Exon 1B Research Articles

The Roles of RUNX1 in Human Hematopoiesis and Megakaryopoiesis Revealed By Genome-Targeted Human iPSCs and an Improved Hematopoietic Differentiation Model

The RUNX1 gene (also called AML1), one of the most mutated genes in acute myeloid leukemia (AML), was first identified decades ago that encodes a key regulatory transcriptional factor. Numerous studies using mouse and zebrafish models show that RUNX1 is essential for definitive hematopoiesis. In mice, its homozygous knock-out (KO) in hematopoietic stem/progenitor cells also causes defects in lymphoid and megakaryocytic (MK) development. However, heterozygous Runx1 gene mutations in laboratory mouse and zebrafish had little effects on development of hematopoietic stem/progenitor cells (HSPCs) or the MK cell lineage. In contrast, heterozygous germline mutations in RUNX1 were found in patients with familial platelet disorder (FPD) with predisposition to AML and MDS. The mechanisms underlying the observed differences between humans and small animal models remain unclear.In the past decade, we and others have utilized human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to investigate human hematopoiesis including the roles of the RUNX1 gene. Using a feeder-free culture system, we generated human CD34+CD45+ HSPCs cells from human ESCs and iPSCs around 11-14 days after embryoid body (EB) formation. The CD34+CD45+ HSPCs were capable to form multiple types of hematopoietic cells such as myeloid, erythroid, and polyploid MK cells (Connelly et al., 2014; Liu et al., 2015). We also reported that human iPSCs derived from FPD patients containing a heterozygous RUNX1 mutation were defective in MK formation, and targeted correction of the mutated RUNX1 allele by genome editing restored the MK potential (Connelly et al., 2014).Since then, we have extended our studies by precise genomic targeting in human wildtype iPSCs to ablate exon 5 that is common in all 3 isoforms of the RUNX1 gene, or exon 1B that is unique to the RUNX1c isoform. Bi-allelic KO of RUNX1 at exon 5 completely abolished the formation of hematopoietic cells at days 11-14 after EB formation. Complete disruption of exon 1B showed little effect, indicating that the RUNX1c isoform is dispensable for definitive hematopoiesis in the presence of the RUNX1a and RUNX1b isoforms transcribed from the downstream P2 promoter. Detailed analysis of EBs at days 6-8 revealed that bi-allelic RUNX1 KO at exon 5 (ablating all 3 isoforms) did not affect the formation of CD34+/CD31+/CD144+ endothelial-like cells. However, the endothelial-to-hematopoietic transition (EHT) was completely blocked and no CD45+ hematopoietic cells emerged from the EHT culture supplemented with hematopoietic cytokines.To elucidate the functions of different RUNX1 isoforms in early steps of human hematopoiesis, we adapted the EHT culture system that uses CD34+ cells isolated from earlier stages of EB formation (day 6-8), before definite hematopoiesis was observable (after day 8, Bai et al., 2015). Two different iPSCs clones with homozygous or bi-allelic RUNX1 KO at exon 5 both failed to form CD45+ hematopoietic cells after EHT culture. Because constitutive transgene expression of RUNX1b or RUNX1c (but not RUNX1a) cDNA in human iPSCs inhibits hematopoietic differentiation, we transduced the day 6 EB cells at the beginning of the EHT culture with RUNX1-expressing vectors. Lentiviral vectors constitutively express RUNX1b or RUNX1c (but not RUNX1a) cDNA partially restored the EHT and hematopoietic (CD45+) cell formation after 4-5 days in EHT culture. We further used a lentiviral vector in which RUNX1c (as an ER fusion protein) can be conditionally activated by 4-HT induction. Induction starting at day 4 and lasting for 3 days rendered the maximal effect of hematopoietic cell formation in the EHT culture using CD34+ cells isolated at day 6 of EB formation.Our data corroborate with limited in vivo data using human fetal tissues on the possible roles of RUNX1 in definitive hematopoiesis. At present we are analyzing iPSC clones with mono-allelic disruption of exon 5 in a wild type iPSC line, comparing to iPSCs derived from FPD patients. The current study of using isogenic human iPSCs will help to understand the roles of RUNX1 in human hematopoiesis and megakaryopoiesis, and offer an amenable system to study the RUNX1 gene functions and downstream target genes. DisclosuresNo relevant conflicts of interest to declare.

TDP-43 functions within a network of hnRNP proteins to inhibit the production of a truncated human SORT1 receptor.

The aggregation and mislocalization of RNA-binding proteins leads to the aberrant regulation of RNA metabolism and is a key feature of many neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. However, the pathological consequences of abnormal deposition of TDP-43 and other RNA-binding proteins remain unclear, as the specific molecular events that drive neurodegeneration have been difficult to identify and continue to be elusive. Here, we provide novel insight into the complexity of the RNA-binding protein network by demonstrating that the inclusion of exon 17b in the SORT1 mRNA, a pathologically relevant splicing event known to be regulated by TDP-43, is also considerably affected by additional RNA-binding proteins, such as hnRNP L, PTB/nPTB and hnRNP A1/A2. Most importantly, the expression of hnRNP A1/A2 and PTB/nPTB is significantly altered in patients with frontotemporal dementia with TDP-43-positive inclusions (FTLD-TDP), indicating that perturbations in RNA metabolism and processing in FTLD-TDP are not exclusively driven by a loss of TDP-43 function. These results also suggest that a comprehensive assessment of the RNA-binding protein network will dramatically advance our current understanding of the role of TDP-43 in disease pathogenesis, as well as enhance both diagnostic and therapeutic capabilities.

Reduced cortical expression of a newly identified splicing variant of the DLG1 gene in patients with early-onset schizophrenia.

The human discs, large homolog 1 gene (DLG1) is mapped to the schizophrenia-susceptibility locus 3q29, and it encodes a scaffold protein that interacts with the N-methyl-D-aspartate receptor presumably dysregulated in schizophrenia. In the current study, we have newly identified a splicing variant of DLG1, which is transcribed from an unreported 95-base-pair exon (exon 3b) and is labeled 3b(+). We investigated the mRNA expression of 3b(+) in the post-mortem dorsolateral prefrontal cortices of patients with psychiatric disorders, obtained from The Stanley Medical Research Institute, and examined the potential association of the expression with the genotype of the single-nucleotide polymorphism (SNP) rs3915512 located within exon 3b. A real-time quantitative reverse transcriptase-polymerase chain reaction revealed that the mRNA levels of 3b(+) were significantly reduced in patients with early-onset schizophrenia (onset at <18 years old, P=0.0003) but not in those with non-early-onset schizophrenia, early-onset or non-early-onset bipolar disorder or in the controls. Furthermore, the genotype at the rs3915512 SNP was closely associated with the levels of 3b(+) mRNA expression. It is inferred that the T allele fails to meet the exonic splicing enhancer consensus, thus resulting in skipping of exon 3b, leading to the expression of 3b(−) (the previously known DLG1 variant) but not 3b(+). Because all the subjects with early-onset schizophrenia in the current study possess the T/T genotype, the reduced level of the DLG1 3b(+) transcript may be involved in the susceptibility and/or pathophysiology of early-onset schizophrenia.

Abstract P061: Selective Deletion of Intracellular Renin in the Brain Causes Hypertension and Elevated Sympathetic Nervous System Activity

Renin expression is regulated by two distinct promoter-1st exon combinations that target renin either for secretion (exon 1a for secreted renin) or cytoplasmic retention (exon 1b for intracellular renin, icREN). We developed icREN knockout (KO) mice by selectively deleting exon 1b. icREN KO mice are essentially brain-specific knockouts of icREN because icREN is predominantly expressed in the brain. Notably, systolic blood pressure measured by telemetry was increased in icREN KO mice (130±2 mmHg, n=8 vs 122±2 mmHg in controls, n=7, P&lt;0.01). The low- to high-frequency ratio (LF/HF) derived from power spectral analysis of heart rate variability, a parameter of sympathetic nerve activity (SNA), was increased in icREN KO mice (KO: 1.24±0.21, n=7 vs control: 0.70±0.11, n=7, P&lt;0.05). Body weight (BW) was normal in icREN KO mice compared to controls, but the BW gain and fat accumulation induced by high fat diet (HFD) were attenuated in male icREN KO mice (BW at 16 wks of HFD- KO: 36.8±1.2 g, n=8 vs control: 41.9±1.4 g, n=9; relative fat mass at 14 wks of HFD- KO: 27.7±1.7%, n=8 vs control: 34.4±2.3%, n=9, both P&lt;0.05). The resting metabolic rate measured by respirometry was increased in icREN KO mice (0.156±0.005 kcal/h, n=46, P&lt;0.05) vs controls (0.145±0.003 kcal/h, n=53), whereas food consumption and absorbed calories were not different. We previously reported that the brain renin-angiotensin system facilitates renal SNA (RSNA) response to acute intracerebroventricular (ICV) injection of leptin. Interestingly, the RSNA response to ICV leptin was greater in icREN KO mice (KO: 214±40 % baseline, n=5 vs control: 114±18 % baseline, n=10, P&lt;0.01). AT1a receptor mRNA was upregulated in the paraventricular nucleus of icREN KO mice (P&lt;0.05). Chronic ICV injection of losartan not only abolished the elevated blood pressure in icREN KO mice, but reduced it to below baseline in controls (systolic blood pressure, 111±3 mmHg in KO, n=5; 124±4 mmHg in controls, n=6). These data suggest that icREN deletion increases the activity of brain renin-angiotensin system and elevates blood pressure and metabolic rate through sympathetic activation. We conclude that this novel icREN isoform contributes to cardiovascular and metabolic control possibly as part of an inhibitory neural circuit.

The RNA-binding protein Sam68 regulates expression and transcription function of the androgen receptor splice variant AR-V7.

Castration-resistant (CR) prostate cancer (PCa) partly arises due to persistence of androgen receptor (AR) transcriptional activity in the absence of cognate ligand. An emerging mechanism underlying the CRPCa phenotype and predicting response to therapy is the expression of the constitutively-active AR-V7 splice variant generated by AR cryptic exon 3b inclusion. Here, we explore the role of the RNA-binding protein (RBP) Sam68 (encoded by KHDRBS1), which is over-expressed in clinical PCa, on AR-V7 expression and transcription function. Using a minigene reporter, we show that Sam68 controls expression of exon 3b resulting in an increase in endogenous AR-V7 mRNA and protein expression in RNA-binding-dependent manner. We identify a novel protein-protein interaction between Sam68 and AR-V7 mediated by a common domain shared with full-length AR, and observe these proteins in the cell nucleoplasm. Using a luciferase reporter, we demonstrate that Sam68 co-activates ligand-independent AR-V7 transcriptional activity in an RNA-binding-independent manner, and controls expression of the endogenous AR-V7-specific gene target UBE2C. Our data suggest that Sam68 has separable effects on the regulation of AR-V7 expression and transcriptional activity, through its RNA-binding capacity. Sam68 and other RBPs may control expression of AR-V7 and other splice variants as well as their downstream functions in CRPCa.

Novel FGFR1 mutations in Kallmann syndrome and normosmic idiopathic hypogonadotropic hypogonadism: evidence for the involvement of an alternatively spliced isoform

To determine the prevalence of fibroblast growth factor receptor 1 (FGFR1) mutations and their predicted functional consequences in patients with idiopathic hypogonadotropic hypogonadism (IHH). Cross-sectional study. Multicentric. Fifty unrelated patients with IHH (21 with Kallmann syndrome and 29 with normosmic IHH). None. Patients were screened for mutations in FGFR1. The functional consequences of mutations were predicted by in silico structural and conservation analysis. Heterozygous FGFR1 mutations were identified in six (12%) kindreds. These consisted of frameshift mutations (p.Pro33-Alafs*17 and p.Tyr654*) and missense mutations in the signal peptide (p.Trp4Cys), in the D1 extracellular domain (p.Ser96Cys) and in the cytoplasmic tyrosine kinase domain (p.Met719Val). A missense mutation was identified in the alternatively spliced exon 8A (p.Ala353Thr) that exclusively affects the D3 extracellular domain of FGFR1 isoform IIIb. Structure-based and sequence-based prediction methods and the absence of these variants in 200 normal controls were all consistent with a critical role for the mutations in the activity of the receptor. Oligogenic inheritance (FGFR1/CHD7/PROKR2) was found in one patient. Two FGFR1 isoforms, IIIb and IIIc, result from alternative splicing of exons 8A and 8B, respectively. Loss-of-function of isoform IIIc is a cause of IHH, whereas isoform IIIb is thought to be redundant. Ours is the first report of normosmic IHH associated with a mutation in the alternatively spliced exon 8A and suggests that this disorder can be caused by defects in either of the two alternatively spliced FGFR1 isoforms.

A novel GYPB-A-B hybrid gene responsible for Ss and MN typing discrepancies.

Transfusion support of S-s- patients is very challenging but can now be alleviated by genotyping mutations in the GYPB gene to predict their U- or U+(var) phenotype. However, the phenotype predicted by genotyping does not always correspond to the observed phenotype of red blood cells (RBCs), which requires further investigation to avoid such a typing discrepancy in the future. In this study, we elucidated the case of an S-s- female patient of African origin who was predicted to be S+s- by our genotyping platform. Long-range polymerase chain reaction (PCR) amplification and extended Sanger sequencing were required. The Ss typing discrepancy in the proband resulted from a converted GYPB allele that encodes neither S nor s due to the replacement of Exon B4 of GYPB by the homologous Exon A4 of GYPA. In this novel GYPB-A-B hybrid gene, the GYPA segment actually starts just downstream of Exon B2, causing a MN typing discrepancy too. While the proband's RBCs were M+N-, the genotyping predicted the M+N+ phenotype. The reported GYPB-A-B hybrid gene constitutes a limitation for the accurate prediction of the MN and Ss phenotypes by current genotyping methods. A PCR assay was therefore developed to detect its presence.

Three promoters with different tissue specificity and pathogen inducibility express the toll-like-receptor 2 (TLR2)-encoding gene in cattle

Toll-like-receptor 2 (TLR2) is a dominant receptor for perceiving presence of bacterial pathogens. The promoter controlling its tissue specific and infection induced expression in cattle was unknown. We structurally defined with 5′-RACE experiments three promoters (P1–3) controlling TLR2 expression in udder, liver and other tissues of cows suffering from E. coli mastitis. P1 is 5′-adjacent to exon 1 as defined by the prototypical TLR2 cDNA sequence. Exon 1 is spliced to the protein-encoding exon 2. P2 and P3 reside in intron 1, express exon 1A and exon 1B, respectively which are each spliced to exon 2. Infection induced massively (>30-fold) activity of P1 and P2, but not of P3 in udders and also somewhat in liver. However, the GC-rich housekeeping promoter P3 expressed exon1B in many tissues providing the wealth of TLR2-encoding transcripts. Similar induction data were obtained after challenging primary cultures of mammary epithelial cells (pbMEC) with E. coli. Reporter gene analyses in pbMEC and the liver cell line HepG2 collectively validated that P1 and constructs containing segments from P2/P3 are in principle capable to drive gene expression. Our structural data provide the basis for more detailed molecular analyses of the infection and tissue specific regulation of TLR2 expression.

ROLE OF BREAK CLUSTER REGION (BCR) - ABELSON MURINE LEUKIMIA (ABL) EXAMINATION IN CHRONIC MYELOGENOUS LEUKEMIA (CML)

Chronic myelogenous leukemia (CML) is a clonal bone marrow stem cell disorder associated with a characteristic chromosomal translocation called the Philadelphia chromosome which caused a proliferation of mature granulocytes (neutrophils, eosinophils and basophils) and their precursors, increasing unregulated growth of predominantly myeloid cells in the bone marrow and its accumulation in the blood. As myeloproliferative disease, Chronic Myelogenous Leukemia or CML is a malignancy of the sixth-highest, reaching 15% of all blood malignancies in adults with an incidence of 1.1 per 100,000 population (Ugroseno, 2012). The CML diagnosis is made based on a presence of Philadelphia chromosome due to the existence of a reciprocal translocation of chromosomes 9 and chromosome 22 t (9.22), and raises the fusion of Break Cluster Region (BCR) gene of chromosome 22 on band q11 by Abelson Murine Leukemia (ABL). The fused BCR-ABL gene has BCR sequences of different length, so it produces a protein that has a different molecular weight. Despite having different length of BCR sequences, however, the length of fuses ABL gene sequence is constant. Associated with this different BCR sequence length are the three variations of the BCR-ABL gene fusion. The first variation is a Major Break Cluster (M-BCR), the BCR gene break is found in exon 2 in e13-E14 region. This type of CML is the fusion of BCR exon b2 or b3 to ABL exon a2, forming two major transcripts of the b2a2 or b3a2, which has a protein product with 210 kD weight or referred to as p210. The second variation is Minor BCR (m-BCR), which has e1a2 fusion. CML with BCR-ABL gene fusion of this type has a protein product with a molecular weight of 190 kDa or called p190. The third variation is micro-BCR (m-BCR), with BCR gene break between exons E19 and e20b that form mRNA transcripts e19a2, with BCR-ABL protein P230. This fused gene can be detected with qualitative multiplex PCR.

Alternative transcripts of the SERPINA1 gene in alpha-1 antitrypsin deficiency.

BackgroundSERPINA1 is the gene for alpha-1 antitrypsin (AAT), an acute phase protein with anti-protease and immunoregulatory activities. Mutations in SERPINA1 gene cause AAT deficiency and predispose individuals to early-onset emphysema and liver diseases. Expression of the SERPINA1 gene is regulated by different promoters and alternative splicing events among non-coding exons 1A, 1B and 1C.MethodsWe have developed three quantitative PCR (QT-PCR) assays (1A, 1B and 1C). These assays were applied for the analysis of SERPINA1 alternative transcripts in: (1) 16 human tissues and (2) peripheral blood leukocytes from 33 subjects with AAT mutations and 7 controls.ResultsTissue-specific expression was found for the SERPINA1 transcripts. The 1A transcripts were mainly expressed in leukocytes and lung tissue while those detected with the 1B assay were highly restricted to leukocytes. Only 1B transcripts significantly correlated with serum AAT levels. The 1C transcripts were specifically found in lung, liver, kidney and pancreas. Furthermore, the expression of transcripts was related to AAT genotypes. While deficient variants of AAT had no pronounced effect on the transcript expression, null alleles were associated with significant reduction of different transcripts.ConclusionsThe possibility to discriminate between SERPINA1 alternative splicing products will help us to understand better the regulation of SERPINA1 gene and its association with SERPINA1 mutations-related diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0585-y) contains supplementary material, which is available to authorized users.

Characterization of the doublesex gene within the Culex pipiens complex suggests regulatory plasticity at the base of the mosquito sex determination cascade.

BackgroundThe doublesex gene controls somatic sexual differentiation of many metazoan species, including the malaria mosquito Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti (Diptera: Culicidae). As in other studied dipteran dsx homologs, the gene maintains functionality via evolutionarily conserved protein domains and sex-specific alternative splicing. The upstream factors that regulate splicing of dsx and the manner in which they do so however remain variable even among closely related organisms. As the induction of sex ratio biases is a central mode of action in many emerging molecular insecticides, it is imperative to elucidate as much of the sex determination pathway as possible in the mosquito disease vectors.ResultsHere we report the full-length gene sequence of the doublesex gene in Culex quinquefasciatus (Cxqdsx) and its male and female-specific isoforms. Cxqdsx maintains characteristics possibly derived in the Culicinae and present in the Aedes aegypti dsx gene (Aeadsx) such as gain of exon 3b and the presence of Rbp1 cis-regulatory binding sites, and also retains presumably ancestral attributes present in Anopheles gambiae such as maintenance of a singular female-specific exon 5. Unlike in Aedes aegypti, we find no evidence for intron gain in the female transcript(s), yet recover a second female isoform generated via selection of an alternate splice donor. Utilizing next-gen sequence (NGS) data, we complete the Aeadsx gene model and identify a putative core promoter region in both Aeadsx and Cxqdsx. Also utilizing NGS data, we construct a full-length gene sequence for the dsx homolog of the northern house mosquito Culex pipiens form pipiens (Cxpipdsx). Analysis of peptide evolutionary rates between Cxqdsx and Cxpipdsx (both members of the Culex pipiens complex) shows the male-specific portion of the transcript to have evolved rapidly with respect to female-specific and common regions.ConclusionsAs in other studied insects, doublesex maintains sex-specific splicing and conserved doublesex/mab-3 domains in the mosquitoes Culex quinquefasciatus and Cx. pipiens. The cis-regulated splicing of Cxqdsx does not appear to follow either currently described mosquito model (for An. gambiae and Ae. aegypti); each of the three mosquito genera exhibit evidence of unique cis-regulatory mechanisms. The male-specific dsx terminus exhibits rapid peptide evolutionary rates, even among closely related sibling species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0386-1) contains supplementary material, which is available to authorized users.

Rare pseudoautosomal copy-number variations involving SHOX and/or its flanking regions in individuals with and without short stature.

Pseudoautosomal region 1 (PAR1) contains SHOX, in addition to seven highly conserved non-coding DNA elements (CNEs) with cis-regulatory activity. Microdeletions involving SHOX exons 1-6a and/or the CNEs result in idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). Here, we report six rare copy-number variations (CNVs) in PAR1 identified through copy-number analyzes of 245 ISS/LWD patients and 15 unaffected individuals. The six CNVs consisted of three microduplications encompassing SHOX and some of the CNEs, two microduplications in the SHOX 3'-region affecting one or four of the downstream CNEs, and a microdeletion involving SHOX exon 6b and its neighboring CNE. The amplified DNA fragments of two SHOX-containing duplications were detected at chromosomal regions adjacent to the original positions. The breakpoints of a SHOX-containing duplication resided within Alu repeats. A microduplication encompassing four downstream CNEs was identified in an unaffected father-daughter pair, whereas the other five CNVs were detected in ISS patients. These results suggest that microduplications involving SHOX cause ISS by disrupting the cis-regulatory machinery of this gene and that at least some of microduplications in PAR1 arise from Alu-mediated non-allelic homologous recombination. The pathogenicity of other rare PAR1-linked CNVs, such as CNE-containing microduplications and exon 6b-flanking microdeletions, merits further investigation.

TheCYP2C19Intron 2 Branch Point SNP is the Ancestral Polymorphism Contributing to the Poor Metabolizer Phenotype in Livers withCYP2C19*35andCYP2C19*2Alleles

CYP2C19 rs12769205 alters an intron 2 branch point adenine leading to an alternative mRNA in human liver with complete inclusion of intron 2 (exon 2B). rs12769205 changes the mRNA reading frame, introduces 87 amino acids, and leads to a premature stop codon. The 1000 Genomes project (http://browser.1000genomes.org/index.html) indicated rs12769205 is in linkage disequilibrium with rs4244285 on CYP2C19*2, but found alone on CYP2C19*35 in Blacks. Minigenes containing rs12769205 transfected into HepG2 cells demonstrated this single nucleotide polymorphism (SNP) alone leads to exon 2B and decreases CYP2C19 canonical mRNA. A residual amount of CYP2C19 protein was detectable by quantitative proteomics with tandem mass spectrometry in CYP2C19*2/*2 and *1/*35 liver microsomes with an exon 2 probe. However, an exon 4 probe, downstream from rs12769205, but upstream of rs4244285, failed to detect CYP2C19 protein in livers homozygous for rs12769205, demonstrating rs12769205 alone can lead to complete loss of CYP2C19 protein. CYP2C19 genotypes and mephenytoin phenotype were compared in 104 Ethiopians. Poor metabolism of mephenytoin was seen in persons homozygous for both rs12769205 and rs4244285 (CYP2C19*2/*2), but with little effect on mephenytoin disposition of CYP2C19*1/*2, CYP2C19*1/*3, or CYP2C19*1/*35 heterozygous alleles. Extended haplotype homozygosity tests of the HapMap Yorubans (YRI) showed both haplotypes carrying rs12769205 (CYP2C19*35 and CYP2C19*2) are under significant natural selection, with CYP2C19*35 having a higher relative extended haplotype homozygosity score. The phylogenetic tree of the YRI CYP2C19 haplotypes revealed rs12769205 arose first on CYP2C19*35 and that rs4244285 was added later, creating CYP2C19*2. In conclusion, rs12769205 is the ancestral polymorphism leading to aberrant splicing of CYP2C19*35 and CYP2C19*2 alleles in liver.

Two polymorphisms in the Cx40 promoter are associated with hypertension and left ventricular hypertrophy preferentially in men

Background: Lack of connexin40, a gap junction protein expressed in endothelial and renin-producing cells, results in hypertension and cardiac hypertrophy in mice due to unleashed renin production caused by disruption of the pressure-induced feedback inhibition. We analysed human GJA5 consisting of two exons (exon1A or 1B and exon2) in a selected cohort identified by a single nucleotide polymorphism (SNP) in the GJA5 intron for polymorphisms and putative association with hypertension and left ventricular hypertrophy (LVH). Methods: Individuals carrying a SNP in the intron of GJA5 (rs791295) were selected from the MONICA/KORA cohort (n = 1677) and searched for GJA5 polymorphisms. We accessed DNA of 178 probands, of which 26 suffered from LVH, 112 were hypertensive and 29 normotensive (unknown: 11). Results: Sequencing of the GJA5 coding region did not reveal alterations suggesting the expression of functional connexin40 in all probands. Sequencing of the upstream region of transcript 1A including exon1A revealed two previously described linked SNPs (rs35594137 −44G>A; rs11552588 + 71A>G) at an increased frequency. Moreover, the rare genotype was significantly associated with hypertension and LVH with a preponderance in men. Functional analysis in a reporter gene assay verified promoter activity, however, it was unchanged by the identified SNPs after expressing respective reporter constructs in HeLa and human endothelial cells. Conclusion: We suggest to consider the −44G>A SNP upstream of the connexin40 transcript 1A indeed as a risk factor for hypertension in men. However, the underlying mechanisms remain unclear but animal data suggest that renin-producing cells may be involved and contribute to hypertension.

Novel APC promoter and exon 1B deletion and allelic silencing in three mutation-negative classic familial adenomatous polyposis families.

BackgroundThe overwhelming majority (approximately 80%) of individuals with classic familial adenomatous polyposis (FAP) exhibit mutations in the coding sequence of the adenomatous polyposis coli (APC) tumor suppressor gene. Families without detectable APC mutations are unable to benefit from the use of genetic testing for clinical management of this autosomal dominant syndrome.MethodsWe used exome sequencing and linkage analysis, coupled with second-generation sequencing of the APC locus including non-coding regions to investigate three APC mutation-negative classical FAP families.ResultsWe identified a novel ~11 kb deletion localized 44 kb upstream of the transcription start site of APC that encompasses the APC 1B promoter and exon. This deletion was present only in affected family members of one kindred with classical FAP. Furthermore, this same deletion with identical breakpoints was found in the probands of two additional APC mutation-negative classical FAP kindreds. Phasing analysis of single nucleotide polymorphisms (SNPs) around the deletion site in the three probands showed evidence of a shared haplotype, suggesting a common founder deletion in the three kindreds. SNP analysis within the coding sequence of APC, revealed that this ~11 kb deletion was accompanied by silencing of one of the APC alleles in blood-derived RNA of affected individuals.ConclusionsThese results support the causal role of a novel promoter deletion in FAP and suggest that non-coding deletions, identifiable using second-generation sequencing methods, may account for a significant fraction of APC mutation-negative classical FAP families.

Identification of Novel Crk-associated Substrate (p130Cas) Variants with Functionally Distinct Focal Adhesion Kinase Binding Activities

Elevated levels of p130(Cas) (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1 gene) are associated with aggressiveness of breast tumors. Following phosphorylation of its substrate domain, p130(Cas) promotes the integration of protein complexes involved in multiple signaling pathways and mediates cell proliferation, adhesion, and migration. In addition to the known BCAR1-1A (wild-type) and 1C variants, we identified four novel BCAR1 mRNA variants, generated by alternative first exon usage (1B, 1B1, 1D, and 1E). Exons 1A and 1C encode for four amino acids (aa), whereas 1D and 1E encode for 22 aa and 1B1 encodes for 50 aa. Exon 1B is non-coding, resulting in a truncated p130(Cas) protein (Cas1B). BCAR1-1A, 1B1, and variant 1C mRNAs were ubiquitously expressed in cell lines and a survey of human tissues, whereas 1B, 1D, and 1E expression was more restricted. Reconstitution of all isoforms except for 1B in p130(Cas)-deficient murine fibroblasts induced lamellipodia formation and membrane ruffling, which was unrelated to the substrate domain phosphorylation status. The longer isoforms exhibited increased binding to focal adhesion kinase (FAK), a molecule important for migration and adhesion. The shorter 1B isoform exhibited diminished FAK binding activity and significantly reduced migration and invasion. In contrast, the longest variant 1B1 established the most efficient FAK binding and greatly enhanced migration. Our results indicate that the p130(Cas) exon 1 variants display altered functional properties. The truncated variant 1B and the longer isoform 1B1 may contribute to the diverse effects of p130(Cas) on cell biology and therefore will be the target of future studies.

Intracellular Renin in the Brain Contributes to Cardiovascular and Metabolic Control

Renin gene expression is regulated by two distinct promoter‐first exon combinations that target renin either for secretion (exon 1a initiates secreted renin) or cytoplasmic retention (exon 1b initiates intracellular renin, icREN). Interestingly, icREN is expressed predominantly in the brain. We selectively deleted exon 1b and developed icREN KO mice to examine the role of icREN in cardiovascular and metabolic control. Systolic blood pressure in the light phase as measured by telemetry was increased in KO mice (126±2 mmHg, n=8 vs 116±2 mmHg, n=7, P&lt;0.01). The low‐ to high‐frequency ratio (LF/HF) derived from power spectral analysis of heart rate variability was increased in KO mice (1.24±0.21, n=7 vs 0.70±0.11, n=7, P&lt;0.05). Body weight was normal in KO mice fed normal chow. However, high fat diet‐induced obesity was attenuated in male KO mice (body weight, 32.4±1.3 g, n=5 vs 36.6±1.4 g, n=9; relative fat mass, 26.1±2.3 %, n=5 vs 34.4±2.3 %, n=9, both P&lt;0.05). The resting metabolic rate measured by respirometry was increased in KO mice (0.156±0.005 kcal/h, n=46, P&lt;0.05) vs controls (0.145±0.003 kcal/h, n=53). We previously reported that the brain renin‐angiotensin system facilitates renal sympathetic nerve activity (RSNA) response to acute intracerebroventricular injection of leptin. Interestingly, the RSNA response to leptin was greater in KO mice compared to controls (214±40 % baseline, n=5 vs 114±18 % baseline, n=10, P&lt;0.01). Further, in KO mice, AT1a receptor mRNA was upregulated in the paraventricular nucleus (P&lt;0.05). These data suggest that this novel icREN isoform contributes to cardiovascular and metabolic control possibly through the modulation of AT1a receptor expression in the brain.

Roles of hepatic glucokinase in intertissue metabolic communication: Examination of novel liver-specific glucokinase knockout mice

Glucokinase is expressed principally in pancreatic β-cells and hepatocytes, and catalyzes the phosphorylation of glucose to glucose-6-phosphate, a rate-limiting step of glycolysis. To better understand the roles of hepatic glucokinase, we generated Gck knockout mice by ablating liver-specific exon 1b. The knockout mice exhibited impaired glucose tolerance, decreased hepatic glycogen content, and reduced Pklr and Fas gene expression in the liver, indicating that hepatic glucokinase plays important roles in glucose metabolism. It has also been reported that hepatic glucokinase regulates the expression of thermogenesis-related genes in brown adipose tissue (BAT) and insulin secretion in response to glucose. However, the liver-specific Gck knockout mice displayed neither altered expression of thermogenesis-related genes in BAT nor impaired insulin secretion by β-cells under a normal chow diet. These results suggest that chronic suppression of hepatic glucokinase has a small influence on intertissue (liver-to-BAT as well as liver-to-β-cell) metabolic communication.

Molecular characterization of cbfβ gene and identification of new transcription variants: Implications for function

The CBFβ gene encodes a transcription factor that, in combination with CBFα (also called Runx, runt-related transcription factor) regulates expression of several target genes. CBFβ interacts with all Runx family members, such as RUNX2, a regulator of bone-related gene transcription that contains a conserved DNA-binding domain. CBFβ stimulates DNA binding of the Runt domain, and is essential for most of the known functions of RUNX2.A comparative analysis of the zebrafish cbfβ gene and protein, and of its orthologous identified homologous proteins in different species indicates a highly conserved function. We cloned eleven zebrafish cbfβ gene transcripts, one resulting in the known Cbfβ protein (with 187 aa), and three additional variants resulting from skipping exon 5a (resulting in a protein with 174 aa) or exon 5b (resulting in a protein with 201 aa), both observed for the first time in zebrafish, and a completely novel isoform containing both exon 5a and 5b (resulting in a protein with 188 aa). Functional analysis of these isoforms provides insight into their role in regulating gene transcription. From the other variants two are premature termination Cbfβ forms, while the others show in-frame exon-skipping causing changes in the Cbfβ domain that may affect its function.

Deletions of RDINK4/ARF enhancer in gastrinomas and nonfunctioning pancreatic neuroendocrine tumors.

The presence of an enhancer element, RD (RD), in the prominent INK4-ARF locus provides a novel en bloc mechanism to simultaneously regulate the transcription of p15, p14ARF, and p16 genes. However, knowledge about RD alterations and its potential contributions to cancer progression remains limited. In this study, we aimed to evaluate the incidence of RD alterations in pancreatic tumors. DNAs from 14 gastrinomas and 6 nonfunctioning pancreatic neuroendocrine tumors were subjected to quantitative real-time polymerase chain reaction-based assays to determine deletions in p15, p14ARF, and p16 (both exons 1 and 2). RD was frequently deleted in gastrinomas and nonfunctioning pancreatic neuroendocrine tumors with an incidence of 30% (6/20 samples). In comparison, the incidences of deletions of p15 (exon 1), p14ARF (exon 1β), and p16 (exon 1α) are 10% (2/20 samples), 10% (2/20 samples), and 45% (9/20 samples), respectively. Whereas some RD deletion events arose from deletions of the entire INK4-ARF locus, RD deletions in some specimens seemed to be independent of genetic alterations in any of the p15, p14ARF, and p16 genes. Our results strongly support that the deletion of RD may represent a novel mechanism to simultaneously downregulate p15, p14ARF, and p16, thus contributing to the development of human pancreatic cancers.