Wu, Yanhong

Background and Aim: Cardioembolic stroke from non-valvular atrial fibrillation (NVAF) begins with the development of left atrial appendage thrombus (LAAT). Although nearly 15% of patients with NVAF have LAAT by transesophageal echocardiography, the annual stroke rate is 5% on average. The overall aim of this project is to identify variables associated with embolic propensity of LAAT. Materials and Methods: LAAT specimens were obtained during cardiac surgery (MAZE procedure; n=26). Embolized thrombi (n=51) were obtained during surgical embolectomy from lower (n=36) and upper extremities (n=11) and mesenteric arteries (n=4). Platelet-rich regions within sectioned thrombi were sampled by punch biopsy from which total RNA was extracted and analyzed for expression of 5 platelet specific genes: A1 domain of factor XIII, (F13A1); integrin α 2b β 3 (ITGA2B); glycoprotein IX (GP9), platelet factor 4 (PF4), glycoprotein Ib (GP1BB). Gene expression was quantified using TaqMan MGB-probe based quantitative real-time polymerase chain reaction normalized to β 2 macroglobulin using cDNAs reverse transcribed from total RNA. Results: Factor XIII gene expression (Figure ) was significantly lower in embolic (mean fold change 0.08) compared to left atrial thrombi in situ (mean fold change 0.28; p=0.03). Expression of other genes did not show significant change by embolic status. Conclusions: Variable Factor XIII gene expression in thrombi generated during NVAF may in part explain the propensity to embolization. Whereas Factor XIII stabilizes thrombi by crosslinking fibrin without influencing atrial attachment, this data suggests that embolization occurs through fragmentation.

AbstractBackgroundCurrently, there is no biologically based rationale for drug selection in migraine prophylactic treatment.MethodsTo investigate the genetic variation underlying treatment response to verapamil prophylaxis, we selected 225 patients from a longitudinally established, deeply phenotyped migraine database (N = 5983), and collected uninterrupted quantitated verapamil treatment response data and DNA for these 225 cases. We recorded the number of headache days in the four weeks preceding treatment with verapamil and for four weeks, following completion of a treatment period with verapamil lasting at least five weeks. Whole‐exome sequencing (WES) was applied to a discovery cohort consisting of 21 definitive responders and 14 definitive non‐responders, and the identified single nucleotide polymorphisms (SNPs) showing significant association were genotyped in a separate confirmation cohort (185 verapamil treated patients). Statistical analysis of the WES data from the discovery cohort identified 524 SNPs associated with verapamil responsiveness (p < 0.01); among them, 39 SNPs were validated in the confirmatory cohort (n = 185) which included the full range of response to verapamil from highly responsive to not responsive.ResultsFourteen SNPs were confirmed by both percentage and arithmetic statistical approaches. Pathway and protein network analysis implicated myo‐inositol biosynthetic and phospholipase‐C second messenger pathways in verapamil responsiveness, emphasizing the earlier pathogenic understanding of migraine. No association was found between genetic variation in verapamil metabolic enzymes and treatment response.ConclusionOur findings demonstrate that genetic analysis in well‐characterized subpopulations can yield important pharmacogenetic information pertaining to the mechanism of anti‐migraine prophylactic medications.

Abstract Background Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with poor overall survival rates. The genetics of PTCLs are poorly understood. We have described t(6;14)(p25.3;q11.2) translocations in PTCLs involving the T-cell receptor-alpha (TRA@) and interferon regulatory factor-4 (IRF4) genes and overexpressing IRF4 protein (also called MUM1). IRF4/MUM1 is a lymphoid transcription factor involved in differentiation and growth, the expression of which is tightly regulated in normal T cells. However, nearly half of PTCLs constitutively express IRF4/MUM1 and this expression is associated with adverse overall survival in several PTCL subtypes. Because IRF4/TRA@ translocations are rare among PTCLs (<1%), we investigated the presence of other genetic abnormalities of IRF4 in PTCLs and examined their association with IRF4/MUM1 protein expression. Methods Studies were performed on paraffin tissue sections of PTCL specimens diagnosed by standard WHO criteria. IRF4/MUM1 immunohistochemical staining (IHC) was performed using the MUM1p antibody and considered positive if nuclear staining was present in >30% of tumor cell nuclei. Fluorescence in situ hybridization (FISH) was performed using breakapart probes constructed from bacterial artificial chromosome DNA that hybridized to loci flanking the IRF4 locus on 6p25.3. Cases with 3 or more fusion signals were considered to have extra copies of the IRF4 gene. In a subset of cases, PCR and Sanger sequencing was performed on DNA extracted from frozen PTCL tumor tissue with primer sets that amplified exons 1 to 10 of the IRF4 gene. Data were analyzed using the chi-square test or t test. Results PTCLs from 277 patients (175 M:102 F; median age, 61 y) were examined by both IHC and FISH. The subtype distribution was: PTCL, not otherwise specified (NOS), 93; anaplastic large cell lymphoma (ALCL), 89; angioimmunoblastic T-cell lymphoma (AITL), 27; cutaneous T-cell lymphoma (CTCL), 43; and cytotoxic PTCL, 25. IRF4/MUM1 was positive in 116 cases (42%). Extra copies of IRF4 were identified in 18 PTCLs (7%), 83% of which were IRF4/MUM1 protein-positive. In contrast, 39% of cases without extra copies of IRF4 were IRF4/MUM1 protein-positive (p=0.0002). The mean percentage of tumor cells with IRF4/MUM1 staining was 69% in cases with extra copies of IRF4, compared to 30% in cases without extra copies (p=0.0001). Extra copies of IRF4 were seen in 13% of ALCLs, 4% each of PTCLs, NOS and cytotoxic PTCLs, 2% of CTCLs, and 0% of AITLs (p=0.02). Two IRF4/MUM1-positive cases had an IRF4/TRA@ translocation (1%). We selected 30 IRF4/MUM1-positive PTCLs for Sanger sequencing. In this subset, we identified a previously undescribed non-synonymous variant in exon 5 (S309R) in 1 case (3%) without an extra copy of IRF4. Conclusions Extra copies of IRF4 are seen in ∼7% of PTCLs, are significantly associated with IRF4/MUM1 expression, and are preferentially observed in ALCLs. Both IRF4/TRA@ translocations and IRF4 mutations are rare in PTCLs. These data suggest that genetic abnormalities of the IRF4 gene – specifically extra copies of IRF4 – may contribute to IRF4/MUM1 expression in some PTCLs. Since IRF4/MUM1 expression is seen in ∼42% of PTCLs, these data also imply the existence of other regulatory mechanisms. Characterizing these mechanisms may be important clinically, since IRF4/MUM1 has been proposed as a therapeutic target in human cancers but drugs that directly inhibit its function are currently not available. Disclosures: No relevant conflicts of interest to declare.

Perineuriomas are rare nerve sheath tumors, divided into intraneural and extraneural (soft tissue) types. Intraneural perineuriomas frequently contain TRAF7 mutations, and rarely, chr22q12 deletions. While chr22q losses can occur in soft tissue perineuriomas, comprehensive high-resolution molecular profiling has not been reported in these tumors and TRAF7 status is unknown. We used whole-exome sequencing and OncoScan single nucleotide polymorphism (SNP) array to evaluate 14 soft tissue perineuriomas. Thirteen cases showed 2 or more chromosomal abnormalities, composed primarily of large deletions. Recurrent chr22q deletions, containing the NF2 locus (n=6) and the previously unreported finding of chr17q deletions, with the NF1 locus (n=4) were frequent events and were mutually exclusive in all but1 case. In addition, 5 cases had varying chr2 deletions; and 4 cases had chr6 deletions. A chr10 deletion (previously reported in the sclerosing variant of soft tissue perineurioma) was observed in one case and another case had chr7 chromothripsis as the sole chromosomal abnormality. No TRAF7 mutations or alterations were identified in any case and no other evaluated gene (MAF<0.0001) had recurrent, deleterious mutations in >2 cases. The molecular genetic profiles showed no association with patient sex, age, tumoral histology or anatomic site. OncoScan SNP array analysis was performed on 10 cases and showed high concordance with the whole exome data, validating the large-scale deletions, duplications, and chr7 chromothripsis findings. In soft tissue perineuriomas, recurrent 22q12 deletions (with NF2) and 17q11 deletions (with NF1) appear to be mutually exclusive events, and alterations in NF1 or NF2 likely contribute to perineurioma pathogenesis, similar to other nerve sheath tumors. Moreover, the lack of TRAF7 mutations in soft tissue perineuriomas indicates divergent pathogenetic mechanisms from those of intraneural perineuriomas.

AbstractIntroductionReticulated platelet (RP) content is increased in nonvalvular atrial fibrillation (NVAF). The purpose of this study was to determine if platelet content, morphology, and RP proportion are modulated by platelet genes.Methods and resultsExpression of six platelet‐predominate genes impacting platelet formation and release, platelet count, and RP content was assessed in NVAF patients before and 3–4 months after pulmonary veins isolation (PVI) and compared to normal sinus rhythm (NSR) controls. RNA from isolated platelets was reverse‐transcribed assayed against selected genes utilizing real‐time qPCR, and expressed as mean cycle threshold (ΔCt) using beta‐2‐microglobulin as endogenous control. RP content was assessed by flow cytometry. A fourfold lower expression of CFL1 gene coding for nonmuscle cofilin (7.8 ± 0.9 vs. 5.7 ± 1.6, P < 0.001) and twofold lower expression of four other genes were associated with similar platelet counts but fourfold higher (28.7+7.0 vs. 6.7+5.4, P < 0.001) RP content (%) in 97 NVAF cases compared to 51 NSR controls. Three to 4 months after PVI, RP decreased by 28%, while CFL1 gene expression increased over twofold but TUBA4A gene expression decreased almost twofold; NFE2 and MYL6 gene expression remained unchanged.ConclusionsNVAF is associated with notable downregulation of genes directing platelet production and size but increased RP content. PVI impacts the expression of many of these genes, implying a direct relationship between atrial fibrillation and platelet biogenesis.

AbstractAimsPlatelets retain cytoplasmic messenger RNA and are capable of protein biosynthesis. Several diseases are known to impact the platelet transcriptome but the effect of non‐valvular atrial fibrillation (NVAF) on platelet RNA transcript is essentially unknown. The aim of this study was to evaluate the impact of NVAF on platelet RNA transcript by measuring platelet genes expression in consecutive NVAF patients before and 3‐4 months after pulmonary vein isolation (PVI) and compared to normal sinus rhythm controls (NSR).Methods and ResultsRNA from isolated platelets were reverse transcribed, assayed against 15 genes using real‐time qPCR, and expressed as mean cycle threshold (ΔCt) using beta‐2‐microglobulin as endogenous control. Expression of all evaluated genes, except cathepsin A gene, was significantly lower (higher ΔCt) in 103 NVAF patients compared to 55 NSR controls. Insulin‐like growth factor binding protein acid labile subunit gene (IGFALS) had expression more than 16 fold‐lower (17.0±2.8 vs 12.5±3.8, P<.001), follow by genes encoding for prostacyclin receptor, and for von Willebrand factor which had fourfold lower expression compared to NSR controls. Gender, type of atrial fibrillation, heart failure, hypertension, prior stroke, diabetes mellitus, and atherosclerosis were associated with different gene expression. Following PVI, expression of four genes significantly increased, particularly IGFALS gene (increased 256‐fold) and ADAMT gene increased 16‐fold); expression of three genes significantly decreased, and expression of eight genes has not changed.ConclusionsPlatelets are capable to respond to the circulatory environment of NVAF by altering transcript and changing prothrombotic status. This shows platelet potential for molecular “reprogramming” possibly induced by flow disturbances of NVAF.

Key PointsThe NF-κB subunits p52 and RelB increase IRF4 promoter activity and expression in PTCL cells. A positive feedback loop involving CD30, NF-κB, and IRF4 drives PTCL cell proliferation and can be blocked by NF-κB inhibitors.