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Introduction The RhoA/ROCK calcium-sensitizing pathway plays a role in clitoral contractility, and therefore we deemed interesting to investigate its involvement in the vagina. Objective The aim of this study was to investigate the sex steroid regulation of the vagina contractility through the RhoA/ROCK pathway, using a validated animal model. Methods Ovariectomized Sprague-Dawley rats (OVX) were treated with 17β-estradiol (E2), testosterone (T), with or without letrozole (T+L), and compared with intact animals. Contractility studies were performed on vaginal strips to test the effect of ROCK inhibitor Y-27632 and NO synthase inhibitor L-NAME. In vaginal tissues, ROCK1 immunolocalization was investigated, mRNA expression was analyzed by semi-quantitative RT-PCR, and RhoA membrane translocation was evaluated by Western blot (Wb) analysis. Finally, rat smooth muscle cells (rvSMCs) were isolated from distal vagina of intact animals and the quantification of RhoA inhibitory protein RhoGDI was performed by Wb after stimulation with NO-donor SNP, with or without the soluble guanylate cyclase inhibitor ODQ or the PKGR1 inhibitor KT5823 administration. Results The results show that ROCK1 was immunolocalized in the smooth muscle bundles and in the blood vessel wall of vagina, while a weak positivity was detected in the epithelium. Y-27632 induced a dose-dependent relaxation of noradrenaline pre-contracted vaginal strips, decreased by OVX and completely restored by E2, while T and T+L further decreased it even below OVX level increased it. Accordingly, in Wb analysis, compared to controls, OVX significantly induced RhoA activation, as reveled by its membrane translocation, with T reverting it at a level significantly lower than in controls. This effect was not exerted by E2. Abolishing the NO formation via L-NAME increased Y-27632 responsiveness in the OVX+T group; L-NAME had only a partial effect in controls whilst it did not modulate Y-27632 responsiveness in OVX. Finally, stimulation of rvSMCs with SNP significantly increased the RhoGDI protein expression, an effect counteracted by ODQ and, partially, by the protein kinase inhibitor KT5823 incubation. Conclusions In conclusion, in vagina androgen administration in OVX functionally decreases RhoA/ROCK activity by hampering RhoA membrane translocation and plays a critical role in the inhibitory mechanism of the smooth muscle distal vagina contractility. Accordingly, androgens, by inhibiting the RhoA/ROCK pathway, could positively contribute to vaginal smooth muscle relaxation, favoring the sexual intercourse. Disclosure No

Purpose Benign Prostatic Hyperplasia (BPH) is a result of prostate inflammation, frequently occurring in metabolic syndrome (MetS). Low testosterone is common in MetS. A randomized clinical trial was designed to evaluate if 24 weeks of testosterone therapy (TTh) in BPH men with MetS and low testosterone improve urinary symptoms and prostate inflammation. Methods One-hundred-twenty men with MetS waitlisted for BPH surgery were enrolled. They were categorized into normal testosterone (TT ≥ 12 nmol/L and cFT ≥ 225 pmol/L; n = 48) and testosterone deficient (TD) (TT n = 72) then randomized to testosterone gel 2% (5 g/daily) or placebo for 24 weeks. At baseline and follow-up, questionnaires for urinary symptoms and trans-rectal ultrasound were performed. Prostate tissue was collected for molecular and histopathological analyses. Results No differences in the improvement of urinary symptoms were found between TTh and placebo (OR [95% CI] 0.96 [0.39; 2.37]). In TD + TTh, increase in prostate but not adenoma volume was observed (2.64 mL [0.07; 5.20] and 1.82 mL [− 0.46; 0.41], respectively). Ultrasound markers of inflammation were improved. In a subset of 61 men, a hyper-expression of several pro-inflammatory genes was found in TD + placebo when compared with normal testosterone. TTh was able to counteract this effect. For 80 men, the inflammatory infiltrate was higher in TD + placebo than in normal testosterone (0.8 points [0.2; 1.4]) and TD + TTh men (0.9 points [0.2; 1.5]). Conclusions Twenty-four weeks of TTh in TD men with BPH and MetS improves ultrasound, molecular and histological proxies of prostate inflammation. This does not result in symptom improvement.

Purpose Female sexual response involves a complex interplay between neurophysiological mechanisms and the nitric oxide (NO)-mediated relaxation of clitoris and vagina. The aim of this study was to evaluate sex steroids regulation of the relaxant pathway in vagina, using a validated animal model. Methods Subgroups of OVX Sprague–Dawley rats were treated with 17β-estradiol, testosterone, or testosterone and letrozole, and compared with a group of intact animals. Masson’s trichrome staining was performed for morphological evaluation of the distal vaginal wall, in vitro contractility studies investigated the effect of OVX and in vivo treatments on vaginal smooth muscle activity. RNA from vaginal tissue was analyzed by semi-quantitative RT-PCR. Results Immunohistochemical analysis showed that OVX induced epithelial and smooth muscle structural atrophy, testosterone and testo + letrozole increased the muscle bundles content and organization without affecting the epithelium while 17β-estradiol mediated the opposite effects. In vitro contractility studies were performed on noradrenaline pre-contracted vaginal strips from each experimental group. Acetylcholine (0.001–10 µM) stimulation induced a concentration-dependent relaxation, significantly reduced by NO-synthase inhibitor L-NAME and by guanylate cyclase inhibitor ODQ. OVX resulted in a decreased responsiveness to acetylcholine, restored by testosterone, with or without letrozole, but not by 17β-estradiol. OVX sensitivity to the NO-donor SNP was higher than in the control. Vardenafil, a PDE5 inhibitor, enhanced SNP effect in OVX + testosterone as well as in control, as supported by RNA expression analysis. Conclusions Our study demonstrates that testosterone improves the NO-mediated smooth muscle vaginal cells relaxation confirming its role in maintaining the integrity of muscular relaxant machinery.

Introduction Female sexual response is a complex phenomenon that involves multiple psychophysiological factors. Sex steroids play a fundamental role in the mechanisms of regulation of sexual arousal, modulating the sexual response and preserving the integrity and functionality of the vaginal tissue. For this reason, imbalances in the sexual environment of steroids in women favor the onset of important pathophysiological conditions such as hypoactive sexual desire disorder (HSDD) and genitourinary syndrome of menopause (GSM). Previous research has focused primarily on the effects of estrogen supplementation on vaginal physiology, but only few studies evaluated the effects of androgen administration, particularly on the vaginal smooth muscle compartment. The calcium sensitizing pathway of RhoA/ROCK may play a role in regulating vaginal contractility but, at present, the regulatory mechanisms of RhoA/ROCK signaling in the female genitourinary tract have yet to be fully elucidated. In particular, no specific research has been conducted on the effects of testosterone (T) on the contractility mechanism of smooth muscle in the vagina. Objective The aim of this study was to evaluate the sex steroid regulation on the distal vagina contractility mechanism, mediated by the RhoA/ROCK pathway, using a validated animal model of female ovariectomized rats. Methods Ovariectomized Sprague-Dawley rats (OVX) were treated with 17β-estradiol (OVX+E), testosterone (OVX+T), or testosterone and letrozole (OVX+T+L) and compared with an intact animals’ group (CTRL). In vitro contractility studies were performed on noradrenaline (NA) pre-contracted vaginal strips to investigate the effect of OVX and in vivo treatments on vaginal smooth muscle activity. The mRNA from vagina tissues isolated from each experimental group was analyzed by semi-quantitative RT-PCR, and membrane translocation of RhoA was evaluated by Western blot analysis. Results Y-27632 induced a dose-dependent relaxation on NA pre-contracted vaginal strips that was significantly decreased by OVX; T, with or without L, further decreased OVX-induced hyporesponsiveness to Y-27632, whilst it was fully restored up to control level by 17β-estradiol (E) supplementation. Accordingly, gene expression analysis indicated that OVX decreased the expression of RhoA/ROCK pathway markers, which was normalized by E. However, at variance with contractility results, androgen receptor (AR) activation upregulated, rather than downregulating, RhoA/ROCK expression. In Western blot analysis, OVX induced activation and membrane translocation of the active form of RhoA as compared to controls. Interestingly, T reverted this effect by reducing RhoA translocation to membrane to a level even significantly below the control one. This effect was not exerted by E administration. Considering that AR activation increases NO signaling (as measured by Prkg1 mRNA expression), we tested the effect of abolishing NO formation through the NO synthase inhibitor L-NAME. Pre-incubation with L-NAME substantially increased Y-27632 responsiveness in the OVX +T group and partially in the CTRL one and, but not in the OVX group. Conclusions This study confirms that in vagina estrogens increase the expression of the calcium-sensitizing pathway RhoA/ROCK, while androgen administration functionally decreases RhoA/ROCK activity, by hampering RhoA translocation to the membrane, a mechanism which is most probably driven by NO. Disclosure No

Benign Prostatic Hyperplasia (BPH) is a result of prostate inflammation, frequently occurring in metabolic syndrome (MetS). Low testosterone is common in MetS. A randomized clinical trial was designed to evaluate if 24 weeks of testosterone therapy (TTh) in BPH men with MetS and low testosterone improve urinary symptoms and prostate inflammation.One-hundred-twenty men with MetS waitlisted for BPH surgery were enrolled. They were categorized into normal testosterone (TT ≥ 12 nmol/L and cFT ≥ 225 pmol/L; n = 48) and testosterone deficient (TD) (TT 12 nmol/L and/or cFT 225 pmol/L; n = 72) then randomized to testosterone gel 2% (5 g/daily) or placebo for 24 weeks. At baseline and follow-up, questionnaires for urinary symptoms and trans-rectal ultrasound were performed. Prostate tissue was collected for molecular and histopathological analyses.No differences in the improvement of urinary symptoms were found between TTh and placebo (OR [95% CI] 0.96 [0.39; 2.37]). In TD + TTh, increase in prostate but not adenoma volume was observed (2.64 mL [0.07; 5.20] and 1.82 mL [- 0.46; 0.41], respectively). Ultrasound markers of inflammation were improved. In a subset of 61 men, a hyper-expression of several pro-inflammatory genes was found in TD + placebo when compared with normal testosterone. TTh was able to counteract this effect. For 80 men, the inflammatory infiltrate was higher in TD + placebo than in normal testosterone (0.8 points [0.2; 1.4]) and TD + TTh men (0.9 points [0.2; 1.5]).Twenty-four weeks of TTh in TD men with BPH and MetS improves ultrasound, molecular and histological proxies of prostate inflammation. This does not result in symptom improvement.

Female sexual response involves a complex interplay between neurophysiological mechanisms and the nitric oxide (NO)-mediated relaxation of clitoris and vagina. The aim of this study was to evaluate sex steroids regulation of the relaxant pathway in vagina, using a validated animal model.Subgroups of OVX Sprague-Dawley rats were treated with 17β-estradiol, testosterone, or testosterone and letrozole, and compared with a group of intact animals. Masson's trichrome staining was performed for morphological evaluation of the distal vaginal wall, in vitro contractility studies investigated the effect of OVX and in vivo treatments on vaginal smooth muscle activity. RNA from vaginal tissue was analyzed by semi-quantitative RT-PCR.Immunohistochemical analysis showed that OVX induced epithelial and smooth muscle structural atrophy, testosterone and testo + letrozole increased the muscle bundles content and organization without affecting the epithelium while 17β-estradiol mediated the opposite effects. In vitro contractility studies were performed on noradrenaline pre-contracted vaginal strips from each experimental group. Acetylcholine (0.001-10 µM) stimulation induced a concentration-dependent relaxation, significantly reduced by NO-synthase inhibitor L-NAME and by guanylate cyclase inhibitor ODQ. OVX resulted in a decreased responsiveness to acetylcholine, restored by testosterone, with or without letrozole, but not by 17β-estradiol. OVX sensitivity to the NO-donor SNP was higher than in the control. Vardenafil, a PDE5 inhibitor, enhanced SNP effect in OVX + testosterone as well as in control, as supported by RNA expression analysis.Our study demonstrates that testosterone improves the NO-mediated smooth muscle vaginal cells relaxation confirming its role in maintaining the integrity of muscular relaxant machinery.

Introduction Female sexual response is a result of a complex interplay between central and peripheral mechanisms and the nitric oxide (NO)-mediated relaxation of clitoris and distal vagina vascular bed leads to clitoris tumescence and vagina lubrification. Objective The aim of this study was to evaluate sex steroids regulation of the relaxant/contractile pathway in vagina, using a validated animal model of ovariectomized (OVX) Sprague-Dawley rats. Methods Subgroups of OVX rats were treated with 17β-estradiol (10 mg/kg/die), testosterone (T, 30 mg/kg/week), or testosterone and letrozole (2.5 mg/kg/die) for 6 weeks. The experimental groups were compared with a group of intact rats. In vitro contractility studies were carried out in order to investigate the effect of OVX and in vivo treatments on vaginal smooth muscle activity. RNA from vaginal tissue was analyzed by semi-quantitative RT-PCR. Results In vitro contractility studies were performed on noradrenaline pre-contracted distal vaginal strips, isolated from each experimental group. Acetylcholine (Ach; 0.001-10 µM) stimulation induced a concentration-dependent relaxation, which was significantly reduced by NO-synthase inhibitor L-NAME (100 µM) and by guanylate cyclase inhibitor ODQ (1 µM). OVX resulted in a decreased responsiveness to Ach, restored by T supplementation, with or without letrozole, but not by 17β-estradiol. Vaginal strips sensitivity to the NO-donor SNP (1 nM - 100 µM) was higher in OVX than in the control. Vardenafil (100 nM), a PDE5 inhibitor, enhanced SNP effect in OVX+T as well as in control, suggesting that T positively modulates PDE5, as supported by RNA expression analysis. Conclusions In vitro contractility studies and mRNA expression analysis highlight the positive regulation of T on Ach- and NO-dependent muscle relaxation by modulating the NO/cGMP/PKG pathways.Our data demonstrate that testosterone improves the NO-mediated vascular smooth muscle vaginal cells relaxation confirming its critical role in maintaining the integrity of muscular relaxant machinery. Our study confirms the decisive role played by androgens, rather than estrogens, in maintaining the functionality and integrity of the vagina. This study might provide support for the use of androgens in the treatment of genitourinary syndrome of menopause (GSM) and/or sexual arousal disorders related to ageing related-hormonal deficiency. Disclosure Work supported by industry: no.

Introduction The human vagina is extremely sensitive to hormonal changes occurring during the woman's lifetime, in particular the decline in estrogens that characterizes the menopause is known to induce a range of genital and sexual symptoms (dryness, burning, irritation, lack of lubrication, discomfort, pain), known as “genitourinary syndrome of menopause” (GSM). However, this reduction in estrogen levels is also accompanied by a progressive ageing-dependent decline in androgens for which the role of androgens in the development and treatment of GSM has received more attention. It is known that peripheral tissues compensate for circulating hormonal imbalances through the in-situ production of small quantities of sex hormones deriving from inactive precursors metabolized by specific cellular enzymes. This non-classical production and local activity of hormones is defined as “intracrinology”. Objective The aim of the study was to investigate whether the human vagina possesses the specific enzymatic machinery to convert weak androgen precursors, such as DHEA, into active androgens [Δ4-androstenedione (A), testosterone (T) and dihydrotestosterone (DHT)] as already described in other species. Methods In this study, a well established model of human smooth muscle cells (hSMCs), isolated from vagina tissues of women undergoing surgery for benign gynecological diseases, was used. Semi-quantitative RT-PCR was employed in order to evaluate the mRNA expression of the steroidogenic markers in hSMCs and corrispective tissue. The production of androgens was quantified in hSMCs medium by liquid chromatography tandem-mass spectrometry (LC-MS/MS). Results In vaginal tissues, AR mRNA was significantly less expressed than estrogen receptor α (ERα), whereas in hvSMCs, its mRNA expression was higher than progestin and both estrogen receptors. In hvSMCs and in vaginal tissue, when compared to ovaries, the mRNA expression of pro-androgenic steroidogenic enzymes (HSD3β1/β2, HSD17β3/β5), along with 5α-reductase isoforms and sulfo-transferase, resulted as being more abundant. In addition, enzymes involved in androgen inactivation were less expressed than in the ovaries. The LC-MS/MS analysis revealed that, in hvSMCs, short term DHEA supplementation increased Δ4-androstenedione levels in spent medium, while increasing testosterone (T) and dihydrotestosterone (DHT) secretion after longer incubation. Finally, androgenic signaling activation was evaluated through AR-dependent marker mRNA expression, after DHEA and T stimulation. Conclusions This study provides evidence for the presence of steroidogenic machinery in the human vagina, supporting, for the first time, the construct of intracrinology in this organ, emphasizing that the vagina is an androgen-dependent organ. These observations suggest the potential mechanism and resulting action of intravaginally administered DHEA, recently indicated for the treatment of GSM and open new perspectives for the treatment of this common and bothersome condition. Disclosure Work supported by industry: no.

Inherited ectopia lentis (EL) is most commonly caused by Marfan syndrome (MFS), a multisystemic disorder caused by mutations in FBN1. Historically the diagnosis for patients with EL who have no systemic features of MFS is isolated EL (IEL). However, the Ghent nosology for MFS was updated in 2010 and made some important alterations. In particular, patients with EL and a FBN1 mutation are now categorically diagnosed with MFS, if their mutation has previously been described with aortic dilation/dissection. This carries significant systemic implications, as many patients previously diagnosed with IEL are now reclassified. We provide a review of all published cases of IEL caused by FBN1 mutations over the last 20 years to assess what impact the new Ghent nosology has on these. Indeed, 57/123 probands (46.3%) are now classified as MFS according to the revised Ghent nosology and 37/96 mutations (38.5%) reported to cause isolated EL have also been found in patients with aortic dilation/dissection. These findings suggest that EL caused by mutations in FBN1 is actually part of a spectrum of fibrillinopathies with MFS, and the term 'IEL' should be avoided in such cases.

Background: Marfan syndrome (MFS), inherited as an autosomal dominant trait, typically affects the cardiovascular, skeletal, and ocular systems. Ectopia lentis (EL) is a clinical manifestation of MFS, with stretching or disruption of the lenticular zonular filaments, leading to displacement of the lenses. EL, with or without minor skeletal changes, exists as an independent autosomal dominant phenotype linked to the same FBN1 locus. Methods: A consecutive series of 11 patients, affected predominantly by EL, was analysed for FBN1 mutations using PCR, SSCA, and sequencing. Results: Six mutations were identified, of which three are novel and one is recurrent in two patients, thus establishing a mutation incidence in this group of 7/11 (63%). Conclusion: The FBN1 variants reported are clustered in the first 15 exons of the gene, while FBN1 mutations reported in the literature are distributed throughout the entire length of the gene. A different type of FBN1 mutation presents in this group of patients, compared with MFS, with arginine to cysteine substitutions appearing frequently.

BPH and LUTS have been associated to obesity, hypogonadism, and metabolic syndrome (MetS). MetS-induced prostate and bladder alterations, including inflammation and tissue remodeling, have been related to a low-testosterone and high-estrogen milieu. In addition to ERs, GPR30/GPER is able to mediate several estrogenic non-genomic actions.Supplementing a subgroup of MetS rabbits with tamoxifen, we analyzed the in vivo effects on MetS-induced prostate and bladder alterations. The effects of selective ER/GPER ligands and GPER silencing on prostate inflammation were also studied in vitro using hBPH cells.ERα, ERβ, and PR expression was upregulated in MetS bladder, where tamoxifen decreased ERα and PR expression, further stimulating ERβ. In addition, tamoxifen-dosing decreased MetS-induced overexpression of inflammatory and tissue remodeling genes. In prostate, sex steroid receptors, pro-inflammatory and pro-fibrotic genes were upregulated in MetS. However, tamoxifen did not affect them and even increased COX-2. In hBPH cells, 17β-estradiol increased IL-8 secretion, an effect blunted by co-treatment with GPER antagonist G15 but not by ER antagonist ICI 182,780, which further increased it. GPER agonist G1 dose-dependently (IC50 = 1.6 nM) induced IL-8 secretion. In vitro analysis demonstrated that GPER silencing reverted these stimulatory effects.GPER can be considered the main mediator of estrogen action in prostate, whereas in bladder the mechanism appears to rely on ERα, as indicated by in vivo experiments with tamoxifen dosing. Limiting the effects of the MetS-induced estrogen action via GPER could offer new perspectives in the management of BPH/LUTS, whereas tamoxifen dosing showed potential benefits in bladder.

Ectopia lentis (EL) is genetically heterogeneous with both autosomal-dominant and -recessive forms. The dominant disorder can be caused by mutations in FBN1, at the milder end of the type-1 fibrillinopathies spectrum. Recently in a consanguineous Jordanian family, recessive EL was mapped to locus 1q21 containing the ADAMTSL4 gene and a nonsense mutation was found in exon 11 (c.1785T>G, p.Y595X). In this study, 36 consecutive probands with EL who did not fulfill the Ghent criteria for MFS were screened for mutations in FBN1 and ADAMTSL4. Causative FBN1 mutations were identified in 23/36 (64%) of probands while homozygous or compound heterozygous ADAMTSL4 mutations were identified in 6/12 (50%) of the remaining probands. Where available, familial screening of these families confirmed the mutation co-segregated with the EL phenotype. This study confirms that homozygous mutations in ADAMTSL4 are associated with autosomal-recessive EL in British families. Furthermore; the first compound heterozygous mutation is described resulting in a PTC and a missense mutation in the PLAC (protease and lacunin) domain. The identification of a causative mutation in ADAMTSL4 may allow the exclusion of Marfan syndrome in these families and guide the clinical management, of particular relevance in young children affected by EL. © 2010 Wiley-Liss, Inc.

International audience; Mutations in the FBN1 gene cause Marfan syndrome (MFS) and have been associated with a wide range of milder overlapping phenotypes. A proportion of patients carrying a FBN1 mutation does not meet diagnostic criteria for MFS, and are diagnosed with "other type I fibrillinopathy." In order to better describe this entity, we analyzed a subgroup of 146 out of 689 adult propositi with incomplete "clinical" international criteria (Ghent nosology) from a large collaborative international study including 1,009 propositi with a pathogenic FBN1 mutation. We focused on patients with only one major clinical criterion, [including isolated ectopia lentis (EL; 12 patients), isolated ascending aortic dilatation (17 patients), and isolated major skeletal manifestations (1 patient)] or with no major criterion but only minor criteria in 1 or more organ systems (16 patients). At least one component of the Ghent nosology, insufficient alone to make a minor criterion, was found in the majority of patients with isolated ascending aortic dilatation and isolated EL. In patients with isolated EL, missense mutations involving a cysteine were predominant, mutations in exons 24-32 were underrepresented, and no mutations leading to a premature truncation were found. Studies of recurrent mutations and affected family members of propositi with only one major clinical criterion argue for a clinical continuum between such phenotypes and classical MFS. Using strict definitions, we conclude that patients with FBN1 mutation and only one major clinical criterion or with only minor clinical criteria of one or more organ system do exist but represent only 5% of the adult cohort.

International audience; Mutations in the FBN1 gene cause Marfan syndrome (MFS) and a wide range of overlapping phenotypes. The severe end of the spectrum is represented by neonatal MFS, the vast majority of probands carrying a mutation within exons 24-32. We previously showed that a mutation in exons 24-32 is predictive of a severe cardiovascular phenotype even in non-neonatal cases, and that mutations leading to premature truncation codons are under-represented in this region. To describe patients carrying a mutation in this so-called 'neonatal' region, we studied the clinical and molecular characteristics of 198 probands with a mutation in exons 24-32 from a series of 1013 probands with a FBN1 mutation (20%). When comparing patients with mutations leading to a premature termination codon (PTC) within exons 24-32 to patients with an in-frame mutation within the same region, a significantly higher probability of developing ectopia lentis and mitral insufficiency were found in the second group. Patients with a PTC within exons 24-32 rarely displayed a neonatal or severe MFS presentation. We also found a higher probability of neonatal presentations associated with exon 25 mutations, as well as a higher probability of cardiovascular manifestations. A high phenotypic heterogeneity could be described for recurrent mutations, ranging from neonatal to classical MFS phenotype. In conclusion, even if the exons 24-32 location appears as a major cause of the severity of the phenotype in patients with a mutation in this region, other factors such as the type of mutation or modifier genes might also be relevant.

From a large series of 1009 probands with pathogenic FBN1 mutations, data for 320 patients

International audience; BACKGROUND: The diagnosis of Marfan syndrome (MFS) is usually initially based on clinical criteria according to the number of major and minor systems affected following international nosology. The number of FBN1 mutation carriers, at risk of aortic complications who would not be properly diagnosed based only on clinical grounds, is of growing importance owing to the increased availability of molecular screening. The aim of the study was to identify patients who should be considered for FBN1 mutation screening. METHODS: Our international series included 1009 probands with a known FBN1 mutation. Patients were classified as either fulfilling or not fulfilling "clinical" criteria. In patients with unfulfilled "clinical" criteria, we evaluated the percentage of additional patients who became positive for international criteria when the FBN1 mutation was considered. The aortic risk was evaluated and compared in patients fulfilling or not fulfilling the "clinical" international criteria. RESULTS: Diagnosis of MFS was possible on clinical grounds in 79% of the adults, whereas 90% fulfilled the international criteria when including the FBN1 mutation. Corresponding figures for children were 56% and 85%, respectively. Aortic dilatation occurred later in adults with unfulfilled "clinical criteria" when compared to the Marfan syndrome group (44% vs 73% at 40 years, p

To determine whether the functional −174 G/C interleukin-6 gene polymorphism is a risk factor for the development of cystoid macular oedema (CMO) following routine uncomplicated phacoemulsification surgery in patients with no established risk factors. A total of 40 patients who underwent routine phacoemulsification surgery as part of a randomised controlled trial comparing the use of postoperative steroid drops against a single sub-tenon injection of triamcinolone were genotyped for the IL-6 −174G/C polymorphism. All patients underwent fluorescein angiography at 30 days and anterior chamber flare measurements pre-operatively and at day 1, 7, and 30. Angiographic CMO developed in 14 patients of the 40 studied. 9 out of the 14 patients carried the GG genotype (Fisher's exact test P=0.05, Hazard ratio for GG genotype; 4.05 (1.02–16.00)). There was no difference in flare measurements between the GG and Non-GG (GC/CC) group. The two groups were otherwise well matched in terms of age, sex, phacoemulsification energy used intraoperatively, and proportion of patients receiving postoperative triamcinolone or steroid drops. The −174G/C interleukin-6 promoter gene variant appears to modulate the response to phacoemulsification surgery and to influence the development of postoperative CMO. These data suggest a genetic predisposition to this complication.

Neonatal Marfan syndrome caused by an exon 25 mutation of the Fibrillin-1 gene: We describe a male infant with severe arachnodactyly, hypermobility of the fingers, flexion contractures of elbows, wrists, hips, and knees, microretrognathia, crumpled ears, rockerbottom feet, loose redundant skin, and lens dislocations. Cardiac valve insufficiency and aortic dilatation resulted in cardiac failure, decompensated with digitalisation and death occurred at the age of 4 months. This case represents the severe end of the clinical spectrum of Marfan syndrome, namely neonatal Marfan syndrome. Molecular diagnostic analyses confirmed a de novo exon 25 mutation in the FBN1 gene.

Fibrillin is the major component of extracellular microfibrils. Mutations in the fibrillin gene on chromosome 15 (FBN1) were first described in the heritable connective disorder, Marfan syndrome (MFS). FBN1 has also been shown to harbor mutations related to a spectrum of conditions phenotypically related to MFS, called "type-1 fibrillinopathies." In 1995, in an effort to standardize the information regarding these mutations and to facilitate their mutational analysis and identification of structure/function and phenotype/genotype relationships, we created a human FBN1 mutation database, UMD-FBN1. This database gives access to a software package that provides specific routines and optimized multicriteria research and sorting tools. For each mutation, information is provided at the gene, protein, and clinical levels. This tool is now a worldwide reference and is frequently used by teams working in the field; more than 220,000 interrogations have been made to it since January 1998. The database has recently been modified to follow the guidelines on mutation databases of the HUGO Mutation Database Initiative (MDI) and the Human Genome Variation Society (HGVS), including their approved mutation nomenclature. The current update shows 559 entries, of which 421 are novel. UMD-FBN1 is accessible at www.umd.be/. We have also recently developed a FBN1 polymorphism database in order to facilitate diagnostics.

Objective: To report a family with Marfan's syndrome in whom a myopathy was associated with respiratory failure; muscle biopsies from affected individuals were examined to determine whether there were abnormalities in fibrillin. Methods: 21 family members underwent detailed clinical examination, including neurological and pulmonary assessment. Muscle biopsies in the most severely affected cases were immunostained using monoclonal antibodies to specific fibrillin components. Genomic DNA from all 21 members was analysed for mutations in the fibrillin gene, FBN1, on 15q21. Results: 13 individuals had a C4621T base change in exon 37 of the FBN1 gene, which in four cases segregated with muscle weakness or evidence of respiratory muscle dysfunction or both. Their muscle biopsies revealed an abnormality in fibrillin immunoreactivity. Conclusions: Abnormalities in fibrillin can be detected in muscle biopsies from patients with Marfan's syndrome who have myopathy. This pedigree, with a point mutation in FBN1, also draws attention to the potential for respiratory failure associated with myopathy.

Marfan syndrome (MFS) is a multisystemic disease associated with mutations in the fibrillin-1 gene. Most of the reported mutations are missense substitutions mainly affecting the epidermal growth factor (EGF)-like protein domain structure and the calcium-binding (cb) site. The aim of our study was to investigate the correlation between fibrillin-1 frameshift mutations and the clinical phenotype in patients affected by MFS. In 48 out of 66 Marfan patients a pathogenetic mutation was found. We detected novel mutations causing premature termination codon in exons 19, 37, 40 and 41 of four Italian patients. The first mutation in exon 19 (cbEGF #8 domain) results in a clinical phenotype involving mainly the skeletal and cardiovascular systems. Interestingly, we noticed that, while mutations in exons 37 and 41 (eight cysteine domains #4 and #5) are milder, the mutation in exon 40 (cbEGF #24 domain) is more severe and causes major cardiovascular involvement with thoracic and abdominal aortic aneurysms. It is noteworthy that the degree of the severity in the phenotype of one of our patients and another from the literature carrying a mutation in exon 41 could be explained with alterations in mRNA expression.