6B in biological samples, even in the case of mixed infections. Our study confirms the wide prevalence of HHV-6B and highlights the potential greater neuropathogenic role of HHV-6A in immunocompromised patients and young infants.

DNA Primers↗

PubMed2025

Genetic study of von Willebrand factor antigen levels ≤ 50 IU/dL identifies variants associated with increased risk of von Willebrand disease and bleeding.

BACKGROUND: von Willebrand disease (VWD) is a common inherited bleeding disorder caused by low levels or activity of circulating von Willebrand factor (VWF). Genetic susceptibility to VWF antigen (VWF:Ag) below normal (≤ 50 IU/dL) in the general population is underexplored. OBJECTIVES: To identify genetic variants influencing VWF:Ag levels ≤ 50 IU/dL. METHODS: We performed a genome-wide association study in 926 cases with VWF:Ag levels ≤ 50 IU/dL and 12 846 controls from 7 studies from the Trans-Omics for Precision Medicine program. We then examined whether significant genome-wide findings were also associated with clinical diagnosis of VWD in 5 biobanks with 708 VWD cases and 1 286 069 controls, and with 6 bleeding and thrombotic disorders in FinnGen. RESULTS: Variants at 2 loci were associated (P < 5 × 10-9) with VWF:Ag levels ≤ 50 IU/dL: ABO and VWF. The VWF index variant, p.Tyr1584Cys, is a rare (0.22%) missense variant with odds ratio (OR) of 78.58, while the ABO index variant is a common intronic variant with a smaller effect (OR = 2.52). Notably, both VWF (OR = 7.16) and ABO (OR = 1.57) variants were also associated (P < .025) with diagnosed VWD. Among p.Tyr1584Cys heterozygotes, the penetrance of VWF:Ag levels ≤ 50 IU/dL was 24.2% and the penetrance of diagnosed VWD was 0.3%. p.Tyr1584Cys was associated (P < .0042) with increased odds of heavy menstrual bleeding (OR = 1.27), iron deficiency anemia (OR = 1.55), and intrapartum hemorrhage (OR = 2.20), but decreased odds of deep vein thrombosis (OR = 0.54). CONCLUSIONS: Although there are currently conflicting interpretations of pathogenicity p.Tyr1584Cys, our results suggest that it is a low penetrance pathogenic variant that contributes to VWF:Ag levels ≤ 50 IU/dL, bleeding, and VWD.

Humans↗

PubMed2006

Human papillomavirus type 16 variant assignment by pyrosequencing.

Polymorphisms in human papillomavirus type 16 (HPV16) result in variants from the prototype sequence which can be designated according to geographic distribution and are broadly classified as European (E), African (Af), Asian (As), or Asian-American (AA). Detection of variants has been used to distinguish persistent HPV16 infection from re-infection in natural history studies, and variants have been associated with an increased risk of cervical disease in some populations. Variant determination usually relies on conventional Sanger sequencing of regions of the viral genome, with the major variant group assignments requiring the sequencing of only seven polymorphic sites spread over a 242-bp region of the E6 gene. We applied pyrosequencing to facilitate rapid sequencing and enable the simultaneous detection of multiple variants. A single-stranded template for pyrosequencing was prepared by amplifying a 314-bp fragment (nt 75-388) with a biotin at the 5'-end of the reverse primer to facilitate strand separation and purification. Polymorphisms at the nucleotide sites 109, 131, 132, 143, 145, 178 and 350 were determined in three separate sequencing reactions, one of which was a multiplex format. Pyrosequencing of 97 HPV16-positive exfoliated cervical samples confirmed the Sanger sequencing results; however pyrosequencing identified additional variants in several samples containing mixed variants.

Base Sequence↗

PubMed2026

Simultaneous quantitative detection of multiple low-frequency variants by high-dynamic-range capillary electrophoresis.

Sensitive and quantitative detection of low-frequency variants across multiple loci is critical for nucleic acid-based diagnostics, yet clinical implementation requires a balance among sensitivity, multiplexing capacity, cost, and operational simplicity. We previously developed a high-dynamic-range capillary electrophoresis system capable of detecting variants at allele frequencies below 1%; however, its application was limited to single-locus analysis. Here, we expanded this platform to multiplex detection by incorporating mobility-shift strategies into the assay design. This approach enabled simultaneous analysis of 15 hotspot variants across three clinically relevant loci: KRAS codons 12 and 13 and GNAS codon 201. Validation using synthetic oligonucleotides, formalin-fixed paraffin-embedded tissue, and liquid specimens demonstrated high quantitative accuracy over clinically relevant variant allele frequency ranges, with measured values closely matching expected values (R2 > 0.97). The assay showed high concordance with targeted amplicon sequencing and digital polymerase chain reaction for all variants at variant allele frequencies ≥1%, while also detecting selected variants below this threshold. Collectively, these results establish a multiplexed high-dynamic-range capillary electrophoresis assay for simultaneous, quantitative detection of low-frequency variants, offering a scalable and cost-effective approach for disease-focused gene panels in clinical laboratory settings.

HiDy↗

PubMed2004

Terpenoid aldehyde formation and lysigenous gland storage sites in cotton: variant with mature glands but suppressed levels of terpenoid aldehydes.

A new cotton variant with reduced levels of terpenoid aldehydes (sesquiterpenoids and sesterterpenoids (heliocides)) was isolated from the progeny of hemizygous cotton (Gossypium hirsutum cv. Coker 312) transformed with antisense (+)-delta-cadinene synthase cDNA. Southern analysis of leaf DNA digested with HindIII, Pst or KpnI restriction endonucleases did not detect any antisense cdn1-C1 DNA in the genome of the variant. The gossypol content in the seed of the variant was markedly lower than in the seed of T1 antisense plants. Eighty-nine percent of the variant seed had a 71.1% reduction in gossypol and the foliage of the variant plants showed a 70% reduction in gossypol and a 31% reduction in heliocides. Compared to non-transformed plants there was no reduction in the number of lysigenous glands in the seed of the variant. The cotton variant shows uncoupling of terpenoid aldehyde synthesis and gland formation. The cotton variant may have resulted from somaclonal variation occurring in the callus tissue during the transformation-regeneration process.

Aldehydes↗

PubMed2005

Human papillomavirus type 18 variant lineages in United States populations characterized by sequence analysis of LCR-E6, E2, and L1 regions.

While HPV 16 variant lineages have been well characterized, the knowledge about HPV 18 variants is limited. In this study, HPV 18 nucleotide variations in the E2 hinge region were characterized by sequence analysis in 47 control and 51 tumor specimens. Fifty of these specimens were randomly selected for sequencing of an LCR-E6 segment and 20 samples representative of LCR-E6 and E2 sequence variants were examined across the L1 region. A total of 2770 nucleotides per HPV 18 variant genome were considered in this study. HPV 18 variant nucleotides were linked among all gene segments analyzed and grouped into three main branches: Asian-American (AA), European (E), and African (Af). These three branches were equally distributed among controls and cases and when stratified by Hispanic and non-Hispanic ethnicities. Among invasive cervical cancer cases, no significant differences in the three HPV variant branches were observed among ethnic groups or when stratified by histopathology (squamous vs. adenocarcinoma). The Af branch showed the greatest nucleotide variability when compared to the HPV 18 reference sequence and was more closely related to HPV 45 than either AA or E branches. Our data also characterize nucleotide and amino acid variations in the L1 capsid gene among HPV 18 variants, which may be relevant to vaccine strategies and subsequent studies of naturally occurring HPV 18 variants. Several novel HPV 18 nucleotide variations were identified in this study.

Base Sequence↗

PubMed2004

Human papillomavirus type 16 E6, E7, and L1 variants in cervical cancer in Indonesia, Suriname, and The Netherlands.

OBJECTIVE: Human papillomavirus type 16 (HPV 16) has several intratypic variants, and some are associated with enhanced oncogenic potential. For risk determination as well as for future vaccine development, knowledge about variants is important. Regarding the geographical distribution of HPV variants and the lack of data from Indonesia and Suriname, we studied the prevalence of HPV 16 variants in cervical cancer in these high incidence countries. Data were compared with The Netherlands, a low-risk country. METHODS: DNA samples from 74 formalin-fixed paraffin-embedded HPV 16-positive cervical carcinomas from Indonesia (Java, N = 22), Suriname (N = 25), and The Netherlands (N = 27) were amplified using primers specific for the E6, E7, and part of the L1 regions. Products were sequenced and analyzed. RESULTS: A specific Javanese variant, with mutations 666A in E7 and 6826T in L1, was found in 73% of the Indonesian samples, 56% having an additional mutation in the E6 open reading frame (ORF; 276G), giving the predicted amino acid change N58S. This Javanese variant was also found in three Surinamese samples, which reflects what could be expected from migration of Javanese people to Surinam. Other non-European variants were identified in Indonesian, Surinamese, and Dutch samples in 14%, 28%, and 19%, respectively. CONCLUSION: The majority of the HPV 16-positive cervical cancers in Indonesia are caused by a specific intratypic variant that was rarely found before in other countries.

Base Sequence↗

PubMed2002

Assessment of the effect of hemoglobin variants on routine HbA1c measurements by electrospray ionization mass spectrometry.

We applied electrospray ionization mass spectrometry (ESI-MS) to identify hemoglobin (Hb) variants, and to assess the effect of the variants on routine measurements of a glycated Hb, HbA1c. Over the past 8 years, we have diagnosed 83 cases, including 42 kinds of variant Hb, using MS as the main technology. Of these variants, 3 were new, and 9 were the first cases in Japan. Some abnormal Hbs cause diseases, and most cause erroneous values of HbA1c measured by various methods. ESI-MS was also successfully used for the accurate determination of glycated Hb. We and other groups proposed methods to examine glycated Hb by ESI-MS using enzyme-digested peptides, or intact globin without enzyme digestion. Using the peptide method, we clarified the extent of discrepancies among the HbA1c values measured by conventional methods and accurate values for samples containing various Hb variants identified by the MS method. We applied the globin method to measure the ratio of the glycated component of a variant chain and that of a normal chain obtained from the same erythrocytes. Although the glycation degree on most variant chains was similar to that on normal chains obtained from the same erythrocytes, the content of the glycated component of a particular variant beta-chain was approximately three times as much as that of the normal beta-chain. Abnormal Hbs cause erroneous values for HbA1c to various extents with commercial measurement methods, and MS may offer an unrivaled strategy to correct these errors.

Amino Acid Substitution↗

PubMed1998

Abnormal expression of CD44 variants in the exfoliated cells in the feces of patients with colorectal cancer.

BACKGROUND & AIMS: Recent investigations have shown that CD44 variant exons are frequently overexpressed in human colorectal adenocarcinoma. The aim of this study was to investigate abnormal expression of the CD44 gene in exfoliated cells from patients with colorectal cancer. METHODS: Exfoliated cells in feces from 25 patients with colorectal cancer before and after surgery and from 15 healthy volunteers were analyzed. CD44 standard, variant 6, and variant 10 messenger RNA (mRNA) expressions were examined in the exfoliated cells in feces by using reverse-transcription polymerase chain reaction followed by Southern hybridization with exon-specific probes. RESULTS: CD44 standard mRNA was detected in all samples before and after surgery and in all healthy volunteers. CD44 variant 6 and variant 10 mRNA were detected in 17 of 25 patients (68%) and 15 of 25 patients (60%), respectively, in individual feces obtained before surgery. CD44 variant 6 mRNA and variant 10 mRNA were detected in postoperative samples in 3 of 25 patients (12%) and 7 of 25 patients (28%), respectively. Fifteen of 17 patients who were positive for CD44v6 based on preoperative fecal samples became negative after surgery (88.2%). Similarly, 12 of 15 patients who were CD44v10 positive in preoperative fecal samples were negative postoperatively (80%). CONCLUSIONS: These results suggest that analysis of CD44 variant expression in the exfoliated cells in feces can provide a noninvasive diagnostic test for colorectal cancer.

Adult↗

PubMed2002

Amyloidogenic unfolding intermediates differentiate sheep prion protein variants.

Sheep is a unique example among mammalian species to present a strong correlation between genotype and prion disease susceptibility phenotype. Indeed a well-defined set of PrP polymorphisms at positions 136, 154 and 171 (sheep numbering) govern scrapie susceptibility, ranging from very high susceptibility for V136-R154-Q171 variant (VRQ) to resistance for A136-R154-R171 variant (ARR). To get better insight into the molecular mechanisms of scrapie susceptibility/resistance, the unfolding pathways of the different full-length recombinant sheep prion protein variants were analysed by differential scanning calorimetry in a wide range of pH. In the pH range 4.5-6.0, thermal unfolding occurs through a reversible one-step process while at pH <4.5 and >6.0 unfolding intermediates are formed, which are stable in the temperature range 65-80 degrees C. While these general behaviours are shared by all variants, VRQ and ARQ (susceptibility variants) show higher thermal stability than AHQ and ARR (resistance variants) and the formation of their unfolding intermediates requires higher activation energy than in the case of AHQ and ARR. Furthermore, secondary structures of the unfolding intermediates differentiate variants: ARR unfolding intermediate exhibits random coil structure, contrasting with the beta-sheet structure of VRQ and ARQ unfolding intermediates. The rate of the unfolding intermediate formation allows us to classify genetic variants along increasing scrapie susceptibility at pH 4.0, VRQ and ARQ rates being the highest. Rather poor correlation is observed at pH 7.2. Upon cooling, these intermediates refold into stable species, which are rich in beta-type secondary structures and, as revealed by thioflavin T fluorescence and electron microscopy, share amyloid characteristics. These results highlight the prion protein plasticity genetically modulated in sheep, and might provide a molecular basis for sheep predisposition to scrapie taking into account both thermodynamic stability and transconformation rate of prion protein.

Amyloid↗

PubMed1997

Differential effects of novel protamine variants on myocyte contractile function with left ventricular failure.

BACKGROUND: Protamine administration can cause left ventricular (LV) dysfunction, which may have clinical significance in the setting of congestive heart failure (CHF). Protamine variants have recently been constructed with heparin reversal capacity similar to protamine. The purpose of this study was to examine the potential differential effects of these protamine variants on isolated myocyte contractile function in normal myocytes and in myocytes after the development of CHF. METHODS: Contractile function was measured by means of computer-aided videomicroscopy in myocytes from five normal pigs and five pigs with CHF induced by rapid pacing (240 beats/min for 3 weeks). Myocyte contractility was examined in the presence of 40 micrograms/ml native protamine or one of three protamine variants: (1) reduced charge (+18) and lysine substituted for arginine; (2) lysine-substituted variant with glutamic acid substituted for the initial proline; or (3) arginine-rich peptide with a terminal arginine-glycine-aspartic acid (RGD) amino acid sequence. RESULTS: In the presence of native protamine, myocyte percent shortening fell from baseline in both the normal (2.86 +/- 0.15 versus 4.58 +/- 0.08, p < 0.05) and the CHF groups (1.01 +/- 0.06 versus 2.07 +/- 0.05, p < 0.05). With both of the lysine-substituted protamine variants, percent shortening fell from baseline in the normal group (3.42 +/- 0.20 for arginine and 3.74 +/- 0.20 for glutamic acid versus 4.58 +/- 0.08, p < 0.05), and was unchanged in the CHF group (1.94 +/- 0.13 versus 2.07 +/- 0.05, p = 0.34 for arginine; and 1.96 +/- 0.10 versus 2.07 +/- 0.05, p = 0.31, for glutamic acid). However, with the arginine/RGD variant, percent shortening fell from baseline in both the normal (2.86 +/- 0.23 versus 4.58 +/- 0.08, p < 0.05) and the CHF groups (1.32 +/- 0.10 versus 2.07 +/- 0.05, p < 0.05). CONCLUSIONS: Specific changes in the primary and secondary structures of protamine had different effects on myocyte contractile function. Furthermore, the negative effects of lysine-substituted protamine variants on myocyte contractility were less pronounced in both CHF and normal myocytes. Thus protamine variants may be of clinical use, particularly in the setting of preexisting LV dysfunction.

Animals↗

PubMed1998

Biological behavior and cell properties of new AKR lymphoma malignancy variants.

The AKR lymphoma-leukemia is a T lymphocyte neoplasm, most suitable as a model for human T cell malignancies. We have been interested in the process of tumor progression in the AKR lymphoma system. In the present study, two newly isolated variants, the TAU-42 and TAU-44, were characterized with respect to their biological behavior, by comparing them to a previously studied low-malignancy variant, the TAU-39. While the TAU-44 variant formed large s.c. local tumors, the TAU-42 variant formed only small growths or none at all. The TAU-42 lymphoma was found to have the highest malignant potential: it displayed very marked dissemination to spleen, lymph nodes, liver and lungs. The TAU-44 variant had an intermediate degree of metastatic potential but presented a predilection for spread to lymph nodes and spleen and was sometimes found to metastasize to peculiar organs, such as heart and pancreas. Cells derived from the different lymphoma variants varied in their immunophenotype: the highly malignant variant cells expressed more CD4 antigen than the low-malignancy one. The opposite was observed with regard to CD8. The variant cells also differed in their migrating capacity, the more malignant one exhibiting a higher motile activity. Studies on the tumor progression model of AKR lymphoma might contribute to the elucidation of the features determining the aggressiveness of T lymphocytic malignancies.

Animals↗

PubMed2001

Identification of a prostaglandin E2 receptor splice variant and its expression in rat tissues.

The intercellular signalling actions of the lipid mediators, the eicosanoids, are transduced by a family of seven transmembrane domain receptors. Members of this receptor family with high affinity for PGE2 are termed EP receptors. There are four known EP receptor genes that are transcribed to generate EP1, EP2, EP3 and EP4 receptors. Two of these receptor transcripts, EP1 and EP3, are further modified by RNA splicing to give multiple receptor isoforms. The EP3 receptor is known to have multiple splice variants in human (9 variants), cow (4 variants), mouse (3 variants) and rat (3 variants). In the rat the three EP3 splice variants differ in the sequence of the intracellular C-terminus. We have identified a fourth splice variant of the rat prostaglandin EP3 receptor that has a greatly truncated intracellular C-terminus when compared to the other EP3 receptor isoforms. Using nested RT-PCR we have shown that this novel splice variant is strongly expressed in rat brain and is also found in spinal cord, kidney and spleen.

Alternative Splicing↗

PubMed2004

Insulinomas and expression of an insulin splice variant.

BACKGROUND: Insulinomas are beta-cell tumours characterised by uncontrolled insulin secretion even in the presence of hypoglycaemia. However, the mechanisms allowing such excessive insulin secretion are not known. Insulin secretion can occur only when the beta-cell insulin stores have been replenished by insulin biosynthesis, which is mainly controlled by translation. Such specific translational regulation often involves the 5' untranslated region. We have identified an insulin splice variant in isolated human pancreatic islets of non-diabetic donors that retains 26 bp of intron 1 and thereby changes the 5' untranslated region, but leaves the coding region unchanged. This splice variant has increased translation efficiency in vitro and in vivo compared with native insulin mRNA. However, splice variant expression is less than 1% of native insulin mRNA in normal islets. METHODS: To test whether this splice variant is involved in insulin production by human insulinomas, we extracted RNA from nine laser-captured surgical insulinoma samples and from isolated islets of nine donors who did not have diabetes. We then determined the ratio of splice variant to native insulin mRNA by quantitative real-time RT-PCR. FINDINGS: The mean ratio of the splice variant to native insulin mRNA was increased more than 50-fold in insulinomas compared with normal islets, and this difference was present in all nine human insulinomas. Overexpression of the splice variant therefore seems to be a general characteristic of insulinomas and is estimated to contribute about 90% to insulin synthesis by these tumours. INTERPRETATION: Overexpression of the insulin splice variant with increased translation efficiency in insulinomas might explain how these tumours maintain high levels of insulin synthesis and secretion leading to hyperinsulinaemia-the hallmark of this disease.

Alternative Splicing↗