Genetic assessment of familial and early-onset Parkinson's disease in a Greek population.

BACKGROUND AND PURPOSE: Although the first mutation associated with Parkinson's disease (PD) was identified several years ago in the alpha-synuclein (SNCA) gene in families of Greek and Italian ancestry, a more systematic study of this and other known PD mutations has not been performed in the Greek population. METHODS: A genetic analysis in 111 familial or sporadic with early-onset (≤50 years, EO) PD patients was performed for the presence of the A53T SNCA mutation. In separate subgroups of these patients, further mutations in the SNCA, LRRK2, Parkin, PINK1 and DJ-1 genes were searched for. Additionally, a subgroup of familial cases was analysed for mutations in the glucocerebrosidase (GBA) gene. RESULTS: In total, five patients (4.5% of our whole population) were identified with the A53T SNCA mutation, two with a heterozygote dosage mutation and one with a heterozygote point mutation in the Parkin gene, and seven patients (10.3% of our familial cohort) with GBA gene mutations. CONCLUSIONS: The A53T mutation in the SNCA gene, although uncommon, does represent a cause of PD in the Greek population, especially of familial EOPD with autosomal dominant inheritance. GBA mutations in the familial cohort tested here were as common as in a cohort of sporadic cases previously examined from the same centres. For the remainder of the genes, genetic defects that could definitively account for the disease were not identified. These results suggest that further Mendelian traits that lead to PD in the Greek population remain to be identified.

Prognostic value of EZH2, paxillin expression and DNA ploidy of breast adenocarcinoma: correlation to pathologic predictors.

PURPOSE: The objective of this study was to examine the association of EZH2 and paxillin expression and DNA ploidy status with pathological parameters of breast cancer, aiming to correlate tumor phenotype with its malignant behavior. METHODS: EZH2 and paxillin expression and DNA ploidy were evaluated in imprint smear samples obtained from 105 breast tumors after surgical removal. RESULTS: Increased expression of paxillin was associated with p53 expression (p=0.005), Ki-67 expression (p=0.018) and EZH2 expression (p<0.0001). EZH2 expression correlated with estrogen receptor (ER) and progesterone receptor (PR) status (p=0.01 and p=0.035, respectively), and expression of p53 and Ki-67 (p=0.007 and p<0.0001, respectively). Aneuploid tumors were significantly correlated with poor differentiation (p=0.000), stage of disease (p=0.000), size of the primary tumor (p=0.015), presence of nodal metastasis (p=0.001), ER status (p=0.008), cerbB2 status (p=0.012), and expression of Ki-67 (p=0.001) and EGFR (p=0.018). Multivariate analysis of ploidy results using paxillin and EZH2 expression as dependent variables revealed that aneuploid tumors were associated with disease stage and grade of differentiation, cerbB2 expression and EZH2 expression. CONCLUSION: Our results show that aneuploid tumors, EZH2 expression and paxillin expression correlate with more aggressive phenotype of breast cancer.

Prasugrel versus high dose clopidogrel to overcome early high on clopidogrel platelet reactivity in patients with ST elevation myocardial infarction.

OBJECTIVE: There is a paucity of data regarding the early effectiveness of the proposed 600 mg clopidogrel loading dose (LD) on platelet reactivity (PR) in ST elevation myocardial infarction (STEMI) patients. If high on-treatment platelet reactivity (HTPR) is present, prasugrel reloading and subsequent maintenance dose (MD), might offer faster and stronger platelet inhibition than high clopidogrel MD. METHODS: In 93 STEMI patients treated by primary percutaneous coronary intervention we assessed PR using the VerifyNow P2Y12 platelet function test, 2 h following 600 mg LD of clopidogrel. All the 60 (64.5 %) patients exhibiting HTPR (defined as PR ≥ 235 P2Y12 reaction units), were randomized to 1 of 2 therapeutic strategies: reloading with prasugrel 60 mg/10 mg MD or high (150 mg) clopidogrel MD. RESULTS: The primary endpoint of PR at 24 h post randomization was lower in the prasugrel compared to the clopidogrel group (51.3, 25.7-77.0 versus 242.4, 215.8-268.9 P2Y12 reaction units, least square estimates, 95 % confidence intervals, p < 0.001). PR at 2 h and 5 days post randomization was lower in the prasugrel compared to the clopidogrel group (117.2, 70.9-163.4 and 101.6, 70.1-133.2 least square mean difference, 95 % confidence intervals, p < 0.001 for both). At all the time points of PR assessment, HTPR rates were lower in prasugrel than in clopidogrel group. CONCLUSIONS: HTPR is commonly observed early post 600 mg clopidogrel LD in STEMI patients. In this case, prasugrel 60 mg LD/10 mg MD provides faster and stronger platelet inhibition than a high clopidogrel MD regimen.

The value of TOP2A, EZH2 and paxillin expression as markers of aggressive breast cancer: relationship with other prognostic factors.

INTRODUCTION: The immunocytochemical expression of topoisomerase II alpha (TOP2A), enhancer of zeste homologue 2 (EZH2) and paxillin has recently gained increasing attention. Although previous studies have commented on the clinical usefulness of these markers, their role remains controversial. AIM: The purpose of the study was to investigate the expression of TOP2A, EZH2 and paxillin in relation to classic prognostic parameters and their significance as prognostic markers in imprints of resected breast carcinomas. METHODS: Imprint smears from 55 patients who underwent surgical treatment for primary carcinoma in our department between 2005 and 2006 were studied immunocytochemically with the use of TOP2A, EZH2 and paxillin antibodies. RESULTS: The expression of TOP2A correlated with higher histologic grade, tumor size and negative PR expression. High intensity staining for EZH2 expression was associated with higher histologic grade, negative ER and PR expression and positive Ki-67 expression. The expression of paxillin showed no correlation with estrogen/progesterone and HER2 expression nor with tumor grade and stage. CONCLUSION: Our data indicate that TOP2A and EZH2 expression are related to a more aggressive tumor phenotype. The expression of paxillin failed to correlate with any of the studied clinicopathologic factors. Further studies are needed to verify these results.

The prognostic value of PTEN, p53, and beta-catenin in endometrial carcinoma: a prospective immunocytochemical study.

Mutations of the PTEN, p53, and beta-catenin genes are the most frequent molecular defects in endometrial carcinomas. The aim of this study was to investigate their prognostic significance in this form of cancer. Imprint smears were obtained from 80 fresh endometrial tumor specimens and studied immunocytochemically for the expression of PTEN, p53, and beta-catenin proteins. The staining pattern was correlated with several well-established prognostic parameters, including 5-year survival. Positive staining of p53 was significantly correlated with increased stage (P < 0.0001), lymph node metastases (P = 0.001), and a nonendometrioid histology (P = 0.001). On the contrary, positive beta-catenin expression was significantly associated with decreased stage (P = 0.002), decreased grade (P = 0.007), and a negative lymph node status (P = 0.023). PTEN positivity was correlated with decreased stage (P = 0.002) and negative lymph nodes (P = 0.008). All the three markers affected survival significantly in univariate analysis but only beta-catenin had an independent prognostic impact. An independent prognostic significance was also shown for PTEN in the stage I subgroup of patients. The results of our study indicate that loss of beta-catenin expression is a strong and independent predictor of an unfavorable outcome in patients with endometrial carcinoma. Loss of PTEN may also be associated with a worse prognosis in patients with early-stage disease.

Gene transfer into human hematopoietic progenitor cells with an episomal vector carrying an S/MAR element.

Episomally maintained self-replicating systems present attractive alternative vehicles for gene therapy applications. Recent insights into the ability of chromosomal scaffold/matrix attachment regions (S/MARs) to mediate episomal maintenance of genetic elements allowed the development of a small circular episomal vector that functions independently of virally encoded proteins. In this study, we investigated the potential of this vector, pEPI-eGFP, to mediate gene transfer in hematopoietic progenitor cell lines and primary human cells. pEPI-eGFP was episomally maintained and conferred sustained eGFP expression even in nonselective conditions in the human cell line, K562, as well as in primary human fibroblast-like cells. In contrast, in the murine erythroleukemia cell line, MEL, transgene expression was silenced through histone deacetylation, despite the vector's episomal persistence. Hematopoietic semisolid cell colonies derived from transfected human cord blood CD34(+) cells expressed eGFP, albeit at low levels. After 4 weeks, the vector is retained in approximately 1% of progeny cells. Our results provide the first evidence that S/MAR-based plasmids can function as stable episomes in primary human cells, supporting long-term transgene expression. However, they do not display universal behavior in all cell types.

Genetic modification of hematopoietic stem cells with nonviral systems: past progress and future prospects.

Serious unwanted complications provoked by retroviral gene transfer into hematopoietic stem cells (HSCs) have recently raised the need for the development and assessment of alternative gene transfer vectors. Within this context, nonviral gene transfer systems are attracting increasing interest. Their main advantages include low cost, ease of handling and large-scale production, large packaging capacity and, most importantly, biosafety. While nonviral gene transfer into HSCs has been restricted in the past by poor transfection efficiency and transient maintenance, in recent years, biotechnological developments are converting nonviral transfer into a realistic approach for genetic modification of cells of hematopoietic origin. Herein we provide an overview of past accomplishments in the field of nonviral gene transfer into hematopoietic progenitor/stem cells and we point at future challenges. We argue that episomally maintained self-replicating vectors combined with physical methods of delivery show the greatest promise among nonviral gene transfer strategies for the treatment of disorders of the hematopoietic system.

Prognostic value of DNA analysis of prostate adenocarcinoma: correlation to clinicopathologic predictors.

The ability to accurately predict tumor behavior and patient survival is a problem in managing patients with prostate cancer. DNA ploidy provides important information for the evaluation of the prognosis of prostate cancer. The aim of this study was to investigate the DNA ploidy in imprints from prostate adenocarcinomas in a group of 70 patients in relation to Gleason score, tumor differentiation, stage and PSA serum levels. The DNA content was studied in Feulgen-stained imprint smears through the image analysis technique using a SAMBA 2005 Image analyzer. According to our measurements, a strong correlation was observed between DNA ploidy status and tumor differentiation (p<0.001). A statistically significant difference was found between DNA aneuploidy and increased pretreatment PSA serum levels (>4 ng/ml) (p<0.001), as well as between ploidy pattern and stage of the disease (p<0.001). Our results conclude that DNA ploidy status appears to be an additional marker in the field of prognosis of prostatic adenocarcinoma and could provide useful information on the potential behavior of prostate cancer.

Evaluation of microbiological quality of coastal waters in Greece.

To evaluate the microbiological water quality of bathing sites along the Achaia coastline (south western Greece), a survey was conducted to determine the concentration of faecal bacterial and phage indicators as well as the presence of human viruses. Seawater samples (234) were collected from nine bathing sites on the Achaia coastline and were analysed for the presence of: total coliforms, faecal coliforms, faecal streptococci, Escherichia coli, somatic coliphages, F-RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, enteroviruses, adenoviruses and hepatitis A viruses. Most of the bacteriological analysis results were in accordance with the European Union standards. In all sites, bacteriophages were detected occasionally. Enteroviruses and adenoviruses were detected in 24 samples (10.26%) and 37 samples (15.81%) respectively. No samples were positive for the presence of hepatitis A virus. The overall data indicates that bathing sites are impacted by human faecal material. Both bacterial indicators and phages have low predictive capability for the presence of human viruses in coastal waters. None of the environmental parameters analysed was strongly related to the presence of the indicator organisms and viruses. Appropriate and effective administrative measures that should be taken into account may be considered in order to improve water quality and reduce public health risk.

Clinical characteristics of the alpha-synuclein mutation (G209A)-associated Parkinson's disease in comparison with other forms of familial Parkinson's disease in Greece.

An Ala53Thr mutation of the alpha-synuclein has been recently identified as a rare cause of familial Parkinson's disease (fPD). In the present study, the clinical characteristics of Parkinson's disease (PD) patients with Ala53Thr alpha-synuclein mutation (alpha-synPD) were compared with fPD patients without any known mutation. An investigator blinded to the results of the genetic analysis examined 15 alpha-synPD patients and 43 consecutive fPD patients. Demographic data, age at onset of the illness, duration of the disease and modality of presentation were collected. Segregation ratios for both sexes in individuals at risk of developing alpha-synPD were estimated. The Unified Parkinson's disease rating scale (UPDRS) was also completed. The 15 alpha-synPD patients were matched for duration of the disease and age at onset with 15 of the 43 fPD patients (MfPD). Comparisons were also made between 14 patients belonging to three multicase families with patterns of inheritance similar to alpha-synPD. The alpha-synPD patients were significantly younger (mean difference 11.8 years) and showed the first sign of the disease earlier in life (mean difference 12.7 years) as compared with the fPD patients. Tremor at onset was present in only one (6.7%) of the alpha -synPD patients compared with 18 (41.9%) of the fPD patients (P = 0.01). At the time of examination rigidity, postural instability, orthostatic hypotension and the overall clinical severity did not differ significantly either between alpha-synPD and fPD or between alpha-synPD and MfPD groups. Nevertheless, some clinically relevant trends concerning the psychiatric symptoms and complications of therapy were recognized. The overall clinical severity and the progression of the disease in patients with alpha-synPD did not differ from that of the fPD patients. The alpha-synPD patients presented the illness at a younger age and also had lower prevalence of tremor when compared with the fPD patients.

LCR-mediated, long-term tissue-specific gene expression within replicating episomal plasmid and cosmid vectors.

Locus control regions (LCRs) are transcriptional regulatory elements, which possess a dominant chromatin remodelling and transcriptional activating capability conferring full physiological levels of expression on a gene linked in cis, when integrated into the host cell genome. Using the human beta-globin LCR (betaLCR) as a model, we show that this class of control element can drive high levels of tissue-specific gene expression in stably transfected cultured cells from within an Epstein-Barr virus-based plasmid REV. Furthermore, a 38-kb betaLCR minilocus-REV cosmid vector was efficiently retained and maintained therapeutic levels of beta-globin transgene expression in the absence of drug selective pressure over a 2-month period of continuous culture equivalent to at least 60 generations. This demonstrates for the first time the feasibility of using REVs for gene therapy of the haemoglobinopathies. Importantly, our results demonstrate that as in the case of integrated transgenes, expression from within REVs is prone to silencing but that the inclusion of the betaLCR prevented this repression of gene function. Therefore, appropriate control elements to provide and maintain tissue-specific gene expression, as well as the episomal status of REVs is a crucial feature in vector design. Our data suggest that LCRs can contribute to this vital function.

Clinical phenotype in patients with alpha-synuclein Parkinson's disease living in Greece in comparison with patients with sporadic Parkinson's disease.

OBJECTIVE: An Ala53Thr mutation of the alpha-synuclein gene has been recently identified as a rare cause of autosomal Parkinson's disease (PD). The clinical characteristics of 15 patients with PD living in Greece with the Ala53Thr alpha-synuclein mutation (alpha-synPD) were compared with patients with sporadic Parkinson's disease (sPD). METHODS: An investigator, blind to the results of the genetic analysis, examined 15 patients with alpha-synPD and 52 consecutive patients with sPD. Demographic data, age at onset of the illness, modality of presentation, and duration of PD were collected. The unified Parkinson's disease rating scale, the Hoehn and Yahr scale, and the Schwab-England scale were completed. The patients with alpha-synPD were matched for duration of disease with 32 of the 52 patients with sporadic PD (MsPD group). RESULTS: Patients with the alpha-synuclein mutation were significantly younger (mean 7.6 years), showed the first sign of the disease significantly earlier in life (mean 10.8 years), and had significantly longer duration of the disease compared with patients with sPD. Tremor at onset of the disease was present in only one (6.7%) of the patients with alpha-synPD, whereas it was present in 32 (61.5%) of the patients with sPD (p=0.0006). During the course of the disease one patient in the alpha-synPD group went on to develop tremor compared with six patients in the sPD group. Rigidity, bradykinesia, postural instability, orthostatic hypotension, intellectual impairment, depression, complications of therapy, and clinical severity of the disease at the time of examination did not differ significantly between patients with alpha-synPD and those with sPD, or between patients with alpha-synPD and the MsPD group. CONCLUSION: The younger age at onset of the illness, the much lower prevalence of tremor, and the longer duration of the disease characterise the clinical phenotype in this sample of patients with alpha-synPD. The other symptoms and signs of the illness did not seem to differentiate the patients with alpha-synPD from those with sPD.

Mutation screening of the Wolfram syndrome gene in psychiatric patients.

Wolfram syndrome, a rare autosomal recessive neurodegenerative disorder, was originally described as a combination of familial juvenile-onset diabetes mellitus and optic atrophy. It was later demonstrated that Wolfram syndrome patients were highly prone to psychiatric disorders. Mutations in exon 8 of the Wolfram syndrome gene account for 88% of the patients with Wolfram syndrome. To examine whether the gene responsible for causing Wolfram syndrome is involved in psychiatric disorders, we screened exon 8 of the Wolfram syndrome gene for mutations in 119 patients with schizophrenia, one patient with schizoaffective disorder, 12 patients with bipolar disorder and 15 patients with major depression, using sequence analysis. In Wolfram syndrome patients, this gene has been shown to have primarily nonsense or frameshift mutations, which would result in a premature truncation of the protein. None of the psychiatric patients screened in this study carried these types of mutations. We identified, however, 24 new variations whose significance remains to be determined.

Nitric oxide synthase expression, enzyme activity and NO production during angiogenesis in the chick chorioallantoic membrane.

In order to elucidate further the role of nitric oxide (NO) as an endogenous antiangiogenic mediator, mRNA expression of inducible nitric oxide synthase (iNOS), enzyme activity and production of NO were determined in the chick chorioallantoic membrane (CAM), an in vivo model of angiogenesis. In this model, maximum angiogenesis is reached between days 9 - 12 of chick embryo development. After that period, vascular density remains constant. Inducible NO synthase (iNOS) mRNA expression, determined by reverse transcriptase polymerase chain reaction (RT - PCR), increased from the 8th day reaching a maximum (70% increase) at days 10 - 11. NO synthase activity, determined as citrulline formation in the presence of calcium, also increased from day 8 reaching a maximum around day 10 (100% increase). Similar results were obtained in the absence of calcium suggesting that the NOS determined was the inducible form. Nitric oxide production, determined as nitrites, increased from day 8 reaching a maximum around day 10 (64% increase) and remaining stable at day 13. Finally, the bacterial lipopolysaccharide LPS (which activates transcriptionally iNOS), inhibited dose dependently angiogenesis in the CAM. These results in connection with previous findings from this laboratory, showing that NO inhibits angiogenesis in the CAM, suggest that increases in iNOS expression, enzyme activity and NO production closely parallel the progression of angiogenesis in the CAM, thus providing an endogenous brake to control this process. British Journal of Pharmacology (2000) 129, 207 - 213

An in vitro system for expression analysis of mutations of the beta-globin gene: validation and application to two mutations in the 5' UTR.

We describe the setting up of an in vitro expression system for the analysis of mutations of the beta-globin gene. The system is based on the stable transfection of a normal or mutated beta-globin gene into mouse erythroleukaemia (MEL) cells. The expression construct contains an Agamma gene as an internal control and both globin genes are under the control of the HS2 element of the beta LCR. The system enables analysis of transcription, RNA processing and transport, as well as mRNA stability. With non-mutant genes, high-level expression of both beta and Agamma genes is seen and both mRNAs are stable. The system was validated by comparing the expression of the beta654 thalassaemia splicing mutation in MEL cells with its well-characterized expression in vivo. The level of the initial transcript, the proportion of abnormally spliced mRNA and its instability during erythroid cell maturation were all faithfully reproduced. The system was used to examine the mechanism by which two mutations in the beta-globin 5' untranslated region (5' UTR) result in beta thalassaemia. Surprisingly, the mechanism appeared to differ in the two cases, with the C-G substitution at position +33 affecting transcription, whereas the -T deletion at position +10 resulted in a translational defect. The stably transfected MEL cells, with an internal control and an endogenous enhancer, appear to be a valid and realistic experimental model, superior to transient expression studies. This system should find wide application in the analysis of the effects and mechanisms of gene inactivation in mutations affecting the beta-globin as well as other genes.

cDNA cloning of the translocon associated protein beta-subunit in the chick cerebellum.

The 'translocon associated protein' is a tetrameric complex residing in the translocation sites, in which nascent polypeptides pass through the endoplasmic reticulum membrane. The beta subunit of this complex is a single spanning membrane protein, as deduced from cDNAs deriving from canine or human epithelial tissues. We have isolated and analysed a cDNA clone of the beta subunit from chick nervous tissue, namely cerebellum. Its deduced protein sequence is 91% homologous to both the canine and the human protein sequences, showing that the molecule is highly conserved.

Mutation in the alpha-synuclein gene identified in families with Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder with a lifetime incidence of approximately 2 percent. A pattern of familial aggregation has been documented for the disorder, and it was recently reported that a PD susceptibility gene in a large Italian kindred is located on the long arm of human chromosome 4. A mutation was identified in the alpha-synuclein gene, which codes for a presynaptic protein thought to be involved in neuronal plasticity, in the Italian kindred and in three unrelated families of Greek origin with autosomal dominant inheritance for the PD phenotype. This finding of a specific molecular alteration associated with PD will facilitate the detailed understanding of the pathophysiology of the disorder.