A complex medical phenotype in a patient with triplication of 2q12.3 to 2q13 characterized with oligonucleotide array CGH.

We report an adult female with a left polycystic kidney, patent ductus arteriosus, left streak ovary, bicornuate uterus and deafness who presented with infertility. She has an intrachromosomal triplication of bands 2q12.3 to 2q13, with inversion of the central segment, which arose de novo from a paternal interchomosomal event. The triplication contains 68 known genes within the 7.28 Mb of DNA between base pairs 107,140,721 and 114,416,131. All intrachromosomal triplications are rare and, while partial duplications of 2q have been previously described, this patient is a unique surviving case of a triplication of proximal 2q.

X chromosome loss and ageing.

The detection of a low level 45,X cell line during routine cytogenetic analysis in an adult female can be difficult to interpret. In the absence of recent information regarding loss of the X chromosome and ageing, we undertook a prospective study. A total of 19,650 cells from 655 females aged from birth to 80 years were screened cytogenetically. The frequency of X chromosome loss ranged from 0.07% at age <16 years to 7.3% at >65 years of age and showed a highly significant quadratic relationship between X chromosome loss and ageing (P < or = 0.00001). We have produced a graphic representation that provides a minimum baseline age-related rate of X chromosome loss. This should assist diagnostic cytogenetics laboratories to determine the significance of 45,X cell lines detected in women of all ages. We also compared the frequency of 45,X cells in women referred with at least one spontaneous abortion with those referred for other reasons and found no significant difference. Thus, in our population, an excess of 45,X cells is not associated with pregnancy loss.

Subtelomeric rearrangements: results from a study of selected and unselected probands with idiopathic mental retardation and control individuals by using high-resolution G-banding and FISH.

The cause of mental retardation, present in approximately 3% of the population, is unexplained in the majority of cases. Recent reports have suggested that cryptic telomeric rearrangements resulting in segmental aneuploidy and gene-dosage imbalance might represent a significant cause of idiopathic mental retardation (IMR). Two groups of patients with unexplained developmental delay (unselected and selected) and a group of control individuals have been investigated to determine the frequency of submicroscopic telomeric rearrangements associated with IMR and the frequency within the normal population. In contrast to current thinking, our data have shown that true cryptic telomeric rearrangements are not a significant cause of IMR. No fully cryptic abnormalities were detected in our IMR groups, although a semi-cryptic unbalanced telomeric translocation was identified in one selected patient by high-resolution G-band analysis. This abnormality was confirmed and characterised by fluorescence in situ hybridisation (FISH) with telomere-specific probes. A further 13 cytogenetically detected subtle terminal rearrangements were characterised by using multi-telomere FISH. Seven of these had previously been reported as normal, three of which were shown to be interstitial deletions. These cases illustrate the importance of high-resolution analysis to exclude subtle but cytogenetically visible abnormalities prior to subtelomere FISH screening when determining the frequency of cryptic telomeric rearrangements. Unexpectedly, two cryptic telomeric abnormalities were detected among our control individuals, suggesting that submicroscopic telomeric abnormalities may be a not uncommon finding in the general population. Hence, our data have important implications when defining the significance of cryptic telomeric rearrangements detected during screening programmes.

Receptor binding studies of soft anticholinergic agents.

Receptor binding studies were performed on 24 soft anticholinergic agents and 5 conventional anticholinergic agents using 4 cloned human muscarinic receptor subtypes. The measured pK(i) values of the soft anticholinergic agents ranged from 6.5 to 9.5, with the majority being in the range of 7.5 to 8.5. Strong correlation was observed between the pK(i) s determined here and the pA 2 values measured earlier in guinea pig ileum contraction assays. The corresponding correlation coefficients (r2) were 0.80, 0.73, 0.81, and 0.78 for pK(i) (m1), pK(i) (m2), pK(i) (m3), and pK(i) (m4), respectively. Quantitative structure-activity relationship (QSAR) studies were also performed, and good characterization could be obtained for the soft anticholinergics containing at least 1 tropine moiety in their structure. For these compounds, the potency as measured by the pK i values was found to be related to geometric, electronic, and lipophilicity descriptors. A linear regression equation using ovality (O(e)), dipole moment (D), and a calculated log octanol-water partition coefficient (QLogP) gave reasonably good descriptions (r = 0.88) for the pK(i) (m3) values.

The phenotypic manifestations of interstitial duplications of proximal 15q with special reference to the autistic spectrum disorders.

This study investigated the phenotypic manifestations of interstitial duplications of chromosome 15 that involve the Prader-Willi/Angelman syndrome critical region (PWACR). Twenty-one affected individuals from six families were evaluated in detail, using standardized and semi-standardized measures of intelligence, psychopathology, and physical anomalies. Special attention was placed on determining the prevalence of autism spectrum disorders as well as the relationship between the parental origin of the duplication and the phenotypic effects. Assessments of the affected individuals were compared with evaluations of the unaffected relatives from the same families. Results indicated that duplications in the region were associated with variable degrees of intellectual impairments and motor coordination problems. Four of the subjects received a diagnosis of pervasive developmental disorder. Three of these cases were probands and only one met criteria for classic autism. There was very little evidence of the duplication cosegregating with autism spectrum disorder diagnosis. Paternally inherited duplications were significantly less likely to give rise to phenotypic effects. The findings indicate that duplications in the PWACR give rise to developmental delay but not necessarily autism spectrum disorders. They also suggest that phenotypic expression is dependent on the parental origin of the duplication and implicate maternally active genes in the pathogenesis of the developmental impairments. Further research will be required to clarify the range and basis of the phenotypic manifestations.

Adenosine kinase inhibitors. 1. Synthesis, enzyme inhibition, and antiseizure activity of 5-iodotubercidin analogues.

Adenosine receptor agonists produce a wide variety of therapeutically useful pharmacologies. However, to date they have failed to undergo successful clinical development due to dose-limiting side effects. Adenosine kinase inhibitors (AKIs) represent an alternative strategy, since AKIs may raise local adenosine levels in a more site- and event-specific manner and thereby elicit the desired pharmacology with a greater therapeutic window. Starting with 5-iodotubercidin (IC50 = 0.026 microM) and 5'-amino-5'-deoxyadenosine (IC50 = 0.17 microM) as lead inhibitors of the isolated human AK, a variety of pyrrolo[2,3-d]pyrimidine nucleoside analogues were designed and prepared by coupling 5-substituted-4-chloropyrrolo[2,3-d]pyrimidine bases with ribose analogues using the sodium salt-mediated glycosylation procedure. 5'-Amino-5'-deoxy analogues of 5-bromo- and 5-iodotubercidins were found to be the most potent AKIs reported to date (IC50S < 0.001 microM). Several potent AKIs were shown to exhibit anticonvulsant activity in the rat maximal electric shock (MES) induced seizure assay.

Soft cannabinoid analogues as potential anti-glaucoma agents.

Cannabinoids have intraocular pressure (IOP) lowering effects, thus, they have a therapeutic potential in the treatment of glaucoma. Unfortunately, in the same time, they show CNS and cardiovascular effects as well. Our aim was to develop a safer, cannabinoid type anti-glaucoma agent, a topically applied soft analogue, that has local, but no systemic effect. The lead compound chosen was a nitrogen-containing cannabinoid analogue that was shown to have IOP lowering activity. A full library of possible soft drugs was generated and the structures were ranked based on the closeness of calculated properties to those of the lead compound. The lead compound has been synthesized, and a preliminary pharmacological study was performed. The structure-activity relationship and pharmacological results indicate a good possibility for the development of a safe, soft anti-glaucoma agent.

Investigation of a cryptic interstitial duplication involving the Prader-Willi/Angelman syndrome critical region.

A 3-year-old female referred with developmental delay, hypotonia and seizures was found to have a cryptic interstitial duplication of the Prader-Willi/Angelman critical region (PWACR). Her clinical features form part of a common phenotype characteristic of PWACR duplications including developmental delay, behavioural problems and speech difficulties. Microsatellite analysis showed that the duplication had arisen de novo, was maternal in origin and involved the entire 4-Mb PWACR between the common deletion breakpoints. The existence of cryptic rearrangements emphasises the need for molecular tests alongside conventional cytogenetics when investigating abnormalities involving this imprinted region.

Xp deletions associated with autism in three females.

We report eight females with small deletions of the short arm of the X chromosome, three of whom showed features of autism. Our results suggest that there may be a critical region for autism in females with Xp deletions between the pseudoautosomal boundary and DXS7103. We hypothesise that this effect might be due either to the loss of function of a specific gene within the deleted region or to functional nullisomy resulting from X inactivation of the normal X chromosome.

Neurofibromatosis pseudogene amplification underlies euchromatic cytogenetic duplications and triplications of proximal 15q.

Cytogenetically visible interstitial duplications of proximal 15q, which lack the Prader-Willi Angelman critical region (PWACR) frequently segregate in families without phenotypic effect, but the nature of the extra euchromatin has remained unclear. We used comparative genome hybridisation to confirm that the extra material in a cytogenetic triplication originated from proximal 15q. A PAC clone containing sequences specific for the type-1 neurofibromatosis (NF-1) pseudogenes, which map to 15q11.2, hybridised along the length of the enlarged region between the PWACR and the centromere. Computerised measurement of the fluorescent signal from the enlarged and normal chromosomes gave an average ratio of 9.85:1, consistent with amplification. In a second family, an amplified P1-4 signal co-segregated with a cytogenetic duplication and the average ratio between amplified and normal signals in the proband was 8.22:1. Ratios in noncarrier family members and control individuals were close to unity in most cases, but significantly greater than one in at least one instance. Our results provide a novel explanation for cytogenetic variation in 15q11.2. They also suggest that NF-1 pseudogene copy number may be polymorphic in the normal population, and that high copy numbers can produce G bands which do not reflect those of the normal constitutional karyotype.

Inherited interstitial duplications of proximal 15q: genotype-phenotype correlations.

We present the cytogenetic, molecular cytogenetic, and molecular genetic results on 20 unrelated patients with an interstitial duplication of the proximal long arm of chromosome 15. Multiple probes showed that the Prader-Willi/Angelman critical region (PWACR) was included in the duplication in 4/20 patients, each ascertained with developmental delay. The duplication was also found in two affected but not in three unaffected sibs of one of these patients. All four probands had inherited their duplication from their mothers, three of whom were also affected. Two of the affected mothers also carried a maternally inherited duplication, whereas the duplication in the unaffected mother and in an unaffected grandmother was paternal in origin, raising the possibility of a parental-origin effect. The PWACR was not duplicated in the remaining 16 patients, of whom 4 were referred with developmental delay. In the 14 families for which parental samples were available, the duplication was inherited with equal frequency from a phenotypically normal parent, mother or father. Comparative genomic hybridization undertaken on two patients suggested that proximal 15q outside the PWACR was the origin of the duplicated material. The use of PWACR probes discriminates between a large group of duplications of no apparent clinical significance and a smaller group, in which a maternally derived PWACR duplication is consistently associated with developmental delay and speech difficulties but not with overt features of either Prader-Willi syndrome or Angelman syndrome.

Small terminal deletions of the long arm of chromosome 2: two new cases.

We report on 2 girls with small de novo terminal deletions of the long arm of chromosome 2 and breakpoints within q37. Four cases with similar or more extensive deletions have been previously reported in full. Hypotonia and psychomotor retardation were the only manifestations common to all 6 cases. The phenotype associated with small terminal 2q deletions is variable and clearly not always as mild as indicated in previous reports. The abnormality may also be more common than has been assumed.

Adenosinergic modulation of homocysteine-induced seizures in mice.

Homocysteine thiolactone (HTL) elicits seizures in mice at a dose of 850 mg/kg (95-100% of animals) with an average latency time of 19.5 min. These seizures are reversed by both 5' N-ethylcarboximide adenosine (NECA) and flunitrazepam, with respective ED50 doses of 0.025 and 0.20 mg/kg. NECA was approximately four-fold more potent as an inhibitor of HTL-induced seizures than of seizures induced by pentylenetetrazol (PTZ, 75 mg/kg). Flunitrazepam was equipotent in both seizure paradigms. The purine precursor 5-amino-4-imidazole carboxamide riboside, (AICAr), although virtually ineffective against PTZ-induced seizures at doses greater than 1 g/kg, was able to inhibit HTL-induced seizures with an ED50 of approximately 350 mg/kg. The anticonvulsant effect of AICAr was dose and time dependent. The anticonvulsant potency of AICAr was increased by simultaneous administration of the adenosine uptake blocker Mioflazine, whereas the central nervous system (CNS)-impermeable adenosine uptake blocker dipyridamole had no effect. The ability of AICAr to permeate the blood-brain barrier (BBB) is limited (less than 1%) and may explain its low potency as an anticonvulsant. AICAr also has very low potency at brain adenosine A1 and A2 receptors as well as adenosine uptake sites (IC50 greater than 10(-3) M), suggesting that its anticonvulsant properties are not mediated by direct action at these sites. The results indicate that AICAr does have frank anticonvulsant effects and further suggest that HTL-induced seizures may represent a useful paradigm for evaluation of adenosinergic agents. AICAr or more potent derivatives thereof may represent a new class of anticonvulsants with the ability to target seizure foci selectively.

Synthesis and antifertility activity of 3,9-dihydroxy-5,6,6a alpha,6b beta,11,12,12a beta,12b alpha-octahydrodibenzo[a,g]biphenylene, a structural relative of diethylstilbestrol.

The title diphenol, 1a, was synthesized from p,p'-dihydroxy-alpha-truxillic acid and shown to be active as an oral postcoital antifertility agent in rats: ED100 = 100 (micrograms/kg)/day. The oral uterotropic potency was estimated to be 16% of that of diethylstilbestrol (95% confidence limits of potency 8--35%). The structure of the diphenol, 1a, was confirmed by single-crystal X-ray analysis of the dimethyl ether.