Deregulation of apoptosis-related genes is associated with PRV1 overexpression and JAK2 V617F allele burden in Essential Thrombocythemia and Myelofibrosis

Background: Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34(+) hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters. Results: By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34(+) cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34(+) cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-XL and BCLW. In contrast, pro-apoptotic BID and BIMEL expression were downregulated in ET leukocytes. Increased BCL-XL protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly. Conclusions: Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.

A novel HLA-E allele, E*01:03:05, identified in two Brazilian individuals

Brazilian National Research Council (CNPq/Brazil)

Variable Na(v)1.5 protein expression from the wild-type allele correlates with the penetrance of cardiac conduction disease in the Scn5a(+/-) mouse model

BACKGROUND: Loss-of-function mutations in SCN5A, the gene encoding Na(v)1.5 Na+ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a(+/-) mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A. METHODOLOGY/PRINCIPAL FINDINGS: Based on ECG, 10-week-old Scn5a(+/-) mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS< or = 18 ms; QRS in wild-type littermates: 10-18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na+ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a(+/-) mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a(+/-) mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A-mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a(+/-) mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a(+/-) mice had similar Na(v)1.5 mRNA but higher Na(v)1.5 protein expression, and moderately larger I(Na) current than severely affected Scn5a(+/-) mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a(+/-) mice than in mildly affected ones. CONCLUSIONS: Scn5a(+/-) mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a(+/-) mice, phenotype severity correlates with wild-type Na(v)1.5 protein expression.

A novel allele of FILAMENTOUS FLOWER reveals new insights on the link between inflorescence and floral meristem organization and flower morphogenesis

Background The Arabidopsis FILAMENTOUS FLOWER (FIL) gene encodes a YABBY (YAB) family putative transcription factor that has been implicated in specifying abaxial cell identities and thus regulating organ polarity of lateral organs. In contrast to double mutants of fil and other YAB genes, fil single mutants display mainly floral and inflorescence morphological defects that do not reflect merely a loss of abaxial identity. Recently, FIL and other YABs have been shown to regulate meristem organization in a non-cell-autonomous manner. In a screen for new mutations affecting floral organ morphology and development, we have identified a novel allele of FIL, fil-9 and characterized its floral and meristem phenotypes. Results The fil-9 mutation results in highly variable disruptions in floral organ numbers and size, partial homeotic transformations, and in defective inflorescence organization. Examination of meristems indicates that both fil-9 inflorescence and floral meristems are enlarged as a result of an increase in cell number, and deformed. Furthermore, primordia emergence from these meristems is disrupted such that several primordia arise simultaneously instead of sequentially. Many of the organs produced by the inflorescence meristems are filamentous, yet they are not considered by the plant as flowers. The severity of both floral organs and meristem phenotypes is increased acropetally and in higher growth temperature. Conclusions Detailed analysis following the development of fil-9 inflorescence and flowers throughout flower development enabled the drawing of a causal link between multiple traits of fil-9 phenotypes. The study reinforces the suggested role of FIL in meristem organization. The loss of spatial and temporal organization of fil-9 inflorescence and floral meristems presumably leads to disrupted cell allocation to developing floral organs and to a blurring of organ whorl boundaries. This disruption is reflected in morphological and organ identity aberrations of fil-9 floral organs and in the production of filamentous organs that are not perceived as flowers. Here, we show the role of FIL in reproductive meristem development and emphasize the potential of using fil mutants to study mersitem organization and the related effects on flower morphogenesis.

Alcohol dehydrogenase 1C*1 allele is a genetic marker for alcohol-associated cancer in heavy drinkers

Chronic alcohol consumption is associated with an increased risk for upper aerodigestive tract cancer and hepatocellular carcinoma. Increased acetaldehyde production via alcohol dehydrogenase (ADH) has been implicated in the pathogenesis. The allele ADH1C*1 of ADH1C encodes for an enzyme with a high capacity to generate acetaldehyde. So far, the association between the ADH1C*1 allele and alcohol-related cancers among heavy drinkers is controversial. ADH1C genotypes were determined by polymerase chain reaction and restriction fragment length polymorphism in a total of 818 patients with alcohol-associated esophageal (n=123), head and neck (n=84) and hepatocellular cancer (n=86) as well as in patients with alcoholic pancreatitis (n=117), alcoholic liver cirrhosis (n=217), combined liver cirrhosis and pancreatitis (n=17) and in alcoholics without gastrointestinal organ damage (n=174). The ADH1C*1 allele and genotype ADH1C*1/1 were significantly more frequent in patients with alcohol-related cancers than that in individuals with nonmalignant alcohol-related organ damage. Using multivariate analysis, ADH1C*1 allele frequency and rate of homozygosity were significantly associated with an increased risk for alcohol-related cancers (p<0.001 in all instances). The odds ratio for genotype ADH1C*1/1 regarding the development of esophageal, hepatocellular and head and neck cancer were 2.93 (CI, 1.84-4.67), 3.56 (CI, 1.33-9.53) and 2.2 (CI, 1.11-4.36), respectively. The data identify genotype ADH1C*1/1 as an independent risk factor for the development of alcohol-associated tumors among heavy drinkers, indicating a genetic predisposition of individuals carrying this genotype.

The common G-allele of interleukin-18 single-nucleotide polymorphism is a genetic risk factor for atopic asthma. The SAPALDIA Cohort Study

BACKGROUND: IL-18 is a pleiotrophic cytokine involved in both, T-helper type 1 (Th1) and Th2 differentiation. Recently genetic variants in the IL-18 gene have been associated with increased risk of atopy and asthma. OBJECTIVE: To examine the relationship of a genetic, haplotype-tagging promotor variant -137G/C in the IL-18 gene with atopic asthma in a large, well-characterized and population-based study of adults. METHODS: Prospective cohort study design was used to collect interview and biological measurement data at two examination time-points 11 years apart. Multivariate logistic regression analysis was used to assess the association of genotype with asthma and atopy. RESULTS: The G-allele of the IL-18 promotor variant (-137G/C) was associated with a markedly increased risk for the prevalence of physician-diagnosed asthma with concomitant skin reactivity to common allergens. Stratification of the asthma cases by skin reactivity to common allergens revealed an exclusive association of IL-18 -137 G-allele with an increased prevalence of atopic asthma (adjusted odds ratio (OR): 3.63; 95% confidence interval: (1.64-8.02) for GC or GG carriers vs. CC carriers), and no according association with asthma and concomitant negative skin reactivity (adjusted OR: 1.13; 0.66-1.94). The interaction between IL-18 -137G/C genotype and positive skin prick test was statistically significant (P=0.029). None of 74 incident asthma cases with atopy at baseline exhibited the CC genotype. CONCLUSION: Our results strongly suggest that this variant of the IL-18 gene is an important genetic determinant involved in the development of atopic asthma.

Short communication: duplication in the 5'-flanking region of the beta-lactoglobulin gene is linked to the BLG A allele

beta-Lactoglobulin (beta-LG) is the major whey protein in the milk of cows and other ruminants. It is well established that the predominant genetic variants beta-LG A and B are differentially expressed. Extensive investigation of the genetic variation in the promoter region of the BLG gene revealed the existence of specific haplotypes associated with the A and B variants. However, the genetic basis for the differentially expressed BLG A and B alleles is still elusive. In this study additional genetic variation further upstream in the 5'-flanking region of the BLG gene was identified, including 6 single nucleotide substitutions, a single nucleotide deletion, and a 7-bp duplication. Comparison of DNA sequences showed that the investigated 5'-flanking region is highly conserved between ruminants, and the duplication g.-1885_-1879dupCTCTCGC and the substitution g.-1888A>G are only found in the BLG A and D alleles in cattle. The cytosine at position g.-1957 and the thymines at positions g.-2008 and g.-2049 are only found in BLG B alleles of cattle. It is suggested that the described genetic variability contributes to the differential allelic expression of the BLG gene.

Ligation dependent allele specific quantification (LASQ) of CFTR cDNA on the LightCycler using MLPA hybridization probes

BACKGROUND: As for Cystic Fibrosis (CF) and many other hereditary diseases there is still a lack in understanding the relationship between genetic (e.g. allelic) and phenotypic diversity. Therefore methods which allow fine quantification of allelic proportions of mRNA transcripts are of high importance. METHODS: We used either genomic DNA (gDNA) or total RNA extracted from nasal cells as starting nucleic acid template for our assay. The subjects included in this study were 9 CF patients compound heterozygous for the F508del mutation and each one F508del homozygous and one wild type homozygous respectively. We established a novel ligation based quantification method which allows fine quantification of the allelic proportions of ss and ds CFTR cDNA. To verify reliability and accuracy of this novel assay we compared it with semiquantitative fluorescent PCR (SQF-PCR). RESULTS: We established a novel assay for allele specific quantification of gene expression which combines the benefits of the specificity of the ligation reaction and the accuracy of quantitative real-time PCR. The comparison with SQF-PCR clearly demonstrates that LASQ allows fine quantification of allelic proportions. CONCLUSION: This assay represents an alternative to other fine quantitative methods such as ARMS PCR and Pyrosequencing.

MEP1A allele for meprin A metalloprotease is a susceptibility gene for inflammatory bowel disease

The MEP1A gene, located on human chromosome 6p (mouse chromosome 17) in a susceptibility region for inflammatory bowel disease (IBD), encodes the alpha-subunit of metalloproteinase meprin A, which is expressed in the intestinal epithelium. This study shows a genetic association of MEP1A with IBD in a cohort of ulcerative colitis (UC) patients. There were four single-nucleotide polymorphisms in the coding region (P=0.0012-0.04), and one in the 3'-untranslated region (P=2 x 10(-7)) that displayed associations with UC. Moreover, meprin-alpha mRNA was decreased in inflamed mucosa of IBD patients. Meprin-alpha knockout mice exhibited a more severe intestinal injury and inflammation than their wild-type counterparts following oral administration of dextran sulfate sodium. Collectively, the data implicate MEP1A as a UC susceptibility gene and indicate that decreased meprin-alpha expression is associated with intestinal inflammation in IBD patients and in a mouse experimental model of IBD.

Systematic Functional Analysis of BicD Serine Phosphorylation and Intragenic Suppression of a Female Sterile Allele of BicD

Protein phosphorylation is involved in posttranslational control of essentially all biological processes. Using mass spectrometry, recent analyses of whole phosphoproteomes led to the identification of numerous new phosphorylation sites. However, the function of most of these sites remained unknown. We chose the Drosophila Bicaudal-D protein to estimate the importance of individual phosphorylation events. Being involved in different cellular processes, BicD is required for oocyte determination, for RNA transport during oogenesis and embryogenesis, and for photoreceptor nuclei migration in the developing eye. The numerous roles of BicD and the available evidence for functional importance of BicD phosphorylation led us to identify eight phosphorylation sites of BicD, and we tested a total of 14 identified and suspected phosphoserine residues for their functional importance in vivo in flies. Surprisingly, all these serines turned out to be dispensable for providing sufficient basal BicD activity for normal growth and development. However, in a genetically sensitized background where the BicD(A40V) protein variant provides only partial activity, serine 103 substitutions are not neutral anymore, but show surprising differences. The S103D substitution completely inactivates the protein, whereas S103A behaves neutral, and the S103F substitution, isolated in a genetic screen, restores BicD(A40V) function. Our results suggest that many BicD phosphorylation events may either be fortuitous or play a modulating function as shown for Ser(103). Remarkably, amongst the Drosophila serines we found phosphorylated, Ser(103) is the only one that is fully conserved in mammalian BicD.