Novel pericentric inversion inv(9)(p23q22.3) in unrelated individuals with fertility problems in the Southeast European population.

Pericentric inversions are among the known polymorphisms detected in the general population at a frequency of 1-2%. Despite their generally benign nature, pericentric inversions affect the reproductive potential of carriers by increasing the risk for unbalanced live-born offspring, miscarriages, or other fertility problems. Here we present a novel large pericentric inversion of chromosome 9, inv(9)(p23q22.3), detected in 30 heterozygote carriers, 24 from seven apparently unrelated families and 6 isolated patients, where the probands were mainly referred for fertility and prenatal problems. The inversion carries a significant risk for recombinant abnormal chromosomes, as in two families one supernumerary rec(9)dup(9p) and one rec(9)dup(9q) were identified, leading to neonatal death and miscarriage, respectively. The inversion carriers were identified by three different laboratories in Greece, Cyprus and Germany respectively, however all carriers have Southeast European origin. The inversion appears to be more frequent in the Greek population, as the majority of the carriers were identified in Greece. We were able to determine that the inversion is identical in all individuals included in the study by applying a combination of several methodologies, such as karyotype, fluorescence in situ hybridization (FISH), chromosomal microarrays (CMA) and haplotype analysis. In addition, haplotype analysis supports that the present inversion is identical by descent (IBD) inherited from a single common ancestor. Our results are, therefore, highly indicative of a founder effect of this inversion, presumably reflecting an event that was present in a small number of individuals that migrated to the current Southeast Europe/Northern Greece from a larger population.

A de novo 2.9 Mb interstitial deletion at 13q12.11 in a child with developmental delay accompanied by mild dysmorphic characteristics.

BACKGROUND: Proximal deletions in the 13q12.11 region are very rare. Much larger deletions including this region have been described and are associated with complex phenotypes of mental retardation, developmental delay and various others anomalies. RESULTS: We report on a 3-year-old girl with a rare 2.9 Mb interstitial deletion at 13q12.11 due to a de novo unbalanced t(13;14) translocation. She had mild mental retardation and relatively mild dysmorphic features such as microcephaly, flat nasal bridge, moderate micrognathia and clinodactyly of 5(th) finger. Molecular karyotyping revealed a deletion on the long arm of chromosome 13 as involving sub-bands 13q12.11, a deletion of about 2.9 Mb. DISCUSSION: The clinical application of array-CGH has made it possible to detect submicroscopical genomic rearrangements that are associated with varying phenotypes.The description of more patients with deletions of the 13q12.11 region will allow a more precise genotype-phenotype correlation.

Characterization of a prenatally assessed de novo supernumerary minute ring chromosome 20 in a phenotypically normal male.

BACKGROUND: The heterogeneous group of small supernumerary marker chromosomes (sSMCs) presents serious counseling problems, especially if they are present de novo and diagnosed prenatally. The incidence has been estimated at 1 in 1000 prenatal samples. We present a case of mosaic sSMC diagnosed prenatally after amniocentesis. The sSMC was characterized by various molecular cytogenetic techniques and determined to be a r(20) chromosome. After genetic counseling, the parents decided to continue the pregnancy, and a boy with minor phenotypic variants was born after 39 weeks of pregnancy. The case is compared with four other cases of prenatally detected r(20) mosaicism. RESULTS: Here we describe a 3 months old male child with normal pre- and postnatal development and with a de novo ring supernumerary marker chromosome in amniocytes cultures. Using new fluorescence in situ hybridization (FISH) techniques, three distinguishable sSMCs (cryptic mosaicism), all derived from chromosome 20, were observed, including ring and minute chromosomes. This heterogeneity was impossible to detect by the conventional G-banding technique or conventional FISH technique that were used before the application of new FISH techniques (subcentromere-specific multicolor-FISH [subcenM-FISH]) and a probe, specific for the 20p12.2 band. The sSMC present in 25% of the cells was present as r(20)(::p12.2~12.3->q11.1::)5/r(20;20)(::p12.1->q11.1::q11.1 >p12.1::)2/min(20;20)(:p12.1->q11.1::q11.1->p12.1:)1. The final karyotype was 47,XY,+r(20)[25%]/46,XY[75%]. CONCLUSION: We emphasize the importance of application of molecular cytogenetics in a prenatally diagnostic laboratory and description of more cases to enable a better genetic counseling and risk evaluation.

Interstitial cells and phasic activity in the isolated mouse bladder.

OBJECTIVE: To describe the distribution of interstitial cells (ICs, defined as cells which show an increase in cGMP in response to nitric oxide, NO) in the isolated mouse bladder, and changes in phasic contractile activity after exposure to a NO donor. MATERIALS AND METHODS: The whole bladder was removed from 17 female mice, killed by cervical dislocation. For immunohistochemistry (six mice) the bladder was incubated in carboxygenated Krebs' solution at 36 degrees C, containing 1 mm of the phosphodiesterase inhibitor isobutyl-methyl-xanthine. Individual pieces of tissue were exposed to 100 microm of the NO donor diethylamine NONOate for 10 min; control tissues remained in Krebs' solution. Tissues were then fixed in 4% paraformaldehyde and processed for cGMP immunohistochemistry. Bladder pressure was measured in bladders from 11 mice; the bladders were cannulated via the urethra and suspended in a heated chamber containing carboxygenated Tyrode solution at 33-35 degrees C and intravesical pressure recorded. All drugs were added to the solution bathing the abluminal surface. RESULTS: NO induced an increase in cGMP in cells in the outer layers of the bladder wall, forming two distinct types based on their location; cells lying on the surface of the muscle bundles (surface muscle ICs) and cells within the muscle bundles (intramuscular ICs). Cholinergic nerve fibres were identified by the expression of vesicular acetylcholine transporter and neuronal NO synthase (nNOS). Choline acetyltransferase- and nNOS-positive nerves also had high cGMP levels in response to 100 microm diethylamine NONOate. In vitro exposure of an isolated whole unstimulated bladder to 100 microm diethylamine NONOate had no effect on resting bladder pressure. When whole bladders were exposed to muscarinic stimulation (30-100 nm arecaidine) there was an initial large transient rise in pressure followed by complex phasic changes in pressure. Adding 100 microm diethylamine NONOate abolished this phasic activity. Interestingly, the phasic activity was inhibited midway between the peak and trough of a phasic cycle. Such a pattern of inhibition might reflect the complexity of the phasic activity involving both excitatory and inhibitory components. CONCLUSIONS: These data show the presence of NO/cGMP-sensitive ICs in the outer muscle layers of the mouse bladder. Activating these cells alters the pattern of muscarinic-induced phasic activity. We suggest that the role of the ICs in the outer muscle layers is to generate and modulate phasic activity. If so, then this is the first report of a functional role for ICs in the bladder.

Location of interstitial cells and neurotransmitters in the mouse bladder.

INTRODUCTION: To investigate whether interstitial cells (ICs) are present in the adult mouse bladder, and what transmitters characterize adjacent nerve fibres, as ICs in human and guinea-pig bladder lie close to nerve fibres but transmitters present in these nerves have not yet been reported. MATERIALS AND METHODS: Sections of the bladder wall from 12 adult male mice (six each, aged 3-4 or 18-24 months) were incubated in carboxygenated Krebs' solution containing isobutyl-methyl-xanthene (1 mm), followed by the nitric oxide (NO) donor diethylamino-NONOate; control tissues remained in Krebs' solution. Samples were fixed in 4% paraformaldehyde and processed for immunofluorescence histochemistry for cGMP, neuronal NO synthase (nNOS), vesicular acetylcholine transferase (VAChT), calcitonin gene-related polypeptide (CGRP) and protein gene product (PGP) 9.5. ICs were identified as non-neuronal cells of appropriate morphology manifesting an increase in cGMP after exposure to the NO donor. RESULTS: ICs were apparent in the outer muscle, but not the inner muscle or suburothelial region. nNOS- and CGRP-immunoreactive fibres were close to and alongside IC processes. In contrast, nerve fibres containing VAChT were only occasionally found close to ICs and rarely running alongside them. ICs showed no immunoreactivity to c-kit. There was no overt difference in IC cell distribution between young and aged adult specimens. Older mice showed patchy denervation of the detrusor, but ICs were not specifically affected. CONCLUSIONS: ICs are confined to the outer part of the bladder wall in the mouse and may receive peptidergic and nitrergic innervation, which might serve to modulate their putative functional role. Alterations in the overall IC population do not appear to underlie ageing-related changes in lower urinary tract function.

Bladder volume alters cholinergic responses of the isolated whole mouse bladder.

PURPOSE: The isolated bladder expresses autonomous activity, which may contribute to the generation of lower urinary tract sensation or pathophysiology. We evaluated how the effect of a cholinergic agonist on autonomous activity alters with increasing volume and in the presence of substances known to modulate functional bladder capacity. MATERIALS AND METHODS: The bladder of 22 adult female C57 black mice were mounted in whole organ tissue baths. Recordings of intravesical pressure were performed under standardized conditions at different bladder volumes. RESULTS: At low volume the muscarinic agonist arecaidine elicited an initial peak response, which subsided to a sustained steady state pressure. At high volume phasic pressure fluctuations were also apparent. An M2-receptor antagonist caused a significantly greater decrease in peak and steady state responses than in pressure fluctuations. An M3-receptor antagonist decreased all 3 components. Alpha, beta-methylene adenosine triphosphate markedly decreased fluctuations, in contrast to norepinephrine, which eliminated the steady state response while preserving fluctuations. CONCLUSIONS: The response to cholinergic stimulation of the isolated bladder has 3 components. The initial tonic peak response increases with bladder distention and it is inhibited by M2 and M3 muscarinic receptor antagonists. The tonic steady state response does not vary with bladder volume and it is inhibited by M2 and M3-receptor antagonists, and by beta3-adrenergic receptor agonists. Phasic fluctuations are minimal at low bladder volume, and with alpha, beta-methylene adenosine triphosphate or an M3-receptor antagonist. Thus, the response to cholinergic stimulation varies with bladder volume. It can be differentially modulated by muscarinic antagonists and also by agents acting through nonmuscarinic receptors.

Volume-induced effects on the isolated bladder: a possible local reflex.

OBJECTIVES: To: (i) determine the effects of changing intravesical volume on autonomous activity in the isolated whole bladder of the guinea pig; (ii) identify the mechanisms which might contribute to induced changes; and (iii) explore the idea that changes in bladder volume which affect phasic activity are part of a local reflex operating within the bladder wall. MATERIALS AND METHODS: Bladders were isolated from female guinea pigs, cannulated via the urethra and maintained in vitro in Tyrode's solution. The intravesical pressure (IVP) was monitored and drugs added to the bathing solution. RESULTS: The isolated unstimulated bladder containing 500-600 microL of fluid generates small (1-2 cm H2O) phasic rises in IVP, i.e. autonomous activity. When the bladder volume was increased, autonomous activity increased. In the presence of muscarinic agonists (100 nmol/L arecaidine and carbachol 100 nmol/L) autonomous activity is augmented, giving rise to large (>10 cm H2O) phasic rises in IVP. When the volume was increased, both the amplitude and frequency of the transients increased. When the bladder volume was reduced there was a period of marked inhibition of phasic activity. To explore the mechanisms underlying these changes the possible involvement of local neural reflexes was explored. The neurotoxin tetrodotoxin had no effect on the volume-induced changes. Sensory nerves are insensitive to tetrodotoxin and thus to assess their possible contribution bladders were exposed to capsaicin (10 micromol/L) to stimulate and eliminate sensory fibres; capsaicin caused complex changes in phasic activity, i.e. an initial increase, a secondary slowing and decrease, followed by a period of recovering amplitude and increased frequency. These changes suggest actions of the sensory nerves on the phasic mechanism indicative of a local axonal reflex. Once the phasic activity had returned to levels before capsaicin, changes in bladder volume still produced increases in activity and inhibition after the volume decrease. Interstitial cells (cells capable of increasing cGMP) are found in the bladder wall; to assess their possible role in the volume-induced changes, bladders were treated with 30 micromol/L ODQ, an inhibitor of guanyl cyclase, for 30-60 min. The volume-induced rise in frequency was little affected but the inhibition seen on volume reduction was reduced. CONCLUSIONS: These results show that there are components in the bladder wall which respond to distension by affecting phasic activity. This stimulus/response may reflect a volume 'reflex' within the bladder wall, consisting of excitatory and inhibitory components. This local reflex does not appear to involve directly motor or sensory nerves, although the latter can affect phasic activity, and their actions may represent a further reflex mechanism in the bladder wall. The possible involvement of guanyl cyclase in the volume-induced inhibition may indicate a role for interstitial cells. The physiological role of these mechanisms as a component of a motor/sensor system in the bladder wall is discussed.