Reciprocal effect of Waardenburg syndrome mutations on DNA binding by the Pax-3 paired domain and homeodomain

The Pax-3 protein contains two DNA-binding domains, a paired domain and a homeodomain. Mutations in Pax-3 cause Waardenburg syndrome (WS) in humans and the mouse Splotch (Sp) phenotype. In the Sp-delayed mouse, a mutation in the Pax-3 paired domain (G9R) abrogates the DNA-binding activity of both the paired domain and the homeodomain, suggesting that they may functionally interact. To investigate this possibility further, we have analyzed the DNA-binding properties of additional point mutants in the Pax-3 paired domain and homeodomain that occur in WS patients (F12L, N14H, G15S, P17L, R23L, G48A, S51F and G66D in the paired domain, V47F and R53G in the homeodomain), the Pax-1 un mutation (G15A) and a substitution associated with Peters' anomaly in the PAX-6 gene (R23G). Within the paired domain, seven of 10 mutations were found to abrogate DNA-binding by the paired domain. Remarkably, these seven mutations also affected DNA binding by the homeodomain, causing either a complete loss (P17L and G66D), a reduction (R23G, R23L, G15S and G15A) or an increase in DNA-binding activity (N14H). In addition, the effect of paired domain mutations occurred at the level of monomer formation by the homeodomain, while the dimerization potential of this domain seemed unaffected in mutants where it could be analyzed. Furthermore, while both homeodomain mutations were found to abolish DNA binding by this domain, the R53G mutation also abrogated DNA binding by the paired domain. The important observation that independent mutations in either domain can affect DNA binding by the other in the intact Pax- 3 protein strongly suggests that the two domains are not functionally independent but bind DNA through cooperative interactions. Modeling the deleterlous mutations on the three-dimensional structure of the paired domain of Drosophila Prd shows that these mutations cluster at the DNA interface, thus suggesting that a series of DNA contacts are essential for DNA binding by both the paired domain and the homeodomain of Pax-3.      

Identification and characterization of aquaporin-2 water channel mutations causing nephrogenic diabetes insipidus with partial vasopressin response

Congenital nephrogenic diabetes insipidus (NDI) is a rare disease caused most often by mutations in the vasopressin V2 receptor (AVPR2). We studied a family which included a female patient with NDI with symptoms dating from infancy. The patient responded to large doses of desmopressin (dDAVP) which decreased urine volume from 10 to 4 I/day. Neither the parents nor the three sisters were polyuric. The patient was found to be a compound heterozygote for two novel recessive point mutations in the aquaporin-2 (AQP2) gene: L22V in exon 1 and C181W in exon 3. Residue Cys181 in AQP2 is the site for inhibition of water permeation by mercurial compounds and is located near to the NPA motif conserved in all aquaporins. Osmotic water permeability (Pf) in Xenopus oocytes injected with cRNA encoding C181W-AQP2 was not increased over water control, while expression of L22V cRNA increased the Pf to approximately 60% of that for wild-type AQP2. Co-injection of the mutant cRNAs with the wild-type cRNA did not affect the function of the wild-type AQP2. Immunolocalization of AQP2-transfected CHO cells showed that the C181W mutant had an endoplasmic reticulum-like intracellular distribution, whereas L22V and wild-type AQP2 showed endosome and plasma membrane staining. Water permeability assays showed a high Pf in cells expressing wild-type and L22V AQP2. This study indicates that AQP2 mutations can confer partially responsive NDI.      

Prognostic factors for the success rate of embryo freezing

To find some prognostic factors for the outcome of frozen-thawed cycles, we have retrospectively analysed all frozen pre-embryos that were thawed during 1993 and 1994 at two in-vitro fertilization (IVF) units in Sweden. Supernumerary pre-embryos were frozen from 551 oocyte retrievals and these resulted in 660 frozen-thawed cycles which lead to 623 thawed embryo transfers. The outcome of these transfers was 137 clinical pregnancies with a pregnancy rate of 22% per frozen-thawed embryo transfers. Women <40 years of age had a higher birth rate than those > or =40 years, 19 and 5% respectively (P < 0.01). Transfers with two and three pre-embryos resulted in pregnancy rates of 23 and 27%, respectively, compared with 14% for transfer of one embryo. A pregnancy resulting from the initial embryo transfers had a predictive value for results of the subsequent frozen-thawed cycle. Embryo grade and cleavage stage at the time of freezing was important for the survival of the frozen-thawed pre-embryos. The pregnancy rate was not influenced by the cleavage stage, but a tendency toward a lower pregnancy rate was seen for the embryos with lower grading. To conclude, cryopreservation seems to be beneficial in women <40 years of age, who have supernumerary pre-embryos of good quality for freezing and of which at least two can be transferred.      

Significance of subcentimetre 18F-FDG PET/CT pulmonary abnormality in patients with known extrapulmonary malignancy

The significance of a subcentimetre ¹⁸F-FDG PET/CT pulmonary abnormality in a patient with known extrapulmonary primary malignancy can have a major impact on the clinical management of the patient. The clinician’s reliance on the semi-quantitative and qualitative PET/CT analysis of the abnormality has, at times, led to untoward diagnostic problems, given the limited spatial resolution of PET for a small volume lesion performed as part of the standard PET/CT study. This paper highlights a case each of an FDG-positive and an FDG-negative focal pulmonary abnormality in a combined PET/CT study of patients with known extrapulmonary malignancy. © 2010 Biomedical Imaging and Intervention Journal. All rights reserved.     

A pilot methodology study for the photographic assessment of post‐inflammatory hyperpigmentation in patients treated with tretinoin

Background Post‐inflammatory hyperpigmentation (PIH) is a common occurrence in patients with acne vulgaris, particularly in those with skin of colour. Aims A previous study has demonstrated the benefit of tretinoin (retinoic acid) in the treatment of PIH; however, there is currently no standard protocol to evaluate change in PIH following treatment. Based on these findings, we performed a pilot, exploratory, blinded, intraindividual‐controlled methodology study that consisted of a photographic assessment protocol with facial mapping. Materials and methods The study was based on a secondary analysis of a phase 4, community‐based trial of 544 acne patients who were treated with tretinoin gel microsphere 0.04% or 0.1%. Only patients with Fitzpatrick types III–V (skin of colour) were included in the study; subjects with Fitzpatrick skin type VI were excluded because the photographic assessment did not allow for proper evaluation. Results Despite the small number of subjects evaluated (n = 25), the results revealed consistent assessment of improvement in PIH between two independent graders (weighted κ = 0.84). Conclusion Further study with a larger population is recommended to validate the accuracy of this method.     

Ossicular erosions in the dry ear: CT diagnosis

Erosions of the ossicular chain that occurred as a complication of noncholesteatomatous chronic otitis media were studied with computed tomography (CT) in 55 patients. The incus (particularly the long and lenticular processes) was the ossicle most commonly involved (50 cases). Coronal and axial CT sections were complementary in the diagnosis of these erosions. Fibrous replacement of the incudostapedial articulation was diagnosed in four cases on axial CT scans when an unusually wide joint was present.