Isoforms In Patients Research Articles

Expression of the growth hormone receptor isoforms and its correlation with the metabolic profile in morbidly obese subjects.

Given the lipolytic effect of GH and its potential role in determining adipose tissue distribution, we evaluated the expression of the GH hormone receptor (GHR) isoforms in patients with morbid obesity seeking associations with metabolic parameters. 262 morbidly obese subjects (mean age 42.5 ± 11 years, 75% women) underwent PCR-genotyping of the exon 3 GHR polymorphism. In 17 of these subjects, who proved to be heterozygous for the exon 3 genotype (+3/-3), subcutaneous and visceral adipose tissue was obtained during bariatric surgery; total RNA was extracted, reversely transcribed, and the different isoforms of the GHR (exon 3 containing and lacking flGHR as well as the trGHR) were PCR-amplified using specific primers. 27% were +3/+3 homozygous, 20% -3/-3 homozygous and 53% were +3/-3 heterozygous. Compared to subjects homozygous for the +3 genotype, homozygous and heterozygous carriers of the -3 genotype were significantly heavier and tended to have a higher HOMA 2-IR. Expression of the flGHR and trGHR mRNA was demonstrated in all evaluated samples of subcutaneous and visceral adipose tissue from the 17 patients. The exon 3+ isoform was expressed in all adipose tissue samples, whereas only six subjects expressed the 3- isoform as well. The only distinctive feature of these six patients was a higher HbA1c. The heterozygous GHR +3/-3 genotype is more prevalent in subjects with morbid obesity. Patients expressing the exon +3 and exon -3 isoforms in adipose tissue had a higher HbA1c, than those expressing only the exon -3 isoform.

Autism spectrum disorders are prevalent among patients with dystrophinopathies.

Recent studies have reported a higher prevalence of autism spectrum disorders among patients with dystrophinopathies. The aim of this study was to investigate the prevalence of autism spectrum disorder (ASD) among those with dystrophinopathies. The possible role of dystrophin isoforms in patients was also explored. Fifty-six patients recruited from Toneyama National Hospital were included in this study (mean age = 12.9years, SD = 5.2years). Autistic symptoms were evaluated using the Pervasive Developmental Disorders/Autism Spectrum Disorders Rating Scale (PARS), a clinician rating scale. Eleven patients (19.6%; 95% confidence interval 10.2-32.4) met the criteria for ASD based on their PARS scores. Patients were separated into two groups based on the cumulative loss of dystrophin isoforms predicted from the mutation location. The prevalence of ASD was examined between these groups. Infantile and current autistic symptoms did not differ between the groups, except on one subscale of the PARS. This study revealed that there was a high prevalence of ASD in patients with dystrophinopathies.

The protective effect and diagnostic performance of NOX-5 in Crimean-Congo haemorrhagic fever patients.

Crimean-Congo haemorrhagic fever (CCHF) is an acute viral haemorrhagic disease. Reactive oxygen species that are mainly generated by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) enzyme family have a pivotal role in the pathophysiology of many diseases. The serum levels of NOX isoforms in patients with CCHF have yet to be assessed. This prospective study was conducted at Cumhuriyet University, Turkey. Only patients with CCHF confirmed by the National Reference Virology Laboratory were enrolled in the study. The study subjects comprised 67 CCHF patients and 70 healthy control subjects. The quantitative sandwich ELISA technique was used for the determination of serum NOX 1, 2, 4 and 5. Higher median median NOX-1 (P=0.001) and NOX-5 (P<0.001) levels were found in patients compared to the control group. Higher median serum NOX-5 levels were found in the low-grade disease group compared to the intermediate-high disease group according to two different severity scores (P=0.003). Negative correlations were also found between the serum NOX-5 levels and the severity scores [(P<0.05, r=-0.259), (P<0.01, r=-0.417)]. The area under the curve (AUC) values for the NOX-1 and NOX-5 were 0.67 (confidence interval: 0.58-0.75) and 0.99 (confidence interval: 0.95-1.00), respectively. Lower NOX-5 levels were found in patients receiving thrombocyte suspension (P=0.004)Conclusions. NOX-5 may have a protective effect on CCHF patients and the measurement of serum NOX-5 levels may be used as a novel biochemical test in the diagnosis of CCHF.

Serum apolipoprotein A2 isoforms in autoimmune pancreatitis

Recently, apolipoprotein A2 (apoA2) isoforms have been reported as candidate serum/plasma biomarkers of pancreatic cancer. However, the distribution of apoA2 isoforms in patients with autoimmune pancreatitis (AIP) has not been investigated yet. In this study, we evaluated the distribution of serum apoA2 isoforms; i.e., homodimer apoA2-ATQ/ATQ, heterodimer apoA2-ATQ/AT, and homodimer apoA2-AT/AT, in AIP patients and healthy volunteers (HV) using enzyme-linked immunosorbent assays, and the clinical characteristics and serum levels of each apoA2 isoform in 32 AIP patients and 38 HV were investigated. The calculated apoA2-ATQ/AT levels of the AIP patients were significantly lower than those of the HV, which agreed with results obtained for patients with pancreatic cancer. Interestingly, most of the AIP patients exhibited high levels of apoA2-ATQ along with low levels of apoA2-AT, indicating that the processing of the C-terminal regions of apoA2 dimer was inhibited in the AIP patients. This specific distribution of serum apoA2 isoforms might provide important information about the disease states of AIP patients and aid the differential diagnosis of AIP versus pancreatic cancer.

Upregulation of an antiangiogenic VEGFA165b isoform in patients with acute myocardial infarction

Upregulation of an antiangiogenic VEGFA165b isoform in patients with acute myocardial infarction

Dynamics and implications of circulating anti-angiogenic VEGF-A165b isoform in patients with ST-elevation myocardial infarction

Angiogenesis is crucial to restore microvascular perfusion in the jeopardized myocardium in the weeks following reperfused ST-segment elevation myocardial infarction (STEMI). (VEGF)-A165b, an anti-angiogenic factor, has been identified as a regulator of vascularization; however, it has not been previously implicated in acute myocardial infarction. We sought to investigate the dynamics of circulating VEGF-A165b and its association with cardiac magnetic resonance-derived infarct size and left ventricular ejection fraction (LVEF). 50 STEMI patients and 23 controls were included. Compared with control individuals, serum VEGF-A165b was elevated in STEMI patients prior to primary percutaneous coronary intervention (PCI). Following PCI, serum VEGF-A165b increased further, reaching a maximum level at 24 h and decreased one month after reperfusion. VEGF-A165b levels at 24 h were associated with a large infarct size and inversely related to LVEF. VEGF-A165b expression was increased in myocardial infarct areas from patients with previous history of AMI. An ex vivo assay using serum from STEMI patients showed that neutralization of VEGF-A165b increased tubulogenesis. Overall, the study suggests that VEGF-A165b might play a deleterious role after AMI as an inhibitor of angiogenesis in the myocardium. Accordingly, neutralization of VEGF-A165b could represent a novel pro-angiogenic therapy for reperfusion of myocardium in STEMI.

P031 Identification of alternative splicing in transcript isoforms in patients with ulcerative colitis: effects of disease duration

P031 Identification of alternative splicing in transcript isoforms in patients with ulcerative colitis: effects of disease duration

Tumor Suppressor Gene P29ing4 is Overexpressed and Induces a CD8 T Effector Cell Response in Human Renal Cell Carcinoma

Objective: ING1 and ING4 are identified as candidate tumor suppressor genes acting in regulation of DNA damage responses and apoptosis through modulation of p53. Their defective function promotes tumor growth in melanoma and breast cancer. Our aim was to determine the overexpression of relevant p33ING1b and p29ING4 isoforms in patients with Renal Cell Cancer (RCC). Methods: Peripheral Blood Mononuclear Cells (PBMCs) from tumor patients (Robson stage I-IV) were stimulated with overlapping peptides of p33ING1b/p29ING4 and results were compared with expression profiles in primary tumors. Results: Early-stage and late-stage tumors demonstrated upregulated ING-isoform gene and protein expression. Early cancers were characterized by increased CD8 and IFN-γ protein and gene expression. Significant p33ING1b and p29ING4 tumor-specific CD8 T effector cell responses from PBMCs of the analyzed tumor patients were observed. Interestingly, peptide sequences p33ING1b (aa259-268) and p29ING4 (aa149-158) elicited significant IFN-γ responses indicative for anti-tumor immune responses while IL-2 responses were detected only for p29ING4 (aa149-158), suggesting inducible T effector cell responses. Conclusion: T effector cell analysis against p29ING4 (aa149-158) suggests a promising candidate for in vivo induction of tumor-reactive CD8 T effector cells in patients with renal cell cancer.

Association of BIM Deletion Polymorphism and BIM-γ RNA Expression in NSCLC with EGFR Mutation.

This pilot study assessed the association of BIM deletion polymorphism and BIM RNA isoform in patients with EGFR-positive non-small cell lung cancer (NSCLC). The study included 33 patients with EGFR-positive NSCLC treated with gefitinib. BIM deletion polymorphism and BIM RNA isoform (EL/L/S/γ) were determined by polymerase chain reaction (PCR). BIM-γ expression was significantly higher in patients with BIM deletion polymorphism than among those without BIM deletion polymorphism inside tumors (p=0.038) and around tumors (p=0.0024). Relative BIM-γ expression was significantly higher in patients with BIM deletion polymorphism than among those without BIM deletion polymorphism (p=0.0017). Patients with BIM-γ had significantly shorter progression-free survival than those without BIM-γ (median: 304 vs. 732 days; p=0.023). Expression of BIM-γ mRNA and BIM deletion polymorphism were strongly associated. BIM-γ overexpression may have a role in apoptosis related to EGFR-tyrosine kinase inhibitor.

Prevalence of LPA single nucleotide polymorphisms and isoforms in patients enrolled in a phase 2 IONIS-APO(a)Rx clinical trial

Prevalence of LPA single nucleotide polymorphisms and isoforms in patients enrolled in a phase 2 IONIS-APO(a)Rx clinical trial

The Presence of Interleukin 18 Binding Protein Isoforms in Chinese Patients with Rheumatoid Arthritis

Objective: Human IL-18BP gene encodes at least four distinct isoforms (IL18BPa-d) derived by alternative splicing. Their presence in RA local inflammation is not yet examined. This study aimed to determine the messenger transcript and protein levels of IL-18BP isoforms in patients with Rheumatoid Arthritis (RA). Materials: The study comprises 65 rheumatic patients, 22 Osteoarthritis (OA), and 40 sex and age matched normal controls (NC). Peripheral blood mononuclear cells (PBMCs) and synovial fluids mononuclear cells (SFMCs) were prepared by using Ficoll-Hypaque procedure. The expression and presence of different isoforms were determined by using real-time PCR and ELISA respectively. Results: IL-18BP messenger transcript has been extremely expressed in synovial fluids (SF) and synovial tissues (ST) of RA patients compared to OA patients (p < 0.001). IL-18BP auto-antibodies were noticed in RA plasma and SF (41.7%; 37.9%), in OA-SF (9.0%), and in plasma of NC (4.0%). Comparable to different isoforms, isoform “c” showed significant local expression (p < 0.001) in RA-SFMC and systematic expression (p < 0.001) between RA- and NC-PBMCs, isoform “a” was least expressed. Isoform “c” and “d” proteins were solely detected by western blot in RA. Conclusions: This study emphasizes the local existence of isoform “c” and “d”, and the possible presence of autoantibodies against IL-18BPa in RA patients, which made a pea for further investigation, putting in place their actual role.

NKp30 isoforms in patients with chronic hepatitis C virus infection.

Natural killer (NK) cells play an important role in virus infection, their action being regulated by several activating and inhibitory receptors. The NKp30 activating receptor and its isoforms have recently emerged as important determinants of efficient NK cell responses. We determined the relative proportions of NKp30 isoforms in patients with chronic hepatitis C virus (HCV) infection and healthy donors (HD). NK cell function (degranulation and cytokine production) and correlations with clinical parameters were assessed following unsupervised hierarchical clustering of patients according to isoform expression. NKp30 receptor expression on NK cells and all isoforms were reduced in HCV-infected patients. Patients were clustered into two groups: the HCV-1 group had similar isoform expression to the HD group, whereas the HCV-2 group had lower expression. The latter showed a better functional activity, and a higher proportion of the activating a isoform and of the NKp30 isoform a/c ratio compared with the HCV-1 cluster. There was a positive correlation between the activating a isoform and liver stiffness and an inverse relationship between the immunosuppressive c isoform and the fibrosis 4 score, suggesting a potentially important role of NKp30 isoforms in influencing liver damage and ensuing fibrosis.

The potential role of estrogen receptor β2 in breast cancer

Endocrine therapy is provided to all patients with estrogen receptor (ER)-positive breast cancer, but only a subset of them derives clinical benefit. The discovery of ERβ and its five isoforms added another layer of complexity in the regulation of estrogen activity in breast cancer cells. Two large retrospective studies showed conflicting results with regard to the prognostic value of the different ERβ isoforms in patients treated with tamoxifen in an adjuvant setting. This study tested the hypothesis that ERβ1 and, or ERβ2 are correlated with clinical outcome. We identified patients with breast cancer who had undergone surgery at Bucheon St. Mary's Hospital, the Catholic University of Korea, between January 2004 and March 2006. We evaluated 101 consecutive cases for ERβ1 and ERβ2 expression using immunohistochemical staining and obtained other clinicopathology by reviewing medical records. ERβ1 was expressed in 81.2% (79 of 97) and ERβ2 was expressed in 50.5% (51 of 101) of primary breast cancer tissues. Disease-free survival (DFS) and overall survival (OS) of patients with cancers expressing ERβ2 was significantly worse. Moreover, in subgroup analysis according to the tamoxifen treatment, ERβ2 expression was significantly associated with shorter DFS of tamoxifen-treated patients. This study indicates that breast cancer with ERβ2 expression was associated with worse DFS and OS, especially in tamoxifen treated patients. Our results suggest a role for ERβ2 as an independent prognostic marker and might serve as a new therapeutic target.

HER2Δ16 expression in HER2-positive breast cancer.

31 Background: HER2Δ16 isoform was detected in breast cancer and showed similar increased transforming activity to somatic mutation or deletion activated rat HER2 oncogene with 89% of HER2-positive breast cancer with this mutation developing local lymph node positive disease progression. This is the first study characterizing and determining the prevalence of HER2Δ16 isoform in patients from a NAPBC-accredited cancer center. Methods: 95 HER2-positive breast cancer tissue samples from 2002 to 2008 were obtained from Akron General Medical Center. Total RNA from cancer cells were isolated and RT-PCR was performed using primers to amplify the wild-type HER2 and HER2Δ16 isoform. DNA products from RT-PCR were then loaded in agarose gel electrophoresis with the HER2Δ16 isoform band being shorter compared to the wild type HER2. Results: Preliminary results from our ongoing study showed 39% (12 of 31) of our HER2 samples has HER2Δ16 isoform. Out of the 12 HER2Δ16 isoform samples, 17% are Stage I, 50% are Stage II, 25% are Stage III, and 8% are Stage IV breast cancer at diagnosis. Patient age ranges from 34 to 87, with 50% of the HER2Δ16 isoform patients having lymph–node positive disease at the time of diagnosis. This is in contrast to patients with HER2 wild type for which 26% (5 of 19) are diagnosed with Stage I disease and 42% have lymph node positive disease at the time of diagnosis. Conclusions: Transgenic mice that overexpressed rat HER2 oncogene with small extracellular domain deletion promoted transforming activity of this protein. HER2Δ16 isoform is detected in 39% of HER2 positive breast cancer based on our preliminary results with 50% having lymph node–positive breast cancer on initial diagnosis. Immunohistochemistry and FISH could not distinguish the wild type HER2 from the HER2Δ16 isoform. Further study is recommended for possible prognosticating utility of HER2Δ16 isoform in breast cancer.

Expression pattern of synaptic vesicle protein 2 (SV2) isoforms in patients with temporal lobe epilepsy and hippocampal sclerosis

Synaptic vesicle proteins 2 (SV2) are neuronal vesicles membrane glycoproteins that appear as important targets in the treatment of partial and generalized epilepsies. Therefore, we analysed the expression of SV2 isoforms in the hippocampus of patients with temporal lobe epilepsy (TLE). SV2A, SV2B and SV2C immunostaining and QuantiGene branched DNA assay were performed on biopsies from 31 consecutive TLE patients with mesial temporal sclerosis (MTS) and compared with 10 autopsy controls. SV2 expression was further compared with Timm's staining, and synaptophysin, Zinc transporter 3 (ZnT3), dynorphin, vesicular glutamate transporter 1 (VGLUT1) and vesicular GABA transporter (VGAT) expression. In TLE patients, SV2A and SV2B expression was decreased in areas of synaptic loss. SV2C, which is weakly expressed or absent in the hippocampus of controls, was overexpressed in 10/11 cases with classical MTS1A and mossy fibre sprouting but not in cases with other types of MTS. SV2C staining was located in the inner molecular layer of the dentate gyrus and colocalized with dynorphin, ZnT3 and VGLUT1, suggesting selective expression in presynaptic glutamatergic Zn(2+) -rich terminals of abnormal sprouting fibres. SV2 expression patterns correlated with histological subtypes of MTS, but not with clinical features or therapeutic regimens in this patient cohort. In classical MTS1A, the expression of SV2 isoforms is altered with a marked decrease of SV2A and SV2B paralleling synaptic loss and a selective increase of SV2C in sprouting mossy fibres. These findings suggest a different physiology of sprouting synapses and the possibility to target them with SV2C-specific strategies.

Reduced C9orf72 protein levels in frontal cortex of amyotrophic lateral sclerosis and frontotemporal degeneration brain with the C9ORF72 hexanucleotide repeat expansion

An intronic G4C2 hexanucleotide repeat expansion in C9ORF72 is a major cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Several mechanisms including RNA toxicity, repeat-associated non-AUG translation mediated dipeptide protein aggregates, and haploinsufficiency of C9orf72 have been implicated in the molecular pathogenesis of this disorder. The aims of this study were to compare the use of two different Southern blot probes for detection of repeat expansions in an amyotrophic lateral sclerosis and frontotemporal lobar degeneration pathological cohort and to determine the levels of C9orf72 transcript variants and protein isoforms in patients versus control subjects. Our Southern blot studies identified smaller repeat expansions (250–1800 bp) that were only detectable with the flanking probe highlighting the potential for divergent results using different Southern blotting protocols that could complicate genotype–phenotype correlation studies. Further, we characterize a new C9orf72 antibody and show for the first time decreased C9orf72 protein levels in the frontal cortex from patients with a pathological hexanucleotide repeat expansion. These data suggest that a reduction in C9orf72 protein may be a consequence of the disease.

Small RNA sequencing-microarray analyses in Parkinson leukocytes reveal deep brain stimulation-induced splicing changes that classify brain region transcriptomes

MicroRNAs (miRNAs) are key post transcriptional regulators of their multiple target genes. However, the detailed profile of miRNA expression in Parkinson's disease, the second most common neurodegenerative disease worldwide and the first motor disorder has not been charted yet. Here, we report comprehensive miRNA profiling by next-generation small-RNA sequencing, combined with targets inspection by splice-junction and exon arrays interrogating leukocyte RNA in Parkinson's disease patients before and after deep brain stimulation (DBS) treatment and of matched healthy control volunteers (HC). RNA-Seq analysis identified 254 miRNAs and 79 passenger strand forms as expressed in blood leukocytes, 16 of which were modified in patients pre-treatment as compared to HC. 11 miRNAs were modified following brain stimulation 5 of which were changed inversely to the disease induced changes. Stimulation cessation further induced changes in 11 miRNAs. Transcript isoform abundance analysis yielded 332 changed isoforms in patients compared to HC, which classified brain transcriptomes of 47 PD and control independent microarrays. Functional enrichment analysis highlighted mitochondrion organization. DBS induced 155 splice changes, enriched in ubiquitin homeostasis. Cellular composition analysis revealed immune cell activity pre and post treatment. Overall, 217 disease and 74 treatment alternative isoforms were predictably targeted by modified miRNAs within both 3′ and 5′ untranslated ends and coding sequence sites. The stimulation-induced network sustained 4 miRNAs and 7 transcripts of the disease network. We believe that the presented dynamic networks provide a novel avenue for identifying disease and treatment-related therapeutic targets. Furthermore, the identification of these networks is a major step forward in the road for understanding the molecular basis for neurological and neurodegenerative diseases and assessment of the impact of brain stimulation on human diseases.

Abnormal expression of PI3K isoforms in patients with tobacco-related oral squamous cell carcinoma

BackgroundThe phosphatidylinositol 3-kinase (PI3K) signaling regulates several cellular functions such as motility, proliferation, angiogenesis and survival. MethodsSince there is no information on expression of PI3K isoforms in oral cancer, we studied the expression of different isoforms of PI3K (p110α, p110γ, PI3K-C2, Vps34p and p85α) in tumor samples and PBMC by RT and q-RTPCR and serum levels of PI3K p110α by SPR and ELISA techniques in 108 patients with tobacco-related oral squamous cell carcinoma (OSCC) and 46 normal subjects. ResultsWe observed significantly higher PI3K p110α (p<0.0001) and lower (p<0.0001) vesicular sorting protein 34p (Vps34p) mRNA both in PBMC and tissue samples of oral cancer patients as compared to the normal controls. Other PI3K isoforms did not show such change. Circulating PI3K p110α levels were higher in patients (p<0.0001) as compared to healthy subjects, the SPR data showed direct correlation with advancing stage of the disease. PI3K p110α was overexpressed in tumor samples but not in the normal buccal mucosa. ConclusionsUpregulation of circulating PI3K p110α isoform and its direct correlation with increasing tumor load in OSCC patients indicates that it may be a significant prognostic indicator and a suitable target for therapeutic/chemo-preventive strategies for tobacco-related OSCC.

Differential expression of the Bcl-2 and Bax isoforms in CD19 positive B-lymphocytes isolated from patients diagnosed with chronic lymphocytic leukaemia

To examine the relative gene expression levels of the anti-apoptotic Bcl-2α and β isoforms and the pro-apoptotic Baxα and β isoforms in patients with chronic lymphocytic leukaemia (CLL) and healthy controls (HC). Peripheral blood was obtained from 36 patients diagnosed with CLL and 10 HC. CD19 B-lymphocytes were isolated using an antibody coupled magnetic bead isolation system; from these cells the total RNA was isolated and purified. The relative levels of gene expression were examined by quantitative real-time polymerase chain reaction (qReTi-PCR) using primers specific for each isoform. Bcl-2α and Baxα are expressed at higher levels than their β-isoforms in CLL and HC. Bcl-2α, Bcl-2β and Baxβ expression is increased in CLL while Bax-α is expressed at similar levels to HC. The Bcl-2α/Bcl-2β ratio is similar in CLL and HC. The Bcl-2α/Baxα ratio is increased in CLL when compared with HC. Bcl-2α and Baxα appear to be the dominant anti- and pro-apoptotic isoforms in CLL. The Bcl-2α/Baxα ratio is increased in CLL while the Bcl-2α/Bcl-2β ratio is similar to HC.

Separation and study of the range of plasminogen isoforms in patients with prostate cancer

Using affinity chromatography, two-dimensional electrophoresis, and MALDI-TOF mass spectrometry, plasminogen isoforms were separated and identified in blood plasma. Healthy donors and patients with prostate cancer in various stages of development were included in the studied sample. With the development of prostate cancer, four additional specific plasminogen isoforms are registered in blood plasma; they are characterized by lower molecular weights and higher pI values compared to isoforms found in the control group.