Natalia Cucu

Determination of S-Adenosylmethionine and S-Adenosylhomocysteine from Human Blood Samples by HPLC-FL

Abstract S-adenosylmethionine plays an important role in many biochemical reactions, including human health maintenance and disease prevention. This paper proposes a simplified, rapid, and easy high performance liquid chromatography (HPLC) with fluorimetric detection method of S-adenosylmethionine and its product, S-adenosylhomocysteine. Determination of S-adenosylmethionine and S-adenosylhomocysteine was by HPLC with fluorimetric detection by the formation of the fluorescent 1,N 6-ethanoderivatives of these compounds, which showed a linear concentration range of 310−6–310−8 molL−1 for S-adenosylmethionine and 510−7–510−9for S-adenosylhomocysteine; the detection limits of the method ranged in the nanomolar domain. The method applied to real plasma samples lead to values of 60.1±19.6 for S-adenosylmethionine and 24±14.1 nmolL−1 for S-adenosylhomocysteine.

Comparative molecular approaches in Prader-Willi syndrome diagnosis.

Prader-Willi and Angelman syndromes are two distinct neurogenetic disorders caused by chromosomal deletions, uniparental disomy or loss of the imprinted gene expression in the 15q11-q13 region. PWS results from the lack of the paternally expressed gene contribution in the region. The aim of our study was to compare a new molecular approach based on the quantification of the expression of non-imprinted bi-allelic gene (NIPA1 and OCA2) with in house MS-PCR and the MS-MLPA test. Blood samples were collected from 12 patients, clinical criteria positives for Prader-Willi syndrome. DNA and RNA samples were isolated from white blood cells. Epigenetic changes at SNRPN gene locus were evaluated by MS-PCR technique. The expression levels of two non-imprinted genes (NIPA1 and OCA2) were evaluated in qReal-Time PCR, in order to identify type 1 and type 2 deletions. SALSA MS-MLPA kit ME028 was used to detect copy number changes and to analyze CpG islands methylation of the 15q11 region. MS-MLPA test confirmed that 8/12 patients presented different types of deletion at the SNRPN gene level (promoter, introns, and exons) and 4/8 displayed type 1 or type 2 deletion. In children with 15q11-13 deletions, the decreased level of NIPA1and OCA2 gene expression is related to chromosomal abnormality in the investigated area. The deletions were confirmed by MS-MLPA analysis, thus recommending NIPA1 and OCA2 gene expression as an alternate method to investigate deletions.

Prader–Willi Syndrome, from Molecular Testing and Clinical Study to Diagnostic Protocols

Prader-Willi syndrome (PWS) is a complex and fascinating human disease, whose patophisiological characteristics are still the targets of research in teams that can afford multidisciplinary approaches for seeking the link between the genetic, epigenetic and phenotypic aspects. The genetic complexity of the PWS chromosomal region, 15q11-q13, relies on the multiple clustered imprinted genes, alternative splice variants, gene duplications and variant copies, that control the epigenetic phenomenon of the imprinting itself. These DNA and transcriptome levels are matched by the wide variety of phenotypes that involve multiple organ systems and the complexity of brain functions influenced by the expression of the PWS critical genes. In this review a general description of the clinical diagnostic criteria will be linked with the most recent knowledge described for the structure of the 15 critical chromosomal region and candidate genes, as well as the model mechanisms explaining the interaction of the cisand transgenetic factors and the epigenetic ones during the establishment and maintenance of the imprinting marks that define the parental characteristic contribution to the critical genes expression. This review aims at explaining the criteria of molecular diagnosis and genetic counceling based on the techniques that are currently used and that will be used in the future approaches for the improvement of the diagnosis and treatment schemes. PWS has been initially linked with its main characteristic phenotype, the obesity, and therefore was the first described genetic human obesity syndrome. The main etiology of this disease included: gross hyperphagia, hypogonadism and growth hormone deficiency, indicating hypothalamic dysfunction. A neurodegenerative aspect was also appreciated as a major contributor to the complex PWS phenotype. Recent epidemiological study proved that PWS is a rare disease with an estimated incidence of about 1 in 25 000 births, and a population prevalence of about 1 in 50 000 (Buiting, 2010). An interesting feature linked with the diagnosis and the treatment impact is that this syndrome develops during late development of neonate. Initially, the signs like hypotonia had not suggested a suspicion for PWS, nor the consideration of further clinical and molecular cytogenetic investigations, until

P3-420: Critical “OMES” in aging and neurodegenerative pathology. From theory to practice

Background: The presentation is centered on the notions issued in the recently growing field of OMICS. Methods: Taking into account that the ‘omics approach’ is not at all a sophisticated “translation” of the usual language in esoteric terms, but a newly proposed methodological tool striving to put a new conceptual order in the scientific thinking, and that the core of ‘omics’ is to perform an integrative attempt able to reveal the intimate core of that field and to drive it to practice improvement, the definition and pursuit of critical ‘omes’ in aging and neurodegenerative pathology could be a useful methodological attempt. Results: We present some partial results obtained by us in two nationally founded research projects, in which we are trying to investigate, on cohorts of helthy elderly by comparison with age matched patients, the relationships of some methylome and genome/epigenome particularities with the nutriomic and sociomic characteristics of that cohorts. Conclusions: We agree and comment the idea that the integrative pursuit of different neuromes and of their interplay with certain environmental (ecomes, nutriomes, chronomes) and inner organism omes such as metabolome,