The consensus sequence of FAMLF alternative splice variants is overexpressed in undifferentiated hematopoietic cells

The familial acute myeloid leukemia related factor gene (FAMLF) was previously identified from a familial AML subtractive cDNA library and shown to undergo alternative splicing. This study used real-time quantitative PCR to investigate the expression of the FAMLF alternative-splicing transcript consensus sequence (FAMLF-CS) in peripheral blood mononuclear cells (PBMCs) from 119 patients with de novo acute leukemia (AL) and 104 healthy controls, as well as in CD34+cells from 12 AL patients and 10 healthy donors. A 429-bp fragment from a novel splicing variant of FAMLF was obtained, and a 363-bp consensus sequence was targeted to quantify total FAMLF expression. Kruskal-Wallis, Nemenyi, Spearman's correlation, and Mann-Whitney U-tests were used to analyze the data. FAMLF-CS expression in PBMCs from AL patients and CD34+ cells from AL patients and controls was significantly higher than in control PBMCs (P<0.0001). Moreover,FAMLF-CS expression in PBMCs from the AML group was positively correlated with red blood cell count (rs=0.317, P=0.006), hemoglobin levels (rs=0.210, P=0.049), and percentage of peripheral blood blasts (rs=0.256, P=0.027), but inversely correlated with hemoglobin levels in the control group (rs=–0.391, P<0.0001). AML patients with high CD34+ expression showed significantly higherFAMLF-CS expression than those with low CD34+ expression (P=0.041). Our results showed thatFAMLF is highly expressed in both normal and malignant immature hematopoietic cells, but that expression is lower in normal mature PBMCs.

Splice variants of the human ZC3H14 gene generate multiple isoforms of a zinc finger polyadenosine RNA binding protein

The human ZC3H14 gene encodes an evolutionarily conserved Cys(3)His zinc finger protein that binds specifically to polyadenosine RNA and is thus postulated to modulate post-transcriptional gene expression. Expressed sequence tag (EST) data predicts multiple splice variants of both human and mouse ZC3H14. Analysis of ZC3H14 expression in both human cell lines and mouse tissues confirms the presence of multiple alternatively spliced transcripts. Although all of these transcripts encode protein isoforms that contain the conserved C-terminal zinc finger domain, suggesting that they could all bind to polyadenosine RNA, they differ in other functionally important domains. Most of the alternative transcripts encode closely related proteins (termed isoforms 1, 2. 3, and 3short) that differ primarily in the inclusion of three small exons, 9, 10, and 11, resulting in predicted protein isoforms ranging from 82 to 64 kDa. Each of these closely related isoforms contains predicted classical nuclear localization signals (cNLS) within exons 7 and 11. Consistent with the presence of these putative nuclear targeting signals, these ZC3H14 isoforms are all localized to the nucleus. In contrast, an additional transcript encodes a smaller protein (34 kDa) with an alternative first exon (isoform, 4). Consistent with the absence of the predicted cNLS motifs located in exons 7 and 11, ZC3H14 isoform 4 is localized to the cytoplasm. Both EST data and experimental data suggest that this variant is enriched in testes and brain. Using an antibody that detects endogenous ZC3H14 isoforms 1-3 reveals localization of these isoforms to nuclear speckles. These speckles co-localize with the splicing factor, SC35, suggesting a role for nuclear ZC3H14 in mRNA processing. Taken together, these results demonstrate that multiple transcripts encoding several ZC3H14 isoforms exist in vivo. Both nuclear and cytoplasmic ZC3H14 isoforms could have distinct effects on gene expression mediated by the common Cys(3)His zinc finger polyadenosine RNA binding domain. (C) 2009 Elsevier B.V. All rights reserved.

Using gene-network landscape to dissect genotype effects of TCF7L2 genetic variant on diabetes and cardiovascular risk

Vaquero AR, Ferreira NE, Omae SV, Rodrigues MV, Teixeira SK, Krieger JE, Pereira AC. Using gene-network landscape to dissect genotype effects of TCF7L2 genetic variant on diabetes and cardiovascular risk. Physiol Genomics 44: 903-914, 2012. First published August 7, 2012; doi:10.1152/physiolgenomics.00030.2012.-The single nucleotide polymorphism (SNP) within the TCF7L2 gene, rs7903146, is, to date, the most significant genetic marker associated with Type 2 diabetes mellitus (T2DM) risk. Nonetheless, its functional role in disease pathology is poorly understood. The aim of the present study was to investigate, in vascular smooth muscle cells from 92 patients undergoing aortocoronary bypass surgery, the contribution of this SNP in T2DM using expression levels and expression correlation comparison approaches, which were visually represented as gene interaction networks. Initially, the expression levels of 41 genes (seven TCF7L2 splice forms and 40 other T2DM relevant genes) were compared between rs7903146 wild-type (CC) and T2DM-risk (CT + TT) genotype groups. Next, we compared the expression correlation patterns of these 41 genes between groups to observe if the relationships between genes were different. Five TCF7L2 splice forms and nine genes showed significant expression differences between groups. RXR alpha gene was pinpointed as showing the most different expression correlation pattern with other genes. Therefore, T2DM risk alleles appear to be influencing TCF7L2 splice form's expression in vascular smooth muscle cells, and RXR alpha gene is pointed out as a treatment target candidate for risk reduction in individuals with high risk of developing T2DM, especially individuals harboring TCF7L2 risk genotypes.

An unusual splice defect in the mitofusin 2 gene (MFN2) is associated with degenerative axonopathy in Tyrolean Grey cattle

Tyrolean Grey cattle represent a local breed with a population size of approximately 5000 registered cows. In 2003, a previously unknown neurological disorder was recognized in Tyrolean Grey cattle. The clinical signs of the disorder are similar to those of bovine progressive degenerative myeloencephalopathy (weaver syndrome) in Brown Swiss cattle but occur much earlier in life. The neuropathological investigation of an affected calf showed axonal degeneration in the central nervous system (CNS) and femoral nerve. The pedigrees of the affected calves suggested a monogenic autosomal recessive inheritance. We localized the responsible mutation to a 1.9 Mb interval on chromosome 16 by genome-wide association and haplotype mapping. The MFN2 gene located in this interval encodes mitofusin 2, a mitochondrial membrane protein. A heritable human axonal neuropathy, Charcot-Marie-Tooth disease-2A2 (CMT2A2), is caused by MFN2 mutations. Therefore, we considered MFN2 a positional and functional candidate gene and performed mutation analysis in affected and control Tyrolean Grey cattle. We did not find any non-synonymous variants. However, we identified a perfectly associated silent SNP in the coding region of exon 20 of the MFN2 gene. This SNP is located within a putative exonic splice enhancer (ESE) and the variant allele leads to partial retention of the entire intron 19 and a premature stop codon in the aberrant MFN2 transcript. Thus we have identified a highly unusual splicing defect, where an exonic single base exchange leads to the retention of the preceding intron. This splicing defect represents a potential explanation for the observed degenerative axonopathy. Marker assisted selection can now be used to eliminate degenerative axonopathy from Tyrolean Grey cattle.

NMDA receptor variant responses to conantokins

Excitotoxicity may have role in neuronal death in many disorders including Alzheimer disease. Sensitivity of a cell to excitotoxicity may depend on its subtype of NMDA receptors. A drug that selectively reduced such overstimulation could limit susceptibility to damage. We examined the pharmacology of NMDA receptor subtypes in response to the agonists glutamate and glycine, the modulator spermine, and the antagonists conantokin-G and its Ala(7) analogue in Xenopus oo¨ cytes. Cells were injected with capped RNA coding for NMDA NR1 and NR2 subunits. Membrane currents induced by rapid application of agonists were recorded under two-electrode voltageclamp. Conantokins were bath-applied to give cumulative concentration responses. Spermine gave slightly different shifts in glutamate affinity when different NR1 splice variants were combined with NR2A subunits. In the presence of spermine, both an increase and a decrease in affinity for glutamate were seen with differing subunit combinations that could not be explained by the absence or presence of the N-terminal 23-amino-acid insert.

EAAT2 splice variants in Alzheimer disease

Regional atrophy caused by neuronal loss is a characteristic of Alzheimer Disease (AD). Excitatory amino acid transporter-2 (EAAT2) is the major carrier responsible for clearing glutamate from the synaptic cleft in mammalian CNS. A localized attenuation of glutamate transport via reduced expression of functional forms of EAAT2 might contribute to regional excitotoxicity. The EAAT2 gene spans over 100 kb and encodes a 12-kb message. Several groups have identified alternative splice variants of EAAT2 in human brain tissue. These variants can affect transport by altering wild-type EAAT2 protein expression, localization, or transport efficiency. Alternative EAAT2 mRNA transcripts reportedly elicit a dominant-negative effect on glutamate uptake in cell culture. A 50% reduction in the expression in AD cortex of the truncated EAAT2 C-terminal isoform, EAAT2b, has been reported. We obtained cerebral cortex tissue, under informed written consent from the next of kin, from pathologically confirmed control, AD, and non-AD dementia cases. We aimed to determine the distribution and expression patterns of EAAT2 subtypes in susceptible and spared brain regions. We detected five alternate transcripts of EAAT2, two of which had not previously been reported. The relative contributions of novel variants, wild-type EAAT2, and previously discovered splice variants was investigated using Real-time PCR in AD, non-AD dementia, and age-matched control cortex. Our aim is to survey the relationship between these expression patterns and those of markers such as tau, GFAP, and b-amyloid, and to assess the correlation between variant-transporter expression and the level of cell loss.

Tumor-related splicing variant RAC1b in colorectal cancer: regulation and role in tumorigenesis

Purpose: Alternative splicing of the small GTPase RAC1 generates RAC1b, a hyperactivated variant that is overexpressed in a subtype of colorectal tumors. The objective of our studies is to understand the molecular regulation of this alternative splicing event and how it contributes to tumorigenesis. Experimental description: The regulation of the RAC1b splicing event in human colon cell lines was dissected using a transfected RAC1 minigene and the role of upstream regulating protein kinases through an RNA interference approach. The functional properties of the RAC1b protein were characterized by experimental modulation of Rac1b levels in colon cell lines. Results: The RAC1b protein results from an in-frame inclusion of an additional alternative exon encoding 19 amino acids that change the regulation and signaling properties of the protein. RAC1b is a hyperactive variant that exists predominantly in the GTP-bound active conformation in vivo and promotes cell cycle progression and cell survival through activation of the transcription factor NF-κB. RAC1b overexpression functionally cooperates with the oncogenic mutation in BRAF-V600E to sustain colorectal tumor cell survival. The splicing factor SRSF1 was identified to bind an exonic splice enhancer element in the alternative exon and acts as a prime regulator of Rac1b alternative splicing in colorectal cells. SRSF1 is controlled by upstream protein kinase SRPK1, the inhibition or depletion of which led to reduced SRSF1 phosphorylation and nuclear translocation with a concomitant reduction in RAC1b levels. As further SRSF1-regulating pathways we discovered kinase GSK3 and a cyclooxygenase independent effect of the non-steroidal anti-inflammatory drug ibuprofen. Conclusions: Expression of tumor-related RAC1b in colorectal cancer depends critically on SRSF1 for the observed deregulation of alternative splicing during tumorigenesis and is controlled by upstream protein kinases that can be pharmacologically targeted.

Tubular Variant Of Basal Cell Adenoma Shares Immunophenotypical Features With Normal Intercalated Ducts And Is Closely Related To Intercalated Duct Lesions Of Salivary Gland.

The morphological criteria for identification of intercalated duct lesions (IDLs) of salivary glands have been defined recently. It has been hypothesised that IDL could be a precursor of basal cell adenoma (BCA). BCAs show a variety of histological patterns, and the tubular variant is the one that presents the strongest resemblance with IDLs. The aim of this study was to analyse the morphological and immunohistochemical profiles of IDLs and BCAs classified into tubular and non-tubular subtypes, to determine whether or not IDL and tubular BCA represent distinct entities. Eight IDLs, nine tubular BCAs and 19 non-tubular BCAs were studied. All tubular BCAs contained IDL-like areas, which represented 20-70% of the tumour. In non-tubular BCA, IDL-like areas were occasional and small (<5%). One patient presented IDLs, tubular BCAs and IDL/tubular BCA combined lesions. Luminal ductal cells of IDLs and tubular BCAs exhibited positivity for CK7, lysozyme, S100 and DOG1. In the non-tubular BCA group, few luminal cells exhibited such an immunoprofile; they were mainly CK14-positive. Basal/myoepithelial cells of IDLs, tubular BCAs and non-tubular BCAs were positive for CK14, calponin, α-SMA and p63; they were more numerous in BCA lesions. IDL, tubular BCA and non-tubular BCA form a continuum of lesions in which IDLs are related closely to tubular BCA. In both, the immunoprofile of luminal and myoepithelial cells recapitulates the normal intercalated duct. The difference between the adenoma-like subset of IDLs and tubular BCA rests mainly on the larger numbers of myoepithelial cells in the latter. Our findings indicate that at least some BCAs can arise via IDLs.

Frequency Of The Allelic Variant C.1150t > C In Exon 10 Of The Fibroblast Growth Factor Receptor 3 (fgfr3) Gene Is Not Increased In Patients With Pathogenic Mutations And Related Chondrodysplasia Phenotypes.

Mutations in the FGFR3 gene cause the phenotypic spectrum of FGFR3 chondrodysplasias ranging from lethal forms to the milder phenotype seen in hypochondroplasia (Hch). The p.N540K mutation in the FGFR3 gene occurs in ∼70% of individuals with Hch, and nearly 30% of individuals with the Hch phenotype have no mutations in the FGFR3, which suggests genetic heterogeneity. The identification of a severe case of Hch associated with the typical mutation c.1620C > A and the occurrence of a c.1150T > C change that resulted in a p.F384L in exon 10, together with the suspicion that this second change could be a modulator of the phenotype, prompted us to investigate this hypothesis in a cohort of patients. An analysis of 48 patients with FGFR3 chondrodysplasia phenotypes and 330 healthy (control) individuals revealed no significant difference in the frequency of the C allele at the c.1150 position (p = 0.34). One patient carrying the combination `pathogenic mutation plus the allelic variant c.1150T > C' had a typical achondroplasia (Ach) phenotype. In addition, three other patients with atypical phenotypes showed no association with the allelic variant. Together, these results do not support the hypothesis of a modulatory role for the c.1150T > C change in the FGFR3 gene.

Variant Rh Alleles And Rh Immunisation In Patients With Sickle Cell Disease.

Alloimmunisation is a major complication in patients with sickle cell disease (SCD) receiving red blood cell (RBC) transfusions and despite provision of Rh phenotyped RBC units, Rh antibodies still occur. These antibodies in patients positive for the corresponding Rh antigen are considered autoantibodies in many cases but variant RH alleles found in SCD patients can also contribute to Rh alloimmunisation. In this study, we characterised variant RH alleles in 31 SCD patients who made antibodies to Rh antigens despite antigen-positive status and evaluated the clinical significance of the antibodies produced. RHD and RHCE BeadChip™ from BioArray Solutions and/or amplification and sequencing of exons were used to identify the RH variants. The serological features of all Rh antibodies in antigen-positive patients were analysed and the clinical significance of the antibodies was evaluated by retrospective analysis of the haemoglobin (Hb) levels before and after transfusion; the change from baseline pre-transfusion Hb and the percentage of HbS were also determined. We identified variant RH alleles in 31/48 (65%) of SCD patients with Rh antibodies. Molecular analyses revealed the presence of partial RHD alleles and variant RHCE alleles associated with altered C and e antigens. Five patients were compound heterozygotes for RHD and RHCE variants. Retrospective analysis showed that 42% of antibodies produced by the patients with RH variants were involved in delayed haemolytic transfusion reactions or decreased survival of transfused RBC. In this study, we found that Rh antibodies in SCD patients with RH variants can be clinically significant and, therefore, matching patients based on RH variants should be considered.

Pharmacogenetics of OATP Transporters Reveals That SLCO1B1 c.388A > G Variant Is Determinant of Increased Atorvastatin Response

Aims: The relationship between variants in SLCO1B1 and SLCO2B1 genes and lipid-lowering response to atorvastatin was investigated. Material and Methods: One-hundred-thirty-six unrelated individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). They were genotyped with a panel of ancestry informative markers for individual African component of ancestry (ACA) estimation by SNaPshot (R) and SLCO1B1 (c.388A>G, c.463C>A and c.521T>C) and SLCO2B1 (-71T>C) gene polymorphisms were identified by TaqMan (R) Real-time PCR. Results: Subjects carrying SLCO1B1 c.388GG genotype exhibited significantly high low-density lipoprotein (LDL) cholesterol reduction relative to c.388AA+c.388AG carriers (41 vs. 37%, p = 0.034). Haplotype analysis revealed that homozygous of SLCO1B1*15 (c.521C and c.388G) variant had similar response to statin relative to heterozygous and non-carriers. A multivariate logistic regression analysis confirmed that c.388GG genotype was associated with higher LDL cholesterol reduction in the study population (OR: 3.2, CI95%: 1.3-8.0, p < 0.05). Conclusion: SLCO1B1 c.388A>G polymorphism causes significant increase in atorvastatin response and may be an important marker for predicting efficacy of lipid-lowering therapy.

ABO genotyping in leukemia patients reveals new ABO variant alleles

The ABO blood group is the most important blood group system in transfusion medicine and organ transplantation. To date, more than 160 ABO alleles have been identified by molecular investigation. Almost all ABO genotyping studies have been performed in blood donors and families and for investigation of ABO subgroups detected serologically. The aim of the present study was to perform ABO genotyping in patients with leukemia. Blood samples were collected from 108 Brazilian patients with chronic myeloid leukemia (N = 69), chronic lymphoid leukemia (N = 13), acute myeloid leukemia (N = 15), and acute lymphoid leukemia (N = 11). ABO genotyping was carried out using allele specific primer polymerase chain reaction followed by DNA sequencing. ABO*001 was the most common allele found, followed by ABO*022 and by ABO*A103. We identified 22 new ABO*(variants) in the coding region of the ABO gene in 25 individuals with leukemia (23.2%). The majority of ABO variants was detected in O alleles (15/60.0%). In 5 of 51 samples typed as blood group O (9.8%), we found non-deletional ABO*O alleles. Elucidation of the diversity of this gene in leukemia and in other diseases is important for the determination of the effect of changes in an amino acid residue on the specificity and activity of ABO glycosyltransferases and their function. In conclusion, this is the first report of a large number of patients with leukemia genotyped for ABO. The findings of this study indicate that there is a high level of recombinant activity in the ABO gene in leukemia patients, revealing new ABO variants.

TCF7L2 variant genotypes and type 2 diabetes risk in Brazil: significant association, but not a significant tool for risk stratification in the general population

Background: Genetic polymorphisms of the TCF7L2 gene are strongly associated with large increments in type 2 diabetes risk in different populations worldwide. In this study, we aimed to confirm the effect of the TCF7L2 polymorphism rs7903146 on diabetes risk in a Brazilian population and to assess the use of this genetic marker in improving diabetes risk prediction in the general population. Methods: We genotyped the single nucleotide polymorphisms (SNP) rs7903146 of the TCF7L2 gene in 560 patients with known coronary disease enrolled in the MASS II (Medicine, Angioplasty, or Surgery Study) Trial and in 1,449 residents of Vitoria, in Southeast Brazil. The associations of this gene variant to diabetes risk and metabolic characteristics in these two different populations were analyzed. To access the potential benefit of using this marker for diabetes risk prediction in the general population we analyzed the impact of this genetic variant on a validated diabetes risk prediction tool based on clinical characteristics developed for the Brazilian general population. Results: SNP rs7903146 of the TCF7L2 gene was significantly associated with type 2 diabetes in the MASS-II population (OR = 1.57 per T allele, p = 0.0032), confirming, in the Brazilian population, previous reports of the literature. Addition of this polymorphism to an established clinical risk prediction score did not increased model accuracy (both area under ROC curve equal to 0.776). Conclusion: TCF7L2 rs7903146 T allele is associated with a 1.57 increased risk for type 2 diabetes in a Brazilian cohort of patients with known coronary heart disease. However, the inclusion of this polymorphism in a risk prediction tool developed for the general population resulted in no improvement of performance. This is the first study, to our knowledge, that has confirmed this recent association in a South American population and adds to the great consistency of this finding in studies around the world. Finally, confirming the biological association of a genetic marker does not guarantee improvement on already established screening tools based solely on demographic variables.

EVOLUTION OF DISPARITY BETWEEN THE REGULAR AND VARIANT PHLOEM IN BIGNONIEAE (BIGNONIACEAE)

Premise of the study: The phloem is a plant tissue with a critical role in plant nutrition and signaling. However, little is still known about the evolution of this tissue. In lianas of the Bignoniaceae, two distinct types of phloem coexist: a regular and a variant phloem. The cells associated with these two phloem types are known to be anatomically different; however, it is still unclear what steps were involved in the evolution of such differences. Methods: Here we studied the anatomical development of the regular and variant phloem in representatives of all 21 genera of Bignonieae and used a phylogenetic framework to investigate the timing of changes associated with the evolution of each phloem type. Key results: We found that the variant phloem always appears in a determinate location, between the leaf orthostichies. Furthermore, the variant phloem was mostly occupied by very wide sieve tubes and generally included a higher concentration of fibers, indicating an increase in conduction and mechanical support. On the other hand, the regular phloem included much more parenchyma, more and wider rays, and tiny sieve tubes that resembled terminal sieve tubes from plants with seasonal formation of vascular tissues; these findings suggest reduced conduction and higher storage capacity in the regular phloem. Conclusions: Overall, differences between the regular and variant phloem increased over time, leading to further specialization in conduction in the variant phloem and an increase in storage specialization in the regular phloem.

LATENT RANK CHANGE DETECTION FOR ANALYSIS OF SPLICE-JUNCTION MICROARRAYS WITH NONLINEAR EFFECTS

Alternative splicing of gene transcripts greatly expands the functional capacity of the genome, and certain splice isoforms may indicate specific disease states such as cancer. Splice junction microarrays interrogate thousands of splice junctions, but data analysis is difficult and error prone because of the increased complexity compared to differential gene expression analysis. We present Rank Change Detection (RCD) as a method to identify differential splicing events based upon a straightforward probabilistic model comparing the over-or underrepresentation of two or more competing isoforms. RCD has advantages over commonly used methods because it is robust to false positive errors due to nonlinear trends in microarray measurements. Further, RCD does not depend on prior knowledge of splice isoforms, yet it takes advantage of the inherent structure of mutually exclusive junctions, and it is conceptually generalizable to other types of splicing arrays or RNA-Seq. RCD specifically identifies the biologically important cases when a splice junction becomes more or less prevalent compared to other mutually exclusive junctions. The example data is from different cell lines of glioblastoma tumors assayed with Agilent microarrays.