PhD student of Dietology
|Institution||Università degli Studi della Campania Luigi Vanvitelli|
|Address||Institute of Genetics and Biophysics "Adriano Buzzati-Traverso"|
|Institution||Università degli Studi della Campania Luigi Vanvitelli|
|Address||Institute of Genetics and Biophysics "Adriano Buzzati-Traverso"|
The N-oxygenation of amines by the human flavin-containing monooxygenase (form 3) (FMO3) represents an important means for the conversion of lipophilic nucleophilic heteroatom-containing compounds into more polar and readily excreted products. In healthy individuals, virtually all Trimethylamine (TMA) are metabolized to Trimethylamine N-oxide (TMAO). Several single nucleotide polymorphisms (SNPs) of the FMO3 gene have been described and result in an enzyme with decreased or abolished functional activity for TMA N-oxygenation thus leading to TMAU, or fish-like odor syndrome. Three coding region variants, c. G472A (p.E158K) in exon 4, c. G769A (p.V257M) in exon 6, and c.A923G (p.E308G) in exon 7, are common polymorphisms identified in all population examined so far and are associated with normal or slightly reduced TMA N-oxygenation activity. However, simultaneous occurrence of 158K and 308G variants results in a more pronounced decrease in FMO3 activity. A fourth polymorphism, c. G1424A (p.G475D) in exon 9, less common in the general population, was observed in individuals suffering severe or moderate trimethylaminuria. The aim of this study was to determine the allelic and genotypic distributions of these four FMO3 variants in 528 healthy individuals collected from the Sicilian and Sardinian populations together with haplotype and linkage analyses. Finally, we present data on the genotype-phenotype correlation by ESI-MS/MS TMA/TMAO urinary determination in 158KK/308EG individuals. Variant 158K shows the same frequency in Sicilian and Sardinian populations while variant 257M was not observed in the Sardinian sampling. No significant differences were found for 308G and 475D variants among two populations. Cis-linkage between 158K and 308G was confirmed with the compound variant (158K-308G) being found in a proportion of 0.9% and 0.3% of Sicilian subjects, and 0.01% and 0.5% in Sardinian population. Urinary determination of TMA/TMAO ratio in 158KK/308EG individuals showed a considerable reduction in FMO3 activity although they do not show the classical features of trimethylaminuria as a strong body odor and breath. Our data support the conclusion that trimethylaminuria is not always accompanied by a fish-like odor, despite the coexistence in the same individual of the two variants 158K and 308G, and other factors account for the expression of that phenotype.
Severe combined immunodeficiencies (SCIDs) represent a group of distinct congenital disorders affecting either cell-mediated or humoral immunity, which lead invariably to severe and life-threatening infections. The different forms of SCID are currently classified according to the presence or absence of T, B, and NK cells. This greatly helps define the site of the blockage during the differentiation process. Even though SCID patients share common clinical features, such as opportunistic infections and failure to thrive, irrespective of the underlying pathogenetic mechanism, the discovery of new causative gene alterations led to identify novel complex clinical phenotypes, sometimes associated to extrahematopoietic manifestations. In a few cases, the presenting signs may be peculiar to that specific form and physicians should be alerted in recognizing such complex phenotypes, in order to avoid delay in the diagnostic procedures. The aim of this review is to alert care-givers to take into account also the less frequent clinical features and novel pathogenic mechanisms to direct the functional and molecular studies toward a certain genetic alteration.
Glutamate is the principal excitatory neurotransmitter in the central nervous system which acts by the activation of either ionotropic (AMPA, NMDA and kainate receptors) or G-protein coupled metabotropic receptors. Glutamate is widely accepted to play a major role in the path physiology of migraine as implicated by data from animal and human studies. Genes involved in synthesis, metabolism and regulation of both glutamate and its receptors could be, therefore, considered as potential candidates for causing/predisposing to migraine when mutated.
Idiopathic scoliosis (IS) is a largely diffused disease in human population but its pathogenesis is still unknown. There is a relationship between scoliotic phenotype and the patient age, since in the early stage the pathology shows a ratio of 50% between male and female teenagers. During puberty the sex ratio is 8.4/1 (female/male), suggesting a sex-conditioned manifestation of the disease. Genetic inheritance of idiopathic scoliosis is still unclear although some authors claim for its X-linked dominant inheritance. There is large agreement in considering the IS as a sex-conditioned disease, in terms of steroid content and their receptor activity, although no evidence has been found yet. The blood content of 17beta-estradiol in teenagers with IS shows lower levels than teenagers of the same age without IS. Also testosterone and progesterone content are lower in IS girls with respect to the control girls. Furthermore, we extracted DNA from white blood cells of IS patients and their relatives until the third generation in order to examine estrogen receptor alpha polymorphisms, considering this tool a plausible molecular marker for IS prognosis. In this respect, we identified four polymorphisms in the exons encoding for the steroid binding domain and two other in the trans-activation domain. Our results show a clear relationship with clinical manifestation of IS.
The HG is a compound tubulo-alveolar gland located in the orbital cavity of the majority of vertebrates. In the golden hamster it shows a clear cut sexual dimorphism in both morphological and biochemical parameters such as cell types, protein pattern, lipid metabolism, porphyrin content, steroid hormone receptor expression. In a previous study we found that in primary culture of male hamster Harderian gland (HG), androgens (A) increase the MHG07 (male Harderian gland) expression and this effect is abrogated by both flutamide and cycloheximide. The present study represents a deeper analysis on MHG07 regulation by other members of steroid/thyroid hormone superfamily. Estrogens (E) impair the stimulatory effect of A and after the addition of a pure anti-estrogen, ICI 164,384, the negative effect of E is abrogated. Dexamethasone (Dex), used alone or in combination with A negatively affect the MHG07 expression. Also T(3) increases the expression of MHG07 mRNA. Progesterone (P) does not affect the expression of MHG07 mRNA. The use of cycloheximide abrogates the effect of steroids, suggesting that the latter act through their own receptors. Dose-response experiments show that low steroid concentrations (10(-12)M) are sufficient to affect the MHG07 expression. It is argued that the expression of MHG07 is under a highly coordinate relationship between androgen, estrogen, glucocorticoid, retinoic acid and thyroid hormones.
Cavernous vascular malformations may affect brain and out-of-brain tissues. In most cases, cerebral cavernous malformations (CCMs) involve the brain alone, and are rarely associated with skin hemangiomas, spinal cord, retinal, hepatic or vertebral lesions. CCMs can cause seizures, intracranial and spinal haemorrhages, focal neurological deficits, and migraine-like headaches. After collecting CCM families of Italian origin and investigating the genetic basis of the disorder we disclosed two novel molecular variations in the KRIT1 and MGC4607 genes. We found a novel CCM1 gene mutation (Q66X) in a family with apparently asymptomatic old-aged mutation carriers and patients who either had skin angiomas alone or the full association of cerebral, spinal, and skin lesions. In this family we report the highest variability in mutation penetrance so far described, including the presence of CCM in one subject since birth (surgery at 19 months of age), a condition to our knowledge so far unreported. In a CCM2 affected family, we also report a novel causative mutation, (54_55delAC) in exon 2 of the MGC4607 gene, that produces a truncated protein containing only 22 amino acids. These data describe novel CCM mutations associated with a particularly high variability of the penetrance causing, in some cases, reduced expression of clinical symptoms and sporadic cases with apparent negative family history. Hence they emphasize the importance of DNA-based diagnostics and genetic counseling to identify unaffected mutation carriers subjects, even at advanced age.
Vitamin A and its principal biologically active derivative, retinoic acid (RA), play a fundamental role in diverse processes, such as proliferation, differentiation, morphogenesis, metabolism and apoptosis of many types of cells. In addition, RA has been shown to be involved in the regulation of testicular function. These effects are mediated by interaction with two families of nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), each with three subtypes alpha, beta and gamma. The physiological involvement of retinoids in testicular function has been conducted mainly in mammals. Recently, we found that exogenous all-trans-retinoic acid impairs spermatogenesis and enhance testicular germ cell apoptosis in the lizard, Podarcis sicula, a seasonal breeder. To further investigate the role of retinoic acid in lizard, we focus this work principally on the characterization of lizard retinoic acid receptors (alpha, beta and gamma isoforms). RARalpha is 2720 bp long with a putative ORF between 699 and 2133. A Kozac sequence is present at 696 and a putative poly-adenilation site is present in position 2612. The RARalpha sequence shares 87% homology with mouse RARalpha mRNA while it has 76 and 80% homology with lizard RARbeta and gamma mRNAs. RARbeta is 2478 bp long showing a putative ORF between 196 and 1543. A canonical Kozac sequence is present at 193 and a putative poly-adenilation site is present at 2294. RARbeta shares 91% homology with mouse RARbeta mRNA and has 76% homology with both RARalpha and gamma. RARgamma is 2416bp long. With a putative ORF between 444 and 1818. A Kozac sequence is present at 441 and a putative poly-adenilation site is present at 2288. RARgamma shares 86% homology with mouse RARbeta mRNA and has 80 and 76% homology with both RARalpha and beta respectively. It is worth to note that, as in mouse, the 5'UTR of all isoforms is TATA and CAAT less. Both Northern blot and PCR analyses indicate that lizard testis expresses only RARalpha and RARbeta mRNAs, while RARgamma mRNA transcript was not found. In the period analysed, RARbeta was expressed during the gonadal full activity (May) and RARalpha was present in the post-reproductive period (August). During the autumnal recrudescence (October) RARalpha and RARbeta are co-expressed and, as indicated by quantitative PCR analysis, RARbeta mRNA levels are lower than RARalpha ones. Thus, the appearance and abundance of each receptor correspond to a specific phase of lizard reproductive cycle, allow us to hypothesize that each RAR subtype could play a specific role in the regulation of spermatogenetic activity. The results of the present study show, for the first time, the characterization of RAR mRNAs in the testis of lizard P. sicula, whose expression is related to the different phase of reproductive cycle. Moreover, the gamma form, is principally expressed in the skin during the March-July period, having probably a role in regulating skin homeostasis and colour livery, which are important factor in mating during the reproductive cycle.
In mammals, retinoic acid is involved in the regulation of testicular function by interaction with two families of nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR). Among RAR isoforms, the testicular cells of the lizard were found to express only RARalpha (3.7 kb) and RARbeta (3.4 kb) mRNAs, as reported here. In this study, the effects of exogenous all-trans-retinoic acid (atRA) on spermatogenesis of a non-mammalian seasonal reproducer were investigated. Daily intraperitoneal injections of atRA or atRA plus testosterone (atRA+T) were given for 2 weeks to adult males of the lizard Podarcis sicula. In animals treated with atRA, the seminiferous tubules were markedly reduced in cross-area. The seminiferous epithelium collapse was responsible for a sensible reduction in the number of germ cells and disruption in normal epithelial organization. In comparison, in atRA+T-treated lizards the loss of germinal cells was significantly less. The loss of germ cells observed in both experimental groups results from an induction of apoptotic process, as revealed by TUNEL analysis. Although low in number, apoptotic germ cells were also observed in the control groups (saline- and T-treated lizard), where the main germ cells undergoing apoptosis are primary spermatocytes (most frequently) and some spermatogonia. In conclusion, it is shown here that retinoic acid has deleterious effects on lizard spermatogenesis, causing a severe depletion of seminiferous epithelium, probably via induction of apoptotic processes. These effects are not completely inhibited by simultaneous administration of testosterone, although this hormone, once injected, is able to stimulate spermatogenesis and protect germinal cells from apoptotic cell death.
The steroid/thyroid hormone receptors are members of a very large family of nuclear-activated transcription factors. These receptors play a crucial role in most biological function, including regulation of development, metabolism, behaviour and reproduction. Among androgen receptor (AR), we have recently demonstrated that its expression in the Harderian gland (HG) of the male hamster is under a well-co-ordinated cross-talk between various steroid hormone receptors. Here, are presented data on the sequence of hamster AR promoter region (5'UTR) and the molecular tools of its regulation. The 5'UTR is 1585 bp. The promoter region shows various responsive elements. Two putative CREM elements are present at -71 and -1576 bp. A putative retinoic acid responsive element is present at -1476 bp. An androgen/glucocorticoid responsive element is present at -473 bp. A putative thyroid hormone-responsive element at -381 bp and an estrogen responsive element at -230 bp. Also, a homopurinic stretch is evident between -1199 and -1118. Furthermore, Sp1 sites are also spread along the sequence. As well as for human, mouse, rat and pig, the hamster lacks the canonical promoter TATA and CCAAT boxes. Gel retardation experiments confirm the presence of active responsive elements for AR, estrogen receptor, glucocorticoid receptor and thyroid hormone receptor. Previous data on the regulation of expression of AR by other members of steroid/thyroid hormone receptors well correlate with sequence analysis and gel retardation experiments. Thus, androgens, thyroid hormone, stimulate the AR transcription, while synthetic glucocorticoid (Dex) and estrogen are potent inhibitors of AR expression. The comparison of hamster AR promoter sequence with other AR promoter shows an 89, 82, 84 and 84% identity with human, rat, mouse and pig AR promoter, respectively. These results, in the light of the extreme plasticity of hamster HG, suggest that the comparative study of expression and regulation of AR gene in the HG of the hamster offers a useful tool to approach the normal and pathological phenotype in human.
The androgen receptor (AR) must be considered a transcription factor belonging to the steroid-thyroid hormones receptor superfamily. Previous results gained from the Harderian gland, a tubulo-alveolar gland located in the orbital cavity of the golden hamster, indicate that Harderian gland cells express mRNAs encoding for androgen, glucocorticoid, thyroid hormone (T(3)), and estrogen receptors, respectively. Since in other systems, these receptors have been related to the expression of the androgen receptor, we have studied the regulation of AR expression in primary cultures of the male hamster Harderian gland. Our in vitro experiments show that androgen, and thyroid hormones increase the expression of AR. Retinoic acids also show a positive effect on AR expression, while exposure to glucocorticoid or estrogen blocks AR expression. Since these steroids differently modulate AR expression, our results must be considered in the context of multi-hormonal control of gene expression that could act through cross-talk between members of the steroid-thyroid hormones.